US20060183196A1

US20060183196A1 - Polynucleotide encoding a novel cysteine protease of the calpain superfamily, CAN-12, and variants thereof

Info

Publication number: US20060183196A1
Application number: US11/407,134
Authority: US
Inventors: Jian Chen; John Feder; Thomas Nelson; Steven Seiler; Roy Vaz; Franck Duclos
Original assignee: Individual
Current assignee: Individual
Priority date: 2001-04-03
Filing date: 2006-04-19
Publication date: 2006-08-17
Also published as: AU2002303218A1; WO2002088303A3; EP1567642A2; AU2002303218A8; EP1567642A4; WO2002088303A2; US7186564B2; CA2443393A1; US20030114373A1

Abstract

The present invention provides novel polynucleotides encoding CAN-12 polypeptides, fragments and homologues thereof. The present invention also provides polynucleotides encoding variants of CAN-12 polypeptides, CAN-12v1 and CAN-12v2. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel CAN-12, CAN-12v1, and CAN-12v2 polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides, particularly neuro- and musculo-degenerative conditions. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.

Description

This application claims benefit to provisional application U.S. Ser. No. 60/281,253 filed Apr. 3, 2001; to provisional application U.S. Ser. No. 60/288,768, filed May 4, 2001; to provisional application U.S. Ser. No. 60/296,180, filed Jun. 6, 2001; to provisional application U.S. Ser. No. 60/300,620, filed Jun. 25, 2001. The entire teachings of the referenced applications are incorporated herein by reference.

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Cysteine or thiol proteases contain a reactive sulphydral moiety activated by an adjacent histidine. Hydrolysis of the substrates peptide bond is initiated when the cysteine sulfur attacks the carbon in the peptide bond forming a thiol-enzyme intermediate, liberating the amino portion of the peptide. The thiol-enzyme intermediate is hydrolyzed by water releasing the substrates C-terminus and restoring the enzyme. There are over 20 some families of cysteine proteases. [Rawlings N. D., & Barrett A. J. Families of cysteine peptidases. Methods in Enzymol. 244 461-486 (1994)]. The present invention relates to a thiol protease of the C2 family that includes the calpain superfamily.
Calpains are calcium-activated intracellular neutral cysteine proteases (EC 3.4.22.17) (for reviews see Sorimachi et al., Structure and physiological function of calpains. Biochem J. 328:721-32, 1997; Carafoli E and Molinari M. Calpain: a protease in search of a function? Biochem Biophys Res Commun 247:193-203, 1998). Some calpains are expressed ubiquitously while others are tissue-specific. μ-Calpain and m-calpains appear in all tissues, p94 is skeletal muscle specific while nCL-2 is stomach specific. (Sorimachi et al., Structure and physiological function of calpains. Biochem J. 328:721-32, 1997). The best characterized are μ-calpain and m-calpains which consist of two subunits. An 80 kDa large subunit contains both Ca²⁺ binding sites and the catalytic activity and small 30 kDa subunit with a separate set of Ca²⁺ binding sites. All the proteolytic activity is contained in the larger subunit of both μ- and m-calpain. In the presence of PEG or chaperones the large subunit is catalytically activated in the absence of the smaller subunit. Other calpains, for example nCL-2 and p94, are proteolytically active monomers with homology to the μ-calpain and m-calpains large subunit.
The large (catalytic) subunit has four domains (I-IV)(Hosfield et al., Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation. EMBO J 18:6880-9, 1999; Strobl et al., The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium. Proc Natl Acad Sci USA. 97:588-92, 2000). The N-terminus (domain I) contains an alpha helical region and a site of autocatalytic cleavage. Domain II contains the catalytically active domain with the active site amino acids (m-calpain residues Cys105, His262, & Asn286). Domain III contains the linker between the Ca²⁺ binding domain (in domain IV) and links Ca²⁺ binding to proteolytic activity. Domain IV contains a calmodulin-like Ca²⁺ binding regions with EF hands. p94 (also called calpain 3) is similarly organized with domains I-IV, but, also contains a proline-rich N-terminus and two unique insertion loops (IS1 and IS2). nCL-2 is also active as a large monomer with domains I-IV; however, a splice variant (nCL-2′) lacks domains III & IV, but maintains proteolytic activity.
Calpains are responsible for limited intracellular proteolytic cleavage, as opposed to complete proteolytic digestion. The proteolysis modifies protein function both specifically and irreversibly. Numerous proteins have been identified as calpain substrates (Carafoli E and Molinari M Calpain: a protease in search of a function? Biochem Biophys Res Commun 247:193-203, 1998; Hayes et al., Drug News Perspect 11:215-222, 1998). The best-characterized substrates are large cytostructural and/or membrane associated proteins, calmodulin-binding proteins and transcriptional factors. Physiologically significant substrates for calpain include kinases, phosphatases, channel proteins and cytoskeletal proteins that link transmembrane receptors to the membrane skeleton. Proteolytic modification of these proteins may have fundamental roles in development, differentiation, and cellular transformation in response to cell signaling, cell-cell and/or cell-extracellular matrix interactions. In platelets, calpain activation appears to be linked to clustering of the integrin receptor aIIb3 (Fox J E On the role of calpain and Rho proteins in regulating integrin-induced signaling. Thromb Haemost 82:385-91, 1999).
Calpains have been implicated in cell signaling through activation of protein kinases and phosphatases (cleaving between regulatory and catalytic domains resulting in changes in activity after hydrolysis) and modulation of their intracellular localization. Calpains have been shown to modify specific enzymes and cytoskeletal proteins as part of calcium-mediated signal pathways. They are also involved in remodeling and disassembling the cytoskeleton, especially where the cytoskeleton attaches to membranes or other subcellular structures.
Several nuclear transcription factors have been suggested as calpain substrates. Calpains are also involved in the progression of cells through the cell cycle (Carafoli E and Molinari M Calpain: a protease in search of a function? Biochem Biophys Res Commun 247:193-203, 1998) in that calpain activity accelerates some cells through the cell cycle by cleavage of p53. Calpain is also thought to play a role in long term potentiation (memory) and rat strains deficient in the endogenous calpain inhibitor, calpastatin, have increased long term potentiation.
Calpains in Disease:
Several diseases have been associated with calpain deficiencies. For example, limb-girdle muscular dystrophy (LGMD) is a group of disorders that primarily cause weakness of the shoulder and pelvic regions. A subtype of LGMD called LGMD2A is caused by defects in the gene for p94 (also called calpain 3) (Richard et al., Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell 81:2740, 1995).
Positional cloning has recently identified single-nucleotide polymorphisms (SNPs) in an intron of the gene coding for calpain-10 that appears to confer insulin resistance in diabetics. Presence of this mutation correlates with reduced levels of calpain 10 in patients susceptible to type II diabetes (Horikawa et al., Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus. Nat Genet. 26:163-75, 2000). The same calpain-10 SNP also correlates with type II diabetes in a high-risk population of Pima Indians (Baier et al., A calpain-10 gene polymorphism is associated with reduced muscle mRNA levels and insulin resistance. J Clin Invest. 106:R69-73, 2000).
Over Activation of Calpain—Ischemic and Traumatic Damage
Intracellular calcium levels and calpain activity are normally tightly regulated. Under stress, such as follows neuronal excitotoxicity, ischemic stroke, hemoragic stroke, hypoxic stress and/or trauma, intracellular calcium levels rise causing inappropriate calpain proteolytic activity. Calpain activity has been implicated in further cell destruction and non-specific calpain inhibitors have been shown to be protective in animal models (Lee et al., Proc. Natl. Acad. Sci. USA, 88:7233-7237, 1991; Wang K K and Yuen P W. Calpain inhibition: an overview of its therapeutic potential. Trends Pharmacol. Sci. 15:412-9, 1994;
Lee, K S, et al., Calcium-activated proteolysis as a therapeutic target in cerebrovascular disease. Annal NY Acad Sci. 825, 95-103, 1997).
Calpains are activated in neurons following ischemia-induced damage in animal models of stroke. (Lee et al., Proc. Natl. Acad. Sci. USA, 88:7233-7237, 1991). Inhibition of calcium-activated proteolysis by means of high doses of (usually non-specific) calpain inhibitors protect against the degeneration of vulnerable hippocampal neurons after ischemia (Rami et al., Brain Research, 609:67-70, 1993; Wang et al., An alpha-mercaptoacrylic acid derivative is a selective nonpeptide cell-permeable calpain inhibitor and is neuroprotective. Proc Natl Acad Sci USA. 93:6687-92, 1996). After an ischemic insult, neuronal death is delayed for hours to days. This time interval represents a potential therapeutic window in which to apply effective therapies to minimize brain damage after stroke.
In addition to neuronal damage, calpains are thought to contribute to cardiac ischemic damage (Iwamoto H et al., Calpain inhibitor-1 reduces infarct size and DNA fragmentation of myocardium in ischemic/reperfused rat heart. J Cardiovasc Pharmacol 33:580-6, 1999) and hepatocyte necrosis during and following anoxia (Arora A S et al., Hepatocellular carcinoma cells resist necrosis during anoxia by preventing phospholipase-mediated calpain activation. J Cell Physiol 167:434-42, 1996).
Neurodegenerative Diseases
Calpains have been implicated in neurodegenerative diseases including, Alzheimer's disease, Multiple sclerosis, Huntington's disease, Parkinson's disease and amyotrophy. Calpain activation is increased during normal aging and a strong case can be made for the involvement of calpain in the abnormal proteolysis underlying the accumulation of plaque and neurofibriles in brain tissue from people who suffered Alzheimer-type dementia (Iwamoto et al., Brain Research, 561:177-180 1991; Nixon et al., Calcium-activated neutral proteinase (calpain) system in aging and Alzheimer's disease. Ann N Y Acad Sci; 747:77-91, 1994; Grynspan et al., Active site-directed antibodies identify calpain II as an early-appearing and pervasive component of neurofibrillary pathology in Alzheimer's disease. Brain Res 763:145-58, 1997). Calpains are significantly activated in human postmortem brain from patients with Alzheimer's disease, and the degree of activation correlated with those regions of the brain showing the greatest amount of degeneration (Saito et al., Proc. Natl. Acad. Sci. USA, 90:2628-2632, 1993). More recently, it has been recognized that in Alzheimer's disease cyclin-dependent kinase 5 (cdk5) and its neuron-specific activator p35 are involved in neurite outgrowth and cortical lamination. Calpain cleavage of p35 produces p25, which accumulates in the brains of patients with Alzheimers disease. Conversion of p35 to p25 causes prolonged activation and mislocalization of cdk5 which hyperphosphorylates tau, disrupts the cytoskeleton and promotes the death (apoptosis) of primary neurons (Lee et al., Neurotoxicity induces cleavage of p35 to p25 by calpain. Nature. 18;405:360-4, 2000). Compounds that inhibit calpain activity could prove useful in reducing or delaying neurodegeneration caused to Alzheimer's disease.
Damage Following Trauma
Traumatic injury also causes calpain activation associated with further cell death, atrophy and shrinkage of the brain. A forceful blow trigger cell damage and increased calpain activity that can cleave structural proteins in the brain for up to weeks afterward (Hayes et al., Potential Contribution of Proteases to Neuronal Damage Drug News & Perspectives 11, 1998).
Calpain activation has also been implicated in spinal cord injury following trauma (for reviews see: Banik et al., Role of calpain and its inhibitors in tissue degeneration and neuroprotection in spinal cord injury. Ann. N. Y. Acad. Sci. 825:120-7 1997; Banik et al., Role of calpain in spinal cord injury: effects of calpain and free radical inhibitors. Ann. N. Y. Acad. Sci. 844:131-7, 1998). Analogous to brain trauma, secondary pathophysiological alterations occur in the traumatized spinal cord well after the initiating insult. These secondary events ultimately cause cell death and tissue damage. Non-specific calpain inhibitors have shown utility in preventing further damage due to spinal chord injury in animal models (Ray et al., Increased calpain expression is associated with apoptosis in rat spinal cord injury: calpain inhibitor provides neuroprotection. Neurochem Res. 25:1191-8, 2000).
These studies indicate the potential utility of calpain inhibitors (especially those calpains located in the spinal cord) in treating traumatic injury resulting from automobile crashes, gunshot wounds, and sports accidents.
Degeneration of Cochlear Tissues Following Noise Exposure
Calpains are activated during acoustic trauma and calpain inhibitors protect against hearing loss caused by noise (Stracher A Calpain inhibitors as therapeutic agents in nerve and muscle degeneration. Ann N Y Acad Sci 884:52-9, 1999).
Inflammation
Calpains also regulate integrin-mediated interaction of T-cells with the extracellular matrix (ECM) and calpain inhibitors prevent acute and chronic inflammation in animal models (Cuzzocrea S et al., Calpain inhibitor I reduces the development of acute and chronic inflammation Am J Pathol 157:2065-79, 2000).
Multiple Sclerosis
Multiple sclerosis is characterized by the progressive loss of the myelin of the brain and spinal cord. In autoimmune demyelinating diseases such as multiple sclerosis and experimental allergic encephalomyelitis, the degradation of myelin proteins results in the destabilization of the myelin sheath. Calpains have been implicated in that calpain degrades all major myelin proteins and increased calpain activity is observed in multiple sclerosis (Shields D C et al., A putative mechanism of demyelination in multiple sclerosis by a proteolytic enzyme, calpain. Proc. Natl. Acad. Sci. USA 96:11486-91, 1999).
Cataract Formation
In the lens, crystallins prevent thermal denaturation and aggregation of other proteins. Crystallins are also substrates for calpains and cataract formation in a rat model of selenite-induced cataract formation is thought to result from calpain activation and cleavage of crystallins (Shearer T R, David L L, Anderson R S, Azuma M. Review of selenite cataract. Curr Eye Res 1992; 11:357-369). In this model the crystallin cleavage could be blocked by calpain inhibitors (Azuma M et al., Cysteine protease inhibitor E64 reduces the rate of formation of selenite cataract in the whole animal. Curr Eye Res 10:657-666, 1991). In a genetic model cataract-prone rats also showed enhanced proteolysis of crystallins and lens cytoskeletin proteins thought to be mediated by calpain (Inomata M et al., Evidence for the involvement of calpain in cataractogenesis,in Shumiya cataract rat (SCR). Biochim Biophys Acta 1362:11-23 1997). Calpain activation is also thought to play a role in cataracts induced by buthionine sulfoximine, calcium ionophore A23187, hydrogen peroxide, diamide, xylose, galactose and streptozotocin (Kadoya et al., Role of calpain in hydrogen peroxide cataract. Curr Eye Res 1993; 12:341-346; David et al., Buthionine sulfoximine induced cataracts in mice contain insolubilized crystallins with calpain II cleavage sites, Exp Eye Res 1994; 59:501-504.). These models of cataract formation in rats suggest that calpain-induced proteolysis is a common underlying mechanism. Fragments of alpha-crystallin, consistent with calpain cleavage, have been also observed in cataractous human lens.
Reovirus Induced Myocarditis
Infection of neonatal mice with reovirus produces histological myocarditis. This is due to a direct viral injury and apoptosis of myocytes. Calpain inhibitors block reovirus-induced apoptosis in vitro and prevented viral-induced induced myocarditis (DeBiasi et al., Calpain inhibition protects against virus-induced apoptotic myocardial injury. Virol 75:351-61, 2001).
The inventors of the present invention describe herein, the polynucleotides corresponding to the full-length novel CAN-12 calpain gene, its encoded polypeptide, in addition to the variants CAN-12v1 and CAN-12v2. Also provided are polypeptide alignments illustrating the strong conservation of the CAN-12, CAN-12v1, and CAN-12v2 polypeptides to known proteases and a model of the active conformation of CAN-12. Based on this strong conservation, the inventors have ascribed the CAN-12, CAN-12v1, and CAN-12v2 polypeptides as having calpain proteolytic activity. Data is also provided illustrating the unique tissue expression profile of the CAN-12 polypeptide in esophagus, lymph node, and spinal cord tissues.
In fact, calpains have been the subject of significant research and development programs designed to identify inhibitors of this disease associated protein class. For example, the following, non-limiting examples of drugs, therapies, or regimens directed to inhibiting calpains are currently known: BDA 410 (Mitsubishi Tokyo); AK 295 (Alkermes; CAS® Registry Number: 160399-35-9, 144231-82-3, and 145731-49-3; (1-(((1-ethyl-3-((3-(4-morpholinyl)propyl)amino)-2,3-dioxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid phenylmethyl ester stereois); AK 275 (Alkermes; CAS® Registry Number: 158798-83-5, and 150519-08-7; N-((phenylmethoxy)carbonyl)-L-leucyl-N-ethyl-L-2-aminobutanamide); inhibitor 1 (University of Indiana; acetyl-leu-leu-norleucinal); calpeptin (University of Indiana; benzyloxycarbonyl-leu-norleucinal); VASOLEX (Cortex); RESTENEX (Cortex); MDL 28170 (Aventis; CBZ-Val-Phe-H); P1 (Sankyo; CASS Registry Number: 128102-74-9, and 128102-75-0; L-phenylalanyl-L-glutaminyl-L-valyl-L-valyl-3-((3-nitro-2-pyridiny 1)dithio)-L-alanylglycinamide); MDL 28170 (Hoechst Marion Roussel); BDA-410 (Mitsubishi-Tokyo); SJA-6017 (Senju; CAS® Registry Number: 190274-534; Butanamide,2-(((4-fluorophenyl)sulfonyl)amino)-N-((1S)-1-formyl-3-methylbutyl.).-3-methyl-, (2S)-); Pharmaprojects No. 5123 (Pfizer; 2-Chloro-acetic acid(3-oxo-4-phenyl-3,4-dihydro-1H-quinoxalin-2-ylidene)hydrazide; WO96-25403); CEP-4143 (Cephalon; WO96-14067); MDL-104903 (Aventis; CAS) Registry Number: 180799-56-8; Carbamic acid,((1S)-1-(((4S,5R)-5-hydroxy-4-(phenylmethyl)-3-oxazolidinyl)carbonyl)-2-methylpropyl)-,phenylmethyl ester)); MDL-28170 (Aventis; CASS Registry Number: 19542-51-9; Alanine, N-(N-carboxy-L-valyl)-3-phenyl-N-benzyl ester, L-); CX-275 (Cortex Pharmaceuticals; PhenylmethylN-((1R)-1-(((1S)-1-ethyl-3-(ethylamino)-2,3-dioxopropyl)amino)carbonyl)-3-methylbutyl)carbamate ); NS 7 (Nippon Shinyaku; 4-(4-Fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride ); Calpain inhibitor 1 (Suntory; N-Acetyl-L-leucinyl-L-leucinyl-L-norleucinal); E 64 (Taisho Pharmaceutical (; CAS®) Registry Number: 66701-25-5); CEP 4143 (Cephalon); SJA 6017 (Senju; N-(4-Fluorophenylsulfonyl)-L-valyl-L-leucinal); The present invention is directed to antagonists specific to the CAN-12, CAN-12v1, and/or CAN-12v2 polypeptides. Modulating the activity of the calpain polypeptides of the present invention may result in fewer toxicities than the drugs, therapies, or regimens presently known to regulate other known calpains.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of CAN-12, CAN-12v1, and CAN-12v2 polypeptides or peptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the CAN-12, CAN-12v1, and CAN-12v2 polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides, particularly activators and inhibitors of the novel CAN-12, CAN-12v1, and CAN-12v2 polypeptides of the present invention.

BRIEF SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid molecules, that comprise, or alternatively consist of, a polynucleotide encoding the CAN-12 protein having the amino acid sequence shown in FIGS. 1A-E (SEQ ID NO:24) or the amino acid sequence encoded by the cDNA clone, CAN-12 (also referred to as protease 5, clone 70).
The present invention provides isolated nucleic acid molecules, that comprise, or alternatively consist of, a polynucleotide encoding the CAN-12+ polypeptide sequence having the amino acid sequence shown in FIGS. 1A-E (SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone, CAN-12+ (also referred to as protease 5, clone 70; +splice amino acids).
The present invention provides isolated nucleic acid molecules, that comprise, or alternatively consist of, a polynucleotide encoding the CAN-12v1 protein having the amino acid sequence shown in FIGS. 8A-C (SEQ ID NO:54) or the amino acid sequence encoded by the cDNA clone, CAN-12v1 (also referred to as protease 5, clone 1e), deposited as ATCC Deposit Number PTA-3434 on Jun. 7, 2001.
The present invention provides isolated nucleic acid molecules, that comprise, or alternatively consist of, a polynucleotide encoding the CAN-12v2 protein having the amino acid sequence shown in FIGS. 9A-C (SEQ ID NO:56) or the amino acid sequence encoded by the cDNA clone, CAN-12v2 (also referred to as protease 5, clone 1e1b-1), deposited as ATCC Deposit Number PTA-3434 on Jun. 7, 2001.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of CAN-12, CAN-12v1, and CAN-12v1 polypeptides or peptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the CAN-12, CAN-12v1, and CAN-12v1 polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.
The invention further provides an isolated CAN-12 polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
The invention further provides an isolated CAN-12v1 polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
The invention further provides an isolated CAN-12v2 polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
The invention also provides a machine readable storage medium which comprises the structure coordinates of CAN-12, including all or any parts conserved calpain regions. Such storage medium encoded with these data are capable of displaying on a computer screen or similar viewing device, a three-dimensional graphical representation of a molecule or molecular complex which comprises said regions or similarly shaped homologous regions.
The invention also provides methods for designing, evaluating and identifying compounds which bind to all or parts of the aforementioned regions. The methods include three dimensional model building (homology modeling) and methods of computer assisted-drug design which can be used to identify compounds which bind or modulate the forementioned regions of the CAN-12 polypeptide. Such compounds are potential inhibitors of CAN-12 or its homologues.
The invention also provides novel classes of compounds, and pharmaceutical compositions thereof, that are useful as inhibitors of CAN-12 or its homologues.
The invention also provides novel classes of compounds, and pharmaceutical compositions thereof, that are useful as inhibitors of CAN-12v1 or its homologues.
The invention also provides novel classes of compounds, and pharmaceutical compositions thereof, that are useful as inhibitors of CAN-12v2 or its homologues.
The invention further relates to a polynucleotide encoding a polypeptide fragment of SEQ ID NO:2, 24, 54, and/or 56, or a polypeptide fragment encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1, 23, 53, and/or 55.
The invention further relates to a polynucleotide encoding a polypeptide domain of SEQ ID NO:2, 24, 54, and/or 56 or a polypeptide domain encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1, 23, 53, and/or 55.
The invention further relates to a polynucleotide encoding a polypeptide epitope of SEQ ID NO:2, 24, 54, and/or 56 or a polypeptide epitope encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1, 23, 53, and/or 55.
The invention further relates to a polynucleotide encoding a polypeptide of SEQ ID NO:2, 24, 54, and/or 56 or the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1, 23, 53, and/or 55, having biological activity.
The invention further relates to a polynucleotide which is a variant of SEQ ID NO:1, 23, 53, and/or 55.
The invention further relates to a polynucleotide which is an allelic variant of SEQ ID NO:1, 23, 53, and/or 55.
The invention further relates to a polynucleotide which encodes a species homologue of the SEQ ID NO:2, 24, 54, and/or 56.
The invention further relates to a polynucleotide which represents the complimentary sequence (antisense) of SEQ ID NO:1, 23, 53, and/or 55.
The invention further relates to a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified herein, wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues.
The invention further relates to an isolated nucleic acid molecule of SEQ ID NO:2, 24, 54, and/or 56, wherein the polynucleotide fragment comprises a nucleotide sequence encoding a calpain protein.
The invention further relates to an isolated nucleic acid molecule of SEQ ID NO:1, 23, 53, and/or 55 wherein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as SEQ ID NO:2, 24, 54, and/or 56 or the polypeptide encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1, 23, 53, and/or 55.
The invention further relates to an isolated nucleic acid molecule of of SEQ ID NO:1, 23, 53, and/or 55 wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO:1, 23, 53, and/or 55 or the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1, 23, 53, and/or 55.
The invention further relates to an isolated nucleic acid molecule of SEQ ID NO:1, 23, 53, and/or 55, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus.
The invention further relates to an isolated polypeptide comprising an amino acid sequence that comprises a polypeptide fragment of SEQ ID NO:2, 24, 54, and/or 56 or the encoded sequence included in the deposited clone.
The invention further relates to a polypeptide fragment of SEQ ID NO:2, 24, 54, and/or 56 or the encoded sequence included in the deposited clone, having biological activity.
The invention further relates to a polypeptide domain of SEQ ID NO:2, 24, 54, and/or 56 or the encoded sequence included in the deposited clone.
The invention further relates to a polypeptide epitope of SEQ ID NO:2, 24, 54, and/or 56 or the encoded sequence included in the deposited clone.
The invention further relates to a full length protein of SEQ ID NO:2, 24, 54, and/or 56 or the encoded sequence included in the deposited clone.
The invention further relates to a variant of SEQ ID NO:2, 24, 54, and/or 56.
The invention further relates to an allelic variant of SEQ ID NO:2, 24, 54, and/or 56.
The invention further relates to a species homologue of SEQ ID NO:2, 24, 54, and/or 56.
The invention further relates to the isolated polypeptide of of SEQ ID NO:2, 24, 54, and/or 56, wherein the full length protein comprises sequential amino acid deletions from either the C-terminus or the N-terminus.
The invention further relates to an isolated antibody that binds specifically to the isolated polypeptide of SEQ ID NO:2, 24, 54, and/or 56.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the polypeptide of SEQ ID NO:2, 24, 54, and/or 56 or the polynucleotide of SEQ ID NO:1, 23, 53, and/or 55.
The invention further relates to a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising the steps of (a) determining the presence or absence of a mutation in the polynucleotide of SEQ ID NO:1, 23, 53, and/or 55; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.
The invention further relates to a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising the steps of (a) determining the presence or amount of expression of the polypeptide of of SEQ ID NO:2, 24, 54, and/or 56 in a biological sample; and diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.
The invention further relates to a method for identifying a binding partner to the polypeptide of SEQ ID NO:2, 24, 54, and/or 56 comprising the steps of (a) contacting the polypeptide of SEQ ID NO:2, 24, 54, and/or 56 with a binding partner; and (b) determining whether the binding partner effects an activity of the polypeptide.
The invention further relates to a gene corresponding to the cDNA sequence of SEQ ID NO:1, 23, 53, and/or 55.
The invention further relates to a method of identifying an activity in a biological assay, wherein the method comprises the steps of expressing SEQ ID NO:1, 23, 53, and/or 55 in a cell, (b) isolating the supernatant; (c) detecting an activity in a biological assay; and (d) identifying the protein in the supernatant having the activity.
The invention further relates to a process for making polynucleotide sequences encoding gene products having altered activity selected from the group consisting of SEQ ID NO:2, 24, 54, and/or 56 activity comprising the steps of (a) shuffling a nucleotide sequence of SEQ ID NO:1, 23, 53, and/or 55, (b) expressing the resulting shuffled nucleotide sequences and, (c) selecting for altered activity selected from the group consisting of SEQ ID NO:2, 24, 54, and/or 56 activity as compared to the activity selected from the group consisting of SEQ ID NO:2, 24, 54, and/or 56 activity of the gene product of said unmodified nucleotide sequence.
The invention further relates to a shuffled polynucleotide sequence produced by a shuffling process, wherein said shuffled DNA molecule encodes a gene product having enhanced tolerance to an inhibitor of any one of the activities selected from the group consisting of SEQ ID NO:2, 24, 54, and/or 56 activity.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid, wherein the medical condition is a gastrointenstinal disorder.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid, wherein the medical condition is a neural disorder.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid, wherein the medical condition is an inflammatory disease.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid, wherein the medical condition is an inflammatory disease where proteases, either directly or indirectly, are involved in disease progression.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid, wherein the medical condition is a degenerative disease wherein proteases, either directly or indirectly, are involved in disease progression.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid, wherein the medical condition is multiple sclerosis.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid, wherein the medical condition is a cancer.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid, wherein the medical condition is a blood disorder.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid, wherein the medical condition is an immune disorder.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid, wherein the medical condition is a hematopoietic disorder.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid, wherein the medical condition is a disorder related to aberrant protease regulation.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid, wherein the medical condition is a condition associated with tissue damage caused by calpain activation, either directly or indirectly.
The invention further relates to a method of identifying a compound that modulates the biological activity of CAN-12, comprising the steps of, (a) combining a candidate modulator compound with CAN-12 having the sequence set forth in one or more of SEQ ID NO:2, 24, 54, and/or 56; and measuring an effect of the candidate modulator compound on the activity of CAN-12.
The invention further relates to a method of identifying a compound that modulates the biological activity of a calpain, comprising the steps of, (a) combining a candidate modulator compound with a host cell expressing CAN-12 having the sequence as set forth in SEQ ID NO:2, 24, 54, and/or 56; and, (b) measuring an effect of the candidate modulator compound on the activity of the expressed CAN-12.
The invention further relates to a method of identifying a compound that modulates the biological activity of CAN-12, comprising the steps of, (a) combining a candidate modulator compound with a host cell containing a vector described herein, wherein CAN-12 is expressed by the cell; and, (b) measuring an effect of the candidate modulator compound on the activity of the expressed CAN-12.
The invention further relates to a method of screening for a compound that is capable of modulating the biological activity of CAN-12, comprising the steps of: (a) providing a host cell described herein; (b) determining the biological activity of CAN-12 in the absence of a modulator compound; (c) contacting the cell with the modulator compound; and (d) determining the biological activity of CAN-12 in the presence of the modulator compound; wherein a difference between the activity of CAN-12 in the presence of the modulator compound and in the absence of the modulator compound indicates a modulating effect of the compound.
The invention further relates to a compound that modulates the biological activity of human CAN-12 as identified by the methods described herein.
The invention also provides a machine readable storage medium which comprises the structure coordinates of CAN-12, including all or any parts conserved calpain regions. Such storage medium encoded with these data are capable of displaying on a computer screen or similar viewing device, a three-dimensional graphical representation of a molecule or molecular complex which comprises said regions or similarly shaped homologous regions.
The invention also provides a machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the structure coordinates of the model CAN-12 according to Table IV or a homologue of said model, wherein said homologue comprises any kind of surrogate atoms that have a root mean square deviation from the backbone atoms of the complex of not more than about 5.0 Å.
The invention also provides a machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the structure coordinates of the model CAN-12 according to Table IV or a homologue of said model, wherein said homologue comprises any kind of surrogate atoms that have a root mean square deviation from the backbone atoms of the complex of not more than about 5.0 Å, preferably not more than about 4.0 Å, or less.
The invention also provides a model comprising all or any part of the model defined by structure coordinates of CAN-12 according to Table IV, or a mutant or homologue of said molecule or molecular complex.
The invention also provides a method for identifying a mutant of CAN-12 with altered biological properties, function, or reactivity, the method comprising one or more of the following steps: (a) use of the model or a homologue of said model according to Table IV, for the design of protein mutants with altered biological function or properties which exhibit any combination of therapeutic effects described herein; and/or (b) use of the model or a homologue of said model, for the design of a protein with mutations in the active site region comprised of the amino acids from about amino acid R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E251 to about amino acid Y255, from about amino acid N277 to about amino acid K281, from about amino acid V327 to about amino acid V330 of SEQ ID NO:2 according to Table IV with altered biological function or properties which exhibit any combination of therapeutic effects described herein.
The invention also provides methods for designing, evaluating and identifying compounds which bind to all or parts of the aforementioned regions. The methods include three dimensional model building (homology modeling) and methods of computer assisted-drug design which can be used to identify compounds which bind or modulate the forementioned regions of the CAN-12 polypeptide. Such compounds are potential inhibitors of CAN-12 or its homologues.
The invention also relates to method for identifying modulators of CAN-12 biological properties, function, or reactivity, the method comprising the step of modeling test compounds that fit spatially into the EF-hand calcium binding region defined by I449-K471 of SEQ ID NO:2 using a homologue or portion thereof or analogue in which the original C, N, and O atoms have been replaced with other elements
The invention also relates to a method of using said structure coordinates as set forth in Table IV to identify structural and chemical features of CAN-12; employing identified structural or chemical features to design or select compounds as potential CAN-12 modulators; employing the three-dimensional structural model to design or select compounds as potential CAN-12 modulators; synthesizing the potential CAN-12 modulators; screening the potential CAN-12 modulators in an assay characterized by binding of a protein to the CAN-12. The invention also relates to said method wherein the potential CAN-12 modulator is selected from a database. The invention further relates to said method wherein the potential CAN-12 modulator is designed de novo. The invention further relates to a method wherein the potential CAN-12 modulator is designed from a known modulator of activity.
The invention also provides a machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the structure coordinates of the model CAN-12v2v2 according to Table V or a homologue of said model, wherein said homologue comprises any kind of surrogate atoms that have a root mean square deviation from the backbone atoms of the complex of not more than 5.0 Å.
The invention also provides a machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the structure coordinates of the model CAN-12v2 according to Table V or a homologue of said model, wherein said homologue comprises any kind of surrogate atoms that have a root mean square deviation from the backbone atoms of the complex of not more than 5.0 Å, preferabbly not more than 4.0 Å, or less
The invention also provides a model comprising all or any part of the model defined by structure coordinates of CAN-12v2 according to Table V, or a mutant or homologue of said molecule or molecular complex.
The invention also provides a method for identifying a mutant of CAN-12v2 with altered biological properties, function, or reactivity, the method comprising one or more of the following steps: (a) use of the model or a homologue of said model according to Table V, for the design of protein mutants with altered biological function or properties which exhibit any combination of therapeutic effects described herein; and/or (b) use of the model or a homologue of said model, for the design of a protein with mutations in the active site region comprised of the amino acids from about amino acid R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E252 to about amino acid Y256, from about amino acid N278 to about amino acid K282, from about amino acid V328 to about amino acid V331 of SEQ ID NO:56 according to Table V with altered biological function or properties which exhibit any combination of therapeutic effects described herein.
The invention also provides methods for designing, evaluating and identifying compounds which bind to all or parts of the aforementioned regions. The methods include three dimensional model building (homology modeling) and methods of computer assisted-drug design which can be used to identify compounds which bind or modulate the forementioned regions of the CAN-12v2 polypeptide. Such compounds are potential inhibitors of CAN-12v2 or its homologues.
The invention also relates to method for identifying modulators of CAN-12v2 biological properties, function, or reactivity, the method comprising the step of modeling test compounds that fit spatially into the EF-hand calcium binding regions defined by amino acids 1565 to K587 of SEQ ID NO:56, using a homologue or portion thereof or analogue in which the original C, N, and O atoms have been replaced with other elements The invention also relates to a method of using said structure coordinates as set forth in Table V to identify structural and chemical features of CAN-12v2; employing identified structural or chemical features to design or select compounds as potential CAN-12v2 modulators; employing the three-dimensional structural model to design or select compounds as potential CAN-12v2 modulators; synthesizing the potential CAN-12v2 modulators; screening the potential CAN-12v2 modulators in an assay characterized by binding of a protein to the CAN-12v2. The invention also relates to said method wherein the potential CAN-12v2 modulator is selected from a database. The invention further relates to said method wherein the potential CAN-12v2 modulator is designed de novo. The invention further relates to a method wherein the potential CAN-12v2 modulator is designed from a known modulator of activity.
The present invention also relates to an isolated polynucleotide consisting of a portion of the human CAN-12 gene consisting of at least 8 bases, specifically excluding Genbank Accession Nos. gi|AL540944, and/or gi|BM554389.
The present invention also relates to an isolated polynucleotide consisting of a nucleotide sequence encoding a fragment of the human CAN-12 protein, wherein said fragment displays one or more functional activities specifically excluding Genbank Accession Nos. gi|AL540944, and/or gi|BM554389.
The present invention also relates to the polynucleotide of SEQ ID NO:1, 23, 53, and/or 55 consisting of at least 10 to 50 bases, wherein said at least 10 to 50 bases specifically exclude the polynucleotide sequence of Genbank Accession Nos. gi|AL540944, and/or gi|BM554389.
The present invention also relates to the polynucleotide of SEQ ID NO:1, 23, 53, and/or 55 consisting of at least 15 to 100 bases, wherein said at least 15 to 100 bases specifically exclude the polynucleotide sequence of Genbank Accession Nos. gi|AL540944, and/or gi|BM554389.
The present invention also relates to the polynucleotide of SEQ ID NO:1, 23, 53, and/or 55 consisting of at least 100 to 1000 bases, wherein said at least 100 to 1000 bases specifically exclude the polynucleotide sequence of Genbank Accession Nos. gi|AL540944, and/or gi|BM554389.
The present invention also relates to an isolated polypeptide fragment of the human CAN-12 protein, wherein said polypeptide fragment does not consist of the polypeptide encoded by the polynucleotide sequence of Genbank Accession Nos. gi|AL540944, and/or gi|BM554389.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

The file of this patent contains at least one Figure executed in color. Copies of this patent with color Figure(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.
FIGS. 1A-E show the polynucleotide sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:24) of the novel human calpain, CAN-12, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence contains a sequence of 4584 nucleotides (SEQ ID NO:1), encoding a polypeptide of about 428 amino acids (SEQ ID NO:24). The polynucleotide sequence of CAN-12 is believed to represent a short splice variant form. As a result, the alternative splicing introduces a stop codon truncating the open reading frame to end at amino acid 428. Additional amino acids beyond amino acid 428 of SEQ ID NO:24 are shown and are represented in bold (beginning at nucleotide 1537 to 1995 of SEQ ID NO:1). These additional amino acids likely corresponded to the polypeptide sequence of alternative splice forms of CAN-12 as evidenced by the presence of the EF-hand calcium binding domain. However, these additional amino acids are not considered to be a part of this splice form (SEQ ID NO:24). Additional splice forms of CAN-12 have been identified and are described herein (CAN-12v1 and CAN-12v2). The CAN-12 polypeptide sequence comprising these additional amino acids is provided as SEQ ID NO:2 to serve as a reference for the CAN-12v1 and CAN12-v2 splice variants. An analysis of the CAN-12 polypeptide determined that it comprised the following features: predicted active site domain amino acids located from about amino acid R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M2 10 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E251 to about amino acid Y255, from about amino acid N277 to about amino acid K281, and/or from about amino acid V327 to about amino acid V330 of SEQ ID NO:24 (FIGS. 1A-E) represented by shading; and a predicted eukaryotic thiol (cysteine) protease active site domain located from about amino acid 90 to about amino acid 111 of SEQ ID NO:24 (FIGS. 1A-E) represented by double underlining. The predicted active site domain amino acids are believed to form the active site binding pocket of the CAN-12 polypeptide and facilitate catalysis of appropriate calpain substrates. The predicted catalytic amino acid residues within the CAN-12 active site are located at amino acid C101, H253, and N277 residues of SEQ ID NO:24 (FIGS. 1A-E) and are denoted by an arrow (“T”). The additional amino acids beyond amino acid 428 of SEQ ID NO:24 depicted in the Figure were predicted to comprise an EF-hand calcium-binding domain located from about amino acid 439 to about amino acid 471 of SEQ ID NO:24 (FIGS. 1A-E) represented by dotted underlining; and a predicted cell attachment sequence located from about amino acid 520 to about amino acid 532 of SEQ ID NO:24 (FIGS. 1A-E) represented in italics. The presence of the eukaryotic thiol (cysteine) protease active site domain in the additional translated amino acids supports the notion that additional splice variants of CAN-12 exist, at least two of which are described herein.
FIGS. 2A-E show the regions of identity and similarity between the encoded CAN-12 (SEQ ID NO:2), CAN-12v1 (SEQ ID NO:54), and CAN-12v2 polypeptides (SEQ ID NO:56) to other calpains, specifically, the human CAN10 protein (type II diabetes linked) (hCAN10; Genbank Accession No: gi|NP_—075574; SEQ ID NO:3); the human CAN5 protein (hCAN5; Genbank Accession No: gi|NP_—004046; SEQ ID NO:4); the large catalytic subunit of the human CALPAIN 1 protein (also referred to as Calcium-Activated Neutral Proteinase, CANP, μ-TYPE) (hCAN1; Genbank Accession No: gi|12408656; SEQ ID NO:5); the large subunit of the human calpain 3 protein (EC 3.4.22.17) (also referred to as CALPAIN L3, CALPAIN P94, Calcium-Activated Neutral Proteinase 3, CANP 3; muscle-specific calcium-activated neutral protease 3 large subunit) (hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6); the large catalytic subunit of the mouse CALPAIN 1 protein (also referred to as Calcium-Activated Neutral Proteinase) (CANP) G-TYPE ) (mCALPAIN1; Genbank Accession No: gi|O88666; SEQ ID NO:7); the mouse CALPAIN LP82 (mLP82; Genbank Accession No: gi|3661585; SEQ ID NO:8); the rat stomach-specific calcium-activated neutral protease large subunit, NCL2 protein (rNCL2; Genbank Accession No: gi|NP_—006606; SEQ ID NO:9); the human CAN11 protein (hCAN11; Genbank Accession No: gi|NP_—008989; SEQ ID NO:10); the human CAN2 protein (hCAN2; Genbank Accession No: gi|4502563; SEQ ID NO:11); and the human CAN9 protein (hCAN9; Genbank Accession No: gi|5729758; SEQ ID NO:12). The alignment was performed using the CLUSTALW algorithm described elsewhere herein. The darkly shaded amino acids represent regions of matching identity. The lightly shaded amino acids represent regions of matching similarity. Lines between residues indicate gapped regions for the aligned polypeptides. The asterisk (“↑”) denotes the characteristic active site cysteine (C101), histidine (H253), and asparagine (N277) residues of calpain proteases. The CAN-12 polypeptide sequence shown (CAN12+; SEQ ID NO:2) includes the additional translated amino acids beyond amino acid 428 of SEQ ID NO:24 (shown in FIGS. 1A-E) to illustrate their identity with the CAN-12v1 and CAN-12v2 splice variants.
FIG. 3 shows a phylogenetic tree organization of various calpain family members with respect to the CAN-12 polypeptide of the present invention. The organization was created using the Vector NTI AlignX algorithm, based upon the CLUSTALW alignment described in FIGS. 2A-E above. As shown, CAN-12 is most closely related, phylogenetically, to the human CAN5 and CAN10 proteins.
FIG. 4 shows an expression profile of the novel human calpain, CAN-12. The figure illustrates the relative expression level of CAN-12 amongst various mRNA tissue sources. As shown, transcripts corresponding to CAN-12 expressed highly in spinal cord. The CAN-12 polypeptide was also expressed significantly in lymph node, thymus, and to a lesser extent, in spleen. Expression data was obtained by measuring the steady state CAN-12 mRNA levels by quantitative PCR using the PCR primer pair provided as SEQ ID NO:21 and 22 as described herein.
FIGS. 5A-B show a table illustrating the percent identity and percent similarity values between the CAN-12+ (SEQ ID NO:2), CAN-12v1 (SEQ ID NO:54), CAN-12v2 (SEQ ID NO:56), and CAN-12 (SEQ ID NO:24) polypeptides of the present invention with other calpains, specifically, the human CAN10 protein (type II diabetes linked) (hCAN10; Genbank Accession No: gi|NP_—075574; SEQ ID NO:3); the human CAN5 protein (hCAN5; Genbank Accession No: gi|NP_—004046; SEQ ID NO:4); the large catalytic subunit of the human CALPAIN 1 protein (also referred to as Calcium-Activated Neutral Proteinase, CANP, μ-TYPE) (hCAN1; Genbank Accession No: gi|12408656; SEQ ID NO:5); the large subunit of the human calpain 3 protein (EC 3.4.22.17) (also referred to as CALPAIN L3, CALPAIN P94, Calcium-Activated Neutral Proteinase 3, CANP 3; muscle-specific calcium-activated neutral protease 3 large subunit) (hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6); the large catalytic subunit of the mouse CALPAIN 1 protein (also referred to as Calcium-Activated Neutral Proteinase) (CANP) μ-TYPE ) (mCALPAIN1; Genbank Accession No: gi|088666; SEQ ID NO:7); the mouse CALPAIN LP82 (mLP82; Genbank Accession No: gi|3661585; SEQ ID NO:8); the rat stomach-specific calcium-activated neutral protease large subunit, NCL2 protein (rNCL2; Genbank Accession No: gi|NP_—006606; SEQ ID NO:9); the human CAN11 protein (hCAN11; Genbank Accession No: gi|NP_—008989; SEQ ID NO:10); the human CAN2 protein (hCAN2; Genbank Accession No: gi|4502563; SEQ ID NO:11); and the human CAN9 protein (hCAN9; Genbank Accession No: gi|5729758; SEQ ID NO:12). The alignment was performed using the CLUSTALW algorithm described elsewhere herein. The darkly shaded amino acids represent regions of matching identity. The lightly shaded amino acids represent regions of matching similarity. Lines between residues indicate gapped regions for the aligned polypeptides.
FIGS. 6 shows a three-dimensional homology model of the CAN-12 polypeptide based upon the homologous structure of a portion of the human m-calpain, also referred to as, CAN2 (hCAN2; Genbank Accession No. gi|4502563; SEQ ID NO:11). The predicted catalytic active site amino acids of the human CAN-12 polypeptide are labeled. The predicted regions of alpha helix structure are represented in magenta; the predicted regions of beta sheet structure are represented in yellow; the predicted regions of flexible loop structure are represented in cyan; the catalytic amino acid residues are shown in a CPK/space filled rendering of the side chain atoms wherein carbon atoms are represented in white, the sulfur atoms are represented in yellow, and the nitrogen atoms are represented in blue. The structural coordinates of the CAN-12 polypeptide are provided in Table IV herein. The homology model of CAN-12 was derived from generating a sequence alignment with the human m-calpain, CAN2 protein (hCAN2; Genbank Accession No. gi|4502563; SEQ ID NO:11) using the Proceryon suite of software (Proceryon Biosciences, Inc. N.Y., N.Y.), and the overall atomic model including plausible sidechain orientations using the program LOOK (V3.5.2, Molecular Applications Group).
FIGS. 8A-C show the polynucleotide sequence (SEQ ID NO:53) and deduced amino acid sequence (SEQ ID NO:54) of the novel human calpain, CAN-12v1, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence contains a sequence of 2095 nucleotides (SEQ ID NO:53), encoding a polypeptide of about 694 amino acids (SEQ ID NO:54). The polynucleotide sequence of CAN-12v1 is believed to represent a novel splice variant of the CAN-12 polynucleotide described herein. An analysis of the CAN-12v1 polypeptide determined that it comprised the following features: predicted active site domain amino acids located from about amino acid R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E252 to about amino acid Y256, from about amino acid N278 to about amino acid K282, and/or from about amino acid V328 to about amino acid V331 of SEQ ID NO:54 (FIGS. 8A-C) represented by shading; a predicted eukaryotic thiol (cysteine) protease active site domain located from about amino acid 90 to about amino acid 111 of SEQ ID NO:54 (FIGS. 8A-C) represented by double underlining; a predicted EF-hand calcium-binding domain located from about amino acid 567 to about amino acid 584 of SEQ ID NO:54 (FIGS. 8A-C) represented by dotted underlining; and a predicted cell attachment sequence located from about amino acid 633 to about amino acid 532 of SEQ ID NO:54 (FIGS. 8A-C) represented in italics. The presence of the eukaryotic thiol (cysteine) protease active site domain, in addition to, the EF-hand calcium binding domain is consistent with the CAN-12v1 polypeptide representing a member of the calpain family of proteases. The predicted active site domain amino acids are believed to form the active site binding pocket of the CAN-12v1 polypeptide and facilitate catalysis of appropriate calpain substrates. The predicted catalytic amino acid residues within the CAN-12v1 active site are located at amino acid C101, H254, and N278 residues of SEQ ID NO:54 (FIGS. 8A-C) and are denoted by an arrow (“↑”).
FIGS. 9A-C show the polynucleotide sequence (SEQ ID NO:55) and deduced amino acid sequence (SEQ ID NO:56) of the novel human calpain, CAN-12v2, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence contains a sequence of 2104 nucleotides (SEQ ID NO:53), encoding a polypeptide of about 694 amino acids (SEQ ID NO:56). The polynucleotide sequence of CAN-12v2 is believed to represent a novel splice variant of the CAN-12 polynucleotide described herein and likely represents the physiologically relevant splice form. An analysis of the CAN-12v2 polypeptide determined that it comprised the following features: predicted active site domain amino acids located from about amino acid R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E252 to about amino acid Y256, from about amino acid N278 to about amino acid K282, and/or from about amino acid V328 to about amino acid V331 of SEQ ID NO:56 (FIGS. 9A-C) represented by shading; a predicted eukaryotic thiol (cysteine) protease active site domain located from about amino acid 90 to about amino acid 111 of SEQ ID NO:56 (FIGS. 9A-C) represented by double underlining; a predicted EF-hand calcium-binding domain located from about amino acid 565 to about amino acid 587 of SEQ ID NO:56 (FIGS. 9A-C) represented by dotted underlining; and a predicted cell attachment sequence located from about amino acid 636 to about amino acid 648 of SEQ ID NO:56 (FIGS. 9A-C) represented by italics. The presence of the eukaryotic thiol (cysteine) protease active site domain, in addition to, the EF-hand calcium binding domain is consistent with the CAN-12v2 polypeptide representing a member of the calpain family of proteases. The predicted active site domain amino acids are believed to form the active site binding pocket of the CAN-12v2 polypeptide and facilitate catalysis of appropriate calpain substrates. The predicted catalytic amino acid residues within the CAN-12v2 active site are located at amino acid C101, H254, and N278 residues of SEQ ID NO:56 (FIGS. 9A-C) and are denoted by an arrow (“↑”).CAN-12v2 is believed to represent the true physioligical form of CAN-12.
FIGS. 10A-B show the regions of identity and similarity between the encoded CAN-12+ (SEQ ID NO:2), CAN-12v1 (SEQ ID NO:54), CAN-12v2 polypeptides (SEQ ID NO:56), and CAN-12 (SEQ ID NO:24) The alignment was performed using the CLUSTALW algorithm described elsewhere herein. The darkly shaded amino acids represent regions of matching identity. The lightly shaded amino acids represent regions of matching similarity. Lines between residues indicate gapped regions for the aligned polypeptides.
FIGS. 11 shows a three-dimensional homology model of the CAN-12v2 polypeptide based upon the homologous structure of a portion of the human m-calpain, also referred to as, CAN2 (hCAN2; Genbank Accession No. gi|4502563; SEQ ID NO:11). The predicted catalytic active site amino acids of the human CAN-12v2 polypeptide are labeled. The predicted regions of alpha helix structure are represented in magenta; the predicted regions of beta sheet structure are represented in yellow; the predicted regions of flexible loop structure are represented in cyan; the catalytic amino acid residues are shown in a CPK/space filled rendering of the side chain atoms wherein carbon atoms are represented in white, the sulfur atoms are represented in yellow, and the nitrogen atoms are represented in blue. The structural coordinates of the CAN-12v2 polypeptide are provided in Table V herein. The homology model of CAN-12v2 was derived from generating a sequence alignment with the human m-calpain, CAN2 protein (hCAN2; Genbank Accession No. gi|4502563; SEQ ID NO:11) using the SYBYL suite of software (Tripos, Inc., St. Louis, Mo.), and the overall atomic model including plausible sidechain orientations using the program COMPOSER (Tripos, Inc., St. Louis, Mo.).
FIG. 12 shows an expanded expression profile of the novel human calpain, CAN-12. The figure illustrates the relative expression level of CAN-12 amongst various mRNA tissue sources. As shown, the CAN-12 polypeptide was expressed at relatively low levels, though predominately in eosphagus, lymph node, and to a lesser extent in other tissues as shown. Expression data was obtained by measuring the steady state CAN-12 mRNA levels by quantitative PCR using the PCR primer pair provided as SEQ ID NO:143 and 144, and Taqman probe (SEQ ID NO:145) as described in Example 6 herein.
FIG. 13 shows an energy graph for the CAN-12.v2 model (see FIG. 11) of the present invention (solid line) and the human m-calpain template (PDB code 1dkv) (dotted line) from which the model was generated. The energy distribution for each protein fold is displayed on the y-axis, while the amino acid residue position of the protein fold is displayed on the x-axis. As shown, the CAN-12.v2 model and 1dkv template have similar energies over the aligned region, suggesting that the structural model of CAN-12.v2 represents a “native-like” conformation of the CAN-12.v2 polypeptide. This graph supports the motif and sequence alignments in confirming that the three-dimensional structure coordinates of CAN-12.v2 are an accurate and useful representation of the structure of the CAN-12.v2 polypeptide.
Table I provides a summary of the novel polypeptides and their encoding polynucleotides of the present invention.
Table II illustrates the preferred hybridization conditions for the polynucleotides of the present invention. Other hybridization conditions may be known in the art or are described elsewhere herein.
Table III provides a summary of various conservative substitutions encompassed by the present invention.
Table IV provides the structural coordinates of the homology model of the CAN-12 polypeptide provided in FIG. 6. A description of the headings are as follows: “Atom No” refers to the atom number within the CAN-12 homology model; “Atom Name” refers to the element whose coordinates are measured, the first letter in the column defines the element; “Residue” refers to the amino acid of the CAN-12 polypeptide within which the atom resides, in addition to the amino acid position in which the atom resides; “X Coord”, “Y Coord”, and “Z Coord” structurally define the atomic position of the element measured in three dimensions.
Table V provides the structural coordinates of the homology model of the CAN-12v2 polypeptide provided in FIG. 11. A description of the headings are as follows: “Atom No” refers to the atom number within the CAN-12v2 homology model; “Atom Name” refers to the element whose coordinates are measured, the first letter in the column defines the element; “Residue” refers to the amino acid of the CAN-12v2 polypeptide within which the atom resides, in addition to the amino acid position in which the atom resides; “X Coord”, “Y Coord”, and “Z Coord” structurally define the atomic position of the element measured in three dimensions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Examples included herein. All references to “CAN-12” shall be construed to apply to CAN-12+, CAN-12, CAN-12v1, and/or CAN-12v2 unless otherwise specified herein.
The invention provides a novel human sequence that encodes a calpain with substantial homology to the large subunits of a variety of known calpains. Calpains affect a variety of cellular processes based upon their involvement in modulating signal transduction. Aberrations in the large subunit polypeptides of calpains have been implicated in a number of diseases and disorders which include, for example, incidence of type II diabetes (Horikawa et al., Nat Genet. 26:163-75 (2000)), limb-girdle muscular dystrophy (Richard et al., Cell 81:27-40 (1995)), ischemia-induced damage in neurons and heart tissue, neurodegnerative disorders such as Alzheimer's disease, Multiple sclerosis, Huntington's disease, Parkinson's disease and amyotrophy, inflammatory disorders, susceptibility to infectious diseases, etc. CAN-12 polynucleotides and polypeptides, including agonists and antagonists thereof are expected to be useful in ameliorating at least some of these disorders. In addition, expression analysis indicates the CAN-12 has strong preferential expression in esophagus, lymph node, spinal cord, and to a lesser extent, in thymus, and spleen. Based on this information, we have provisionally named the gene and protein CAN-12.
In the present invention, “isolated” refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. The term “isolated” does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.
In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
As used herein, a “polynucleotide” refers to a molecule having a nucleic acid sequence contained in SEQ ID NO:1, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:23 or the cDNA contained within the clone(s) deposited with the ATCC. For example, the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5′ and 3′ untranslated sequences, the coding region, with or without a signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a “polypeptide” refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.
In the present invention, the full length sequence identified as SEQ ID NO:1, SEQ ID NO:53, SEQ ID NO:55, and SEQ ID NO:23 was often generated by overlapping sequences contained in one or more clones (contig analysis). A representative clone containing all or most of the sequence for SEQ ID NO:1, SEQ ID NO:53, SEQ ID NO:55, and/or, SEQ ID NO:23 was deposited with the American Type Culture Collection (“ATCC”). As shown in Table I, each clone is identified by a cDNA Clone ID (Identifier) and the ATCC Deposit Number. The ATCC is located at 10801 University Boulevard, Manassas, Va., 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure. The deposited clone is inserted in the pSport1 plasmid (Life Technologies) using the NotI and SalI restriction endonuclease cleavage sites.
Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373, preferably a Model 3700, from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
Using the information provided herein, such as the nucleotide sequence in FIGS. 1A-E (SEQ ID NO:1), a nucleic acid molecule of the present invention encoding the CAN-12 polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in FIGS. 1A-E (SEQ ID NO:1) was discovered in a cDNA library derived from human liver, brain and testis and spleen.
The determined nucleotide sequence of the CAN-12 cDNA in FIGS. 1A-E (SEQ ID NO:1) contains an open reading frame encoding a protein of about 428 amino acid residues, with a deduced molecular weight of about 49.5 kDa. The amino acid sequence of the predicted CAN-12 polypeptide is shown in FIGS. 1A-E (SEQ ID NO:24).
Using the information provided herein, such as the nucleotide sequence in FIGS. 8A-C (SEQ ID NO:53), a nucleic acid molecule of the present invention encoding the CAN-12v1 polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in FIGS. 8A-C (SEQ ID NO:53) was discovered in a cDNA library derived from human liver, brain and testis and spleen.
The determined nucleotide sequence of the CAN-12v1 cDNA in FIGS. 8A-C (SEQ ID NO:53) contains an open reading frame encoding a protein of about 694 amino acid residues, with a deduced molecular weight of about 80.3 kDa. The amino acid sequence of the predicted CAN-12v1 polypeptide is shown in FIGS. 8A-C (SEQ ID NO:54).
Using the information provided herein, such as the nucleotide sequence in FIGS. 9A-C (SEQ ID NO:55), a nucleic acid molecule of the present invention encoding the CAN-12v2 polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in FIGS. 9A-C (SEQ ID NO:55) was discovered in a cDNA library derived from human liver, brain and testis and spleen.
The determined nucleotide sequence of the CAN-12v2 cDNA in FIGS. 9A-C (SEQ ID NO:55) contains an open reading frame encoding a protein of about 697 amino acid residues, with a deduced molecular weight of about 80.6 kDa. The amino acid sequence of the predicted CAN-12v2 polypeptide is shown in FIGS. 9A-C (SEQ ID NO:56).
A “polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:1, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:23, the complement thereof, or the cDNA within the clone deposited with the ATCC. “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65 degree C.
Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37 degree C. in a solution comprising 6×SSPE (20×SSPE 3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg/mi salmon sperm blocking DNA; followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5×SSC).
Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of “polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).
The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)
“A polypeptide having biological activity” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention.)
The term “organism” as referred to herein is meant to encompass any organism referenced herein, though preferably to eukaryotic organisms, more preferably to mammals, and most preferably to humans.
As used herein the terms “modulate” or “modulates” refer to an increase or decrease in the amount, quality or effect of a particular activity, DNA, RNA, or protein. The definition of “modulate” or “modulates” as used herein is meant to encompass agonists and/or antagonists of a particular activity, DNA, RNA, or protein.
The present invention encompasses the identification of proteins, nucleic acids, or other molecules, that bind to polypeptides and polynucleotides of the present invention (for example, in a receptor-ligand interaction). The polynucleotides of the present invention can also be used in interaction trap assays (such as, for example, that described by Ozenberger and Young (Mol Endocrinol., 9(10):1321-9, (1995); and Ann. N. Y. Acad. Sci., 7;766:279-81, (1995)).
The polynucleotide and polypeptides of the present invention are useful as probes for the identification and isolation of full-length cDNAs and/or genomic DNA which correspond to the polynucleotides of the present invention, as probes to hybridize and discover novel, related DNA sequences, as probes for positional cloning of this or a related sequence, as probe to “subtract-out” known sequences in the process of discovering other novel polynucleotides, as probes to quantify gene expression, and as probes for microarrays.
In addition, polynucleotides and polypeptides of the present invention may comprise one, two, three, four, five, six, seven, eight, or more membrane domains.
Also, in preferred embodiments the present invention provides methods for further refining the biological function of the polynucleotides and/or polypeptides of the present invention.
Specifically, the invention provides methods for using the polynucleotides and polypeptides of the invention to identify orthologs, homologs, paralogs, variants, and/or allelic variants of the invention. Also provided are methods of using the polynucleotides and polypeptides of the invention to identify the entire coding region of the invention, non-coding regions of the invention, regulatory sequences of the invention, and secreted, mature, pro-, prepro-, forms of the invention (as applicable).
In preferred embodiments, the invention provides methods for identifying the glycosylation sites inherent in the polynucleotides and polypeptides of the invention, and the subsequent alteration, deletion, and/or addition of said sites for a number of desirable characteristics which include, but are not limited to, augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.
In further preferred embodiments, methods are provided for evolving the polynucleotides and polypeptides of the present invention using molecular evolution techniques in an effort to create and identify novel variants with desired structural, functional, and/or physical characteristics.
The present invention further provides for other experimental methods and procedures currently available to derive functional assignments. These procedures include but are not limited to spotting of clones on arrays, micro-array technology, PCR based methods (e.g., quantitative PCR), anti-sense methodology, gene knockout experiments, and other procedures that could use sequence information from clones to build a primer or a hybrid partner.
Polynucleotides and Polvpeptides of the Invention
Features of the Polypeptide Encoded by Gene No:1
The polypeptide of this gene provided as SEQ ID NO:24 (FIGS. 1A-E), encoded by the polynucleotide sequence according to SEQ ID NO:1 (FIGS. 1A-E), and/or encoded by the polynucleotide contained within the deposited clone, CAN-12, has significant homology at the nucleotide and amino acid level to a number of calpains, which include, for example, the human CAN10 protein (type II diabetes linked) (hCAN10; Genbank Accession No: gi|NP_—075574; SEQ ID NO:3); the human CAN5 protein (hCAN5; Genbank Accession No: gi|NP_—004046; SEQ ID NO:4); the large catalytic subunit of the human CALPAIN 1 protein (also referred to as Calcium-Activated Neutral Proteinase, CANP, μ-TYPE) (HCAN1; Genbank Accession No: gi|12408656; SEQ ID NO:5); the large subunit of the human calpain 3 protein (EC 3.4.22.17) (also referred to as CALPAIN L3, CALPAIN P94, Calcium-Activated Neutral Proteinase 3, CANP 3; muscle-specific calcium-activated neutral protease 3 large subunit) (hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6); the large catalytic subunit of the mouse CALPAIN 1 protein (also referred to as Calcium-Activated Neutral Proteinase) (CANP) μ-TYPE ) (mCALPAIN1; Genbank Accession No: gi|088666; SEQ ID NO:7); the mouse CALPAIN LP82 (nLP82; Genbank Accession No: gi|3661585; SEQ ID NO:8); the rat stomach-specific calcium-activated neutral protease large subunit, NCL2 protein (rNCL2; Genbank Accession No: gi|NP_—006606; SEQ ID NO:9); the human CAN11 protein (hCAN11; Genbank Accession No: gi|NP-008989; SEQ ID NO:10); the human CAN2 protein (hCAN2; Genbank Accession No: gi|4502563; SEQ ID NO:11); and the human CAN9 protein (hCAN9; Genbank Accession No: gi|5729758; SEQ ID NO:12). An alignment of the CAN-12 polypeptide with these proteins is provided in FIGS. 2A-E. Based upon such strong conservation, the inventors have ascribed the CAN-12 polypeptide as having proteolytic activity, preferably calpain activity.
The CAN-12+ (SEQ ID NO:2) polypeptide was determined to have 30.7% identity and 38.0% similarity with the human CAN10 protein (type II diabetes linked) (hCAN10; Genbank Accession No: gi|NP_—075574; SEQ ID NO:3); to have 34.2% identity and 45.4% similarity with the human CAN5 protein (hCAN5; Genbank Accession No: gi|NP_—004046; SEQ ID NO:4); to have 37.9% identity and 47.4% similarity with the large catalytic subunit of the human CALPAIN 1 protein (also referred to as Calcium-Activated Neutral Proteinase, CANP, μ-TYPE) (hCAN1; Genbank Accession No: gi|12408656; SEQ ID NO:5); to have 36.3% identity and 43.6% similarity with the large subunit of the human calpain 3 protein (EC 3.4.22.17) (also referred to as CALPAIN L3, CALPAIN P94, Calcium-Activated Neutral Proteinase 3, CANP 3; muscle-specific calcium-activated neutral protease 3 large subunit) (hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6); to have 39.0% identity and 47.6% similarity with the large catalytic subunit of the mouse CALPAIN 1 protein (also referred to as Calcium-Activated Neutral Proteinase) (CANP) μ-TYPE ) (mCALPAIN1; Genbank Accession No: gi|O88666; SEQ ID NO:7); to have 37.8% identity and 45.3% similarity with the mouse CALPAIN LP82 (mLP82; Genbank Accession No: gi|3661585; SEQ ID NO:8); to have 34.2% identity and 45.4% similarity with the rat stomach-specific calcium-activated neutral protease large subunit, NCL2 protein (rNCL2; Genbank Accession No: gi|NP_—006606; SEQ ID NO:9); to have 40.4% identity and 47.3% similarity with the human CAN11 protein (HCAN11; Genbank Accession No: gi|NP_—008989; SEQ ID NO:10); to have 36.8% identity and 45.8% similarity with the human CAN2 protein (hCAN2; Genbank Accession No: gi|4502563; SEQ ID NO:11); and to have 39.4% identity and 47.2% similarity with the human CAN9 protein (hCAN9; Genbank Accession No: gi|5729758; SEQ ID NO:12). The polypeptide sequence used for these percent identity and similarity values comprised the additional amino acids that extend beyond amino acid 428 of SEQ ID NO:24—specifically, SEQ ID NO:2.
The CAN-12 polypeptide was determined to have 34.3% identity and 42.3% similarity with the human CAN10 protein (type II diabetes linked) (hCAN10; Genbank Accession No: gi|NP_—075574; SEQ ID NO:3); to have 40.5% identity and 51.9% similarity with the human CAN5 protein (hCAN5; Genbank Accession No: gi|NP_—004046; SEQ ID NO:4); to have 44.3% identity and 51.9% similarity with the large catalytic subunit of the human CALPAIN 1 protein (also referred to as Calcium-Activated Neutral Proteinase, CANP, μ-TYPE) (hCAN1; Genbank Accession No: gi|12408656; SEQ ID NO:5); to have 44.9% identity and 51.8% similarity with the large subunit of the human calpain 3 protein (EC 3.4.22.17) (also referred to as CALPAIN L3, CALPAIN P94, Calcium-Activated Neutral Proteinase 3, CANP 3; muscle-specific calcium-activated neutral protease 3 large subunit) (hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6); to have 46.1% identity and 52.7% similarity with the large catalytic subunit of the mouse CALPAIN 1 protein (also referred to as Calcium-Activated Neutral Proteinase) (CANP) μ-TYPE ) (mCALPAIN1; Genbank Accession No: gi|O88666; SEQ ID NO:7); to have 46.2% identity and 53.5% similarity with the mouse CALPAIN LP82 (mLP82; Genbank Accession No: gi|3661585; SEQ ID NO:8); to have 44.6% identity and 51.4% similarity with the rat stomach-specific calcium-activated neutral protease large subunit, NCL2 protein (rNCL2; Genbank Accession No: gi|NP_—006606; SEQ ID NO:9); to have 45.5% identity and 51.7% similarity with the human CAN11 protein (hCAN11; Genbank Accession No: gi|NP_—008989; SEQ ID NO:10); to have 46.2% identity and 53.5% similarity with the human CAN2 protein (hCAN2; Genbank Accession No: gi|4502563; SEQ ID NO:11); and to have 44.3% identity and 51.9% similarity with the human CAN9 protein (hCAN9; Genbank Accession No: gi|5729758; SEQ ID NO:12). The polypeptide sequence used for these percent identity and similarity values was the full-length CAN-12 polypeptide (SEQ ID NO:24).
The human CAN10 protein (type II diabetes linked) (HCAN10; Genbank Accession No: gi|NP _—075574; SEQ ID NO:3) is a human calpain gene that encodes a large calpain subunit. CAN10 is an atypical calpain in that it lacks the calmodulin-like calcium-binding domain and instead has a divergent C-terminal domain. CAN10 is similar in organization to calpains 5 and 6 and is associated with type 2 or non-insulin-dependent diabetes mellitus (NIDDM) and located within the NIDDM1 chromosomal region (Nat. Genet. 26 (2), 163-175 (2000)).
The large subunit of the human calpain 3 protein (EC 3.4.22.17) (also referred to as CALPAIN L3, CALPAIN P94, Calcium-Activated Neutral Proteinase 3, CANP 3; muscle-specific calcium-activated neutral protease 3 large subunit) (hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6) is a muscle-specific member of the calpain large subunit family. Loss of CAPN3 function has been associated with limb-girdle muscular dystrophies type 2A (Cell 81 (1), 2740 (1995)).
The human CAN9 protein (hCAN9; Genbank Accession No: gi|5729758; SEQ ID NO:12) is a calpain that is expressed predominantly in stomach and small intestine and is thought to have specialized functions in the digestive tract, and be associated with gastric cancer. (Biol. Chem. 379 (2), 175-183 (1998); and Jpn. J. Cancer Res. 91 (5), 459-463 (2000)).
As described above, the CAN-12 polypeptide was found to have significant sequence homology with calpains, particularly members of the m-calpain family. A conserved peptide signature of Qx3(G,E)xC(Y,W)x2(S,T,A,G,C)(S,T,A,G,C,V) Qx{3}(G)xC(W)x{2}(A)(A) (referred to as a thiol (cysteine) protease active site domain) common to most calpain family members is found in the protein sequence of CAN-12 from amino acid 90 to amino acid 111 of SEQ ID NO:24 (FIGS. 1A-E). Protein threading and molecular modeling of CAN-12 suggests that CAN-12 has a structural fold similar to representative m-calpains. Moreover, the structural and threading alignments of the present invention suggest that amino acids 101 (“C”), 253 (“H”), and 277 (“N”) of SEQ ID NO:24 (FIGS. 1A-E) may represent the catalytic amino acids within the active site domain. Thus, based upon the sequence and structural homology to known calpains, particularly the presence of the thiol cysteine protease active site domain, the novel CAN-12 is believed to represent a novel human calpain.
In an alternative embodiment, the following polypeptide is encompassed by the present invention: MSLWPPFRCRWKLAPRYSRRASPQQPQQDFEALLAECLRNGCLFEDTSFPATLSSIGS GSLLQKLPPRLQWKRPPELHSNPQFYFARRRRLDLCQGIVGDCWFLAALQALALHQ DILSRVVPLNQSFTEKYAGIFRFWFWHYGNWVPVVIDDRLPVNEAGQLVFVSSTYKN LFWGALLEKAYAKLSGSYEDLQSGQVSEALVDFTGGVTMTINLAEAHGNLWDILIE ATYNRTLIGCQTHSGKILENGLVEGHAYTLTGIRKVTCKHRPEYLVKLRNPWGKVE WKGDWSDSSSKWELLSPKEKILLLRKDNDGEFWMTLQDFKTHFVLLVICKLTPGLLS QEAAQKWTYTMREGRWEKRSTAGGQRQLLQDTFWKNPQFLLSVWRPEEGRRSLRP CSVLVSLLQKPRHRCRKRKPLLAIGFYLYRMNK (SEQ ID NO:24). Polynucleotides encoding these polypeptides are also provided (SEQ ID NO:23).
In confirmation of the strong homology to known calpains, the CAN-12 polypeptide was determined to have several conserved catalytic amino acids at amino acid C101, H253, and N277 of SEQ ID NO:24 (FIGS. 1A-E). As discussed more particularly herein, calpains are a group of structurally diverse, high molecular weight (400 to 500 amino acids) proteins that have a catalytic cysteine amino acid and one or more calcium binding domains. Despite the structural heterogeneity, calpains share some well defmed structural-functional characteristics, particularly in their active site domains.
In preferred embodiments, the CAN-12 polypeptide of the present invention is directed to a polypeptide having structural similarity to calpains.
Based upon the strong homology to members of the calpain family, the CAN-12 polypeptide is expected to share at least some biological activity with calpains, preferably with m-calpain family members, and more preferable to the large subunits of m-calpain family members, in addition to other calpains and calpain subunits referenced herein and/or otherwise known in the art.
Expression profiling designed to measure the steady state mRNA levels encoding the CAN-12 polypeptide showed predominately high expression levels in spinal cord tissue; significantly high expression in lymph node and thymus, and to a lesser extent, in spleen tissue (See FIG. 4).
Expanded analysis of CAN-12 expression levels by TaqMan™ quantitative PCR (see FIG. 6) confirmed that the CAN-12 polypeptide is expressed in the lymph gland. However, the TaqMan™ quantitative PCR determined that the CAN-12 polypeptide is primarily expressed in the eosophagus. In fact, with the exception of the lymph gland, the steady state mRNA level of CAN-12 was approximately 2700 times higher in the esophagus than in all other tissues tested. These data suggest modulators of the CAN-12 polynucleotides and polypeptides may be useful for the treatment, detection, and/or amelioration of the following, non-limiting diseases and disorders associated with the eosphagus: dysphagia, cricoharyngeal incoordination, esophageal carcinoma, esophageal webs, achalasia, symptomatic diffuse esophageal spasm, gastroesophageal reflux, and/or corrosive esophagitis.
The polynucleotides encoding the CAN-12 polypeptide of the present invention were used to determine the chromosomal localization of the calpain12 gene. Polynuclelotides corresponding to CAN-12 (SEQ IID NO:1) were shown to localize to chromosome 2, specifically 2p16-p21. The comparison of the chromosomal location of the calpain12 gene with the location of chromosomal regions which have been shown to be associated with specific diseases or conditions, e.g. by linkage analysis, can be indicative of diseases in which calpain12 may play a role. Interestingly, a whole-genome linkage scan in multiple sclerosis families (Ebers et al. A full genome search in multiple sclerosis. Nature Genet. 13: 472-476, 1996.) identified 5 susceptibility loci on chromosomes 2, 3, 5, 11, and X. In particular, an association was identified with marker D2S119 on chromosome 2 and MS. The localization of the D2S119 marker was further delimeated to 2p16-p21 based on a radiation hybrid linkage map retrieved from an online query at NCBI web site: http:/www.ncbi.nlm.nih.gov/genome/sts/. Since the map of calpain 12 and the susceptibility marker D2S119 overlaps, it is reasonable to postulate that calpain 12 may contribute to MS. Furthermore, the transcription profile of calpain12 indicated a prominent expression in spinal cord, and implication of calpains in MS has been suggested (Shields D C et al. A putative mechanism of demyelination in multiple sclerosis by a proteolytic enzyme, calpain. Proc Natl Acad Sci USA. 96:11486-91.1999).
The CAN-12 polynucleotides and polypeptides of the present invention, including agonists, antagonists, and/or fragments thereof, have uses that include modulating cellular adhesion events, cellular proliferation, and inflammation, in various cells, tissues, and organisms, and particularly in mammalian spinal cord tissue, lymph node, thymus, and spleen tissue, preferably human tissue. CAN-12 polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, may-be useful in diagnosing, treating, prognosing, and/or preventing neural, immune, hematopoietic, and/or proliferative diseases or disorders.
The strong homology to human calpains, particularly m-calpains, combined with the predominate localized expression in esophagus tissue suggests the CAN-12 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing gastrointestinal diseases, particularly esophageal diseases and/or disorders which include the following non-limiting examples: aberrant transport of food bolus from the mouth to the stomach, aberrant prevention of retrograde flow of gastrointestinal contents, aberrant esophageal peristaltic contractions, pyrosis, painful swallowing, reflux esophagitis, esophageal motility disorders, esophageal spasms, diffuse esophageal spasm, atypical chest pain, regurgitation, oropharyngeal paralysis, nasal regurgitation, dysphagia, cricopharyngeal bar, globus pharyngeus, achalasia, motor disorders of the esophageal smooth muscle, scleroderma esophagus, gastroesophageal reflux disease (GERD), esophagitis, Barrett's esophagus, viral esophagitis, Herpes simplex virus mediated viral esophagitis, Varicella-zoster virus mediated viral esophagitis, Cytomegalovirus mediated viral esophagitis, bacterial esophagitis, Lactobacillus mediated bacterial esophagitis, Candida mediated esophagitis, radiation esophagitis, corrosive esophagitis, pill-induced esophagitis, esophagitis associated with mucocutaneous and systemic diseases, diverticula, lower esophageal mucosal ring, lower esophageal muscular ring, hiatal hernia, paraesophageal hernia, esophageal rupture, and/or Mallory-Weiss Syndrome.
Although calpains are typically associated primarily with neurogenerative conditions, their association in gastrointenstinal tissues has precedence. For example, the human CAN9 protein (hCAN9; Genbank Accession No: gi|5729758; SEQ ID NO:12) is predominately expressed in the stomach and small intestine and is thought to be associated with gastric cancers.
The strong homology to human calpains, particularly m-calpains, combined with the predominate expression in spinal cord tissue suggests the CAN-12 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing neural diseases, neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Neurological Diseases”, “Regeneration” and “Hyperproliferative Disorders” sections below, in the Examples, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Alternatively, the strong homology to human calpains, particularly m-calpains, combined with the localized expression in lymph node, thymus, and spleen tissue suggests the CAN-12 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, ameliorating, and/or preventing immune diseases and/or disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, and elsewhere herein. Briefly, the strong expression in immune tissue indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. The CAN-12 polypeptide may also be useful as a preventative agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product may be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
Moreover, the protein would be useful in the detection, treatment, and/or prevention of a variety of vascular disorders and conditions, which include, but are not limited to miscrovascular disease, vascular leak syndrome, aneurysm, stroke, embolism, thrombosis, coronary artery disease, arteriosclerosis, and/or atherosclerosis. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
In addition, antagonists of the CAN-12 polynucleotides and polypeptides may have uses that include diagnosing, treating, prognosing, and/or preventing diseases or disorders related to hyper calpain activity, which may include immune and/or proliferative diseases or disorders, particularly thrombosis, embolism, and other blood disorders. Therapeutic and/or pharmaceutical compositions comprising the CAN-12 polypeptides may be formulated to comprise heparin.
In addition, antagonists of the CAN-12 polynucleotides and polypeptides may have uses that include diagnosing, treating, ameliorating, prognosing, and/or preventing diseases or disorders related to hyper calpain activity, which may include neuronal excitotoxicity, ischemic stroke, hemoragic stroke, hypoxic stress, trauma, cell destruction, spinal cord injury following trauma, degeneration of vulnerable hippocampal neurons after ischemia, reovirus-induced apoptosis, viral-induced induced myocarditis, acute and chronic inflammation, cataract formation, multiple sclerosis, demylenating disorders, acoustic trauma, hearing loss caused by noise, neuronal damage, cardiac ischemic damage, and/or hepatocyte necrosis during and following anoxia
CAN-12 polynucleotides and polypeptides, including fragments and/or antagonsists thereof, may have uses which include modulating development, differentiation, cellular transformation in response to cell signaling, cell-cell and/or cell-extracellular matrix interactions, clustering of the integrin receptor aIIb3, modulating in long term potentiation (memory), modulating neurite outgrowth, modulating cortical lamination activation of protein kinases and phosphatases, remodeling and disassembling the cytoskeleton, cell cycle modulation, in addition, to ameliorating, preventing, and/or treating limb-girdle muscular dystrophy (LGMD), insulin resistance in diabetics, Alzheimer's disease, Multiple sclerosis, Huntington's disease, Parkinson's disease and amyotrophy.
Moreover, CAN-12 polynucleotides and polypeptides, including fragments and agonists thereof, may have uses which include treating, diagnosing, prognosing, and/or preventing hyperproliferative disorders, particularly of the neural and immune systems. Such disorders may include, for example, cancers, and metastatic conditions.
CAN-12 polynucleotides and polypeptides, including fragments and/or antagonsists thereof, may have uses which include identification of modulators of CAN-12 function including antibodies (for detection or neutralization), naturally-occurring modulators and small molecule modulators. Antibodies to domains (including CAN-12 epitopes provided herein) of the CAN-12 protein could be used as diagnostic agents of inflammatory conditions in patients, are useful in monitoring the activation and presence of cognate proteases, and can be used as a biomarker for the protease involvement in disease states and in the evaluation of inhibitors of the cognate protease in vivo.
CAN-12 polypeptides and polynucleotides are useful for diagnosing diseases related to over or under expression of CAN-12 proteins by identifying mutations in the CAN-12 gene using CAN-12 probes, or determining CAN-12 protein or mRNA expression levels. CAN-12 polypeptides are also useful for screening for compounds, which affect activity of the protein. Diseases that can be treated with CAN-12 include, the following, non-limiting examples: neuro-regeneration, neuropathic pain, obesity, anorexia, HIV infections, cancers, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, osteoporosis, angina pectoris, myocardial infarction, psychotic, immune, metabolic, cardiovascular, and neurological disorders.
The predominate expression in neural tissues, combined with the significant expression in a number of other tissues, suggests the CAN-12 polynucleotide and polypeptide of the present invention may be involved in modulating nerve invasion, innervation, nerve maintenance, and potentially myeline sheath maintenance and integrity.
The CAN-12 polynucleotides and polypeptides, including fragments and antagonists thereof, may have uses which include detecting, diagnosing, treating, ameliorating, and/or preventing diseases and disorders of the neural system, particularly Alzheimer's disease, either directly or indirectly, in addition to other neural disorders known in the art or provided in the “Neurological Diseases” section herein, such as modulating nerve invasion, innervation, nerve maintenance, potentially myelin sheath maintenance and integrity, encephalomyelitis, autoimmune encephalomyelitis, human T cell leukemia virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and neuro-inflammatory diseases.
Molecular genetic manipulation of the structure of the active site domain, particularly the predicted catalytic amino acids, and of other functional domains in the calpain family (e.g., active site domain binding pocket) enables the production of calpains with tailor-made activities. Thus, the CAN-12 polypeptides, and fragments thereof, as well as any homologous product resulting from genetic manipulation of the structure, are useful for NMR-based design of modulators of CAN-12 biological activity, and calpains, in general.
CAN-12 polypeptides and polynucleotides have additional uses which include diagnosing diseases related to the over and/or under expression of CAN-12 by identifying mutations in the CAN-12 gene by using CAN-12 sequences as probes or by determining CAN-12 protein or mRNA expression levels. CAN-12 polypeptides may be useful for screening compounds that affect the activity of the protein. CAN-12 peptides can also be used for the generation of specific antibodies and as bait in yeast two hybrid screens to find proteins the specifically interact with CAN-12 (described elsewhere herein).
The CAN-12 polynucleotides and polypeptides, including fragments and agonists thereof, may have uses which include detecting, diagnosing, treating, ameliorating, and/or preventing metabolic diseases and disorders, such as diabetes. Moreover, expressed human CAN-12 may be useful in the detection of patients susceptible to diabetes. Also paradigms that would simulate intracellular CAN-12 activity would be useful in treating diabetes.
The CAN-12 polynucleotides and polypeptides, including fragments thereof, may have uses which include identifying inhibitors of intracellular calpain inhibitors (calpastatins) leading to an effective increase in calpain activity.
Various approaches to detect alterations or allelic variants at the genomic or mRNA level of CAN-12, could be used as a diagnostic for identifying MS patients, or individuals susceptible to have MS. It is likely that the calpain12 gene comprises polymorphic sites (i.e. SNPs), with specific alleles which may be associated with MS or other neurodegenerative disorders, or associated with an increased likelihood of developing these diseases. Therefore, the invention provides the calpain 12 sequence that can be used to design specific primers for the identification of polymorphisms or mutations in calpain12 of patients affected with MS. The presence of a specific allele variant, such as a SNP allele or SNPs haplotype that renders the subject carrying it more susceptible to develop MS or other related diseases could be identified (e.g. a variant in the can12 promoter region that increased transcript levels of can12, or mutations in the coding sequence that increased the stability or half-life of the can12 protein). Other methods such as Northern-blot analysis could be performed to measure transcript levels using a can12 cDNA probe derived from the sequence of the invention.
Although it is believed the encoded polypeptide may share at least some biological activities with human calpains (particularly m-calpains), a number of methods of determining the exact biological function of this clone are either known in the art or are described elsewhere herein. Briefly, the function of this clone may be determined by applying microarray methodology. Nucleic acids corresponding to the CAN-12 polynucleotides, in addition to, other clones of the present invention, may be arrayed on microchips for expression profiling. Depending on which polynucleotide probe is used to hybridize to the slides, a change in expression of a specific gene may provide additional insight into the function of this gene based upon the conditions being studied. For example, an observed increase or decrease in expression levels when the polynucleotide probe used comes from diseased neural tissue, as compared to, normal tissue might indicate a function in modulating neural function, for example. In the case of CAN-12, spinal cord, lymph node, thymus, and/or spleen tissue should be used to extract RNA to prepare the probe.
In addition, the function of the protein may be assessed by applying quantitative PCR methodology, for example. Real time quantitative PCR would provide the capability of following the expression of the CAN-12 gene throughout development, for example. Quantitative PCR methodology requires only a nominal amount of tissue from each developmentally important step is needed to perform such experiments. Therefore, the application of quantitative PCR methodology to refining the biological function of this polypeptide is encompassed by the present invention. In the case of CAN-12, a disease correlation related to CAN-12 may be made by comparing the mRNA expression level of CAN-12 in normal tissue, as compared to diseased tissue (particularly diseased tissue isolated from the following: esophagus, spinal cord, lymph node, thymus, and/or spleen tissue). Significantly higher or lower levels of CAN-12 expression in the diseased tissue may suggest CAN-12 plays a role in disease progression, and antagonists against CAN-12 polypeptides would be useful therapeutically in treating, preventing, and/or ameliorating the disease. Alternatively, significantly higher or lower levels of CAN-12 expression in the diseased tissue may suggest CAN-12 plays a defensive role against disease progression, and agonists of CAN-12 polypeptides may be useful therapeutically in treating, preventing, and/or ameliorating the disease. Also encompassed by the present invention are quantitative PCR probes corresponding to the polynucleotide sequence provided as SEQ ID NO:1 (FIGS. 1A-E).
The function of the protein may also be assessed through complementation assays in yeast. For example, in the case of the CAN-12, transforming yeast deficient in calpain activity, particularly m-calpain activity, and assessing their ability to grow would provide convincing evidence the CAN-12 polypeptide has calpain activity, and possibly m-calpain activity. Additional assay conditions and methods that may be used in assessing the function of the polynucleotides and polypeptides of the present invention are known in the art, some of which are disclosed elsewhere herein.
Alternatively, the biological function of the encoded polypeptide may be determined by disrupting a homologue of this polypeptide in Mice and/or rats and observing the resulting phenotype. Such knock-out experiments are known in the art, some of which are disclosed elsewhere herein.
Moreover, the biological function of this polypeptide may be determined by the application of antisense and/or sense methodology and the resulting generation of transgenic mice and/or rats. Expressing a particular gene in either sense or antisense orientation in a transgenic mouse or rat could lead to respectively higher or lower expression levels of that particular gene. Altering the endogenous expression levels of a gene can lead to the observation of a particular phenotype that can then be used to derive indications on the function of the gene. The gene can be either over-expressed or under expressed in every cell of the organism at all times using a strong ubiquitous promoter, or it could be expressed in one or more discrete parts of the organism using a well characterized tissue-specific promoter (e.g., an esophagus, spinal cord, lymph node, thymus, or spleen specific promoter), or it can be expressed at a specified time of development using an inducible and/or a developmentally regulated promoter.
In the case of CAN-12 transgenic mice or rats, if no phenotype is apparent in normal growth conditions, observing the organism under diseased conditions (neural, immune, hematopoietic diseases or disorders, cancers, etc.) may lead to understanding the function of the gene. Therefore, the application of antisense and/or sense methodology to the creation of transgenic mice or rats to refine the biological function of the polypeptide is encompassed by the present invention.
In preferred embodiments, the following N-terminal CAN-12 deletion polypeptides are encompassed by the present invention: M1-L581, S2-L581, L3-L581, W4-L581, P5-L581, P6-L581, F7-L581, R8-L581, C9-L581, R10-L581, W11-L581, K12-L581, L13-L581, A14-L581, P15-L581, R16-L581, Y17-L581, S18-L581, R19-L581, R20-L581, A21-L581, S22-L581, P23-L581, Q24-L581, Q25-L581, P26-L581, Q27-L581, Q28-L581, D29-L581, F30-L581, E31-L581, A32-L581, L33-L581, L34-L581, A35-L581, E36-L581, C37-L581, L38-L581, R39-L581, N40-L581, G41-L581, C42-L581, L43-L581, F44-L581, E45-L581, D46-L581, T47-L581, S48-L581, F49-L581, P50-L581, A51-L581, T52-L581, L53-L581, S54-L581, S55-L581, I56-L581, G57-L581, S58-L581, G59-L581, S60-L581, L61-L581, L62-L581, Q63-L581, K64-L581, L65-L581, P66-L581, P67-L581, R68-L581, L69-L581, Q70-L581, W71-L581, K72-L581, R73-L581, P74-L581, P75-L581, E76-L581, L77-L581, H78-L581, S79-L581, N80-L581, P81-L581, Q82-L581, F83-L581, Y84-L581, F85-L581, A86-L581, K87-L581, A88-L581, K89-L581, R90-L581, L91-L581, D92-L581, L93-L581, C94-L581, Q95-L581, G96-L581, I97-L581, V98-L581, G99-L581, D100-L581, C101-L581, W102-L581, F103-L581, L104-L581, A105-L581, A106-L581, L107-L581, Q108-L581, A109-L581, L110-L581, A111-L581, L112-L581, H113-L581, Q114-L581, D115-L581, I116-L581, L117-L581, S118-L581, R119-L581, V120-L581, V121-L581, P122-L581, L123-L581, N124-L581, Q125-L581, S126-L581, F127-L581, T128-L581, E129-L581, K130-L581, Y131-L581, A132-L581, G133-L581, I134-L581, F135-L581, R136-L581, F137-L581, W138-L581, F139-L581, W140-L581, H141-L581, Y142-L581, G143-L581, N144-L581, W145-L581, V146-L581, P147-L581, V148-L581, V149-L581, I150-L581, D151-L581, D152-L581, R153-L581, L154-L581, P155-L581, V156-L581, N157-L581, E158-L581, A159-L581, G160-L581, Q161-L581, L162-L581, V163-L581, F164-L581, V165-L581, S166-L581, S167-L581, T168-L581, Y169-L581, K170-L581, N171-L581, L172-L581, F173-L581, W174-L581, G175-L581, A176-L581, L177-L581, L178-L581, E179-L581, K180-L581, A181-L581, Y182-L581, A183-L581, K184-L581, L185-L581, S186-L581, G187-L581, S188-L581, Y189-L581, E190-L581, D191-L581, L192-L581, Q193-L581, S194-L581, G195-L581, Q196-L581, V197-L581, S198-L581, E199-L581, A200-L581, L201-L581, V202-L581, D203-L581, F204-L581, T205-L581, G206-L581, G207-L581, V208-L581, T209-L581, M210-L581, T211-L581, I212-L581, N213-L581, L214-L581, A215-L581, E216-L581, A217-L581, H218-L581, G219-L581, N220-L581, L221-L581, W222-L581, D223-L581, I224-L581, L225-L581, I226-L581, E227-L581, A228-L581, T229-L581, Y230-L581, N231-L581, R232-L581, T233-L581, L234-L581, I235-L581, G236-L581, C237-L581, Q238-L581, T239-L581, H240-L581, S241-L581, G242-L581, K243-L581, I244-L581, L245-L581, E246-L581, N247-L581, G248-L581, L249-L581, V250-L581, E251-L581, G252-L581, H253-L581, A254-L581, Y255-L581, T256-L581, L257-L581, T258-L581, G259-L581, I260-L581, R261-L581, K262-L581, V263-L581, T264-L581, C265-L581, K266-L581, H267-L581, R268-L581, P269-L581, E270-L581, Y271-L581, L272-L581, V273-L581, K274-L581, L275-L581, R276-L581, N277-L581, P278-L581, W279-L581, G280-L581, K281-L581, V282-L581, E283-L581, W284-L581, K285-L581, G286-L581, D287-L581, W288-L581, S289-L581, D290-L581, S291-L581, S292-L581, S293-L581, K294-L581, W295-L581, E296-L581, L297-L581, L298-L581, S299-L581, P300-L581, K301-L581, E302-L581, K303-L581, I304-L581, L305-L581, L306-L581, L307-L581, R308-L581, K309-L581, D310-L581, N311-L581, D312-L581-L581, G313-L581, E314-L581, F315-L581, W316-L581, M317-L581, T318-L581, L319-L581-L581, Q320-L581, D321-L581, F322-L581, K323-L581, T324-L581, H325-L581, F326-L581, V327-L581, L328-L581, L329-L581, V330-L581, I331-L581, C332-L581, K333-L581, L334-L581, T335-L581, P336-L581, G337-L581, L338-L581, L339-L581, S340-L581, Q341-L581, E342-L581, A343-L581, A344-L581, Q345-L581, K346-L581, W347-L581, T348-L581, Y349-L581, T350-L581, M351-L581, R352-L581, E353-L581, G354-L581, R355-L581, W356-L581, E357-L581, K358-L581, R359-L581, S360-L581, T361-L581, A362-L581, G363-L581, G364-L581, Q365-L581, R366-L581, Q367-L581, L368-L581, L369-L581, Q370-L581, D371-L581, T372-L581, F373-L581, W374-L581, K375-L581, N376-L581, P377-L581, Q378-L581, F379-L581, L380-L581, L381-L581, S382-L581, V383-L581, W384-L581, R385-L581, P386-L581, E387-L581, E388-L581, G389-L581, R390-L581, R391-L581, S392-L581, L393-L581, R394-L581, P395-L581, C396-L581, S397-L581, V398-L581, L399-L581, V400-L581, S401-L581, L402-L581, L403-L581, Q404-L581, K405-L581, P406-L581, R407-L581, H408-L581, R409-L581, C410-L581, R411-L581, K412-L581, R413-L581, K414-L581, P415-L581, L416-L581, L417-L581, A418-L581, I419-L581, G420-L581, F421-L581, Y422-L581, L423-L581, Y424-L581, R425-L581, M426-L581, N427-L581, K428-L581, M429-L581, T430-L581, W431-L581, S432-L581, S433-L581, L434-L581, G435-L581, S436-L581, R437-L581, Q438-L581, P439-L581, F440-L581, F441-L581, S442-L581, L443-L581, E444-L581, A445-L581, C446-L581, Q447-L581, G448-L581, I449-L581, L450-L581, A451-L581, L452-L581, L453-L581, D454-L581, L455-L581, N456-L581, A457-L581, S458-L581, G459-L581, T460-L581, M461-L581, S462-L581, I463-L581, Q464-L581, E465-L581, F466-L581, R467-L581, D468-L581, L469-L581, W470-L581, K471-L581, Q472-L581, L473-L581, K474-L581, L475-L581, S476-L581, Q477-L581, K478-L581, V479-L581, F480-L581, H481-L581, K482-L581, Q483-L581, D484-L581, R485-L581, G486-L581, S487-L581, G488-L581, Y489-L581, L490-L581, N491-L581, W492-L581, E493-L581, Q494-L581, L495-L581, H496-L581, A497-L581, A498-L581, M499-L581, R500-L581, E501-L581, A502-L581, G503-L581, R504-L581, H505-L581, R506-L581, K507-L581, S508-L581, W509-L581, S510-L581, C511-L581, G512-L581, H513-L581, T514-L581, R515-L581, A516-L581, G517-L581, C518-L581, T519-L581, L520-L581, I521-L581, R522-L581, Q523-L581, R524-L581, R525-L581, G526-L581, D527-L581, V528-L581, W529-L581, H530-L581, A531-L581, E532-L581, V533-L581, T534-L581, L535-L581, I536-L581, R537-L581, S538-L581, V539-L581, T540-L581, L541-L581, K542-L581, D543-L581, V544-L581, D545-L581, L546-L581, Q547-L581, S548-L581, T549-L581, P550-L581, T551-L581, F552-L581, F553-L581, M554-L581, I555-L581, V556-L581, P557-L581, V558-L581, I559-L581, L560-L581, A561-L581, N562-L581, I563-L581, D564-L581, G565-L581, G566-L581, V567-L581, A568-L581, H569-L581, S570-L581, T571-L581, S572-L581, Y573-L581, L574-L581, and/or I575-L581 of SEQ ID NO:2. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal CAN-12 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein. The invention specifically encompasses the N-terminal deletions from amino acid 1 to amino acid 421 of SEQ ID NO:2.
In preferred embodiments, the following C-terminal CAN-12 deletion polypeptides are encompassed by the present invention: M1-L581, M1-L580, M1-T579, M1-T578, M1-N577, M1-F576, M1-I575, M1-L574, M1-Y573, M1-S572, M1-T571, M1-S570, M1-H569, M1-A568, M1-V567, M1-G566, M1-G565, M1-D564, M1-I563, M1-N562, M1-A561, M1-L560, M1-I559, M1-V558, M1-P557, M1-V556, M1-I555, M1-M554, M1-F553, M1-F552, M1-T551, M1-P550, M1-T549, M1-S548, M1-Q547, M1-L546, M1-D545, M1-V544, M1-D543, M1-K542, M1-L541, M1-T540, M1-V539, M1-S538, M1-R537, M1-I536, M1-L535, M1-T534, M1-V533, M1-E532, M1-A531, M1-H530, M1-W529, M1-V528, M1-D527, M1-G526, M1-R525, M1-R524, M1-Q523, M1-R522, M1-I521, M1-L520, M1-T519, M1-C518, M1-G517, M1-A516, M1-R515, M1-T514, M1-H513, M1-G512, M1-C511, M1-S510, M1-W509, M1-S508, M1-K507, M1-R506, M1-H505, M1-R504, M1-G503, M1-A502, M1-E501, M1-R500, M1-M499, M1-A498, M1-A497, M1-H496, M1-L495, M1-Q494, M1-E493, M1-W492, M1-N491, M1-L490, M1-Y489, M1-G488, M1-S487, M1-G486, M1-R485, M1-D484, M1-Q483, M1-K482, M1-H481, M1-F480, M1-V479, M1-K478, M1-Q477, M1-S476, M1-L475, M1-K474, M1-L473, M1-Q472, M1-K471, M1-W470, M1-L469, M1-D468, M1-R467, M1-F466, M1-E465, M1-Q464, M1-I463, M1-S462, M1-M461, M1-T460, M1-G459, M1-S458, M1-A457, M1-N456, M1-L455, M1-D454, M1-L453, M1-L452, M1-A451, M1-L450, M1-I449, M1-G448, M1-Q447, M1-C446, M1-A445, M1-E444, M1-L443, M1-S442, M1-F441, M1-F440, M1-P439, M1-Q438, M1-R437, M1-S436, M1-G435, M1-L434, M1-S433, M1-S432, M1-W431, M1-T430, M1-M429, M1-K428, M1-N427, M1-M426, M1-R425, M1-Y424, M1-L423, M1-Y422, M1-F421, M1-G420, M1-I419, M1-A418, M1-L417, M1-L416, M1-P415, M1-K414, M1-R413, M1-K412, M1-R411, M1-C410, M1-R409, M1-H408, M1-R407, M1-P406, M1-K405, M1-Q404, M1-L403, M1-L402, M1-S401, M1-V400, M1-L399, M1-V398, M1-S397, M1-C396, M1-P395, M1-R394, M1-L393, M1-S392, M1-R391, M1-R390, M1-G389, M1-E388, M1-E387, M1-P386, M1-R385, M1-W384, M1-V383, M1-S382, M1-L381, M1-L380, M1-F379, M1-Q378, M1-P377, M1-N376, M1-K375, M1-W374, M1-F373, M1-T372, M1-D371, M1-Q370, M1-L369, M1-L368, M1-Q367, M1-R366, M1-Q365, M1-G364, M1-G363, M1-A362, M1-T361, M1-S360, M1-R359, M1-K358, M1-E357, M1-W356, M1-R355, M1-G354, M1-E353, M1-R352, M1-M351, M1-T350, M1-Y349, M1-T348, M1-W347, M1-K346, M1-Q345, M1-A344, M1-A343, M1-E342, M1-Q341, M1-S340, M1-L339, M1-L338, M1-G337, M1-P336, M1-T335, M1-L334, M1-K333, M1-C332, M1-1331, M1-V330, M1-L329, M1-L328, M1-V327, M1-F326, M1-H325, M1-T324, M1-K323, M1-F322, M1-D321, M1-Q320, M1-L319, M1-T318, M1-M317, M1-W316, M1-F315, M1-E314, M1-G313, M1-D312, M1-N311, M1-D310, M1-K309, M1-R308, M1-L307, M1-L306, M1-L305, M1-I304, M1-K303, M1-E302, M1-K301, M1-P300, M1-S299, M1-L298, M1-L297, M1-E296, M1-W295, M1-K294, M1-S293, M1-S292, M1-S291, M1-D290, M1-S289, M1-W288, M1-D287, M1-G286, M1-K285, M1-W284, M1-E283, M1-V282, M1-K281, M1-G280, M1-W279, M1-P278, M1-N277, M1-R276, M1-L275, M1-K274, M1-V273, M1-L272, M1-Y271, M1-E270, M1-P269, M1-R268, M1-H267, M1-K266, M1-C265, M1-T264, M1-V263, M1-K262, M1-R261, M1-I260, M1-G259, M1-T258, M1-L257, M1-T256, M1-Y255, M1-A254, M1-H253, M1-G252, M1-E251, M1-V250, M1-L249, M1-G248, M1-N247, M1-E246, M1-L245, M1-I244, M1-K243, M1-G242, M1-S241, M1-H240, M1-T239, M1-Q238, M1-C237, M1-G236, M1-I235, M1-L234, M1-T233, M1-R232, M1-N231, M1-Y230, M1-T229, M1-A228, M1-E227, M1-I226, M1-L225, M1-I224, M1-D223, M1-W222, M1-L221, M1-N220, M1-G219, M1-H218, M1-A217, M1-E216, M1-A215, M1-L214, M1-N213, M1-I212, M1-T211, M1-M210, M1-T209, M1-V208, M1-G207, M1-G206, M1-T205, M1-F204, M1-D203, M1-V202, M1-L201, M1-A200, M1-E199, M1-S198, M1-V197, M1-Q196, M1-195, M1-S194, M1-Q193, M1-L192, M1-D191, M1-E190, M1-Y189, M1-S188, M1-G187, M1-S186, M1-L185, M1-K184, M1-A183, M1-Y182, M1-A181, M1-K180, M1-E179, M1-L178, M1-L177, M1-A176, M1-G175, M1-W174, M1-F173, M1-L172, M1-N171, M1-K170, M1-Y169, M1-T168, M1-S167, M1-S166, M1-V165, M1-F164, M1-V163, M1-L162, M1-Q161, M1-G160, M1-A159, M1-E158, M1-N157, M1-V156, M1-P155, M1-L154, M1-R153, M1-D152, M1-D151, M1-I150, M1-V149, M1-V148, M1-P147, M1-V146, M1-W145, M1-N144, M1-G143, M1-Y142, M1-H141, M1-W140, M1-F139, M1-W138, M1-F137, M1-R136, M1-F135, M1-I134, M1-G133, M1-A132, M1-Y131, M1-K130, M1-E129, M1-T128, M1-F127, M1-S126, M1-Q125, M1-N124, M1-L123, M1-P122, M1-V121, M1-V120, M1-R119, M1-S118, M1-L117, M1-I116, M1-D115, M1-Q114, M1-H113, M1-L112, M1-A111, M1-L110, M1-A109, M1-Q108, M1-L107, M1-A106, M1-A105, M1-L104, M1-F103, M1-W102, M1-C101, M1-D100, M1-G99, M1-V98, M1-I97, M1-G96, M1-Q95, M1-C94, M1-L93, M1-D92, M1-L91, M1-R90, M1-K89, M1-A88, M1-K87, M1-A86, M1-F85, M1-Y84, M1-F83, M1-Q82, M1-P81, M1-N80, M1-S79, M1-H78, M1-L77, M1-E76, M1-P75, M1-P74, M1-R73, M1-K72, M1-W71, M1-Q70, M1-L69, M1-R68, M1-P67, M1-P66, M1-L65, M1-K64, M1-Q63, M1-L62, M1-L61, M1-S60, M1-G59, M1-S58, M1-G57, M1-I56, M1-S55, M1-S54, M1-L53, M1-T52, M1-A51, M1-P50, M1-F49, M1-S48, M1-T47, M1-D46, M1-E45, M1-F44, M1-L43, M1-C42, M1-G41, M1-N40, M1-R39, M1-L38, M1-C37, M1-E36, M1-A35, M1-L34, M1-L33, M1-A32, M1-E31, M1-F30, M1-D29, M1-Q28, M1-Q27, M1-P26, M1-Q25, M1-Q24, M1-P23, M1-S22, M1-A21, M1-R20, M1-R19, M1-S18, M1-Y17, M1-R16, M1-P15, M1-A14, M1-L13, M1-K12, M1-W11, M1-R10, M1-C9, M1-R8, and/or M1-F7 of SEQ ID NO:2. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal CAN-12 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein. The invention specifically encompasses the C-terminal deletions from amino acid 428 to amino acid 7 of SEQ ID NO:2.
Alternatively, preferred polypeptides of the present invention may comprise polypeptide sequences corresponding to, for example, internal regions of the CAN-12 polypeptide (e.g., any combination of both N— and C-terminal CAN-12 polypeptide deletions) of SEQ ID NO:2. For example, internal regions could be defined by the equation: amino acid NX to amino acid CX, wherein NX refers to any N-terminal deletion polypeptide amino acid of CAN-12 (SEQ ID NO:2), and where CX refers to any C-terminal deletion polypeptide amino acid of CAN-12 (SEQ ID NO:2). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.
The present invention also encompasses immunogenic and/or antigenic epitopes of the CAN-12 polypeptide.
The CAN-12 polypeptides of the present invention were determined to comprise several phosphorylation sites based upon the Motif algorithm (Genetics Computer Group, Inc.). The phosphorylation of such sites may regulate some biological activity of the CAN-12 polypeptide. For example, phosphorylation at specific sites may be involved in regulating the proteins ability to associate or bind to other molecules (e.g., proteins, ligands, substrates, DNA, etc.). In the present case, phosphorylation may modulate the ability of the CAN-12 polypeptide to associate with other polypeptides, particularly the serine protease substrate for CAN-12, or its ability to modulate serine protease function.
The CAN-12 polypeptide was predicted to comprise eleven PKC phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). In vivo, protein kinase C exhibits a preference for the phosphorylation of serine or threonine residues. The PKC phosphorylation sites have the following consensus pattern: [ST]-x-[RK], where S or T represents the site of phosphorylation and ‘x’ an intervening amino acid residue. Additional information regarding PKC phosphorylation sites can be found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem. 161:177-184(1986), and Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem. 260:12492-12499(1985); which are hereby incorporated by reference herein.
In preferred embodiments, the following PKC phosphorylation site polypeptides are encompassed by the present invention: LAPRYSRRASPQQ (SEQ ID NO:27), LNQSFTEKYAGIF (SEQ ID NO:28), VFVSSTYKNLFWG (SEQ ID NO:29), GCQTHSGKILENG (SEQ ID NO:30), GIRKVTCKHRPEY (SEQ ID NO:31), DWSDSSSKWELLS (SEQ ID NO:32), KWELLSPKEKILL (SEQ ID NO:33), QKWTYTMREGRWE (SEQ ID NO:34), EEGRRSLRPCSVL (SEQ ID NO:35), KQLKLSQKVFHKQ (SEQ ID NO:36), and/or LIRSVTLKDVDLQ (SEQ ID NO:37). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the CAN-12 PKC phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The CAN-12 polypeptide has been shown to comprise four glycosylation site according to the Motif algorithm (Genetics Computer Group, Inc.). As discussed more specifically herein, protein glycosylation is thought to serve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.
In preferred embodiments, the following asparagine glycosylation site polypeptide is encompassed by the present invention: RVVPLNQSFTEKYA (SEQ ID NO:38), IEATYNRTLIGCQT (SEQ ID NO:39), ALLDLNASGTMSIQ (SEQ ID NO:40), and/or SYLWNTTLL (SEQ ID NO:41). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of the CAN-12 asparagine glycosylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The CAN-12 polypeptide has been shown to comprise one amidation site according to the Motif algorithm (Genetics Computer Group, Inc.). The precursor of hormones and other active peptides which are C-terminally amidated is always directly followed by a glycine residue which provides the amide group, and most often by at least two consecutive basic residues (Arg or Lys) which generally function as an active peptide precursor cleavage site. Although all amino acids can be amidated, neutral hydrophobic residues such as Val or Phe are good substrates, while charged residues such as Asp or Arg are much less reactive. A consensus pattern for amidation sites is the following: x-G-[RK]-[RK], wherein “X” represents the amidation site. Additional information relating to asparagine glycosylation may be found in reference to the following publications, which are hereby incorporated by reference herein: Kreil G., Meth. Enzymol. 106:218-223(1984); and Bradbury A. F., Smyth D. G., Biosci. Rep. 7:907-916(1987).
In preferred embodiments, the following amidation site polypeptide is encompassed by the present invention: VWRPEEGRRSLRPC (SEQ ID NO:42). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this CAN-12 amidation site polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The CAN-12 polypeptide has been shown to comprise one RGD cell attachment site domain according to the Motif algorithm (Genetics Computer Group, Inc.). The sequence Arg-Gly-Asp, found in fibronectin, is crucial for its interaction with its cell surface receptor, an integrin. What has been called the ‘RGD’ tripeptide is also found in the sequences of a number of other proteins, where it has been shown to play a role in cell adhesion. Non-limiting examples of these proteins are the following: some forms of collagens, fibrinogen, vitronectin, von Willebrand factor (VWF), snake disintegrins, and slime mold discoidins. The ‘RGD’ tripeptide is also found in other proteins where it may serve the same purpose. A consensus pattern for RGD cell attachment sites is the following: R-G-D. Additional information relating to RGD cell attachment site domains may be found in reference to the following publications, which are hereby incorporated by reference herein: Ruoslahti E., Pierschbacher M. D., Cell 44:517-518(1986); and d'Souza S. E., Ginsberg M. H., Plow E. F., Trends Biochem. Sci. 16:246-250(1991).
In preferred embodiments, the following RGD cell attachment site domain polypeptide is encompassed by the present invention: LIRQRRGDVWHAE (SEQ ID NO:43). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this RGD cell attachment site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
In confirmation of the CAN-12 polypeptide being a calpain, it has been shown to comprise one EF-hand calcium-binding domain according to the Motif algorithm (Genetics Computer Group, Inc.). Many calcium-binding proteins belong to the same evolutionary family and share a type of calcium-binding domain known as the EF-hand. This type of domain consists of a twelve residue loop flanked on both side by a twelve residue alpha-helical domain. In an EF-hand loop the calcium ion is coordinated in a pentagonal bipyramidal configuration. The six residues involved in the binding are in positions 1, 3, 5, 7, 9 and 12; these residues are denoted by X, Y, Z, —Y, —X and -Z. The invariant Glu or Asp at position 12 provides two oxygens for liganding Ca (bidentate ligand). Several representative proteins containing EF-hand regions are provided below: For each type of protein, the total number of EF-hand regions known or supposed to exist are provided in parenthesis: Aequorin and Renilla luciferin binding protein (LBP) (Ca=3); Alpha actinin (Ca=2); Calbindin (Ca=4); Calcineurin B subunit (protein phosphatase 2B regulatory subunit) (Ca=4); Calcium-binding protein from Streptomyces erythraeus (Ca=3?); Calcium-binding protein from Schistosoma mansoni (Ca=2?); Calcium-binding proteins TCBP-23 and TCBP-25 from Tetrahymena thermophila (Ca=4?); Calcium-dependent protein kinases (CDPK) from plants (Ca=4); Calcium vector protein from amphoxius (Ca=2); Calcyphosin (thyroid protein p24) (Ca=4?); Calmodulin (Ca=4, except in yeast where Ca=3); Calpain small and large chains (Ca=2); Calretinin (Ca=6); Calcyclin (prolactin receptor associated protein) (Ca=2); Caltractin (centrin) (Ca=2 or 4); Cell Division Control protein 31 (gene CDC3 1) from yeast (Ca=2?); Diacylglycerol kinase (EC 2.7.1.107) (DGK) (Ca=2); FAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) from mammals (Ca=1); Fimbrin (plastin) (Ca=2); Flagellar calcium-binding protein (lf8) from Trypanosoma cruzi (Ca=1 or 2); Guanylate cyclase activating protein (GCAP) (Ca=3); Inositol phospholipid-specific phospholipase C isozymes gamma-1 and delta-1 (Ca=2) [10]; Intestinal calcium-binding protein (ICaBPs) (Ca=2); MIF related proteins 8 (MRP-8 or CFAG) and 14 (MRP-14) (Ca=2); Myosin regulatory light chains (Ca=1); Oncomodulin (Ca=2); Osteonectin (basement membrane protein BM40) (SPARC) and proteins that contains an ‘osteonectin’ domain (QR1, matrix glycoprotein SC1) (Ca=1); Parvalbumins alpha and beta (Ca=2); Placental calcium-binding protein (18a2) (nerve growth factor induced protein 42a) (p9k) (Ca=2); Recoverins (visinin, hippocalcin, neurocalcin, S-modulin) (Ca=2 to 3); Reticulocalbin (Ca=4); S-100 protein, alpha and beta chains (Ca=2); Sarcoplasmic calcium-binding protein (SCPs) (Ca=2 to 3); Sea urchin proteins Spec 1 (Ca=4), Spec 2 (Ca=4?), Lps-1 (Ca=8); Serine/threonine protein phosphatase rdgc (EC 3.1.3.16) from Drosophila (Ca=2); Sorcin V19 from hamster (Ca=2); Spectrin alpha chain (Ca=2); Squidulin (optic lobe calcium-binding protein) from squid (Ca=4); and Troponins C; from skeletal muscle (Ca=4), from cardiac muscle (Ca=3), from arthropods and molluscs (Ca=2).

A consensus pattern for EF hand calcium binding domains is the following:


1 2 3 4 5 6 7 8 9 10 12 13
X Y Z -Y -X -Z
D-x-[DNS]-{ILVFYW}-[DENSTG]-[DNQGHRK]-{GP}-[LIVMC]
-[DENQSTAGC]-x(2)-[DE]-[LIVMFYW],

wherein X, Y, Z, —Y, —X, and -Z are as defined above, and wherein “x” represents any amino acid. Amino acid residues within the consensus at positions 1 (X), 3 (Y) and 12 (-Z) are the most conserved. The 6th residue in an EF-hand loop is in most cases a Gly.

Additional information relating to RGD cell attachment site domains may be found in reference to the following publications, which are hereby incorporated by reference herein: Kawasaki H., Kretsinger R. H., Protein Prof. 2:305490(1995); Kretsinger R. H., Cold Spring Harbor Symp. Quant. Biol. 52:499-510(1987); Moncrief N. D., Kretsinger R. H., Goodman M., J. Mol. Evol. 30:522-562(1990); Nakayama S., Moncrief N. D., Kretsinger R. H., J. Mol. Evol. 34:416-448(1992); Heizmann C. W., Hunziker W., Trends Biochem. Sci. 16:98-103(1991); Kligman D., Hilt D. C., Trends Biochem. Sci. 13:437-443(1988); Strynadka N. C. J., James M. N. G., Annu. Rev. Biochem. 58:951-98(1989); Haiech J., Sallantin J., Biochimie 67:555-560(1985); Chauvaux S., Beguin P., Aubert J.-P., Bhat K. M., Gow L. A., Wood T. M., Bairoch A., Biochem. J. 265:261-265(1990); Bairoch A., Cox J. A., FEBS Lett. 269:454-456(1990).
In preferred embodiments, the following EF-hand calcium binding domain polypeptide is encompassed by the present invention: ILALLDLNASGTMSIQEFRDLWK (SEQ ID NO:44). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this EF-hand calcium binding domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
In further confirmation of the CAN-12 polypeptide being a calpain, it has been shown to comprise one eukaryotic thiol (cysteine) protease active site domain according to the Motif algorithm (Genetics Computer Group, Inc.). Eukaryotic thiol proteases (EC 3.4.22.-) are a family of proteolytic enzymes which contain an active site cysteine. Catalysis proceeds through a thioester intermediate and is facilitated by a nearby histidine side chain; an asparagine completes the essential catalytic triad. Non-limiting examples of proteases which are known to belong to this family are provided below: Vertebrate lysosomal cathepsins B (EC 3.4.22.1), H (EC 3.4.22.16), L (EC 3.4.22.15), and S (EC 3.4.22.27); Vertebrate lysosomal dipeptidyl peptidase I (EC 3.4.14.1) (also known as cathepsin C); Vertebrate calpains (EC 3.4.22.17) (Calpains are intracellular calcium-activated thiol protease that contain both a N-terminal catalytic domain and a C-terminal calcium-binding domain; Mammalian cathepsin K, which seems involved in osteoclastic bone resorption; Human cathepsin O; Bleomycin hydrolase (An enzyme that catalyzes the inactivation of the antitumor drug BLM (a glycopeptide); Plant enzymes: barley aleurain (EC 3.4.22.16), EP-B1/B4; kidney bean EP-C1, rice bean SH-EP; kiwi fruit actinidin (EC 3.4.22.14); papaya latex papain (EC 3.4.22.2), chymopapain (EC 3.4.22.6), caricain (EC 3.4.22.30), and proteinase IV (EC 3.4.22.25); pea turgor-responsive protein 15A; pineapple stem bromelain (EC 3.4.22.32); rape COT44; rice oryzain alpha, beta, and gamma; tomato low-temperature induced, Arabidopsis thaliana A494, RD 1 9A and RD2 1 A; House-dust mites allergens DerP 1 and EurM1; Cathepsin B-like proteinases from the worms Caenorhabditis elegans (genes gcp-1, cpr-3, cpr4, cpr-5 and cpr-6), Schistosoma mansoni (antigen SM31) and Japonica (antigen SJ3 1), Haemonchus contortus (genes AC-1 and AC-2), and Ostertagia ostertagi (CP-1 and CP-3); Slime mold cysteine proteinases CP1 and CP2; Cruzipain from Trypanosoma cruzi and brucei; Throphozoite cysteine proteinase (TCP) from various Plasmodium species; Proteases from Leishmania mexicana, Theileria annulata and Theileria parva; Baculoviruses cathepsin-like enzyme (v-cath); Drosophila small optic lobes protein (gene sol), a neuronal protein that contains a calpain-like domain; Yeast thiol protease BLH1/YCP1I/LAP3; and Caenorhabditis elegans hypothetical protein C06G4.2, a calpain-like protein; Two bacterial peptidases are also part of this family—Aminopeptidase C from Lactococcus lactis (gene pepC), and Thiol protease tpr from Porphyromonas gingivalis.
A consensus pattern for eukaryotic thiol (cysteine) protease active site domains is the following: Q-x(3)-[GE]-x-C-[YW]-x(2)-[STAGC]-[STAGCV], wherein C is the active site residue, and “x” represents any amino acid. The residue in position 4 of the pattern is almost always cysteine; the only exceptions are calpains (Leu), bleomycin hydrolase (Ser) and yeast YCP1 (Ser); while the residue in position 5 of the pattern is always Gly except in papaya protease IV where it is Glu.
An additional consensus pattern for eukaryotic thiol (cysteine) protease active site domains is the following: [LIVMGSTAN]-x-H-[GSACE]-[LWM]-x-[LIVMAT](2)-G-x-[GSADNH], wherein H is the active site residue, and “x” represents any amino acid.
An additional consensus pattern for eukaryotic thiol (cysteine) protease active site domains is the following: [FYCH]-[WI]-[LIVT]-x-[KRQAG]-N-[ST]-W-x(3)-[FYW]-G-x(2)-G-[LFYW]-[L MFYG]-x-[LWMF], wherein N is the active site residue, and “x” represents any amino acid.
Additional information relating to for eukaryotic thiol (cysteine) protease active site domains may be found in reference to the following publications, which are hereby incorporated by reference herein: Dufour E., Biochimie 70:1335-1342(1988); Kirschke H., Barrett A. J., Rawlings N. D., Protein Prof. 2:1587-1643(1995); Shi G.-P., Chapman H. A., Bhairi S. M., Deleeuw C., Reddy V. Y., Weiss S. J., FEBS Lett. 357:129-134(1995); Velasco G., Ferrando A. A., Puente X. S., Sanchez L. M., Lopez-Otin C., J. Biol. Chem. 269:27136-27142(1994); Chapot-Chartier M. P., Nardi M., Chopin M. C., Chopin A., Gripon J. C., Appl. Environ. Microbiol. 59:330-333(1993); Higgins D. G., McConnell D. J., Sharp P. M., Nature 340:604-604(1989); Rawlings N. D., Barrett A. J., Meth. Enzymol. 244:461-486(1994), and http://www.expasy.ch/c gi-bin/lists?peptidas.txt, which are hereby incorporated by reference in their entirety herein.
In preferred embodiments, the following for eukaryotic thiol (cysteine) protease active site domain polypeptide is encompassed by the present invention: RLDLCQGIVGDCWFLAALQALA (SEQ ID NO:45). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this for eukaryotic thiol (cysteine) protease active site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The present invention also provides a three-dimensional homology model of the CAN-12 polypeptide (see FIG. 6) representing amino acids 12 to 524 of CAN-12 (SEQ ID NO:2). As referenced herein, SEQ ID NO:2 comprises additional amino acids that are not part of the CAN-12 polypeptide sequence but have been added to the sequence for reference to the CAN-12 splice variants referenced herein. The inclusion of the additional amino acids also helps for a more complete homology model. A three-dimensional homology model can be constructed on the basis of the known structure of a homologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995, Yuan, et al, 1995). The homology model of the CAN-12 polypeptide, corresponding to amino acid residues 12 to 524 of SEQ ID NO:2, was based upon the homologous structure of CAN2, a m-calpain family member (Strobl et al, 2000; hCAN2; Genbank Accession No. gi|7546423; SEQ ID NO:11) and is defined by the set of structural coordinates set forth in Table IV herein.
The CAN-12 homology model of the present invention may provide one basis for designing rational stimulators (agonists) and/or inhibitors (antagonists) of one or more of the biological functions of CAN-12, or of CAN-12 mutants having altered specificity (e.g., molecularly evolved CAN-12 polypeptides, engineered site-specific CAN-12 mutants, CAN-12 allelic variants, etc.).
Homology models are not only useful for designing rational agonists and/or antagonists, but are also useful in predicting the function of a particular polypeptide. The functional predictions from homology models are typically more accurate than the functional attributes derived from traditional polypeptide sequence homology alignments (e.g., CLUSTALW), particularly when the three dimensional structure of a related polypeptide is known (e.g., m-calpain family member CAN2 protein; Genbank Accession No. gi|7546423; SEQ ID NO:11). The increased prediction accuracy is based upon the fact that homology models approximate the three-dimensional structure of a protein, while homology based alignments only take into account the one dimension polypeptide sequence. Since the function of a particular polypeptide is determined not only by its primary, secondary, and tertiary structure, functional assignments derived solely upon homology alignments using the one dimensional protein sequence may be less reliable. A 3-dimensional model can be constructed on the basis of the known structure of a homologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995, Yuan, et al, 1995).
Prior to developing a homology model, those of skill in the art would appreciate that a template of a known protein, or model protein, must first be identified which will be used as a basis for constructing the homology model for the protein of unknown structure (query template). In the case of the CAN-12 polypeptide of the present invention, the model protein template used in constructing the CAN-12 homology model was the m-calpain family member CAN2 protein; Genbank Accession No. gi|7546423; SEQ ID NO:11).
Identifying a template can be accomplished using pairwise alignment of protein sequences using such programs as FASTA (Pearson, et al 1990) and BLAST (Altschul, et al, 1990). In cases where sequence similarity is high (greater than 30%), such pairwise comparison methods may be adequate for identifying an appropriate template. Likewise, multiple sequence alignments or profile-based methods can be used to align a query sequence to an alignment of multiple (structurally and biochemically) related proteins. When the sequence similarity is low, more advanced techniques may be used. Such techniques, include, for example, protein fold recognition (protein threading; Hendlich, et al, 1990), where the compatibility of a particular polypeptide sequence with the 3-dimensional fold of a potential template protein is gauged on the basis of a knowledge-based potential.
Following the initial sequence alignment, the second step would be to optimally align the query template to the model template by manual manipulation and/or by the incorporation of features specific to the polypeptides (e.g., motifs, secondary structure predictions, and allowed conservations). Preferably, the incorporated features are found within both the model and query template.
The third step would be to identify structurally conserved regions that could be used to construct secondary core structure (Sali, et al, 1995). Loops could be added using knowledge-based techniques, and by performing forcefield calculations (Sali, et al, 1995).
The term “structure coordinates” refers to Cartesian coordinates generated from the building of a homology model. In this invention, the homology model of residues 12 to 524 of CAN-12 (SEQ ID NO:2) was derived from generating a sequence alignment with m-calpain (Strobl et al, 2000; hCAN2; Genbank Accession No. gi|7546423; SEQ ID NO:11) using the COMPOSER suite of software within SYBYL6.6 (Tripos Associates, St. Louis, Mo.) and then generating the backbone and side chain conformations. In the original crystal structure (pdb code 1dkv) as well as the crystal structure reported elsewhere (Hosfield et al, 1999), the active site of the enzyme comprising a cysteine, a histidine and an asparagine residue was not “formed”. The helix that contains the active site C101 was altered by moving the helix down one pitch so that the active site geometry could match that found in Papain (pdb code 1b4). This modified structure of human m-calpain was used as the template for construction of the homology model (illustrated in FIG. 6 herein).
The skilled artisan would appreciate that a set of structure coordinates for a protein represents a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates, as emanate from the generation of similar homology models using different alignment templates (i.e., other than the m-calpain (Strobl et al, 2000; hCAN2; Genbank Accession No. gi|7546423; SEQ ID NO:11), and/or using different methods in generating the homology model, will likely have minor effects on the overall shape. Variations in coordinates may also be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth in Table IV could be manipulated by fractionalization of the structure coordinates; integer additions, or integer subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.
Therefore, various computational analyses are necessary to determine whether a template molecule or a portion thereof is sufficiently similar to all or part of a query template (e.g., CAN-12) in order to be considered the same. Such analyses may be carried out in current software applications, such as SYBYL version 6.6 or INSIGHTII (Molecular Simulations Inc., San Diego, Calif.) version 2000 and as described in the accompanying User's Guides.
Using the superimposition tool in the program SYBYL, comparisons can be made between different structures and different conformations of the same structure. The procedure used in SYBYL to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalencies in these structures; 3) perform a fitting operation; and 4) analyze the results. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); the second structure (i.e., moving structure) is identified as the source structure. The atom equivalency within SYBYL is defined by user input. For the purpose of this invention, we will define equivalent atoms as protein backbone atoms (N, Cα, C and O) for all conserved residues between the two structures being compared. We will also consider only rigid fitting operations. When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atoms is an absolute minimum. This number, given in angstroms, is reported by the SYBYL program. For the purpose of the present invention, any homology model of a CAN-12 that has a root mean square deviation of conserved residue backbone atoms (N, Cα, C, O) of less than 3.0 A when superimposed on the relevant backbone atoms described by structure coordinates listed in Table IV are considered identical. More preferably, the root mean square deviation for the CAN-12 polypeptide is less than 2.0 Å.
The homology model of the present invention is useful for the structure-based design of modulators of the CAN-12 biological function, as well as mutants with altered biological function and/or specificity.
In accordance with the structural coordinates provided in Table IV and the three dimensional homology model of CAN-12, the CAN-12 polypeptide has been shown to comprise a an active site region embodied by the following amino acids: from about amino acid R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E251 to about amino acid Y255, from about amino acid N277 to about amino acid K281, from about amino acid V327 to about amino acid V330 of SEQ ID NO:2 or SEQ ID NO:24 (FIGS. 1A-E). In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids more in either the N— or C-terminal direction of the above referenced amino acids.
Also more preferred are polypeptides comprising all or any part of the CAN-12 active site domain, or a mutant or homologue of said polypeptide or molecular complex. By mutant or homologue of the molecule is meant a molecule that has a root mean square deviation from the backbone atoms of said CAN-12 amino acids of not more than about 4.5 Angstroms, and preferably not more than about 3.5 Angstroms.
In preferred embodiments, the following CAN-12 active site domain polypeptide is encompassed by the present invention: RLDLCQGOVGDCWFLAALQALALHQDILSRVVPLNQSFTEKYAGIFRFWFWHYGNW VPVVIDDRLPVNEAGQLVFVSSTYKNLFWGALLEKAYAKLSGSYEDLQSGQVSEAL VDFTGGVTMTINLAEAHGNLWDILIEATYNRTLIGCQTHSGKILENGLVEGHAYTLT GIRKVTCKHRPEYLVKLRNPWGKVEWKGDWSDSSSKWELLSPKEKILLLRKDNDGE FWMTLQDFKTHFVLLV (SEQ ID NO:46). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of the CAN-12 active site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The present invention also encompasses polypeptides comprising at least a portion of the CAN-12 active site domain (SEQ ID NO:46). Such polypeptides may correspond, for example, to the N— and/or C-terminal deletions of the active site domain.
In preferred embodiments, the following N-terminal CAN-12 active site domain deletion polypeptides are encompassed by the present invention: R1-V241, L2-V241, D3-V241, L4-V241, C5-V241, Q6-V241, G7-V241, I8-V241, V9-V241, G 10-V241, D11-V241, C12-V241, W13-V241, F14-V241, L15-V241, A16-V241, A17-V241, L18-V241, Q19-V241, A20-V241, L21-V241, A22-V241, L23-V241, H24-V241, Q25-V241, D26-V241, I27-V241, L28-V241, S29-V241, R30-V241, V31-V241, V32-V241, P33-V241, L34-V241, N35-V241, Q36-V241, S37-V241, F38-V241, T39-V241, E40-V241, K41-V241, Y42-V241, A43-V241, G44-V241, I45-V241, F46-V241, R47-V241, F48-V241, W49-V241, F50-V241, W51-V241, H52-V241, Y53-V241, G54-V241, N55-V241, W56-V241, V57-V241, P58-V241, V59-V241, V60-V241, I61-V241, D62-V241, D63-V241, R64-V241, L65-V241, P66-V241, V67-V241, N68-V241, E69-V241, A70-V241, G71-V241, Q72-V241, L73-V241, V74-V241, F75-V241, V76-V241, S77-V241, S78-V241, T79-V241, Y80-V241, K81-V241, N82-V241, L83-V241, F84-V241, W85-V241, G86-V241, A87-V241, L88-V241, L89-V241, E90-V241, K91-V241, A92-V241, Y93-V241, A94-V241, K95-V241, L96-V241, S97-V241, G98-V241, S99-V241, Y100-V241, E101-V241, D102-V241, L103-V241, Q104-V241, S105-V241, G106-V241, Q107-V241, V108-V241, S109-V241, E110-V241, A111-V241, L112-V241, V113-V241, D114-V241, F115-V241, T116-V241-, G117-V241, G118-V241, V119-V241, T120-V241, M121-V241, T122-V241, I123-V241, N124-V241, L125-V241, A126-V241, E127-V241, A128-V241, H129-V241, G130-V241, N131-V241, L132-V241, W133-V241, D134-V241, I135-V241, L136-V241, I137-V241, E138-V241, A139-V241, T140-V241, Y141-V241, N142-V241, R143-V241, T144-V241, L145-V241, I146-V241, G147-V241, C148-V241, Q149-V241, T150-V241, H151-V241, S152-V241, G153-V241, K154-V241, I155-V241, L156-V241, E157-V241, N158-V241, G159-V241, L160-V241, V161-V241, E162-V241, G163-V241, H164-V241, A165-V241, Y166-V241, T167-V241, L168-V241, T169-V241, G170-V241, I171-V241, R172-V241, K173-V241, V174-V241, T175-V241, C176-V241, K177-V241, H178-V241, R179-V241, P180-V241, E181-V241, Y182-V241, L183-V241, V184-V241, K185-V241, L186-V241, R187-V241, N188-V241, P189-V241, W190-V241, G191-V241, K192-V241, V193-V241, E194-V241, W195-V241, K196-V241, G197-V241, D198-V241, W199-V241, S200-V241, D201-V241, S202-V241, S203-V241, S204-V241, K205-V241, W206-V241, E207-V241, L208-V241, L209-V241, S210-V241, P211-V241, K212-V241, E213-V241, K214-V241, I215-V241, L216-V241, L217-V241, L218-V241, R219-V241, K220-V241, D221-V241, N222-V241, D223-V241, G224-V241, E225-V241, F226-V241, W227-V241, M228-V241, T229-V241, L230-V241, Q231-V241, D232-V241, F233-V241, K234-V241, and/or T235-V241 of SEQ ID NO:46. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal CAN-12 active site domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal CAN-12 active site domain deletion polypeptides are encompassed by the present invention: R1-V241, R1-L240, R1-L239, R1-V238, R1-F237, R1-H236, R1-T235, R1-K234, R1-F233, R1-D232, R1-Q231, R1-L230, R1-T229, R1-M228, R1-W227, R1-F226, R1-E225, R1-G224, R1-D223, R1-N222, R1-D221, R1-K220, R1-R219, R1-L218, R1-L217, R1-L216, R1-I215, R1-K214, R1-213, R1-K212, R1-P211, R1-S210, R1-L209, R1-L208, R1-E207, R1-W206, R1-K205, R1-S204, R1-S203, R1-S202, R1-D201, R1-S200, R1-W199, R1-D198, R1-G197, R1-K196, R1-W195, R1-E194, R1-V193, R1-K192, R1-G191, R1-W190, R1-P189, R1-N188, R1-R187, R1-L186, R1-K185, R1-V184, R1-L183, R1-Y182, R1-E181, R1-P180, R1-R179, R1-H178, R1-K177, R1-C176, R1-T175, R1-V174, R1-K173, R1-R172, R1-I171, R1-G170, R1-T169, R1-L168, R1-T167, R1-Y166, R1-A165, R1-H164, R1-G163, R1-E162, R1-V161, R1-L160, R1-G159, R1-N158, R1-E157, R1-L156, R1-I155, R1-K154, R1-G153, R1-S152, R1-H151, R1-T150, R1-Q149, R1-C148, R1-G147, R1-I146, R1-L145, R1-T144, R1-R143, R1-N142, R1-Y141, R1-T140, R1-A139, R1-E138, R1-I137, R1-L136, R1-I135, R1-D134, R1-W133, R1-L132, R1-N131, R1-G130, R1-H129, R1-A128, R1-E127, R1-A126, R1-L125, R1-N124, R1-I123, R1-T122, R1-M121, R1-T120, R1-V119, R1-G118, R1-G117, R1-T116, R1-F115, R1-D114, R1-V113, R1-L112, R1-A111, R1-E110, R1-S109, R1-V108, R1-Q107, R1-G106, R1-S105, R1-Q104, R1-L103, R1-D102, R1-E101, R1-Y100, R1-S99, R1-G98, R1-S97, R1-L96, R1-K95, R1-A94, R1-Y93, R1-A92, R1-K91, R1-E90, R1-L89, R1-L88, R1-A87, R1-G86, R1-W85, R1-F84, R1-L83, R1-N82, R1-K81, R1-Y80, R1-T79, R1-S78, R1-S77, R1-V76, R1-F75, R1-V74, R1-L73, R1-Q72, R1-G71, R1-A70, R1-E69, R1-N68, R1-V67, R1-P66, R1-L65, R1-R64, R1-D63, R1-D62, R1-I61, R1-V60, R1-V59, R1-P58, R1-V57, R1-W56, R1-N55, R1-G54, R1-Y53, R1-H52, R1-W51, R1-F50, R1-W49, R1-F48, R1-R47, R1-F46, R1-I45, R1-G44, R1-A43, R1-Y42, R1-K41, R1-E40, R1-T39, R1-F38, R1-S37, R1-Q36, R1-N35, R1-L34, R1-P33, R1-V32, R1-V31, R1-R30, R1-S29, R1-L28, R1-I27, R1-D26, R1-Q25, R1-H24, R1-L23, R1-A22, R1-L21, R1-A20, R1-Q19, R1-L18, R1-A17, R1-A16, R1-L15, R1-F14, R1-W13, R1-C12, R1-D11, R1-G10, R1-V9, R1-I8, and/or R1-G7 of SEQ ID NO:46. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal CAN-12 active site domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
Alternatively, such polypeptides may comprise polypeptide sequences corresponding, for example, to internal regions of the CAN-12 active site domain (e.g., any combination of both N— and C-terminal CAN-12 active site domain deletions) of SEQ ID NO:46. For example, internal regions could be defined by the equation NX to CX, where NX refers to any N-terminal amino acid position of the CAN-12 active site domain (SEQ ID NO:46), and where CX refers to any C-terminal amino acid position of the CAN-12 active site domain (SEQ ID NO:46). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.
In preferred embodiments, the following CAN-12 active site domain amino acid substitutions are encompassed by the present invention: wherein R90 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q. S, T, V, W, or Y; wherein L91 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D92 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L93 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein C94 is substituted with either an A, D, E, F, G, H. I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q95 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein G96 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein 197 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V98 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein G99 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D100 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C101 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W102 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein F103 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L104 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A105 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A106 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L107 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Q108 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein A109 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L110 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A111 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L112 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein H113 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q114 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein D115 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I116 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L117 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein S118 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein R119 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein V120 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein V121 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein P122 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein L123 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein N124 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, P, S, T, V, W, or Y; wherein Q125 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein S126 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein F127 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T128 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein E129 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K130 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y131 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein A132 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G133 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I134 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F135 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R136 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein F137 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W138 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein F139 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W140 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein H141 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y142 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein G143 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N144 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein W145 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein V146 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein P147 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein V148 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein V149 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein I150 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D151 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D152 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R153 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein L154 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein P155 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein V156 is substituted with either an A, C, D, E, F, O, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein N157 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein E158 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A159 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G160 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q161 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein L162 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein V163 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein F164 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V165 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein S166 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein S167 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein T168 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein Y169 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein K170 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N171 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein L172 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein F173 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W174 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein G175 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A176 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L177 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L178 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein E179 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K180 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A181 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y182 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein A183 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K184 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L185 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein S186 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein G187 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, P, S, T, V, W, or Y; wherein S188 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein Y189 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein E190 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D191 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L192 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Q193 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein S194 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein G195 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q196 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein V197 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein S198 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein E199 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A200 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L201 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein V202 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein D203 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F204 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T205 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein G206 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G207 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V208 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein T209 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein M210 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein T211 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein 1212 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N213 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein L214 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A215 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E216 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A217 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H218 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G219 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q. P, S, T, V, W, or Y; wherein N220 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein L221 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein W222 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein D223 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein 1224 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L225 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein I226 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E227 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A228 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T229 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein Y230 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein N231 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein R232 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein T233 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L234 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein 1235 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G236 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C237 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q238 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein T239 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein H240 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S241 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein G242 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K243 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein 1244 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L245 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein E246 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N247 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein G248 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L249 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein V250 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein E251 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G252 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H253 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A254 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y255 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein T256 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L257 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein T258 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein G259 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I260 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R261 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein K262 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V263 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein T264 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein C265 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K266 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H267 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R268 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein P269 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein E270 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y271 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein L272 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein V273 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein K274 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L275 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein R276 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein N277 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein P278 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein W279 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein G280 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K281 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V282 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein E283 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q. R, S, T, V, W, or Y; wherein W284 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein K285 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G286 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D287 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W288 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein S289 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein D290 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S291 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein S292 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein S293 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein K294 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W295 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein E296 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L297 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L298 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein S299 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein P300 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein K301 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E302 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K303 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I304 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L305 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L306 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L307 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein R308 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein K309 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D310 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N311 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein D312 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G313 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E314 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F315 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W316 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein M317 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein T318 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L319 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Q320 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein D321 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F322 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K323 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T324 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein H325 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F326 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V327 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein L328 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L329 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; and/or wherein V330 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y of SEQ ID NO:2 or SEQ ID NO:24, in addition to any combination thereof. The present invention also encompasses the use of these CAN-12 active site domain amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following CAN-12 active site domain conservative amino acid substitutions are encompassed by the present invention: wherein R90 is substituted with either a K, or H; wherein L91 is substituted with either an A, I, or V; wherein D92 is substituted with an E; wherein L93 is substituted with either an A, I, or V; wherein C94 is a C; wherein Q95 is substituted with a N; wherein G96 is substituted with either an A, M, S, or T; wherein 197 is substituted with either an A, V, or L; wherein V98 is substituted with either an A, I, or L; wherein G99 is substituted with either an A, M, S, or T; wherein D100 is substituted with an E; wherein C101 is a C; wherein W102 is either an F, or Y; wherein F103 is substituted with either a W, or Y; wherein L104 is substituted with either an A, I, or V; wherein A105 is substituted with either a G, I, L, M, S, T, or V; wherein A106 is substituted with either a G, I, L, M, S, T, or V; wherein L107 is substituted with either an A, I, or V; wherein Q108 is substituted with a N; wherein A109 is substituted with either a G, I, L, M, S, T, or V; wherein L110 is substituted with either an A, I, or V; wherein A111 is substituted with either a G, I, L, M, S, T, or V; wherein L112 is substituted with either an A, I, or V; wherein H113 is substituted with either a K, or R; wherein Q114 is substituted with a N; wherein D115 is substituted with an E; wherein I116 is substituted with either an A, V, or L; wherein L117 is substituted with either an A, I, or V; wherein S118 is substituted with either an A, G, M, or T; wherein R119 is substituted with either a K, or H; wherein V120 is substituted with either an A, I, or L; wherein V121 is substituted with either an A, I, or L; wherein P122 is a P; wherein L123 is substituted with either an A, I, or V; wherein N124 is substituted with a Q; wherein Q125 is substituted with a N; wherein S126 is substituted with either an A, G, M, or T; wherein F127 is substituted with either a W, or Y; wherein T128 is substituted with either an A, G, M, or S; wherein E129 is substituted with a D; wherein K130 is substituted with either a R, or H; wherein Y131 is either an F, or W; wherein A132 is substituted with either a G, I, L, M, S, T, or V; wherein G133 is substituted with either an A, M, S, or T; wherein I134 is substituted with either an A, V, or L; wherein F135 is substituted with either a W, or Y; wherein R136 is substituted with either a K, or H; wherein F137 is substituted with either a W, or Y; wherein W138 is either an F, or Y; wherein F139 is substituted with either a W, or Y; wherein W140 is either an F, or Y; wherein H141 is substituted with either a K, or R; wherein Y142 is either an F, or W; wherein G143 is substituted with either an A, M, S, or T; wherein N144 is substituted with a Q; wherein W145 is either an F, or Y; wherein V146 is substituted with either an A, I, or L; wherein P147 is a P; wherein V148 is substituted with either an A, I, or L; wherein V149 is substituted with either an A, I, or L; wherein I150 is substituted with either an A, V, or L; wherein D151 is substituted with an E; wherein D152 is substituted with an E; wherein R153 is substituted with either a K, or H; wherein L154 is substituted with either an A, I, or V; wherein P155 is a P; wherein V156 is substituted with either an A, I, or L; wherein N157 is substituted with a Q; wherein E158 is substituted with a D; wherein A159 is substituted with either a G, I, L, M, S, T, or V; wherein G160 is substituted with either an A, M, S, or T; wherein Q161 is substituted with a N; wherein L162 is substituted with either an A, I, or V; wherein V163 is substituted with either an A, I, or L; wherein F164 is substituted with either a W, or Y; wherein V165 is substituted with either an A, I, or L; wherein S166 is substituted with either an A, G, M, or T; wherein S167 is substituted with either an A, G, M, or T; wherein T168 is substituted with either an A, G, M, or S; wherein Y169 is either an F, or W; wherein K170 is substituted with either a R, or H; wherein N171 is substituted with a Q; wherein L172 is substituted with either an A, I, or V; wherein F173 is substituted with either a W, or Y; wherein W174 is either an F, or Y; wherein G175 is substituted with either an A, M, S, or T; wherein A176 is substituted with either a G, I, L, M, S, T, or V; wherein L177 is substituted with either an A, I, or V; wherein L178 is substituted with either an A, I, or V; wherein E179 is substituted with a D; wherein K180 is substituted with either a R, or H; wherein A181 is substituted with either a G, I, L, M, S, T, or V; wherein Y182 is either an F, or W; wherein A183 is substituted with either a G, I, L, M, S, T, or V; wherein K184 is substituted with either a R, or H; wherein L185 is substituted with either an A, I, or V; wherein S186 is substituted with either an A, G, M, or T; wherein G187 is substituted with either an A, M, S, or T; wherein S188 is substituted with either an A, G, M, or T; wherein Y189 is either an F, or W; wherein E190 is substituted with a D; wherein D191 is substituted with an E; wherein L192 is substituted with either an A, I, or V; wherein Q193 is substituted with a N; wherein S194 is substituted with either an A, G, M, or T; wherein G195 is substituted with either an A, M, S. or T; wherein Q196 is substituted with a N; wherein V197 is substituted with either an A, I, or L; wherein S198 is substituted with either an A, G, M, or T; wherein E199 is substituted with a D; wherein A200 is substituted with either a G, I, L, M, S, T, or V; wherein L201 is substituted with either an A, I, or V; wherein V202 is substituted with either an A, I, or L; wherein D203 is substituted with an E; wherein F204 is substituted with either a W, or Y; wherein T205 is substituted with either an A, G, M, or S; wherein G206 is substituted with either an A, M, S, or T; wherein G207 is substituted with either an A, M, S, or T; wherein V208 is substituted with either an A, I, or L; wherein T209 is substituted with either an A, G, M, or S; wherein M210 is substituted with either an A, G, S, or T; wherein T211 is substituted with either an A, G, M, or S; wherein I212 is substituted with either an A, V, or L; wherein N213 is substituted with a Q; wherein L214 is substituted with either an A, I, or V; wherein A215 is substituted with either a G, I, L, M, S, T, or V; wherein E216 is substituted with a D; wherein A217 is substituted with either a G, I, L, M, S, T, or V; wherein H218 is substituted with either a K, or R; wherein G219 is substituted with either an A, M, S, or T; wherein N220 is substituted with a Q; wherein L221 is substituted with either an A, I, or V; wherein W222 is either an F, or Y; wherein D223 is substituted with an E; wherein I224 is substituted with either an A, V, or L; wherein L225 is substituted with either an A, I, or V; wherein I226 is substituted with either an A, V, or L; wherein E227 is substituted with a D; wherein A228 is substituted with either a G, I, L, M, S, T, or V; wherein T229 is substituted with either an A, G, M, or S; wherein Y230 is either an F, or W; wherein N231 is substituted with a Q; wherein R232 is substituted with either a K, or H; wherein T233 is substituted with either an A, G, M, or S; wherein L234 is substituted with either an A, I, or V; wherein I235 is substituted with either an A, V, or L; wherein G236 is substituted with either an A, M, S, or T; wherein C237 is a C; wherein Q238 is substituted with a N; wherein T239 is substituted with either an A, G, M, or S; wherein H240 is substituted with either a K, or R; wherein S241 is substituted with either an A, G, M, or T; wherein G242 is substituted with either an A, M, S, or T; wherein K243 is substituted with either a R, or H; wherein I244 is substituted with either an A, V, or L; wherein L245 is substituted with either an A, I, or V; wherein E246 is substituted with a D; wherein N247 is substituted with a Q; wherein G248 is substituted with either an A, M, S, or T; wherein L249 is substituted with either an A, I, or V; wherein V250 is substituted with either an A, I, or L; wherein E251 is substituted with a D; wherein G252 is substituted with either an A, M, S, or T; wherein H253 is substituted with either a K, or R; wherein A254 is substituted with either a G, I, L, M, S, T, or V; wherein Y255 is either an F, or W; wherein T256 is substituted with either an A, G, M, or S; wherein L257 is substituted with either an A, I, or V; wherein T258 is substituted with either an A, G, M, or S; wherein G259 is substituted with either an A, M, S, or T; wherein 1260 is substituted with either an A, V, or L; wherein R261 is substituted with either a K, or H; wherein K262 is substituted with either a R, or H; wherein V263 is substituted with either an A, I, or L; wherein T264 is substituted with either an A, G, M, or S; wherein C265 is a C; wherein K266 is substituted with either a R, or H; wherein H267 is substituted with either a K, or R; wherein R268 is substituted with either a K, or H; wherein P269 is a P; wherein E270 is substituted with a D; wherein Y271 is either an F, or W; wherein L272 is substituted with either an A, I, or V; wherein V273 is substituted with either an A, I, or L; wherein K274 is substituted with either a R, or H; wherein L275 is substituted with either an A, I, or V; wherein R276 is substituted with either a K, or H; wherein N277 is substituted with a Q; wherein P278 is a P; wherein W279 is either an F, or Y; wherein G280 is substituted with either an A, M, S, or T; wherein K281 is substituted with either a R, or H; wherein V282 is substituted with either an A, I, or L; wherein E283 is substituted with a D; wherein W284 is either an F, or Y; wherein K285 is substituted with either a R, or H; wherein G286 is substituted with either an A, M, S, or T; wherein D287 is substituted with an E; wherein W288 is either an F, or Y; wherein S289 is substituted with either an A, G, M, or T; wherein D290 is substituted with an E; wherein S291 is substituted with either an A, G, M, or T; wherein S292 is substituted with either an A, G, M, or T; wherein S293 is substituted with either an A, G, M, or T; wherein K294 is substituted with either a R, or H; wherein W295 is either an F, or Y; wherein E296 is substituted with a D; wherein L297 is substituted with either an A, I, or V; wherein L298 is substituted with either an A, I, or V; wherein S299 is substituted with either an A, G, M, or T; wherein P300 is a P; wherein K301 is substituted with either a R, or H; wherein E302 is substituted with a D; wherein K303 is substituted with either a R, or H; wherein I304 is substituted with either an A, V, or L; wherein L305 is substituted with either an A, I, or V; wherein L306 is substituted with either an A, I, or V; wherein L307 is substituted with either an A, I, or V; wherein R308 is substituted with either a K, or H; wherein K309 is substituted with either a R, or H; wherein D310 is substituted with an E; wherein N311 is substituted with a Q; wherein D312 is substituted with an E; wherein G313 is substituted with either an A, M, S, or T; wherein E314 is substituted with a D; wherein F315 is substituted with either a W, or Y; wherein W316 is either an F, or Y; wherein M317 is substituted with either an A, G, S, or T; wherein T318 is substituted with either an A, G, M, or S; wherein L319 is substituted with either an A, I, or V; wherein Q320 is substituted with a N; wherein D321 is substituted with an E; wherein F322 is substituted with either a W, or Y; wherein K323 is substituted with either a R, or H; wherein T324 is substituted with either an A, G, M, or S; wherein H325 is substituted with either a K, or R; wherein F326 is substituted with either a W, or Y; wherein V327 is substituted with either an A, I, or L; wherein L328 is substituted with either an A, I, or V; wherein L329 is substituted with either an A, I, or V; and/or wherein V330 is substituted with either an A, I, or L of SEQ ID NO:2 or SEQ ID NO:24 in addition to any combination thereof. Other suitable substitutions within the CAN-12 active site domain are encompassed by the present invention and are referenced elsewhere herein. The present invention also encompasses the use of these CAN-12 active site domain conservative amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
For purposes of the present invention, by “at least a portion of” is meant all or any part of the CAN-12 active site domain defined by the structure coordinates according to Table IV (e.g., fragments thereof). More preferred are molecules comprising all or any parts of the CAN-12 active site domain, according to Table IV, or a mutant or homologue of said molecule or molecular complex. By mutant or homologue of the molecule it is meant a molecule that has a root mean square deviation from the backbone atoms of said CAN-12 amino acids of not more than 4.5 Angstroms, and preferably not more than 3.5 Angstroms.
The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a term that expresses the deviation or variation from a trend or object. For the purposes of the present invention, the “root mean square deviation” defines the variation in the backbone of a protein from the relevant portion of the backbone of the AR portion of the complex as defined by the structure coordinates described herein.
A preferred embodiment is a machine-readable data storage medium that is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex that is defined by the structure coordinates of all of the amino acids in Table IV ± a root mean square deviation from the backbone atoms of those amino acids of not more than 4.0 ANG, preferably 3.0 ANG.
The structure coordinates of a CAN-12 homology model, including portions thereof, is stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery.
Accordingly, in one embodiment of this invention is provided a machine-readable data storage medium comprising a data storage material encoded with the structure coordinates set forth in Table IV.
One embodiment utilizes System 10 as disclosed in WO 98/11134, the disclosure of which is incorporated herein by reference in its entirety. Briefly, one version of these embodiments comprises a computer comprising a central processing unit (“CPU”), a working memory which may be, e.g, RAM (random-access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more cathode-ray tube (“CRT”) display terminals, one or more keyboards, one or more input lines, and one or more output lines, all of which are interconnected by a conventional bidirectional system bus.
Input hardware, coupled to the computer by input lines, may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line. Alternatively or additionally, the input hardware may comprise CD-ROM drives or disk drives. In conjunction with a display terminal, keyboard may also be used as an input device.
Output hardware, coupled to the computer by output lines, may similarly be implemented by conventional devices. By way of example, output hardware may include a CRT display terminal for displaying a graphical representation of a region or domain of the present invention using a program such as QUANTA as described herein. Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use.
In operation, the CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage, and accesses to and from the working memory, and determines the sequence of data processing steps. A number of programs may be used to process the machine-readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein. Specific references to components of the hardware system are included as appropriate throughout the following description of the data storage medium.
For the purpose of the present invention, any magnetic data storage medium which can be encoded with machine-readable data would be sufficient for carrying out the storage requirements of the system. The medium could be a conventional floppy diskette or hard disk, having a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, on one or both sides, containing magnetic domains whose polarity or orientation could be altered magnetically, for example. The medium may also have an opening for receiving the spindle of a disk drive or other data storage device.
The magnetic domains of the coating of a medium may be polarized or oriented so as to encode in a manner which may be conventional, machine readable data such as that described herein, for execution by a system such as the system described herein.
Another example of a suitable storage medium which could also be encoded with such machine-readable data, or set of instructions, which could be carried out by a system such as the system described herein, could be an optically-readable data storage medium. The medium could be a conventional compact disk read only memory (CD-ROM) or a rewritable medium such as a magneto-optical disk which is optically readable and magneto-optically writable. The medium preferably has a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, usually of one side of substrate.
In the case of a CD-ROM, as is well known, the coating is reflective and is impressed with a plurality of pits to encode the machine-readable data. The arrangement of pits is read by reflecting laser light off the surface of the coating. A protective coating, which preferably is substantially transparent, is provided on top of the reflective coating.
In the case of a magneto-optical disk, as is well known, the coating has no pits, but has a plurality of magnetic domains whose polarity or orientation can be changed magnetically when heated above a certain temperature, as by a laser. The orientation of the domains can be read by measuring the polarization of laser light reflected from the coating. The arrangement of the domains encodes the data as described above.
Thus, in accordance with the present invention, data capable of displaying the three dimensional structure of the CAN-12 homology model, or portions thereof and their structurally similar homologues is stored in a machine-readable storage medium, which is capable of displaying a graphical three-dimensional representation of the structure. Such data may be used for a variety of purposes, such as drug discovery.
For the first time, the present invention permits the use of structure-based or rational drug design techniques to design, select, and synthesize chemical entities that are capable of modulating the biological function of CAN-12.
Accordingly, the present invention is also directed to the design of small molecules which imitates the structure of the CAN-12 active site domain (SEQ ID NO:46), or a portion thereof, in accordance with the structure coordinates provided in Table IV. Alternatively, the present invention is directed to the design of small molecules which may bind to at least part of the CAN-12 active site domain (SEQ ID NO:25), or some portion thereof. For purposes of this invention, by CAN-12 active site domain, it is also meant to include mutants or homologues thereof. In a preferred embodiment, the mutants or homologues have at least 25% identity, more preferably 50% identity, more preferably 75% identity, and most preferably 90% identity to SEQ ID NO:46. In this context, the term “small molecule” may be construed to mean any molecule described known in the art or described elsewhere herein, though may include, for example, peptides, chemicals, carbohydrates, nucleic acids, PNAs, and any derivatives thereof.
The three-dimensional model structure of the CAN-12 will also provide methods for identifying modulators of biological function. Various methods or combination thereof can be used to identify these compounds.
For example, test compounds can be modeled that fit spatially into the active site domain in CAN-12 embodied by the sequence from about R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E251 to about amino acid Y255, from about amino acid N277 to about amino acid K281, from about amino acid V327 to about amino acid V330, or some portion thereof, of SEQ ID NO:2 or SEQ ID NO:24 (corresponding to SEQ ID NO:46), in accordance with the structural coordinates of Table IV.
Structure coordinates of the active site domain in CAN-12 defined by the amino acids from about R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E251 to about amino acid Y255, from about amino acid N277 to about amino acid K281, from about amino acid V327 to about amino acid V330 of SEQ ID NO:2 or SEQ ID NO:24, can also be used to identify structural and chemical features. Identified structural or chemical features can then be employed to design or select compounds as potential CAN-12 modulators. By structural and chemical features it is meant to include, but is not limited to, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interaction. Alternatively, or in conjunction with, the three-dimensional structural model can be employed to design or select compounds as potential CAN-12 modulators. Compounds identified as potential CAN-12 modulators can then be synthesized and screened in an assay characterized by binding of a test compound to the CAN-12, or in characterizing the ability of CAN-12 to modulate a protease target in the presence of a small molecule. Examples of assays useful in screening of potential CAN-12 modulators include, but are not limited to, screening in silico, in vitro assays and high throughput assays. Finally, these methods may also involve modifying or replacing one or more amino acids at amino acid positions, C101, H253, and/or N277 of SEQ ID NO:2 or SEQ ID NO:24 in accordance with the structure coordinates of Table IV.
However, as will be understood by those of skill in the art upon this disclosure, other structure based design methods can be used. Various computational structure based design methods have been disclosed in the art.
For example, a number computer modeling systems are available in which the sequence of the CAN-12 and the CAN-12 structure (i.e., atomic coordinates of CAN-12 and/or the atomic coordinates of the active site domain as provided in Table IV) can be input. This computer system then generates the structural details of one or more these regions in which a potential CAN-12 modulator binds so that complementary structural details of the potential modulators can be determined. Design in these modeling systems is generally based upon the compound being capable of physically and structurally associating with CAN-12. In addition, the compound must be able to assume a conformation that allows it to associate with CAN-12. Some modeling systems estimate the potential inhibitory or binding effect of a potential CAN-12 modulator prior to actual synthesis and testing.
Methods for screening chemical entities or fragments for their ability to associate with a given protein target are also well known. Often these methods begin by visual inspection of the binding site on the computer screen. Selected fragments or chemical entities are then positioned in the active site domain of CAN-12. Docking is accomplished using software such as INSIGHTII, QUANTA and SYBYL, following by energy minimization and molecular dynamics with standard molecular mechanic forcefields such as CHARMM and AMBER. Examples of computer programs which assist in the selection of chemical fragment or chemical entities useful in the present invention include, but are not limited to, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntz et al. 1982).
Upon selection of preferred chemical entities or fragments, their relationship to each other and CAN-12 can be visualized and then assembled into a single potential modulator. Programs useful in assembling the individual chemical entities include, but are not limited to CAVEAT (Bartlett et al. 1989) and 3D Database systems (artin1992).
Alternatively, compounds may be designed de novo using either an empty active site or optionally including some portion of a known inhibitor. Methods of this type of design include, but are not limited to LUDI (Bohm 1992) and LeapFrog (Tripos Associates, St. Louis Mo.).
In addition, CAN-12 is overall well suited to modern methods including combinatorial chemistry.
Programs such as DOCK (Kuntz et al. 1982) can be used with the atomic coordinates from the homology model to identify potential ligands from databases or virtual databases which potentially bind CAN-12 active site domain, and which may therefore be suitable candidates for synthesis and testing.
Additionally, the three-dimensional homology model of CAN-12 will aid in the design of mutants with altered biological activity.
The following are encompassed by the present invention: a machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the structure coordinates of the model CAN-12 according to Table IV or a homologue of said model, wherein said homologue comprises backbone atoms that have a root mean square deviation from the backbone atoms of the complex of not more than 4.5 Å, preferably not more than 4.0 Å, most preferably not more than 3.5 Å, and even more preferably not more than 3.0 Å; and a machine-readable data storage medium, wherein said molecule is defined by the set of structure coordinates of the model for CAN-12 according to Table IV, or a homologue of said molecule, said homologue having a root mean square deviation from the backbone atoms of said amino acids of not more than 4.5 Å, preferably not more than 4.0 Å, most preferably not more than 3.5 Å, and even more preferably not more than 3.0 Å; a model comprising all or any part of the model defined by structure coordinates of CAN-12 according to Table IV, or a mutant or homologue of said molecule or molecular complex.
In a further embodiment, the following are encompassed by the present invention: a method for identifying a mutant of CAN-12 with altered biological properties, function, or reactivity, the method comprising any combination of steps of: use of the model or a homologue of said model according to Table IV, for the design of protein mutants with altered biological function or properties which exhibit any combination of therapeutic effects provided elsewhere herein; and use of the model or a homologue of said model, for the design of a protein with mutations in the active site domain comprised of the amino acids from about R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E251 to about amino acid Y255, from about amino acid N277 to about amino acid K281, from about amino acid V327 to about amino acid V330 of SEQ ID NO:2 or SEQ ID NO:24 according to Table IV with altered biological function or properties which exhibit any combination of therapeutic effects provided elsewhere herein.
In further preferred embodiments, the following are encompassed by the present invention: a method for identifying modulators of CAN-12 biological properties, function, or reactivity, the method comprising any combination of steps of: modeling test compounds that overlay spatially into the active site domain defined by all or any portion of residues from about R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E251 to about amino acid Y255, from about amino acid N277 to about amino acid K281, from about amino acid V327 to about amino acid V330 of SEQ ID NO:2 or SEQ ID NO:24 and of the three-dimensional structural model according to Table IV, or using a homologue or portion thereof.
The present invention encompasses using the structure coordinates as set forth herein to identify structural and chemical features of the CAN-12 polypeptide; employing identified structural or chemical features to design or select compounds as potential CAN-12 modulators; employing the three-dimensional structural model to design or select compounds as potential CAN-12 modulators; synthesizing the potential CAN-12 modulators; screening the potential CAN-12 modulators in an assay characterized by binding of a protein to the CAN-12; selecting the potential CAN-12 modulator from a database; designing the CAN-12 modulator de novo; and/or designing said CAN-12 modulator from a known modulator activity.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 1 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 4570 of SEQ ID NO: 1, b is an integer between 15 to 4584, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 1, and where b is greater than or equal to a+14.
In one embodiment, a CAN12 polypeptide comprises a portion of the amino sequence depicted in FIGS. 1A-E. In another embodiment, a CAN12 polypeptide comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of the amino sequence depicted in FIGS. 1A-E. In further embodiments, the CAN12 polypeptide does not consist of the sequence ALLEKAYAKL (SEQ ID NO: 141), and/or ALLEKAYAKLSGSYE. (SEQ ID NO: 142).
Features of the Polypeptide Encoded by Gene No:2
The polypeptide of this gene provided as SEQ ID NO:54 (FIGS. 8A-C), encoded by the polynucleotide sequence according to SEQ ID NO:53 (FIGS. 8A-C), and/or encoded by the polynucleotide contained within the deposited clone, CAN-12v1, has significant homology at the nucleotide and amino acid level to a number of calpains, which include, for example, the human CAN10 protein (type II diabetes linked) (hCAN10; Genbank Accession No: gi|NP_—075574; SEQ ID NO:3); the human CAN5 protein (hCAN5; Genbank Accession No: gi|NP_—004046; SEQ ID NO:4); the large catalytic subunit of the human CALPAIN 1 protein (also referred to as Calcium-Activated Neutral Proteinase, CANP, μ-TYPE) (hCAN1; Genbank Accession No: gi|12408656; SEQ ID NO:5); the large subunit of the human calpain 3 protein (EC 3.4.22.17) (also referred to as CALPAIN L3, CALPAIN P94, Calcium-Activated Neutral Proteinase 3, CANP 3; muscle-specific calcium-activated neutral protease 3 large subunit) (hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6); the large catalytic subunit of the mouse CALPAIN 1 protein (also referred to as Calcium-Activated Neutral Proteinase) (CANP) μ-TYPE ) (mCALPAIN1; Genbank Accession No: gi|O88666; SEQ ID NO:7); the mouse CALPAIN LP82 (mLP82; Genbank Accession No: gi|3661585; SEQ ID NO:8); the rat stomach-specific calcium-activated neutral protease large subunit, NCL2 protein (rNCL2; Genbank Accession No: gi|NP_—006606; SEQ ID NO:9); the human CAN11 protein (hCAN11; Genbank Accession No: gi|NP008989; SEQ ID NO:10); the human CAN2 protein (hCAN2; Genbank Accession No: gi|4502563; SEQ ID NO:11); and the human CAN9 protein (hCAN9; Genbank Accession No: gi|5729758; SEQ ID NO:12). An alignment of the CAN-12v1 polypeptide with these proteins is provided in FIGS. 2A-E. Based upon such strong conservation, the inventors have ascribed the CAN-12v1 polypeptide as having proteolytic activity, preferably calpain activity.
The CAN-12v1 polypeptide was determined to have 28.7% identity and 35.6% similarity with the human CAN10 protein (type II diabetes linked) (hCAN10; Genbank Accession No: gi|NP_—075574; SEQ ID NO:3); to have 33.3% identity and 45.1% similarity with the human CAN5 protein (hCAN5; Genbank Accession No: gi|NP-004046; SEQ ID NO:4); to have 38.3% identity and 46.8% similarity with the large catalytic subunit of the human CALPAIN 1 protein (also referred to as Calcium-Activated Neutral Proteinase, CANP, μ-TYPE) (hCAN1; Genbank Accession No: gi|12408656; SEQ ID NO:5); to have 40.4% identity and 49.1% similarity with the large subunit of the human calpain 3 protein (EC 3.4.22.17) (also referred to as CALPAIN L3, CALPAIN P94, Calcium-Activated Neutral Proteinase 3, CANP 3; muscle-specific calcium-activated neutral protease 3 large subunit) (hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6); to have 39.8% identity and 47.8% similarity with the large catalytic subunit of the mouse CALPAIN 1 protein (also referred to as Calcium-Activated Neutral Proteinase) (CANP) μ-TYPE ) (mCALPAIN1; Genbank Accession No: gi|O88666; SEQ ID NO:7); to have 40.6% identity and 48.8% similarity with the mouse CALPAIN LP82 (mLP82; Genbank Accession No: gi|3661585; SEQ ID NO:8); to have 36.3% identity and 44.9% similarity with the rat stomach-specific calcium-activated neutral protease large subunit, NCL2 protein (rNCL2; Genbank Accession No: gi|NP_—006606; SEQ ID NO:9); to have 38.8% identity and 47.4% similarity with the human CAN11 protein (hCAN11; Genbank Accession No: gi|NP_—008989; SEQ ID NO:10); to have 37.9% identity and 47.6% similarity with the human CAN2 protein (hCAN2; Genbank Accession No: gi|4502563; SEQ ID NO:11); and to have 40.7% identity and 49.8% similarity with the human CAN9 protein (hCAN9; Genbank Accession No: gi|5729758; SEQ ID NO:12).
The human CAN10 protein (type II diabetes linked) (hCAN10; Genbank Accession No: gi|NP _—075574; SEQ ID NO:3) is a human calpain gene that encodes a large calpain subunit. CAN10 is an atypical calpain in that it lacks the calmodulin-like calcium-binding domain and instead has a divergent C-terminal domain. CAN10 is similar in organization to calpains 5 and 6 and is associated with type 2 or non-insulin-dependent diabetes mellitus (NIDDM) and located within the NIDDM1 chromosomal region (Nat. Genet. 26 (2), 163-175 (2000)).
The large subunit of the human calpain 3 protein (EC 3.4.22.17) (also referred to as CALPAIN L3, CALPAIN P94, Calcium-Activated Neutral Proteinase 3, CANP 3; muscle-specific calcium-activated neutral protease 3 large subunit) (hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6) is a muscle-specific member of the calpain large subunit family. Loss of CAPN3 function has been associated with limb-girdle muscular dystrophies type 2A (Cell 81 (1), 27-40 (1995)).
The human CAN9 protein (hCAN9; Genbank Accession No: gi|5729758; SEQ ID NO:12) is a calpain that is expressed predominantly in stomach and small intestine and is thought to have specialized functions in the digestive tract, and be associated with gastric cancer. (Biol. Chem. 379 (2), 175-183 (1998); and Jpn. J. Cancer Res. 91 (5), 459-463 (2000)).
As described above, the CAN-12v1 polypeptide was found to have significant sequence homology with calpains, particularly members of the m-calpain family. A conserved peptide signature of Q×3(G,E)×C(Y,W)×2(S,T,A,G,C)(S,T,A,G,C,V) Q×{3}(G)×C(W)×{2}(A)(A) (referred to as a thiol (cysteine) protease active site domain) common to most calpain family members is found in the protein sequence of CAN-12v1 from amino acid 90 to amino acid 111 of SEQ ID NO:54 (FIGS. 1A-C). Protein threading and molecular modeling of CAN-12v1 suggests that CAN-12v1 has a structural fold similar to representative m-calpains. Moreover, the structural and threading alignments of the present invention suggest that-amino acids 101 (“C”), 254 (“H”), and 278 (“N”) of SEQ ID NO:54 (FIGS. 8A-C) may represent the catalytic amino acids within the active site domain. Thus, based upon the sequence and structural homology to known calpains, particularly the presence of the thiol cysteine protease active site domain, the novel CAN-12v1 is believed to represent a novel human calpain.
In confirmation of the strong homology to known calpains, the CAN-12v1 polypeptide was determined to have several conserved catalytic amino acids at amino acid C101, H254, and N278 of SEQ ID NO:54 (FIGS. 8A-C). As discussed more particularly herein, calpains are a group of structurally diverse, high molecular weight (400 to 500 amino acids) proteins that have a catalytic cysteine amino acid and one or more calcium binding domains. Despite the structural heterogeneity, calpains share some well defined structural-functional characteristics, particularly in their active site domains.
In preferred embodiments, the CAN-12v1 polypeptide of the present invention is directed to a polypeptide having structural similarity to calpains.
Based upon the strong homology to members of the calpain family, the CAN-12v1 polypeptide is expected to share at least some biological activity with calpains, preferably with m-calpain family members, and more preferable to the large subunits of m-calpain family members, in addition to other calpains and calpain subunits referenced herein and/or otherwise known in the art.
Expression profiling designed to measure the steady state mRNA levels encoding the CAN-12 polypeptide showed predominately high expression levels in spinal cord tissue; significantly high expression in lymph node and thymus, and to a lesser extent, in spleen tissue (See FIG. 4).
Expanded analysis of CAN-12 expression levels by TaqMan™ quantitative PCR (see FIG. 6) confirmed that the CAN-12 polypeptide is expressed in the lymph gland. However, the TaqMan™ quantitative PCR determined that the CAN-12 polypeptide is primarily expressed in the eosophagus. In fact, with the exception of the lymph gland, the steady state mRNA level of CAN-12 was approximately 2700 times higher in the esophagus than in all other tissues tested. These data suggest modulators of the CAN-12 polynucleotides and polypeptides may be useful for the treatment, detection, and/or amelioration of the following, non-limiting diseases and disorders associated with the eosphagus: dysphagia, cricoharyngeal incoordination, esophageal carcinoma, esophageal webs, achalasia, symptomatic diffuse esophageal spasm, gastroesophageal reflux, and/or corrosive esophagitis.
The polynucleotides encoding the CAN-12 polypeptide of the present invention were used to determine the chromosomal localization of the calpain12 gene. Polynuclelotides corresponding to CAN-12 (SEQ ID NO:1) were shown to localize to chromosome 2, specifically 2p16-p21. The comparison of the chromosomal location of the calpain12 gene with the location of chromosomal regions which have been shown to be associated with specific diseases or conditions, e.g. by linkage analysis, can be indicative of diseases in which calpain12 may play a role. Interestingly, a whole-genome linkage scan in multiple sclerosis families (Ebers et al. A full genome search in multiple sclerosis. Nature Genet. 13: 472-476, 1996.) identified 5 susceptibility loci on chromosomes 2, 3, 5, 11, and X. In particular, an association was identified with marker D2S119 on chromosome 2 and MS, The localization of the D2 S119 marker was further delimeated to 2p16-p21 based on a radiation hybrid linkage map retrieved from an online query at NCBI web site: http:/www.ncbi.nlm.nih.gov/genome/sts/. Since the map of calpain 12 and the susceptibility marker D2S119 overlaps, it is reasonable to postulate that calpain 12 may contribute to MS. Furthermore, the transcription profile of calpain12 indicated a prominent expression in spinal cord, and implication of calpains in MS has been suggested (Shields D C et al. A putative mechanism of demyelination in multiple sclerosis by a proteolytic enzyme, calpain. Proc Natl Acad Sci U S A. 96:11486-91.1999).
The CAN-12v1 polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include modulating cellular adhesion events, cellular proliferation, and inflammation, in various cells, tissues, and organisms, and particularly in mammalian spinal cord tissue, lymph node, thymus, and spleen tissue, preferably human tissue. CAN-12v1 polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, may be useful in diagnosing, treating, prognosing, and/or preventing neural, immune, hematopoietic, and/or proliferative diseases or disorders.
The strong homology to human calpains, particularly m-calpains, combined with the predominate localized expression in esophagus tissue suggests the CAN-12v1 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing gastrointestinal diseases, particularly esophageal diseases and/or disorders which include the following non-limiting examples: aberrant transport of food bolus from the mouth to the stomach, aberrant prevention of retrograde flow of gastrointestinal contents, aberrant esophageal peristaltic contractions, pyrosis, painful swallowing, reflux esophagitis, esophageal motility disorders, esophageal spasms, diffuse esophageal spasm, atypical chest pain, regurgitation, oropharyngeal paralysis, nasal regurgitation, dysphagia, cricopharyngeal bar, globus pharyngeus, achalasia, motor disorders of the esophageal smooth muscle, scleroderma esophagus, gastroesophageal reflux disease (GERD), esophagitis, Barrett's esophagus, viral esophagitis, Herpes simplex virus mediated viral esophagitis, Varicella-zoster virus mediated viral esophagitis, Cytomegalovirus mediated viral esophagitis, bacterial esophagitis, Lactobacillus mediated bacterial esophagitis, Candida mediated esophagitis, radiation esophagitis, corrosive esophagitis, pill-induced esophagitis, esophagitis associated with mucocutaneous and systemic diseases, diverticula, lower esophageal mucosal ring, lower esophageal muscular ring, hiatal hernia, paraesophageal hernia, esophageal rupture, and/or Mallory-Weiss Syndrome.
Although calpains are typically associated primarily with neurogenerative conditions, their association in gastrointenstinal tissues has precedence. For example, the human CAN9 protein (hCAN9; Genbank Accession No: gi|5729758; SEQ ID NO:12) is predominately expressed in the stomach and small intestine and is thought to be associated with gastric cancers.
The strong homology to human calpains, particularly m-calpains, combined with the localized expression in spinal cord tissue suggests the CAN-12v1 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing neural diseases, neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Neurological Diseases”, “Regeneration” and “Hyperproliferative Disorders” sections below, in the Examples, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Alternatively, the strong homology to human calpains, particularly m-calpains, combined with the localized expression in lymph node, thymus, and spleen tissue suggests the CAN-12v1 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, ameliorating, and/or preventing immune diseases and/or disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, and elsewhere herein. Briefly, the strong expression in immune tissue indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. The CAN-12v1 polypeptide may also be useful as a preventative agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product may be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
Moreover, the protein would be useful in the detection, treatment, and/or prevention of a variety of vascular disorders and conditions, which include, but are not limited to miscrovascular disease, vascular leak syndrome, aneurysm, stroke, embolism, thrombosis, coronary artery disease, arteriosclerosis, and/or atherosclerosis. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
In addition, antagonists of the CAN-12v1 polynucleotides and polypeptides may have uses that include diagnosing, treating, prognosing, and/or preventing diseases or disorders related to hyper calpain activity, which may include immune and/or proliferative diseases or disorders, particularly thrombosis, embolism, and other blood disorders. Therapeutic and/or pharmaceutical compositions comprising the CAN-12v1 polypeptides may be formulated to comprise heparin.
In addition, antagonists of the CAN-12v1 polynucleotides and polypeptides may have uses that include diagnosing, treating, ameliorating, prognosing, and/or preventing diseases or disorders related to hyper calpain activity, which may include neuronal excitotoxicity, ischemic stroke, hemoragic stroke, hypoxic stress, trauma, cell destruction, spinal cord injury following trauma, degeneration of vulnerable hippocampal neurons after ischemia, reovirus-induced apoptosis, viral-induced induced myocarditis, acute and chronic inflammation, cataract formation, multiple sclerosis, demylenating disorders, acoustic trauma, hearing loss caused by noise, neuronal damage, cardiac ischemic damage, and/or hepatocyte necrosis during and following anoxia
CAN-12v1 polynucleotides and polypeptides, including fragments and/or antagonsists thereof, may have uses which include modulating development, differentiation, cellular transformation in response to cell signaling, cell-cell and/or cell-extracellular matrix interactions, clustering of the integrin receptor aIIb3, modulating in long term potentiation (memory), modulating neurite outgrowth, modulating cortical lamination activation of protein kinases and phosphatases, remodeling and disassembling the cytoskeleton, cell cycle modulation, in addition, to ameliorating, preventing, and/or treating limb-girdle muscular dystrophy (LGMD), insulin resistance in diabetics, Alzheimer's disease, Multiple sclerosis, Huntington's disease, Parkinson's disease and amyotrophy.
Moreover, CAN-12v1 polynucleotides and polypeptides, including fragments and agonists thereof, may have uses which include treating, diagnosing, prognosing, and/or preventing hyperproliferative disorders, particularly of the neural and immune systems. Such disorders may include, for example, cancers, and metastatic conditions.
CAN-12v1 polynucleotides and polypeptides, including fragments and/or antagonsists thereof, may have uses which include identification of modulators of CAN-12v1 function including antibodies (for detection or neutralization), naturally-occurring modulators and small molecule modulators. Antibodies to domains (including CAN-12v1 epitopes provided herein) of the CAN-12v1 protein could be used as diagnostic agents of inflammatory conditions in patients, are useful in monitoring the activation and presence of cognate proteases, and can be used as a biomarker for the protease involvement in disease states and in the evaluation of inhibitors of the cognate protease in vivo.
CAN-12v1 polypeptides and polynucleotides are useful for diagnosing diseases related to over or under expression of CAN-12v1 proteins by identifying mutations in the CAN-12v1 gene using CAN-12v1 probes, or determining CAN-12v1 protein or mRNA expression levels. CAN-12v1 polypeptides are also useful for screening for compounds, which affect activity of the protein. Diseases that can be treated with CAN-12v1 include, the following, non-limiting examples: neuro-regeneration, neuropathic pain, obesity, anorexia, HIV infections, cancers, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, osteoporosis, angina pectoris, myocardial infarction, psychotic, immune, metabolic, cardiovascular, and neurological disorders.
The predominate expression in neural tissues, combined with the significant expression in a number of other tissues, suggests the CAN-12v1 polynucleotide and polypeptide of the present invention may be involved in modulating nerve invasion, innervation, nerve maintenance, and potentially myeline sheath maintenance and integrity.
The CAN-12v1 polynucleotides and polypeptides, including fragments and antagonists thereof, may have uses which include detecting, diagnosing, treating, ameliorating, and/or preventing diseases and disorders of the neural system, particularly Alzheimer's disease, either directly or indirectly, in addition to other neural disorders known in the art or provided in the “Neurological Diseases” section herein, such as modulating nerve invasion, innervation, nerve maintenance, potentially myelin sheath maintenance and integrity, encephalomyelitis, autoimmune encephalomyelitis, human T cell leukemia virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and neuro-inflammatory diseases.
Molecular genetic manipulation of the structure of the active site domain, particularly the predicted catalytic amino acids, and of other functional domains in the calpain family (e.g., active site domain binding pocket) enables the production of calpains with tailor-made activities. Thus, the CAN-12v1 polypeptides, and fragments thereof, as well as any homologous product resulting from genetic manipulation of the structure, are useful for NMR-based design of modulators of CAN-12v1 biological activity, and calpains, in general.
CAN-12v1 polypeptides and polynucleotides have additional uses which include diagnosing diseases related to the over and/or under expression of CAN-12v1 by identifying mutations in the CAN-12v1 gene by using CAN-12v1 sequences as probes or by determining CAN-12v1 protein or mRNA expression levels. CAN-12v1 polypeptides may be useful for screening compounds that affect the activity of the protein. CAN-12v1 peptides can also be used for the generation of specific antibodies and as bait in yeast two hybrid screens to find proteins the specifically interact with CAN-12v1 (described elsewhere herein).
The CAN-12v1 polynucleotides and polypeptides, including fragments and agonists thereof, may have uses which include detecting, diagnosing, treating, ameliorating, and/or preventing metabolic diseases and disorders, such as diabetes. Moreover, expressed human CAN-12v1 may be useful in the detection of patients susceptible to diabetes. Also paradigms that would simulate intracellular CAN-12v1 activity would be useful in treating diabetes.
The CAN-12v1 polynucleotides and polypeptides, including fragments thereof, may have uses which include identifying inhibitors of intracellular calpain inhibitors (calpastatins) leading to an effective increase in calpain activity.
Various approaches to detect alterations or allelic variants at the genomic or mRNA level of CAN-12v1, could be used as a diagnostic for identifying MS patients, or individuals susceptible to have MS. It is likely that the CAN-12v1 gene comprises polymorphic sites (i.e. SNPs), with specific alleles which may be associated with MS or other neurodegenerative disorders, or associated with an increased likelihood of developing these diseases. Therefore, the invention provides the CAN-12v1 sequence that can be used to design specific primers for the identification of polymorphisms or mutations in CAN-12v1 of patients affected with MS, The presence of a specific allele variant, such as a SNP allele or SNPs haplotype that renders the subject carrying it more susceptible to develop MS or other related diseases could be identified (e.g. a variant in the CAN-12v1 promoter region that increased transcript levels of CAN-i 2v1, or mutations in the coding sequence that increased the stability or half-life of the CAN-12v1 protein). Other methods such as Northern-blot analysis could be performed to measure transcript levels using a CAN-12v1 cDNA probe derived from the sequence of the invention.
Although it is believed the encoded polypeptide may share at least some biological activities with human calpains (particularly m-calpains), a number of methods of determining the exact biological function of this clone are either known in the art or are described elsewhere herein. Briefly, the function of this clone may be determined by applying microarray methodology. Nucleic acids corresponding to the CAN-12v1 polynucleotides, in addition to, other clones of the present invention, may be arrayed on microchips for expression profiling. Depending on which polynucleotide probe is used to hybridize to the slides, a change in expression of a specific gene may provide additional insight into the function of this gene based upon the conditions being studied. For example, an observed increase or decrease in expression levels when the polynucleotide probe used comes from diseased neural tissue, as compared to, normal tissue might indicate a function in modulating neural function, for example. In the case of CAN-12v1, spinal cord, lymph node, thymus, and/or spleen tissue should be used to extract RNA to prepare the probe.
In addition, the function of the protein may be assessed by applying quantitative PCR methodology, for example. Real time quantitative PCR would provide the capability of following the expression of the CAN-12v1 gene throughout development, for example. Quantitative PCR methodology requires only a nominal amount of tissue from each developmentally important step is needed to perform such experiments. Therefore, the application of quantitative PCR methodology to refining the biological function of this polypeptide is encompassed by the present invention. In the case of CAN-12v1, a disease correlation related to CAN-12v1 may be made by comparing the mRNA expression level of CAN-12v1 in normal tissue, as compared to diseased tissue (particularly diseased tissue isolated from the following: esophagus, spinal cord, lymph node, thymus, and/or spleen tissue). Significantly higher or lower levels of CAN-12v1 expression in the diseased tissue may suggest CAN-12v1 plays a role in disease progression, and antagonists against CAN-12v1 polypeptides would be useful therapeutically in treating, preventing, and/or ameliorating the disease. Alternatively, significantly higher or lower levels of CAN-12v1 expression in the diseased tissue may suggest CAN-12v1 plays a defensive role against disease progression, and agonists of CAN-12v1 polypeptides may be useful therapeutically in treating, preventing, and/or ameliorating the disease. Also encompassed by the present invention are quantitative PCR probes corresponding to the polynucleotide sequence provided as SEQ ID NO:53 (FIGS. 8A-C).
The function of the protein may also be assessed through complementation assays in yeast. For example, in the case of the CAN-12v1, transforming yeast deficient in calpain activity, particularly m-calpain activity, and assessing their ability to grow would provide convincing evidence the CAN-12v1 polypeptide has calpain activity, and possibly m-calpain activity. Additional assay conditions and methods that may be used in assessing the function of the polynucleotides and polypeptides of the present invention are known in the art, some of which are disclosed elsewhere herein.
Alternatively, the biological function of the encoded polypeptide may be determined by disrupting a homologue of this polypeptide in Mice and/or rats and observing the resulting phenotype. Such knock-out experiments are known in the art, some of which are disclosed elsewhere herein.
Moreover, the biological function of this polypeptide may be determined by the application of antisense and/or sense methodology and the resulting generation of transgenic mice and/or rats. Expressing a particular gene in either sense or antisense orientation in a transgenic mouse or rat could lead to respectively higher or lower expression levels of that particular gene. Altering the endogenous expression levels of a gene can lead to the observation of a particular phenotype that can then be used to derive indications on the function of the gene. The gene can be either over-expressed or under expressed in every cell of the organism at all times using a strong ubiquitous promoter, or it could be expressed in one or more discrete parts of the organism using a well characterized tissue-specific promoter (e.g., an esophagus, spinal cord, lymph node, thymus, or spleen specific promoter), or it can be expressed at a specified time of development using an inducible and/or a developmentally regulated promoter.
In the case of CAN-12v1 transgenic mice or rats, if no phenotype is apparent in normal growth conditions, observing the organism under diseased conditions (neural, immune, hematopoietic diseases or disorders, cancers, etc.) may lead to understanding the function of the gene. Therefore, the application of antisense and/or sense methodology to the creation of transgenic mice or rats to refine the biological function of the polypeptide is encompassed by the present invention.
In preferred embodiments, the following N-terminal CAN-12v1 deletion polypeptides are encompassed by the present invention: M1-L694, S2-L694, L3-L694, W4-L694, P5-L694, P6-L694, F7-L694, R8-L694, C9-L694, R10-L694, W11-L694, K12-L694, L13-L694, A14-L694, P15-L694, R16-L694, Y17-L694, S18-L694, R19-L694, R20-L694, A21-L694, S22-L694, P23-L694, Q24-L694, Q25-L694, P26-L694, Q27-L694, Q28-L694, D29-L694, F30-L694, E31-L694, A32-L694, L33-L694, L34-L694, A35-L694, E36-L694, C37-L694, L38-L694, R39-L694, N40-L694, G41-L694, C42-L694, L43-L694, F44-L694, E45-L694, D46-L694, T47-L694, S48-L694, F49-L694, P50-L694, A51-L694, T52-L694, L53-L694, S54-L694, S55-L694, I56-L694, G57-L694, S58-L694, G59-L694, S60-L694, L61-L694, L62-L694, Q63-L694, K64-L694, L65-L694, P66-L694, P67-L694, R68-L694, L69-L694, Q70-L694, W71-L694, K72-L694, R73-L694, P74-L694, P75-L694, E76-L694, L77-L694, H78-L694, S79-L694, N80-L694, P81-L694, Q82-L694, F83-L694, Y84-L694, F85-L694, A86-L694, K87-L694, A88-L694, K89-L694, R90-L694, L91-L694, D92-L694, L93-L694, C94-L694, Q95-L694, G96-L694, I97-L694, V98-L694, G99-L694, D100-L694, C101-L694, W102-L694, F103-L694, L104-L694, A105-L694, A106-L694, L107-L694, Q108-L694, A109-L694, L110-L694, A111-L694, L112-L694, H113-L694, Q114,-L694, D115-L694, I116-L694, L117-L694, S118-L694, R119-L694, V120-L694, V121-L694, P122-L694, L123-L694, N124-L694, Q125-L694, S126-L694, F127-L694, T128-L694, E129-L694, K130-L694, Y131-L694, A132-L694, G133-L694, I134-L694, F135-L694, R136-L694, F137-L694, W138-L694, F139-L694, W140-L694, H141-L694, Y142-L694, G143-L694, N144-L694, W145-L694, V146-L694, P147-L694, V148-L694, V149-L694, I150-L694, D151-L694, D152-L694, R153-L694, L154-L694, P155-L694, V156-L694, N157-L694, E158-L694, A159-L694, G160-L694, Q161-L694, L162-L694, V163-L694, F164-L694, V165-L694, S166-L694, S167-L694, T168-L694, Y169-L694, K170-L694, N171-L694, L172-L694, F173-L694, W174-L694, G175-L694, A176-L694, L177-L694, L178-L694, E179-L694, K180-L694, A181-L694, Y182-L694, A183-L694, K184-L694, L185-L694, S186-L694, G187-L694, S188-L694, Y189-L694, E190-L694, D191-L694, L192-L694, Q193-L694, S194-L694, G195-L694, Q196-L694, V197-L694, S198-L694, E199-L694, A200-L694, L201-L694, V202-L694, D203-L694, F204-L694, T205-L694, G206-L694, G207-L694, V208-L694, T209-L694, M210-L694, T211-L694, I212-L694, N213-L694, L214-L694, A215-L694, E216-L694, A217-L694, H218-L694, G219-694, N220-L694, L221-L694, W222-L694, D223-L694, 1224-L694, L225-L694, I226-L694, E227-L694, A228-L694, T229-L694, Y230-L694, N231-L694, R232-L694, T233-L694, L234-L694, I235-L694, G236-L694, C237-L694, Q238-L694, T239-L694, H240-L694, S241-L694, G242-L694, E243-L694, K244-L694, I245-L694, L246-L694, E247-L694, N248-L694, G249-L694, L250-L694, V251-L694, E252-L694, G253-L694, H254-L694, A255-L694, Y256-L694, T257-L694, L258-L694, T259-L694, G260-L694, I261-L694, R262-L694, K263-L694, V264-L694, T265-L694, C266-L694, K267-L694, H268-L694, R269-L694, P270-L694, E271-L694, Y272-L694, L273-L694, V274-L694, K275-L694, L276-L694, R277-L694, N278-L694, P279-L694, W280-L694, G281-L694, K282-L694, V283-L694, E284-L694, W285-L694, K286-L694, G287-L694, D288-L694, W289-L694, S290-L694, D291-L694, S292-L694, S293-L694, S294-L694, K295-L694, W296-L694, E297-L694, L298-L694, L299-L694, S300-L694, P301-L694, K302-L694, E303-L694, K304-L694, I305-L694, L306-L694, L307-L694, L308-L694, R309-L694, K310-L694, D311-L694, N312-L694, D313-L694, G314-L694, E315-L694, F316-L694, W317-L694, M318-L694, T319-L694, L320-L694, Q321-L694, D322-L694, F323-L694, K324-L694, T325-L694, H326-L694, F327-L694, V328-L694, L329-L694, L330-L694, V331-L694, I332-L694, C333-L694, K334-L694, L335-L694, T336-L694, P337-L694, G338-L694, L339-L694, L340-L694, S341-L694, Q342-L694, E343-L694, A344-L694, A345-L694, Q346-L694, K347-L694, W348-L694, T349-L694, Y350-L694, T351-L694, M352-L694, R353-L694, E354-L694, G355-L694, R356-L694, W357-L694, E358-L694, K359-L694, R360-L694, S361-L694, T362-L694, A363-L694, G364-L694, G365-L694, Q366-L694, R367-L694, Q368-L694, L369-L694, L370-L694, Q371-L694, D372-L694, T373-L694, F374-L694, W375-L694, K376-L694, N377-L694, P378-L694, Q379-L694, F380-L694, L381-L694, L382-L694, S383-L694, V384-L694, W385-L694, R386-L694, P387-L694, E388-L694, E389-L694, G390-L694, R391-L694, R392-L694, S393-L694, L394-L694, R395-L694, P396-L694, C397-L694, S398-L694, V399-L694, L400-L694, V401-L694, S402-L694, L403-L694, L404-L694, Q405-L694, K406-L694, P407-L694, R408-L694, H409-L694, R410-L694, C411-L694, R412-L694, K413-L694, R414-L694, K415-L694, P416-L694, L417-L694, L418-L694, A419-L694, I420-L694, G421-L694, F422-L694, Y423-L694, L424-L694, Y425-L694, R426-L694, Y427-L694, H428-L694, D429-L694, D430-L694, Q431-L694, R432-L694, R433-L694, L434-L694, P435-L694, P436-L694, E437-L694, F438-L694, F439-L694, Q440-L694, R441-L694, N442-L694, T443-L694, P444-L694, L445-L694, S446-L694, Q447-L694, P448-L694, D449-L694, R450-L694, F451-L694, L452-L694, K453-L694, E454-L694, K455-L694, E456-L694, V457-L694, S458-L694, Q459-L694, E460-L694, L461-L694, C462-L694, L463-L694, E464-L694, P465-L694, G466-L694, T467-L694, Y468-L694, L469-L694, I470-L694, V471-L694, P472-L694, C473-L694, I474-L694, L475-L694, E476-L694, A477-L694, H478-L694, Q479-L694, K480-L694, S481-L694, E482-L694, F483-L694, V484-L694, L485-L694, R486-L694, V487-L694, F488-L694, S489-L694, R490-L694, K491-L694, H492-L694, I493-L694, F494-L694, Y495-L694, E496-L694, I497-L694, G498-L694, S499-L694, N500-L694, S501-L694, G502-L694, V503-L694, V504-L694, F505-L694, S506-L694, K507-L694, E508-L694, I509-L694, E510-L694, D511-L694, Q512-L694, N513-L694, E514-L694, R515-L694, Q516-L694, D517-L694, E518-L694, F519-L694, F520-L694, T521-L694, K522-L694, F523-L694, F524-L694, E525-L694, K526-L694, H527-L694, P528-L694, E529-L694, I530-L694, N531-L694, A532-L694, V533-L694, Q534-L694, L535-L694, Q536-L694, N537-L694, L538-L694, L539-L694, N540-L694, Q541-L694, and/or M542-L694 of SEQ ID NO:54. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal CAN-12v1 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal CAN-12v1 deletion polypeptides are encompassed by the present invention: M1-L694, M1-L693, M1-T692, M1-T691, M1-N690, M1-F689, M1-I688, M1-L687, M1-Y686, M1-S685, M1-T684, M1-S683, M1-H682, M1-A681, M1-V680, M1-G679, M1-G678, M1-D677, M1-I676, M1-N675, M1-A674, M1-L673, M1-1672, M1-V671, M1-P670, M1-V669, M1-I668, M1-M667, M1-F666, M1-F665, M1-T664, M1-P663, M1-T662, M1-S661, M1-Q660, M1-L659, M1-D658, M1-V657, M1-D656, M1-K655, M1-L654, M1-T653, M1-V652, M1-S651, M1-R650, M1-I649, M1-L648, M1-T647, M1-V646, M1-E645, M1-A644, M1-H643, M1-W642, M1-V641, M1-D640, M1-G639, M1-R638, M1-R637, M1-Q636, M1-R635, M1-I634, M1-L633, M1-T632, M1-C631, M1-G630, M1-A629, M1-R628, M1-T627, M1-H626, M1-G625, M1-C624, M1-S623, M1-W622, M1-S621, M1-K620, M1-R619, M1-H618, M1-R617, M1-616, M1-A615, M1-E614, M1-R613, M1-M612, M1-A611, M1-A610, M1-H609, M1-L608, M1-Q607, M1-E606, M1-W605, M1-N604, M1-L603, M1-Y602, M1-G601, M1-S600, M1-G599, M1-R598, M1-D597, M1-Q596, M1-K595, M1-H594, M1-F593, M1-V592, M1-K591, M1-Q590, M1-S589, M1-L588, M1-K587, M1-L586, M1-Q585, M1-K584, M1-W583, M1-L582, M1-D581, M1-R580, M1-F579, M1-E578, M1-Q577, M1-I576, M1-S575, M1-M574, M1-T573, M1-G572, M1-S571, M1-A570, M1-N569, M1-L568, M1-D567, M1-L566, M1-L565, M1-A564, M1-L563, M1-I562, M1-G561, M1-Q560, M1-C559, M1-A558, M1-E557, M1-L556, M1-S555, M1-F554, M1-F553, M1-P552, M1-Q551, M1-R550, M1-S549, M1-G548, M1-L547, M1-S546, M1-S545, M1-W544, M1-T543, M1-M542, M1-Q541, M1-N540, M1-L539, M1-L538, M1-N537, M1-Q536, M1-L535, M1-Q534, M1-V533, M1-A532, M1-N531, M1-1530, M1-E529, M1-P528, M1-H527, M1-K526, M1-E525, M1-F524, M1-F523, M1-K522, M1-T521, M1-F520, M1-F519, M1-E518, M1-D517, M1-Q516, M1-R515, M1-E514, M1-N513, M1-Q512, M1-D511, M1-E510, M1-I509, M1-E508, M1-K507, M1-S506, M1-F505, M1-V504, M1-V503, M1-G502, M1-S501, M1-N500, M1-S499, M1-G498, M1-I497, M1-E496, M1-Y495, M1-F494, M1-1493, M1-H492, M1-K491, M1-R490, M1-S489, M1-F488, M1-V487, M1-R486, M1-L485, M1-V484, M1-F483, M1-E482, M1-S481, M1-K480, M1-Q479, M1-H478, M1-A477, M1-E476, M1-L475, M1-I474, M1-C473, M1-P472, M1-V471, M1-I470, M1-L469, M1-Y468, M1-T467, M1-G466, M1-P465, M1-E464, M1-L463, M1-C462, M1-L461, M1-E460, M1-Q459, M1-S458, M1-V457, M1-E456, M1-K455, M1-E454, M1-K453, M1-L452, M1-F451, M1-R450, M1-D449, M1-P448, M1-Q447, M1-S446, M1-L445, M1-P444, M1-T443, M1-N442, M1-R441, M1-Q440, M1-F439, M1-F438, M1-E437, M1-P436, M1-P435, M1-L434, M1-R433, M1-R432, M1-Q431, M1-D430, M1-D429, M1-H428, M1-Y427, M1-R426, M1-Y425, M1-L424, M1-Y423, M1-F422, M1-G421, M1-1420, M1-A419, M1-L418, M1-L417, M1-P416, M1-K415, M1-R414, M1-K413, M1-R412, M1-C411, M1-R410, M1-H409, M1-R408, M1-P407, M1-K406, M1-Q405, M1-L404, M1-L403, M1-S402, M1-V401, M1-L400, M1-V399, M1-S398, M1-C397, M1-P396, M1-R395, M1-L394, M1-S393, M1-R392, M1-R391, M1-G390, M1-E389, M1-E388, M1-P387, M1-R386, M1-W385, M1-V384, M1-S383, M1-L382, M1-L381, M1-F380, M1-Q379, M1-P378, M1-N377, M1-K376, M1-W375, M1-F374, M1-T373, M1-D372, M1-Q371, M1-L370, M1-L369, M1-Q368, M1-R367, M1-Q366, M1-G365, M1-G364, M1-A363, M1-T362, M1-S361, M1-R360, M1-K359, M1-E358, M1-W357, M1-R356, M1-G355, M1-E354, M1-R353, M1-M352, M1-T351, M1-Y350, M1-T349, M1-W348, M1-K347, M1-Q346, M1-A345, M1-A344, M1-E343, M1-Q342, M1-S341, M1-L340, M1-L339, M1-G338, M1-P337, M1-T336, M1-L335, M1-K334, M1-C333, M1-I332, M1-V331, M1-L330, M1-L329, M1-V328, M1-F327, M1-H326, M1-T325, M1-K324, M1-F323, M1-D322, M1-Q321, M1-L320, M1-T319, M1-M318, M1-W317, M1-F316, M1-E315, M1-G314, M1-D313, M1-N312, M1-D311, M1-K310, M1-R309, M1-L308, M1-L307, M1-L306, M1-I305, M1-K304, M1-E303, M1-K302, M1-P301, M1-S300, M1-L299, M1-L298, M1-E297, M1-W296, M1-K295, M1-S294, M1-S293, M1-S292, M1-D291, M1-S290, M1-W289, M1-D288, M1-G287, M1-K286, M1-W285, M1-E284, M1-V283, M1-K282, M1-G281, M1-W280, M1-P279, M1-N278, M1-R277, M1-L276, M1-K275, M1-V274, M1-L273, M1-Y272, M1-E271, M1-P270, M1-R269, M1-H268, M1-K267, M1-C266, M1-T265, M1-V264, M1-K263, M1-R262, M1-I261, M1-G260, M1-T259, M1-L258, M1-T257, M1-Y256, M1-A255, M1-H254, M1-G253, M1-E252, M1-V251, M1-L250, M1-G249, M1-N248, M1-E247, M1-L246, M1-I245, M1-K244, and/or M1-E243 of SEQ ID NO:54. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal CAN-12v1 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
Alternatively, preferred polypeptides of the present invention may comprise polypeptide sequences corresponding to, for example, internal regions of the CAN-12v1 polypeptide (e.g., any combination of both N— and C-terminal CAN-12v1 polypeptide deletions) of SEQ ID NO:54. For example, internal regions could be defined by the equation: amino acid NX to amino acid CX, wherein NX refers to any N-terminal deletion polypeptide amino acid of CAN-12v1 (SEQ ID NO:54), and where CX refers to any C-terminal deletion polypeptide amino acid of CAN-12v1 (SEQ ID NO:54). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.
The present invention also encompasses immunogenic and/or antigenic epitopes of the CAN-12v1 polypeptide.
The CAN-12v1 polypeptides of the present invention were determined to comprise several phosphorylation sites based upon the Motif algorithm (Genetics Computer Group, Inc.). The phosphorylation of such sites may regulate some biological activity of the CAN-12v1 polypeptide. For example, phosphorylation at specific sites may be involved in regulating the proteins ability to associate or bind to other molecules (e.g., proteins, ligands, substrates, DNA, etc.). In the present case, phosphorylation may modulate the ability of the CAN-12v1 polypeptide to associate with other polypeptides, particularly the serine protease substrate for CAN-12v1, or its ability to modulate serine protease function.
The CAN-12v1 polypeptide was predicted to comprise eleven PKC phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). In vivo, protein kinase C exhibits a preference for the phosphorylation of serine or threonine residues. The PKC phosphorylation sites have the following consensus pattern: [ST]-x-[RK], where S or T represents the site of phosphorylation and ‘x’ an intervening amino acid residue. Additional information regarding PKC phosphorylation sites can be found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem. 161:177-184(1986), and Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem. 260:12492-12499(1985); which are hereby incorporated by reference herein.
In preferred embodiments, the following PKC phosphorylation site polypeptides are encompassed by the present invention: LAPRYSRRASPQQ (SEQ ID NO:58), LNQSFTEKYAGIF (SEQ ID NO:59), VFVSSTYKNLFWG (SEQ ID NO:60), GIRKVTCKHRPEY (SEQ ID NO:61), DWSDSSSKWELLS (SEQ ID NO:62), KWELLSPKEKILL (SEQ ID NO:63), QKWTYTMREGRWE (SEQ ID NO:64), EEGRRSLRPCSVL (SEQ ID NO:65), VLRVFSRKHIFYE (SEQ ID NO:66), KQLKLSQKVFHKQ (SEQ ID NO:67), and/or LIRSVTLKDVDLQ (SEQ ID NO:68). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the CAN-12v1 PKC phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The CAN-12v1 polypeptide has been shown to comprise four glycosylation site according to the Motif algorithm (Genetics Computer Group, Inc.). As discussed more specifically herein, protein glycosylation is thought to serve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.
In preferred embodiments, the following asparagine glycosylation site polypeptide is encompassed by the present invention: RVVPLNQSFTEKYA (SEQ ID NO:69), IEATYNRTLIGCQT (SEQ ID NO:70), ALLDLNASGTMSIQ (SEQ ID NO:71), and/or SYLIFNTTLL (SEQ ID NO:72). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of the CAN-12v1 asparagine glycosylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The CAN-12v1 polypeptide has been shown to comprise one amidation site according to the Motif algorithm (Genetics Computer Group, Inc.). The precursor of hormones and other active peptides which are C-terminally amidated is always directly followed by a glycine residue which provides the amide group, and most often by at least two consecutive basic residues (Arg or Lys) which generally function as an active peptide precursor cleavage site. Although all amino acids can be amidated, neutral hydrophobic residues such as Val or Phe are good substrates, while charged residues such as Asp or Arg are much less reactive. A consensus pattern for amidation sites is the following: x-G-[RK]-[RK], wherein “X” represents the amidation site. Additional information relating to asparagine glycosylation may be found in reference to the following publications, which are hereby incorporated by reference herein: Kreil G., Meth. Enzymol. 106:218-223(1984); and Bradbury A. F., Smyth D. G., Biosci. Rep. 7:907-916(1987).
In preferred embodiments, the following amidation site polypeptide is encompassed by the present invention: VWRPEEGRRSLRPC (SEQ ID NO:73). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this CAN-12v1 amidation site polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The CAN-12v1 polypeptide has been shown to comprise one RGD cell attachment site domain according to the Motif algorithm (Genetics Computer Group, Inc.). The sequence Arg-Gly-Asp, found in fibronectin, is crucial for its interaction with its cell surface receptor, an integrin. What has been called the ‘RGD’ tripeptide is also found in the sequences of a number of other proteins, where it has been shown to play a role in cell adhesion. Non-limiting examples of these proteins are the following: some forms of collagens, fibrinogen, vitronectin, von Willebrand factor (VWF), snake disintegrins, and slime mold discoidins. The ‘RGD’ tripeptide is also found in other proteins where it may serve the same purpose. A consensus pattern for RGD cell attachment sites is the following: R-G-D. Additional information relating to RGD cell attachment site domains may be found in reference to the following publications, which are hereby incorporated by reference herein: Ruoslahti E., Pierschbacher M. D., Cell 44:517-518(1986); and d'Souza S. E., Ginsberg M. H., Plow E. F., Trends Biochem. Sci. 16:246-250(1991).
In preferred embodiments, the following RGD cell attachment site domain polypeptide is encompassed by the present invention: LIRQRRGDVWHAE (SEQ ID NO:74). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this RGD cell attachment site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
In confirmation of the CAN-12v1 polypeptide being a calpain, it has been shown to comprise one EF-hand calcium-binding domain according to the Motif algorithm (Genetics Computer Group, Inc.). Many calcium-binding proteins belong to the same evolutionary family and share a type of calcium-binding domain known as the EF-hand. This type of domain consists of a twelve residue loop flanked on both side by a twelve residue alpha-helical domain. In an EF-hand loop the calcium ion is coordinated in a pentagonal bipyramidal configuration. The six residues involved in the binding are in positions 1, 3, 5, 7, 9 and 12; these residues are denoted by X, Y, Z, —Y, —X and -Z. The invariant Glu or Asp at position 12 provides two oxygens for liganding Ca (bidentate ligand). Several representative proteins containing EF-hand regions are provided below: For each type of protein, the total number of EF-hand regions known or supposed to exist are provided in parenthesis: Aequorin and Renilla luciferin binding protein (LBP) (Ca=3); Alpha actinin (Ca=2); Calbindin (Ca=4); Calcineurin B subunit (protein phosphatase 2B regulatory subunit) (Ca=4); Calcium-binding protein from Streptomyces erythraeus (Ca=3?); Calcium-binding protein from Schistosoma mansoni (Ca=2?); Calcium-binding proteins TCBP-23 and TCBP-25 from Tetrahymena thermophila (Ca=4?); Calcium-dependent protein kinases (CDPK) from plants (Ca=4); Calcium vector protein from amphoxius (Ca=2); Calcyphosin (thyroid protein p24) (Ca=4?); Calmodulin (Ca=4, except in yeast where Ca=3); Calpain small and large chains (Ca=2); Calretinin (Ca=6); Calcyclin (prolactin receptor associated protein) (Ca=2); Caltractin (centrin) (Ca=2 or 4); Cell Division Control protein 31 (gene CDC31) from yeast (Ca=2?); Diacylglycerol kinase (EC 2.7.1.107) (DGK) (Ca=2); FAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) from mammals (Ca=1); Fimbrin (plastin) (Ca=2); Flagellar calcium-binding protein (1f8) from Trypanosoma cruzi (Ca=1 or 2); Guanylate cyclase activating protein (GCAP) (Ca=3); Inositol phospholipid-specific phospholipase C isozymes gamma-1 and delta-1 (Ca=2) [10]; Intestinal calcium-binding protein (ICaBPs) (Ca=2); MIF related proteins 8 (MRP-8 or CFAG) and 14 (MRP-14) (Ca=2); Myosin regulatory light chains (Ca=1); Oncomodulin (Ca=2); Osteonectin (basement membrane protein BM-40) (SPARC) and proteins that contains an ‘osteonectin’ domain (QR1, matrix glycoprotein SC1) (Ca=1); Parvalbumins alpha and beta (Ca=2); Placental calcium-binding protein (18a2) (nerve growth factor induced protein 42a) (p9k) (Ca=2); Recoverins (visinin, hippocalcin, neurocalcin, S-modulin) (Ca=2 to 3); Reticulocalbin (Ca=4); S-100 protein, alpha and beta chains (Ca=2); Sarcoplasmic calcium-binding protein (SCPs) (Ca=2 to 3); Sea urchin proteins Spec 1 (Ca=4), Spec 2 (Ca=4?), Lps-1 (Ca=8); Serine/threonine protein phosphatase rdgc (EC 3.1.3.16) from Drosophila (Ca=2); Sorcin V19 from hamster (Ca=2); Spectrin alpha chain (Ca-2); Squidulin (optic lobe calcium-binding protein) from squid (Ca=4); and Troponins C; from skeletal muscle (Ca=4), from cardiac muscle (Ca=3), from arthropods and molluscs (Ca=2).

A consensus pattern for EF hand calcium binding domains is the following:

Additional information relating to RGD cell attachment site domains may be found in reference to the following publications, which are hereby incorporated by reference herein: Kawasaki H., Kretsinger R. H., Protein Prof. 2:305-490(1995); Kretsinger R. H., Cold Spring Harbor Symp. Quant. Biol. 52:499-510(1987); Moncrief N. D., Kretsinger R. H., Goodman M., J. Mol. Evol. 30:522-562(1990); Nakayama S., Moncrief N. D., Kretsinger R. H., J. Mol. Evol. 34:416-448(1992); Heizmann C. W., Hunziker W., Trends Biochem. Sci. 16:98-103(1991); Kligman D., Hilt D. C., Trends Biochem. Sci. 13:437-443(1988); Strynadka N. C. J., James M. N. G., Annu. Rev. Biochem. 58:951-98(1989); Haiech J., Sallantin J., Biochimie 67:555-560(1985); Chauvaux S., Beguin P., Aubert J.-P., Bhat K. M., Gow L. A., Wood T. M., Bairoch A., Biochem. J. 265:261-265(1990); Bairoch A., Cox J. A., FEBS Lett. 269:454-456(1990).
In preferred embodiments, the following EF-hand calcium binding domain polypeptide is encompassed by the present invention: WLALLDLNASGTMSIQEFRDLWK (SEQ ID NO:75). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this EF-hand calcium binding domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
In further confirmation of the CAN-12v1 polypeptide being a calpain, it has been shown to comprise one eukaryotic thiol (cysteine) protease active site domain according to the Motif algorithm (Genetics Computer Group, Inc.). Eukaryotic thiol proteases (EC 3.4.22.-) are a family of proteolytic enzymes which contain an active site cysteine. Catalysis proceeds through a thioester intermediate and is facilitated by a nearby histidine side chain; an asparagine completes the essential catalytic triad. Non-limiting examples of proteases which are known to belong to this family are provided below: Vertebrate lysosomal cathepsins B (EC 3.4.22.1), H (EC 3.4.22.16), L (EC 3.4.22.15), and S (EC 3.4.22.27); Vertebrate lysosomal dipeptidyl peptidase I (EC 3.4.14.1) (also known as cathepsin C); Vertebrate calpains (EC 3.4.22.17) (Calpains are intracellular calcium-activated thiol protease that contain both a N-terminal catalytic domain and a C-terminal calcium-binding domain; Mammalian cathepsin K, which seems involved in osteoclastic bone resorption; Human cathepsin O; Bleomycin hydrolase (An enzyme that catalyzes the inactivation of the antitumor drug BLM (a glycopeptide); Plant enzymes: barley aleurain (EC 3.4.22.16), EP-B1/B4; kidney bean EP-C1, rice bean SH-EP; kiwi fruit actinidin (EC 3.4.22.14); papaya latex papain (EC 3.4.22.2), chymopapain (EC 3.4.22.6), caricain (EC 3.4.22.30), and proteinase IV (EC 3.4.22.25); pea turgor-responsive protein 15A; pineapple stem bromelain (EC 3.4.22.32); rape COT44; rice oryzain alpha, beta, and gamma; tomato low-temperature induced, Arabidopsis thaliana A494, RD19A and RD21A; House-dust mites allergens DerP1 and EurM1; Cathepsin B-like proteinases from the worms Caenorhabditis elegans (genes gcp-1, cpr-3, cpr-4, cpr-5 and cpr-6), Schistosoma mansoni (antigen SM31) and Japonica (antigen SJ3 1), Haemonchus contortus (genes AC-1 and AC-2), and Ostertagia ostertagi (CP-1 and CP-3); Slime mold cysteine proteinases CP1 and CP2; Cruzipain from Trypanosoma cruzi and brucei; Throphozoite cysteine proteinase (TCP) from various Plasmodium species; Proteases from Leishmania mexicana, Theileria annulata and Theileria parva; Baculoviruses cathepsin-like enzyme (v-cath); Drosophila small optic lobes protein (gene sol), a neuronal protein that contains a calpain-like domain; Yeast thiol protease BLH1/YCP1/LAP3; and Caenorhabditis elegans hypothetical protein C06G4.2, a calpain-like protein; Two bacterial peptidases are also part of this family—Aminopeptidase C from Lactococcus lactis (gene pepC), and Thiol protease tpr from Porphyromonas gingivalis.
A consensus pattern for eukaryotic thiol (cysteine) protease active site domains is the following: Q-x(3)-[GE]-x-C-[YW]-x(2)-[STAGC]-[STAGCV], wherein C is the active site residue, and “x” represents any amino acid. The residue in position 4 of the pattern is almost always cysteine; the only exceptions are calpains (Leu), bleomycin hydrolase (Ser) and yeast YCP1 (Ser); while the residue in position 5 of the pattern is always Gly except in papaya protease IV where it is Glu.
An additional consensus pattern for eukaryotic thiol (cysteine) protease active site domains is the following: [LIVMGSTAN]-x-H-[GSACE]-[LIVM]-x-[LIVMAT](2)-G-x-[GSADNH], wherein H is the active site residue, and “x” represents any amino acid.
An additional consensus pattern for eukaryotic thiol (cysteine) protease active site domains is the following: [FYCH]-[WI]-[LIVT]-x-[KRQAG]-N-[ST]-W-x(3)-[FYW]-G-x(2)-G-[LFYW]-[LIVMFYG]-x-[LIVMF], wherein N is the active site residue, and “x” represents any amino acid.
Additional information relating to for eukaryotic thiol (cysteine) protease active site domains may be found in reference to the following publications, which are hereby incorporated by reference herein: Dufour E., Biochimie 70:1335-1342(1988); Kirschke H., Barrett A. J., Rawlings N. D., Protein Prof. 2:1587-1643(1995); Shi G.-P., Chapman H. A., Bhairi S. M., Deleeuw C., Reddy V. Y., Weiss S. J., FEBS Lett. 357:129-134(1995); Velasco G., Ferrando A. A., Puente X. S., Sanchez L. M., Lopez-Otin C., J. Biol. Chem. 269:27136-27142(1994); Chapot-Chartier M. P., Nardi M., Chopin M. C., Chopin A., Gripon J. C., Appl. Environ. Microbiol. 59:330-333(1993); Higgins D. G., McConnell D. J., Sharp P. M., Nature 340:604-604(1989); Rawlings N. D., Barrett A. J., Meth. Enzymol. 244:461-486(1994), and http://www.expasy.ch/cgi-bin/lists?peptidas.txt, which are hereby incorporated by reference in their entirety herein.
In preferred embodiments, the following for eukaryotic thiol (cysteine) protease active site domain polypeptide is encompassed by the present invention: RLDLCQGIVGDCWFLAALQALA (SEQ ID NO:76). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this for eukaryotic thiol (cysteine) protease active site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
As referenced elsewhere herein, calpains are organized in domains. As a point of reference, the larger catalytic subunit of the best characterized m-calpain is organized in four domains (I-IV)(Hosfield et al., Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation. EMBO J 18:6880-9, 1999; Strobl et al., The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium. Proc Natl Acad Sci U S A. 97:588-92, 2000). The N-termninal domain I contains an alpha helical region. Domain II contains the catalytic active domain with the active site amino acids. Domain III contains the linker between the Ca2+ binding domain in domain IV to the active site domain II.
The CAN-12v1 calpain of the present invention has the same domain I and II as the CAN-12 calpain, but differ in domains III and IV. The N-terminal domain I consists of residues Met1-Arg20. Domain II of the present calpains (Ala21-Lys333) contain the catalytic active site residues acids (Cys101, His254 and Asn 278). As can be seen in the sequence alignments (FIGS. 2A-E), there is high amino acid sequence homology in the amino acid residues bracketing the active site amino acids. Combined domains I and II of the calpains of the present invention are 42-45% homologous to m-calpain.
The CAN-12v1 calpain of the present invention, have the same domain I and II, although they differ in composition and content of domains III and IV. The CAN-12 and CAN-12v1 calpain contains the linker (domain III) and the C-ternminal domain IV, though the CAN-12v1 calpain is lacking residues Met426, Asn427 and Lys428 of SEQ ID NO54) in the “linker” domain.
The present invention also provides a three-dimensional homology model of the CAN-12 polypeptide (see FIG. 6). Although the CAN-12 polypeptide sequence is different than the CAN-12v1 polypeptide sequence, the fact that domain I and II are substantially the same suggests the homology model of CAN-12 may be used for designing potential ligands (including agonists and/or antagonists) for the CAN-12v1 polypeptide. A three-dimensional homology model can be constructed on the basis of the known structure of a homologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995, Yuan, et al, 1995). The homology model of the CAN-12 polypeptide, corresponding to amino acid residues 12 to 524 of SEQ ID NO:2, was based upon the homologous structure of CAN2, a m-calpain family member (Strobl et al, 2000; hCAN2; Genbank Accession No. gi|7546423; SEQ ID NO:11) and is defined by the set of structural coordinates set forth in Table IV herein.
A description of the headings in Table IV are as follows: “Atom No” refers to the atom number within the CAN-12 homology model; “Atom name” refers to the element whose coordinates are measured, the first letter in the column defines the element; “Residue” refers to the amino acid within which the atom resides, and the provided number after the amino acid refers to the amino acid number of the “residue”; “X Coord”, “Y Coord”, and “Z Coord” structurally define the atomic position of the element measured in three dimensions.
The CAN-12 homology model of the present invention may provide one basis for designing rational stimulators (agonists) and/or inhibitors (antagonists) of one or more of the biological functions of CAN-12v1, or of CAN-12v1 mutants having altered specificity (e.g., molecularly evolved CAN-12v1 polypeptides, engineered site-specific CAN-12v1 mutants, CAN-12v1 allelic variants, etc.).
Homology models are not only useful for designing rational agonists and/or antagonists, but are also useful in predicting the function of a particular polypeptide. The functional predictions from homology models are typically more accurate than the functional attributes derived from traditional polypeptide sequence homology alignments (e.g., CLUSTALW), particularly when the three dimensional structure of a related polypeptide is known (e.g., m-calpain family member CAN2 protein; Genbank Accession No. gi|7546423; SEQ ID NO:11). The increased prediction accuracy is based upon the fact that homology models approximate the three-dimensional structure of a protein, while homology based alignments only take into account the one dimension polypeptide sequence. Since the function of a particular polypeptide is determined not only by its primary, secondary, and tertiary structure, functional assignments derived solely upon homology alignments using the one dimensional protein sequence may be less reliable. A 3-dimensional model can be constructed on the basis of the known structure of a homologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995, Yuan, et al, 1995).
Prior to developing a homology model, those of skill in the art would appreciate that a template of a known protein, or model protein, must first be identified which will be used as a basis for constructing the homology model for the protein of unknown structure (query template). In the case of the CAN-12 polypeptide of the present invention, the model protein template used in constructing the CAN-12 homology model was the m-calpain family member CAN2 protein; Genbank Accession No. gi|7546423; SEQ ID NO:11).
Identifying a template can be accomplished using pairwise alignment of protein sequences using such programs as FASTA (Pearson, et al 1990) and BLAST (Altschul, et al, 1990). In cases where sequence similarity is high (greater than 30%), such pairwise comparison methods may be adequate for identifying an appropriate template. Likewise, multiple sequence alignments or profile-based methods can be used to align a query sequence to an alignment of multiple (structurally and biochemically) related proteins. When the sequence similarity is low, more advanced techniques may be used. Such techniques, include, for example, protein fold recognition (protein threading; Hendlich, et al, 1990), where the compatibility of a particular polypeptide sequence with the 3-dimensional fold of a potential template protein is gauged on the basis of a knowledge-based potential.
Following the initial sequence alignment, the second step would be to optimally align the query template to the model template by manual manipulation and/or by the incorporation of features specific to the polypeptides (e.g., motifs, secondary structure predictions, and allowed conservations). Preferably, the incorporated features are found within both the model and query template.
The third step would be to identify structurally conserved regions that could be used to construct secondary core structure (Sali, et al, 1995). Loops could be added using knowledge-based techniques, and by performing forcefield calculations (Sali, et al, 1995).
The term “structure coordinates” refers to Cartesian coordinates generated from the building of a homology model. In this invention, the homology model of residues 12 to 524 of CAN-12 was derived from generating a sequence alignment with m-calpain (Strobl et al, 2000; hCAN2; Genbank Accession No. gi|7546423; SEQ ID NO:11) using the COMPOSER suite of software within SYBYL6.6 (Tripos Associates, St. Louis, Mo.) and then generating the backbone and side chain conformations. In the original crystal structure (pdb code 1dkv) as well as the crystal structure reported elsewhere (Hosfield et al, 1999), the active site of the enzyme comprising a cysteine, a histidine and an asparagine residue was not “formed”. The helix that contains the active site C101 was altered by moving the helix down one pitch so that the active site geometry could match that found in Papain (pdb code 1b4). This modified structure of human m-calpain was used as the template for construction of the homology model (illustrated in FIG. 6 herein).
The skilled artisan would appreciate that a set of structure coordinates for a protein represents a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates, as emanate from the generation of similar homology models using different alignment templates (i.e., other than the m-calpain (Strobl et al, 2000; hCAN2; Genbank Accession No. gi|7546423; SEQ ID NO:11), and/or using different methods in generating the homology model, will likely have minor effects on the overall shape. Variations in coordinates may also be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth in Table IV could be manipulated by fractionalization of the structure coordinates; integer additions, or integer subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.
Therefore, various computational analyses are necessary to determine whether a template molecule or a portion thereof is sufficiently similar to all or part of a query template (e.g., CAN-12) in order to be considered the same. Such analyses may be carried out in current software applications, such as SYBYL version 6.6 or INSIGHTII (Molecular Simulations Inc., San Diego, Calif.) version 2000 and as described in the accompanying User's Guides.
Using the superimposition tool in the program SYBYL, comparisons can be made between different structures and different conformations of the same structure. The procedure used in SYBYL to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalencies in these structures; 3) perform a fitting operation; and 4) analyze the results. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); the second structure (i.e., moving structure) is identified as the source structure. The atom equivalency within SYBYL is defined by user input. For the purpose of this invention, we will define equivalent atoms as protein backbone atoms (N, Cα, C and O) for all conserved residues between the two structures being compared. We will also consider only rigid fitting operations. When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atoms is an absolute minimum. This number, given in angstroms, is reported by the SYBYL program. For the purpose of the present invention, any homology model of a CAN-12 that has a root mean square deviation of conserved residue backbone atoms (N, Cα, C, O) of less than 3.0 A when superimposed on the relevant backbone atoms described by structure coordinates listed in Table IV are considered identical. More preferably, the root mean square deviation for the CAN-12 polypeptide is less than 2.0 Å.
The homology model of the present invention is useful for the structure-based design of modulators of the CAN-12 biological function, as well as mutants with altered biological function and/or specificity.
In accordance with the structural coordinates provided in Table IV and the three dimensional homology model of CAN-12, the CAN-12v1 polypeptide has been shown to comprise a an active site region embodied by the following amino acids: from about amino acid R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid 1212, from about amino acid G236 to about amino acid H240, from about amino acid E252 to about amino acid Y256, from about amino acid N278 to about amino acid K282, from about amino acid V328 to about amino acid V331 of SEQ ID NO:54 (FIGS. 8A-C). In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids more in either the N— or C-terminal direction of the above referenced amino acids.
Also more preferred are polypeptides comprising all or any part of the CAN-12v1 active site domain, or a mutant or homologue of said polypeptide or molecular complex. By mutant or homologue of the molecule is meant a molecule that has a root mean square deviation from the backbone atoms of said CAN-12 amino acids of not more than about 4.5 Angstroms, and preferably not more than about 3.5 Angstroms.
In preferred embodiments, the following CAN-12v1 active site domain polypeptide is encompassed by the present invention: RLDLCQGIVGDCWFLAALQALALHQDILSRVVPLNQSFTEKYAGIFRFWFWHYGNW VPVVIDDRLPVNEAGQLVFVSSTYKNLFWGALLEKAYAKLSGSYEDLQSGQVSEAL VDFTGGVTMTINLAEAHGNLWDILEATYNRTLIGCQTHSGKILENGLVEGHAYTLT GIRKVTCKHRPEYLVKLRNPWGKVEWKGDWSDSSSKWELLSPKEKILLLRKDNDGE FWMTLQDFKTHFVLLV (SEQ ID NO:92). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of the CAN-12v1 active site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The present invention also encompasses polypeptides comprising at least a portion of the CAN-12 active site domain (SEQ ID NO: 92). Such polypeptides may correspond, for example, to the N— and/or C-terminal deletions of the active site domain.
In preferred embodiments, the following N-terminal CAN-12v1 active site domain deletion polypeptides are encompassed by the present invention: R1-V242, L2-V242, D3-V242, L4-V242, C5-V242, Q6-V242, G7-V242, I8-V242, V9-V242, G10-V242, D11-V242, C12-V242, W13-V242, F14-V242, L15-V242, A16-V242, A17-V242, L18-V242, Q19- V242, A20-V242, L21-V242, A22-V242, L23-V242, H24-V242, Q25-V242, D26-V242, I27-V242, L28-V242, S29-V242, R30-V242, V31-V242, V32-V242, P33-V242, L34-V242, N35-V242, Q36-V242, S37-V242, F38-V242, T39-V242, E40-V242, K41-V242, Y42-V242, A43-V242, G44-V242, I45-V242, F46-V242, R47-V242, F48-V242, W49-V242, F50-V242, W51-V242, H52-V242, Y53-V242, G54-V242, N55-V242, W56-V242, V57-V242, P58-V242, V59-V242, V60-V242, I61-V242, D62-V242, D63-V242, R64-V242, L65-V242, P66-V242, V67-V242, N68-V242, E69-V242, A70-V242, G71-V242, Q72-V242, L73-V242, V74-V242, F75-V242, V76-V242, S77-V242, S78-V242, T79-V242, Y80-V242, K81-V242, N82-V242, L83-V242, F84-V242, W85-V242, G86-V242, A87-V242, L88-V242, L89-V242, E90-V242, K91-V242, A92-V242, Y93-V242, A94-V242, K95-V242, L96-V242, S97-V242, G98-V242, S99-V242, Y100-V242, E101-V242, D102-V242, L103-V242, Q104-V242, S105-V242, G106-V242, Q107-V242, V108-V242, S109-V242, E110-V242, A111-V242, L112-V242, V113-V242, D114-V242, F115-V242, T116-V242, G117-V242, G118-V242, V119-V242, T120-V242, M121-V242, T122-V242, I123-V242, N124-V242, L125-V242, A126-V242, E127-V242, A128-V242, H129-V242, G130-V242, N131-V242, L132-V242, W133-V242, D134-V242, I135-V242, L136-V242, I137-V242, E138-V242, A139-V242, T140-V242, Y141-V242, N142-V242, R143-V242, T144-V242, L145-V242, I146-V242, G147-V242, C148-V242, Q149-V242, T150-V242, H151-V242, S152-V242, G153-V242, E154-V242, K155-V242, I156-V242, L157-V242, E158-V242, N159-V242, G160-V242, L161-V242, V162-V242, E163-V242, G164-V242, H165-V242, A166-V242, Y167-V242, T168-V242, L169-V242, T170-V242, G171-V242, I172-V242, R173-V242, K174-V242, V175-V242, T176-V242, C177-V242, K178-V242, H179-V242, R180-V242, P181-V242, E182-V242, Y183-V242, L184-V242, V185-V242, K186-V242, L187-V242, R188-V242, N189-V242, P190-V242, W191-V242, G192-V242, K193-V242, V194-V242, E195-V242, W196-V242, K197-V242, G198-V242, D199-V242, W200-V242, S201-V242, D202-V242, S203-V242, S204-V242, S205-V242, K206-V242, W207-V242, E208-V242, L209-V242, L210-V242, S211-V242, P212-V242, K213-V242, E214-V242, K215-V242, I216-V242, L217-V242, L218-V242, L219-V242, R220-V242, K221-V242, D222-V242, N223-V242, D224-V242, G225-V242, E226-V242, F227-V242, W228-V242, M229-V242, T230-V242, L231 -V242, Q232-V242, D233-V242, F234-V242, K235-V242, and/or T236-V242 of SEQ ID NO:92. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal CAN-12v1 active site domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal CAN-12v1 active site domain deletion polypeptides are encompassed by the present invention: R1-V242, R1-L241, R1-L240, R1-V239, R1-F238, R1-H237, R1-T236, R1-K235, R1-F234, R 1-D233, R1-Q232, R1-L231, R1-T230, R1-M229, R1-W228, R1-F227, R1-E226, R1-G225, R1-D224, R1-N223, R1-D222, R1-K221, R1-R220, R1-L219, R1-L218, R1-L217, R1-I216, R1-K215, R1-E214, R1-K213, R1-P212, R1-S211, R1-L210, R1-L209, R1-E208, R1-W207, R1-K206, R1-S205, R1-S204, R1-S203, R1-D202, R1-S201, R1-W200, R1-D199, R1-G198, R1-K197, R1-W196, R1-E195, R1-V194, R1-K193, R1-G192, R1-W191, R1-P190, R1-N189, R1-R188, R1-L187, R1-K186, R1-V185, R1-L184, R1-Y183, R1-E182, R1-P181, R1-R180, R1-H179, R1-K178, R1-C177, R1-T176, R1-V175, R1-K174, R1-R173, R1-I172, R1-G171, R1-T170, R1-L169, R1-T168, R1-Y167, R1-A166, R1-H165, R1-G164, R1-E163, R1-V162, R1-L161, R1-G160, R1-N159, R1-E158, R1-L157, R1-I156, R1-K155, R1-E154, R1-G153, R1-S152, R1-H151, R1-T150, R1-Q149, R1-C148, R1-G147, R1-I146, R1-L145, R1-T144, R1-R143, R1-N142, R1-Y141, R1-T140, R1-A139, R1-E138, R1-I137, R1-L136, R1-I135, R1-D134, R1-W133, R1-L132, R1-N131, R1-G130, R1-H129, R1-A128, R1-E127, R1-A126, R1-L125, R1-N124, R1-I123, R1-T122, R1-M121, R1-T120, R1-V119, R1-G118, R1-G117, R1-T116, R1-F115, R1-D114, R1-V113, R1-L112, R1-A111, R1-E110, R1-S109, R1-V108, R1-Q107, R1-G106, R1-S105, R1-Q104, R1-L103, R1-D102, R1-E101, R1-Y100, R1-S99, R1-G98, R1-S97, R1-L96, R1-K95, R1-A94, R1-Y93, R1-A92, R1-K91, R1-E90, R1-L89, R1-L88, R1-A87, R1-G86, R1-W85, R1-F84, R1-L83, R1-N82, R1-K81, R1-Y80, R1-T79, R1-S78, R1-S77, R1-V76, R1-F75, R1-V74, R1-L73, R1-Q72, R1-G71, R1-A70, R1-E69, R1-N68, R1-V67, R1-P66, R1-L65, R1-R64, R1-D63, R1-D62, R1-I61, R1-V60, R1-V59, R1-P58, R1-V57, R1-W56, R1-N55, R1-G54, R1-Y53, R1-H52, R1-W51, R1-F50, R1-W49, R1-F48, R1-R47, R1-F46, R1-I45, R1-G44, R1-A43, R1-Y42, R1-K41, R1-E40, R1-T39, R1-F38, R1-S37, R1-Q36, R1-N35, R1-L34, R1-P33, R1-V32, R1-V31, R1-R30, R1-S29, R1-L28, R1-I27, R1-D26, R1-Q25, R1-H24, R1-L23, R1-A22, R1-L21, R1-A20, R1-Q19, R1-L18, R1-A17, R1-A16, R1-L15, R1-F14, R1-W13, R1-C12, R1-D11, R1-G10, R1-I8, and/or R1-G7 of SEQ ID NO:92. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal CAN-12v1 active site domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
Alternatively, such polypeptides may comprise polypeptide sequences corresponding, for example, to internal regions of the CAN-12v1 active site domain (e.g., any combination of both N— and C-terminal CAN-12v1 active site domain deletions) of SEQ ID NO:92. For example, internal regions could be defined by the equation NX to CX, where NX refers to any N-terminal amino acid position of the CAN-12v1 active site domain (SEQ ID NO:92), and where CX refers to any C-terminal amino acid position of the CAN-12v1 active site domain (SEQ ID NO:92). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.
In preferred embodiments, the following CAN-12v1 active site domain amino acid substitutions are encompassed by the present invention: wherein R90 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein L91 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D92 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L93 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein C94 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q95 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein G96 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein 197 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V98 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein G99 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D100 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C101 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W102 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein F103 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L104 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A105 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A106 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L107 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Q108 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein A109 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L110 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A111 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L112 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein H113 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q114 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein D115 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I116 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L117 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein S118 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein R119 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein V120 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein V121 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein P122 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein L123 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein N124 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein Q125 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein S126 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein F127 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T128 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein E129 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K130 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y131 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein A132 is substituted with either a C, D, E, F, G, H. I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G133 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I134 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F135 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R136 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein F137 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W138 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein F139 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, P, S, T, V, W, or Y; wherein W140 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein H141 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y142 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein G143 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N144 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein W145 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein V146 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein P147 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein V148 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein V149 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein I150 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D151 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D152 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R153 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein L154 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein P155 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein V156 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein N157 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein E158 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A159 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G160 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q161 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein L162 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein V163 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein F164 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V165 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein S166 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein S167 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein T168 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein Y169 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein K170 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N171 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, P, S, T, V, W, or Y; wherein L172 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein F173 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W174 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein G175 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A176 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L177 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L178 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein E179 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K180 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A181 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y182 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein A183 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K184 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L185 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein S186 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein G187 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S188 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein Y189 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein E190 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D191 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L192 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Q193 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein S194 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein G195 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q196 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein V197 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein S198 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein E199 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A200 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L201 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein V202 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein D203 is substituted withiheihr an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F204 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T205 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein G206 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G207 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V208 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein T209 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein M210 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein T211 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein I212 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N213 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein L214 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A215 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E216 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A217 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H218 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G219 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N220 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein L221 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein W222 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein D223 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I224 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L225 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein I226 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E227 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A228 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T229 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein Y230 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein N231 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein R232 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein T233 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L234 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein I235 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, P, S, T, V, W, or Y; wherein G236 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C237 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q238 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein T239 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein H240 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S241 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein G242 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E243 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K244 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I245 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L246 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein E247 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N248 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein G249 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L250 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein V251 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein E252 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G253 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H254 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A255 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y256 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein T257 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L258 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein T259 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein G260 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I261 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R262 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein K263 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V264 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein T265 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein C266 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K267 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H268 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R269 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein P270 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein E271 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y272 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein L273 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein V274 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein K275 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L276 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein R277 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein N278 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein P279 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein W280 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein G281 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K282 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V283 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein E284 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W285 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein K286 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G287 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D288 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W289 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein S290 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein D291 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S292 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein S293 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein S294 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein K295 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W296 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein E297 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L298 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L299 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein S300 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein P301 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein K302 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E303 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K304 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I305 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L306 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L307 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L308 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein R309 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein K310 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D311 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N312 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein D313 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G314 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E315 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F316 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W317 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein M318 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein T319 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L320 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Q321 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein D322 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F323 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K324 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T325 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein H326 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F327 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V328 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein L329 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L330 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, P, S, T, V, W, or Y; and/or wherein V331 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y of SEQ ID NO:54, in addition to any combination thereof. The present invention also encompasses the use of these CAN-12v1 active site domain amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following CAN-12v1 active site domain conservative amino acid substitutions are encompassed by the present invention: wherein R90 is substituted with either a K, or H; wherein L91 is substituted with either an A, I, or V; wherein D92 is substituted with an E; wherein L93 is substituted with either an A, I, or V; wherein C94 is a C; wherein Q95 is substituted with a N; wherein G96 is substituted with either an A, M, S, or T; wherein I97 is substituted with either an A, V, or L; wherein V98 is substituted with either an A, I, or L; wherein G99 is substituted with either an A, M, S, or T; wherein D100 is substituted with an E; wherein C101 is a C; wherein W102 is either an F, or Y; wherein F103 is substituted with either a W, or Y; wherein L104 is substituted with either an A, I, or V; wherein A105 is substituted with either a G, I, L, M, S, T, or V; wherein A106 is substituted with either a G, I, L, M, S, T, or V; wherein L107 is substituted with either an A, I, or V; wherein Q108 is substituted with a N; wherein A109 is substituted with either a G, I, L, M, S, T, or V; wherein L110 is substituted with either an A, I, or V; wherein A111 is substituted with either a G, I, L, M, S, T, or V; wherein L112 is substituted with either an A, I, or V; wherein H113 is substituted with either a K, or R; wherein Q114 is substituted with a N; wherein D115 is substituted with an E; wherein I116 is substituted with either an A, V, or L; wherein L117 is substituted with either an A, I, or V; wherein S118 is substituted with either an A, G, M, or T; wherein R119 is substituted with either a K, or H; wherein V120 is substituted with either an A, I, or L; wherein V121 is substituted with either an A, I, or L; wherein P122 is a P; wherein L123 is substituted with either an A, I, or V; wherein N124 is substituted with a Q; wherein Q125 is substituted with a N; wherein S126 is substituted with either an A, G, M, or T; wherein F127 is substituted with either a W, or Y; wherein T128 is substituted with either an A, G, M, or S; wherein E129 is substituted with a D; wherein K130 is substituted with either a R, or H; wherein Y131 is either an F, or W; wherein A132 is substituted with either a G, I, L, M, S, T, or V; wherein G133 is substituted with either an A, M, S, or T; wherein I134 is substituted with either an A, V, or L; wherein F135 is substituted with either a W, or Y; wherein R136 is substituted with either a K, or H; wherein F137 is substituted with either a W, or Y; wherein W138 is either an F, or Y; wherein F139 is substituted with either a W, or Y; wherein W140 is either an F, or Y; wherein H141 is substituted with either a K, or R; wherein Y142 is either an F, or W; wherein G143 is substituted with either an A, M, S, or T; wherein N144 is substituted with a Q; wherein W145 is either an F, or Y; wherein V146 is substituted with either an A, I, or L; wherein P147 is a P; wherein V148 is substituted with either an A, I, or L; wherein V149 is substituted with either an A, I, or L; wherein I150 is substituted with either an A, V, or L; wherein D151 is substituted with an E; wherein D152 is substituted with an E; wherein R153 is substituted with either a K, or H; wherein L154 is substituted with either an A, I, or V; wherein P155 is a P; wherein V156 is substituted with either an A, I, or L; wherein N157 is substituted with a Q; wherein E158 is substituted with a D; wherein A159 is substituted with either a G, I, L, M, S, T, or V; wherein G160 is substituted with either an A, M, S, or T; wherein Q161 is substituted with a N; wherein L162 is substituted with either an A, I, or V; wherein V163 is substituted with either an A, I, or L; wherein F164 is substituted with either a W, or Y; wherein V165 is substituted with either an A, I, or L; wherein S166 is substituted with either an A, G, M, or T; wherein S167 is substituted with either an A, G, M, or T; wherein T168 is substituted with either an A, G, M, or S; wherein Y169 is either an F, or W; wherein K170 is substituted with either a R, or H; wherein N171 is substituted with a Q; wherein L172 is substituted with either an A, I, or V; wherein F173 is substituted with either a W, or Y; wherein W174 is either an F, or Y; wherein G175 is substituted with either an A, M, S, or T; wherein A176 is substituted with either a G, I, L, M, S, T, or V; wherein L177 is substituted with either an A, I, or V; wherein L178 is substituted with either an A, I, or V; wherein E179 is substituted with a D; wherein K180 is substituted with either a R, or H; wherein A181 is substituted with either a G, I, L, M, S, T, or V; wherein Y182 is either an F, or W; wherein A183 is substituted with either a G, I, L, M, S, T, or V; wherein K184 is substituted with either a R, or H; wherein L185 is substituted with either an A, I, or V; wherein S186 is substituted with either an A, G, M, or T; wherein G187 is substituted with either an A, M, S, or T; wherein S188 is substituted with either an A, G, M, or T; wherein Y189 is either an F, or W; wherein E190 is substituted with a D; wherein D191 is substituted with an E; wherein L192 is substituted with either an A, I, or V; wherein Q193 is substituted with a N; wherein S194 is substituted with either an A, G, M, or T; wherein G195 is substituted with either an A, M, S, or T; wherein Q196 is substituted with a N; wherein V197 is substituted with either an A, I, or L; wherein S198 is substituted with either an A, G, M, or T; wherein E199 is substituted with a D; wherein A200 is substituted with either a G, I, L, M, S, T, or V; wherein L201 is substituted with either an A, I, or V; wherein V202 is substituted with either an A, I, or L; wherein D203 is substituted with an E; wherein F204 is substituted with either a W, or Y; wherein T205 is substituted with either an A, G, M, or S; wherein G206 is substituted with either an A, M, S, or T; wherein G207 is substituted with either an A, M, S, or T; wherein V208 is substituted with either an A, I, or L; wherein T209 is substituted with either an A, G, M, or S; wherein M210 is substituted with either an A, G, S, or T; wherein T211 is substituted with either an A, G, M, or S; wherein I212 is substituted with either an A, V, or L; wherein N213 is substituted with a Q; wherein L214 is substituted with either an A, I, or V; wherein A215 is substituted with either a G, I, L, M, S, T, or V; wherein E216 is substituted with a D; wherein A217 is substituted with either a G, I, L, M, S, T, or V; wherein H218 is substituted with either a K, or R; wherein G219 is substituted with either an A, M, S, or T; wherein N220 is substituted with a Q; wherein L221 is substituted with either an A, I, or V; wherein W222 is either an F, or Y; wherein D223 is substituted with an E; wherein I224 is substituted with either an A, V, or L; wherein L225 is substituted with either an A, I, or V; wherein I226 is substituted with either an A, V, or L; wherein E227 is substituted with a D; wherein A228 is substituted with either a G, I, L, M, S, T, or V; wherein T229 is substituted with either an A, G, M, or S; wherein Y230 is either an F, or W; wherein N231 is substituted with a Q; wherein R232 is substituted with either a K, or H; wherein T233 is substituted with either an A, G, M, or S; wherein L234 is substituted with either an A, I, or V; wherein I235 is substituted with either an A, V, or L; wherein G236 is substituted with either an A, M, S, or T; wherein C237 is a C; wherein Q238 is substituted with a N; wherein T239 is substituted with either an A, G, M, or S; wherein H240 is substituted with either a K, or R; wherein S241 is substituted with either an A, G, M, or T; wherein G242 is substituted with either an A, M, S, or T; wherein E243 is substituted with a D; wherein K244 is substituted with either a R, or H; wherein I245 is substituted with either an A, V, or L; wherein L246 is substituted with either an A, I, or V; wherein E247 is substituted with a D; wherein N248 is substituted with a Q; wherein G249 is substituted with either an A, M, S, or T; wherein L250 is substituted with either an A, I, or V; wherein V251 is substituted with either an A, I, or L; wherein E252 is substituted with a D; wherein G253 is substituted with either an A, M, S, or T; wherein H254 is substituted with either a K, or R; wherein A255 is substituted with either a G, I, L, M, S, T, or V; wherein Y256 is either an F, or W; wherein T257 is substituted with either an A, G, M, or S; wherein L258 is substituted with either an A, I, or V; wherein T259 is substituted with either an A, G, M, or S; wherein G260 is substituted with either an A, M, S, or T; wherein I261 is substituted with either an A, V, or L; wherein R262 is substituted with either a K, or H; wherein K263 is substituted with either a R, or H; wherein V264 is substituted with either an A, I, or L; wherein T265 is substituted with either an A, G, M, or S; wherein C266 is a C; wherein K267 is substituted with either a R, or H; wherein H268 is substituted with either a K, or R; wherein R269 is substituted with either a K, or H; wherein P270 is a P; wherein E271 is substituted with a D; wherein Y272 is either an F, or W; wherein L273 is substituted with either an A, I, or V; wherein V274 is substituted with either an A, I, or L; wherein K275 is substituted with either a R, or H; wherein L276 is substituted with either an A, I, or V; wherein R277 is substituted with either a K, or H; wherein N278 is substituted with a Q; wherein P279 is a P; wherein W280 is either an F, or Y; wherein G281 is substituted with either an A, M, S, or T; wherein K282 is substituted with either a R, or H; wherein V283 is substituted with either an A, I, or L; wherein E284 is substituted with a D; wherein W285 is either an F, or Y; wherein K286 is substituted with either a R, or H; wherein G287 is substituted with either an A, M, S, or T; wherein D288 is substituted with an E; wherein W289 is either an F, or Y; wherein S290 is substituted with either an A, G, M, or T; wherein D291 is substituted with an E; wherein S292 is substituted with either an A, G, M, or T; wherein S293 is substituted with either an A, G, M, or T; wherein S294 is substituted with either an A, G, M, or T; wherein K295 is substituted with either a R, or H; wherein W296 is either an F, or Y; wherein E297 is substituted with a D; wherein L298 is substituted with either an A, I, or V; wherein L299 is substituted with either an A, I, or V; wherein S300 is substituted with either an A, G, M, or T; wherein P301 is a P; wherein K302 is substituted with either a R, or H; wherein E303 is substituted with a D; wherein K304 is substituted with either a R, or H; wherein I305 is substituted with either an A, V, or L; wherein L306 is substituted with either an A, I, or V; wherein L307 is substituted with either an A, I, or V; wherein L308 is substituted with either an A, I, or V; wherein R309 is substituted with either a K, or H; wherein K310 is substituted with either a R, or H; wherein D311 is substituted with an E; wherein N312 is substituted with a Q; wherein D313 is substituted with an E; wherein G314 is substituted with either an A, M, S, or T; wherein E315 is substituted with a D; wherein F316 is substituted with either a W, or Y; wherein W317 is either an F, or Y; wherein M318 is substituted with either an A, G, S, or T; wherein T319 is substituted with either an A, G, M, or S; wherein L320 is substituted with either an A, I, or V; wherein Q321 is substituted with a N; wherein D322 is substituted with an E; wherein F323 is substituted with either a W, or Y; wherein K324 is substituted with either a R, or H; wherein T325 is substituted with either an A, G, M, or S; wherein H326 is substituted with either a K, or R; wherein F327 is substituted with either a W, or Y; wherein V328 is substituted with either an A, I, or L; wherein L329 is substituted with either an A, I, or V; wherein L330 is substituted with either an A, I, or V; and/or wherein V331 is substituted with either an A, I, or L of SEQ ID NO:54 in addition to any combination thereof. Other suitable substitutions within the CAN-12v1 active site domain are encompassed by the present invention and are referenced elsewhere herein. The present invention also encompasses the use of these CAN-12v1 active site domain conservative amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
For purposes of the present invention, by “at least a portion of” is meant all or any part of the CAN-12v1 active site domain corresponding to the analogous amino acids of the CAN-12 active site domain defined by the structure coordinates according to Table IV (e.g., fragments thereof). More preferred are molecules comprising all or any parts of the CAN-12v1 active site domain, corresponding to the analogous amino acids of the CAN-12 active site domain defined by the structure coordinates according to Table IV, or a mutant or homologue of said molecule or molecular complex. By mutant or homologue of the molecule it is meant a molecule that has a root mean square deviation from the backbone atoms of said CAN-12v1 amino acids of not more than 4.5 Angstroms, and preferably not more than 3.5 Angstroms.
The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a term that expresses the deviation or variation from a trend or object. For the purposes of the present invention, the “root mean square deviation” defines the variation in the backbone of a protein from the relevant portion of the backbone of the AR portion of the complex as defined by the structure coordinates described herein.
A preferred embodiment is a machine-readable data storage medium that is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex that is defined by the structure coordinates of all of the amino acids in Table IV ±a root mean square deviation from the backbone atoms of those amino acids of not more than 4.0 ANG, preferably 3.0 ANG.
The structure coordinates of a CAN-12 homology model, including portions thereof, is stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery.
Accordingly, in one embodiment of this invention is provided a machine-readable data storage medium comprising a data storage material encoded with the structure coordinates set forth in Table IV.
One embodiment utilizes System 10 as disclosed in WO 98/11134, the disclosure of which is incorporated herein by reference in its entirety. Briefly, one version of these embodiments comprises a computer comprising a central processing unit (“CPU”), a working memory which may be, e.g, RAM (random-access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more cathode-ray tube (“CRT”) display terminals, one or more keyboards, one or more input lines, and one or more output lines, all of which are interconnected by a conventional bidirectional system bus.
Input hardware, coupled to the computer by input lines, may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line. Alternatively or additionally, the input hardware may comprise CD-ROM drives or disk drives. In conjunction with a display terminal, keyboard may also be used as an input device.
Output hardware, coupled to the computer by output lines, may similarly be implemented by conventional devices. By way of example, output hardware may include a CRT display terminal for displaying a graphical representation of a region or domain of the present invention using a program such as QUANTA as described herein. Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use.
In operation, the CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage, and accesses to and from the working memory, and determines the sequence of data processing steps. A number of programs may be used to process the machine-readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein. Specific references to components of the hardware system are included as appropriate throughout the following description of the data storage medium.
For the purpose of the present invention, any magnetic data storage medium which can be encoded with machine-readable data would be sufficient for carrying out the storage requirements of the system. The medium could be a conventional floppy diskette or hard disk, having a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, on one or both sides, containing magnetic domains whose polarity or orientation could be altered magnetically, for example. The medium may also have an opening for receiving the spindle of a disk drive or other data storage device.
The magnetic domains of the coating of a medium may be polarized or oriented so as to encode in a manner which may be conventional, machine readable data such as that described herein, for execution by a system such as the system described herein.
Another example of a suitable storage medium which could also be encoded with such machine-readable data, or set of instructions, which could be carried out by a system such as the system described herein, could be an optically-readable data storage medium. The medium could be a conventional compact disk read only memory (CD-ROM) or a rewritable medium such as a magneto-optical disk which is optically readable and magneto-optically writable. The medium preferably has a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, usually of one side of substrate.
In the case of a CD-ROM, as is well known, the coating is reflective and is impressed with a plurality of pits to encode the machine-readable data. The arrangement of pits is read by reflecting laser light off the surface of the coating. A protective coating, which preferably is substantially transparent, is provided on top of the reflective coating.
In the case of a magneto-optical disk, as is well known, the coating has no pits, but has a plurality of magnetic domains whose polarity or orientation can be changed magnetically when heated above a certain temperature, as by a laser. The orientation of the domains can be read by measuring the polarization of laser light reflected from the coating. The arrangement of the domains encodes the data as described above.
Thus, in accordance with the present invention, data capable of displaying the three dimensional structure of the CAN-12 homology model, or portions thereof and their structurally similar homologues is stored in a machine-readable storage medium, which is capable of displaying a graphical three-dimensional representation of the structure. Such data may be used for a variety of purposes, such as drug discovery.
For the first time, the present invention permits the use of structure-based or rational drug design techniques to design, select, and synthesize chemical entities that are capable of modulating the biological function of CAN-12v1.
Accordingly, the present invention is also directed to the design of small molecules which imitates the structure of the CAN-12v1 active site domain (SEQ ID NO:92), or a portion thereof, corresponding to the analogous amino acids of the CAN-12 active site domain defined by the structure provided in Table IV. Alternatively, the present invention is directed to the design of small molecules which may bind to at least part of the CAN-12v1 active site domain (SEQ ID NO:92), or some portion thereof. For purposes of this invention, by CAN-12v1 active site domain, it is also meant to include mutants or homologues thereof. In a preferred embodiment, the mutants or homologues have at least 25% identity, more preferably 50% identity, more preferably 75% identity, and most preferably 90% identity to SEQ ID NO:92. In this context, the term “small molecule” may be construed to mean any molecule described known in the art or described elsewhere herein, though may include, for example, peptides, chemicals, carbohydrates, nucleic acids, PNAs, and any derivatives thereof.
The three-dimensional model structure of CAN-12v1, corresponding to the structure coordinates of the analogous amino acids of the CAN-12 polypeptide, will also provide methods for identifying modulators of biological function. Various methods or combination thereof can be used to identify these compounds.
For example, test compounds can be modeled that fit spatially into the active site domain in CAN-12v1 embodied by the sequence from about R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E252 to about amino acid Y256, from about amino acid N278 to about amino acid K282, from about amino acid V328 to about amino acid V331, or some portion thereof, of SEQ ID NO:54 (corresponding to SEQ ID NO:92), in accordance with the structural coordinates of the corresponding amino acids of the CAN-12 polypeptide of Table IV.
Structure coordinates of the active site domain in CAN-12v1 defined by the amino acids from about R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E252 to about amino acid Y256, from about amino acid N278 to about amino acid K282, from about amino acid V328 to about amino acid V331 of SEQ ID NO:54, can also be used to identify structural and chemical features. Identified structural or chemical features can then be employed to design or select compounds as potential CAN-12v1 modulators. By structural and chemical features it is meant to include, but is not limited to, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interaction. Alternatively, or in conjunction with, the three-dimensional structural model can be employed to design or select compounds as potential CAN-12v1 modulators. Compounds identified as potential CAN-12v1 modulators can then be synthesized and screened in an assay characterized by binding of a test compound to the CAN-12v1, or in characterizing the ability of CAN-12v1 to modulate a protease target in the presence of a small molecule. Examples of assays useful in screening of potential CAN-12v1 modulators include, but are not limited to, screening in silico, in vitro assays and high throughput assays. Finally, these methods may also involve modifying or replacing one or more amino acids at amino acid positions, C101, H254, and/or N278 of SEQ ID NO:54 in accordance with the structure coordinates of the corresponding amino acids of CAN-12 as provided in Table IV.
However, as will be understood by those of skill in the art upon this disclosure, other structure based design methods can be used. Various computational structure based design methods have been disclosed in the art.
For example, a number of computer modeling systems are available in which the sequence of the CAN-12v1 and the CAN-12 structure (i.e., atomic coordinates of CAN-12 and/or the atomic coordinates of the active site domain as provided in Table IV, or the corresponding amino acids of CAN-12v1 which are identical to the same region of CAN-12 and for which the same coordinates may be relied thereon) can be input. This computer system then generates the structural details of one or more these regions in which a potential CAN-12v1 modulator binds so that complementary structural details of the potential modulators can be determined. Design in these modeling systems is generally based upon the compound being capable of physically and structurally associating with CAN-12v1. In addition, the compound must be able to assume a conformation that allows it to associate with CAN-12v1. Some modeling systems estimate the potential inhibitory or binding effect of a potential CAN-12v1 modulator prior to actual synthesis and testing.
Methods for screening chemical entities or fragments for their ability to associate with a given protein target are also well known. Often these methods begin by visual inspection of the binding site on the computer screen. Selected fragments or chemical entities are then positioned in the active site domain of CAN-12v1. Docking is accomplished using software such as INSIGHTII, QUANTA and SYBYL, following by energy minimization and molecular dynamics with standard molecular mechanic forcefields such as CHARS and AMBER. Examples of computer programs which assist in the selection of chemical fragment or chemical entities useful in the present invention include, but are not limited to, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntz et al. 1982).
Upon selection of preferred chemical entities or fragments, their relationship to each other and CAN-12v1 can be visualized and then assembled into a single potential modulator. Programs useful in assembling the individual chemical entities include, but are not limited to CAVEAT (Bartlett et al. 1989) and 3D Database systems (Martin 1992).
Alternatively, compounds may be designed de novo using either an empty active site or optionally including some portion of a known inhibitor. Methods of this type of design include, but are not limited to LUDI (Bohm 1992) and LeapFrog (Tripos Associates, St. Louis Mo.).
In addition, CAN-12v1 is overall well suited to modem methods including combinatorial chemistry.
Programs such as DOCK (Kuntz et al. 1982) can be used with the atomic coordinates from the homology model to identify potential ligands from databases or virtual databases which potentially bind CAN-12 active site domain, and which may therefore be suitable candidates for synthesis and testing.
Additionally, the three-dimensional homology model of CAN-12 will aid in the design of mutants with altered biological activity for the CAN-12v1 polypeptide.
The following are encompassed by the present invention: a machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the structure coordinates of the model CAN-12 according to Table IV or a homologue of said model, wherein said homologue comprises backbone atoms that have a root mean square deviation from the backbone atoms of the complex of not more than 4.5 Å, preferably not more than 4.0 Å, most preferably not more than 3.5 Å, and even more preferably not more than 3.0 Å; and a machine-readable data storage medium, wherein said molecule is defined by the set of structure coordinates of the model for CAN-12 according to Table IV, or a homologue of said molecule, said homologue having a root mean square deviation from the backbone atoms of said amino acids of not more than 4.5 Å, preferably not more than 4.0 Å, most preferably not more than 3.5 Å, and even more preferably not more than 3.0 Å; a model comprising all or any part of the model defined by structure coordinates of CAN-12 according to Table IV, or a mutant or homologue of said molecule or molecular complex.
In a further embodiment, the following are encompassed by the present invention: a method for identifying a mutant of CAN-12v1 with altered biological properties, function, or reactivity, the method comprising any combination of steps of: use of the model or a homologue of said model according to Table IV, for the design of protein mutants with altered biological function or properties which exhibit any combination of therapeutic effects provided elsewhere herein; and use of the model or a homologue of said model, for the design of a protein with mutations in the active site domain comprised of the amino acids from about R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E252 to about amino acid Y256, from about amino acid N278 to about amino acid K282, from about amino acid V328 to about amino acid V331 of SEQ ID NO:54 according to the corresponding amino acids of CAN-12 as provided in Table IV with altered biological function or properties which exhibit any combination of therapeutic effects provided elsewhere herein.
In further preferred embodiments, the following are encompassed by the present invention: a method for identifying modulators of CAN-12v1 biological properties, function, or reactivity, the method comprising any combination of steps of: modeling test compounds that overlay spatially into the active site domain defined by all or any portion of residues from about R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E252 to about amino acid Y256, from about amino acid N278 to about amino acid K282, from about amino acid V328 to about amino acid V331 of SEQ ID NO:54 in reference to the corresponding amino acids of CAN-12 according to Table IV, or using a homologue or portion thereof.
The present invention encompasses using the structure coordinates as set forth herein to identify structural and chemical features of the CAN-12v1 polypeptide; employing identified structural or chemical features to design or select compounds as potential CAN-12v1 modulators; employing the three-dimensional structural model to design or select compounds as potential CAN-12v1 modulators; synthesizing the potential CAN-12v1 modulators; screening the potential CAN-12v1 modulators in an assay characterized by binding of a protein to the CAN-12v1; selecting the potential CAN-12v1 modulator from a database; designing the CAN-12v1 modulator de novo; and/or designing said CAN-12v1 modulator from a known modulator activity.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 53 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2081 of SEQ ID NO:53, b is an integer between 15 to 2095, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:53, and where b is greater than or equal to a+14.
In one embodiment, a CAN12.v1 polypeptide comprises a portion of the amino sequence depicted in FIGS. 8A-C. In another embodiment, a CAN12.v1 polypeptide comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of the amino sequence depicted in FIGS. 8A-C. In further embodiments, the CAN12.v1 polypeptide does not consist of the sequence ALLEKAYAKL (SEQ ID NO:141), and/or ALLEKAYAKLSGSYE. (SEQ ID NO:142).
Features of the Polypeptide Encoded by Gene No:3
The polypeptide of this gene provided as SEQ ID NO:56 (FIGS. 9A-C), encoded by the polynucleotide sequence according to SEQ ID NO:55 (FIGS. 9A-C), and/or encoded by the polynucleotide contained within the deposited clone, CAN-12v2, has significant homology at the nucleotide and amino acid level to a number of calpains, which include, for example, the human CAN10 protein (type II diabetes linked) (hCAN10; Genbank Accession No: gi|[NP_—075574; SEQ ID NO:3); the human CAN5 protein (hCAN5; Genbank Accession No: gi|[NP_—004046; SEQ ID NO:4); the large catalytic subunit of the human CALPAIN 1 protein (also referred to as Calcium-Activated Neutral Proteinase, CANP, μ-TYPE) (hCAN1; Genbank Accession No: gi|12408656; SEQ ID NO:5); the large subunit of the human calpain 3 protein (EC 3.4.22.17) (also referred to as CALPAIN L3, CALPAIN P94, Calcium-Activated Neutral Proteinase 3, CANP 3; muscle-specific calcium-activated neutral protease 3 large subunit) (hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6); the large catalytic subunit of the mouse CALPAIN 1 protein (also referred to as Calcium-Activated Neutral Proteinase) (CANP) μ-TYPE ) (mCALPAIN1; Genbank Accession No: gi|088666; SEQ ID NO:7); the mouse CALPAIN LP82 (mLP82; Genbank Accession No: gi|3661585; SEQ ID NO:8); the rat stomach-specific calcium-activated neutral protease large subunit, NCL2 protein (rNCL2; Genbank Accession No: gi|NP-006606; SEQ ID NO:9); the human CAN11 protein (hCAN11; Genbank Accession No: gi|NP_—008989; SEQ ID NO:10); the human CAN2 protein (hCAN2; Genbank Accession No: gi|4502563; SEQ ID NO:11); and the human CAN9 protein (hCAN9; Genbank Accession No: gi|5729758; SEQ ID NO:12). An alignment of the CAN-12v2 polypeptide with these proteins is provided in FIGS. 2A-E. Based upon such strong conservation, the inventors have ascribed the CAN-12v2 polypeptide as having proteolytic activity, preferably calpain activity.
The CAN-12v2 polypeptide was determined to have 28.8% identity and 35.7% similarity with the human CAN10 protein (type II diabetes linked) (hCAN10; Genbank Accession No: gi|NP_—075574; SEQ ID NO:3); to have 33.3% identity and 45.1% similarity with the human CAN5 protein (hCAN5; Genbank Accession No: gi|NP_—004046; SEQ ID NO:4); to have 38.3% identity and 46.6% similarity with the large catalytic subunit of the human CALPAIN 1 protein (also referred to as Calcium-Activated Neutral Proteinase, CANP, μ-TYPE) (hCAN1; Genbank Accession No: gi|12408656; SEQ ID NO:5); to have 41.3% identity and 49.8% similarity with the large subunit of the human calpain 3 protein (EC 3.4.22.17) (also referred to as CALPAIN L3, CALPAIN P94, Calcium-Activated Neutral Proteinase 3, CANP 3; muscle-specific calcium-activated neutral protease 3 large subunit) (hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6); to have 39.6% identity and 47.6% similarity with the large catalytic subunit of the mouse CALPAIN 1 protein (also referred to as Calcium-Activated Neutral Proteinase) (CANP) μ-TYPE ) (mCALPAIN1; Genbank Accession No: gi|088666; SEQ ID NO:7); to have 40.6% identity and 48.8% similarity with the mouse CALPAIN LP82 (mLP82; Genbank Accession No: gi|3661585; SEQ ID NO:8); to have 36.3% identity and 44.9% similarity with the rat stomach-specific calcium-activated neutral protease large subunit, NCL2 protein (rNCL2; Genbank Accession No: gi|NP_—006606; SEQ ID NO:9); to have 38.8% identity and 47.3% similarity with the human CAN11 protein (hCAN11; Genbank Accession No: gi|NP_—008989; SEQ ID NO:10); to have 37.9% identity and 47.3% similarity with the human CAN2 protein (hCAN2; Genbank Accession No: gi|4502563; SEQ ID NO:11); and to have 40.7% identity and 49.8% similarity with the human CAN9 protein (hCAN9; Genbank Accession No: gi|5729758; SEQ ID NO:12).
The human CAN10 protein (type II diabetes linked) (hCAN10; Genbank Accession No: gi|NP _—075574; SEQ ID NO:3)is a human calpain gene that encodes a large calpain subunit. CAN10 is an atypical calpain in that it lacks the calmodulin-like calcium-binding domain and instead has a divergent C-terminal domain. CAN10 is similar in organization to calpains 5 and 6 and is associated with type 2 or non-insulin-dependent diabetes mellitus (NIDDM) and located within the NIDDM1 chromosomal region (Nat. Genet. 26 (2), 163-175 (2000)).
The large subunit of the human calpain 3 protein (EC 3.4.22.17) (also referred to as CALPAIN L3, CALPAIN P94, Calcium-Activated Neutral Proteinase 3, CANP 3; muscle-specific calcium-activated neutral protease 3 large subunit) (hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6) is a muscle-specific member of the calpain large subunit family. Loss of CAPN3 function has been associated with limb-girdle muscular dystrophies type 2A (Cell 81 (1), 27-40 (1995)).
The human CAN9 protein (hCAN9; Genbank Accession No: gi|5729758; SEQ ID NO:12) is a calpain that is expressed predominantly in stomach and small intestine and is thought to have specialized functions in the digestive tract, and be associated with gastric cancer.( Biol. Chem. 379 (2), 175-183 (1998); and Jpn. J. Cancer Res. 91 (5), 459-463 (2000)).
As described above, the CAN-12v2 polypeptide was found to have significant sequence homology with calpains, particularly members of the m-calpain family. A conserved peptide signature of Qx3(G,E)xC(Y,W)x2(S,T,A,G,C)(S,T,A,G,C,V) Qx{3}(G)xC(W)x{2}(A)(A) (referred to as a thiol (cysteine) protease active site domain) common to most calpain family members is found in the protein sequence of CAN-12v2 from amino acid 90 to amino acid 111 of SEQ ID NO:56 (FIGS. 9A-C). Protein threading and molecular modeling of CAN-12v2 suggests that CAN-12v2 has a structural fold similar to representative m-calpains. Moreover, the structural and threading alignments of the present invention suggest that amino acids 101 (“C”), 254 (“H”), and 278 (“N”) of SEQ ID NO:56 (FIGS. 9A-C) may represent the catalytic amino acids within the active site domain. Thus, based upon the sequence and structural homology to known calpains, particularly the presence of the thiol cysteine protease active site domain, the novel CAN-12v2 is believed to represent a novel human calpain.
In confirmation of the strong homology to known calpains, the CAN-12v2 polypeptide was determined to have several conserved catalytic amino acids at amino acid C101, H254, and N278 of SEQ ID NO:56 (FIGS. 9A-C). As discussed more particularly herein, calpains are a group of structurally diverse, high molecular weight (400 to 500 amino acids) proteins that have a catalytic cysteine amino acid and one or more calcium binding domains. Despite the structural heterogeneity, calpains share some well defined structural-functional characteristics, particularly in their active site domains.
In preferred embodiments, the CAN-12v2 polypeptide of the present invention is directed to a polypeptide having structural similarity to calpains.
Based upon the strong homology to members of the calpain family, the CAN-12v2 polypeptide is expected to share at least some biological activity with calpains, preferably with m-calpain family members, and more preferable to the large subunits of m-calpain family members, in addition to other calpains and calpain subunits referenced herein and/or otherwise known in the art.
Expression profiling designed to measure the steady state mRNA levels encoding the CAN-12 polypeptide showed predominately high expression levels in spinal cord tissue; significantly high expression in lymph node and thymus, and to a lesser extent, in spleen tissue (See FIG. 4).
Expanded analysis of CAN-12 expression levels by TaqMan™ quantitative PCR (see FIG. 6) confirmed that the CAN-12 polypeptide is expressed in the lymph gland. However, the TaqMan™ quantitative PCR determined that the CAN-12 polypeptide is primarily expressed in the eosophagus. In fact, with the exception of the lymph gland, the steady state mRNA level of CAN-12 was approximately 2700 times higher in the esophagus than in all other tissues tested. These data suggest modulators of the CAN-12 polynucleotides and polypeptides may be useful for the treatment, detection, and/or amelioration of the following, non-limiting diseases and disorders associated with the eosphagus: dysphagia, cricoharyngeal incoordination, esophageal carcinoma, esophageal webs, achalasia, symptomatic diffuse esophageal spasm, gastroesophageal reflux, and/or corrosive esophagitis.
The polynucleotides encoding the CAN-12 polypeptide of the present invention were used to determine the chromosomal localization of the calpain12 gene. Polynuclelotides corresponding to CAN-12 (SEQ ID NO:1) were shown to localize to chromosome 2, specifically 2p16-p21. The comparison of the chromosomal location of the calpain12 gene with the location of chromosomal regions which have been shown to be associated with specific diseases or conditions, e.g. by linkage analysis, can be indicative of diseases in which calpain12 may play a role. Interestingly, a whole-genome linkage scan in multiple sclerosis families (Ebers et al. A full genome search in multiple sclerosis. Nature Genet. 13: 472-476, 1996.) identified 5 susceptibility loci on chromosomes 2, 3, 5, 11, and X. In particular, an association was identified with marker D2S119 on chromosome 2 and MS. The localization of the D2S119 marker was further delimeated to 2p16-p21 based on a radiation hybrid linkage map retrieved from an online query at NCBI web site: http:/www.ncbi.nlm.nih.gov/genome/sts/. Since the map of calpain 12 and the susceptibility marker D2S119 overlaps, it is reasonable to postulate that calpain 12 may contribute to MS. Furthermore, the transcription profile of calpain12 indicated a prominent expression in spinal cord, and implication of calpains in MS has been suggested (Shields D C et al. A putative mechanism of demyelination in multiple sclerosis by a proteolytic enzyme, calpain. Proc Natl Acad Sci USA. 96:11486-91.1999).
The CAN-12v2 polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include modulating cellular adhesion events, cellular proliferation, and inflammation, in various cells, tissues, and organisms, and particularly in mammalian spinal cord tissue, lymph node, thymus, and spleen tissue, preferably human tissue. CAN-12v2 polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, may be useful in diagnosing, treating, prognosing, and/or preventing neural, immune, hematopoietic, and/or proliferative diseases or disorders.
The strong homology to human calpains, particularly m-calpains, combined with the predominate localized expression in esophagus tissue suggests the CAN-12 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing gastrointestinal diseases, particularly esophageal diseases and/or disorders which include the following non-limiting examples: aberrant transport of food bolus from the mouth to the stomach, aberrant prevention of retrograde flow of gastrointestinal contents, aberrant esophageal peristaltic contractions, pyrosis, painful swallowing, reflux esophagitis, esophageal motility disorders, esophageal spasms, diffuse esophageal spasm, atypical chest pain, regurgitation, oropharyngeal paralysis, nasal regurgitation, dysphagia, cricopharyngeal bar, globus pharyngeus, achalasia, motor disorders of the esophageal smooth muscle, scleroderma esophagus, gastroesophageal reflux disease (GERD), esophagitis, Barrett's esophagus, viral esophagitis, Herpes simplex virus mediated viral esophagitis, Varicella-zoster virus mediated viral esophagitis, Cytomegalovirus mediated viral esophagitis, bacterial esophagitis, Lactobacillus mediated bacterial esophagitis, Candida mediated esophagitis, radiation esophagitis, corrosive esophagitis, pill-induced esophagitis, esophagitis associated with mucocutaneous and systemic diseases, diverticula, lower esophageal mucosal ring, lower esophageal muscular ring, hiatal hernia, paraesophageal hernia, esophageal rupture, and/or Mallory-Weiss Syndrome.
Although calpains are typically associated primarily with neurogenerative conditions, their association in gastrointenstinal tissues has precedence. For example, the human CAN9 protein (hCAN9; Genbank Accession No: gi|5729758; SEQ ID NO:12) is predominately expressed in the stomach and small intestine and is thought to be associated with gastric cancers.
The strong homology to human calpains, particularly m-calpains, combined with the localized expression in spinal cord tissue suggests the CAN-12v2 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing neural diseases, neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Neurological Diseases”, “Regeneration” and “Hyperproliferative Disorders” sections below, in the Examples, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Alternatively, the strong homology to human calpains, particularly m-calpains, combined with the localized expression in lymph node, thymus, and spleen tissue suggests the CAN-12v2 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, ameliorating, and/or preventing immune diseases and/or disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, and elsewhere herein. Briefly, the strong expression in immune tissue indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. The CAN-12v2 polypeptide may also be useful as a preventative agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product may be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
Moreover, the protein would be useful in the detection, treatment, and/or prevention of a variety of vascular disorders and conditions, which include, but are not limited to miscrovascular disease, vascular leak syndrome, aneurysm, stroke, embolism, thrombosis, coronary artery disease, arteriosclerosis, and/or atherosclerosis. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
In addition, antagonists of the CAN-12v2 polynucleotides and polypeptides may have uses that include diagnosing, treating, prognosing, and/or preventing diseases or disorders related to hyper calpain activity, which may include immune and/or proliferative diseases or disorders, particularly thrombosis, embolism, and other blood disorders. Therapeutic and/or pharmaceutical compositions comprising the CAN-12v2 polypeptides may be formulated to comprise heparin.
In addition, antagonists of the CAN-12v2 polynucleotides and polypeptides may have uses that include diagnosing, treating, ameliorating, prognosing, and/or preventing diseases or disorders related to hyper calpain activity, which may include neuronal excitotoxicity, ischemic stroke, hemoragic stroke, hypoxic stress, trauma, cell destruction, spinal cord injury following trauma, degeneration of vulnerable hippocampal neurons after ischemia, reovirus-induced apoptosis, viral-induced induced myocarditis, acute and chronic inflammation, cataract formation, multiple sclerosis, demylenating disorders, acoustic trauma, hearing loss caused by noise, neuronal damage, cardiac ischemic damage, and/or hepatocyte necrosis during and following anoxia.
CAN-12v2 polynucleotides and polypeptides, including fragments and/or antagonsists thereof, may have uses which include modulating development, differentiation, cellular transformation in response to cell signaling, cell-cell and/or cell-extracellular matrix interactions, clustering of the integrin receptor aIIb3, modulating in long term potentiation (memory), modulating neurite outgrowth, modulating cortical lamination activation of protein kinases and phosphatases, remodeling and disassembling the cytoskeleton, cell cycle modulation, in addition, to ameliorating, preventing, and/or treating limb-girdle muscular dystrophy (LGMD), insulin resistance in diabetics, Alzheimer's disease, Multiple sclerosis, Huntington's disease, Parkinson's disease and amyotrophy.
Moreover, CAN-12v2 polynucleotides and polypeptides, including fragments and agonists thereof, may have uses which include treating, diagnosing, prognosing, and/or preventing hyperproliferative disorders, particularly of the neural and immune systems. Such disorders may include, for example, cancers, and metastatic conditions.
CAN-12v2 polynucleotides and polypeptides, including fragments and/or antagonsists thereof, may have uses which include identification of modulators of CAN-12v2 function including antibodies (for detection or neutralization), naturally-occurring modulators and small molecule modulators. Antibodies to domains (including CAN-12v2 epitopes provided herein) of the CAN-12v2 protein could be used as diagnostic agents of inflammatory conditions in patients, are useful in monitoring the activation and presence of cognate proteases, and can be used as a biomarker for the protease involvement in disease states and in the evaluation of inhibitors of the cognate protease in vivo.
CAN-12v2 polypeptides and polynucleotides are useful for diagnosing diseases related to over or under expression of CAN-12v2 proteins by identifying mutations in the CAN-12v2 gene using CAN-12v2 probes, or determining CAN-12v2 protein or mRNA expression levels. CAN-12v2 polypeptides are also useful for screening for compounds, which affect activity of the protein. Diseases that can be treated with CAN-12v2 include, the following, non-limiting examples: neuro-regeneration, neuropathic pain, obesity, anorexia, HIV infections, cancers, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, osteoporosis, angina pectoris, myocardial infarction, psychotic, immune, metabolic, cardiovascular, and neurological disorders.
The predominate expression in neural tissues, combined with the significant expression in a number of other tissues, suggests the CAN-12v2 polynucleotide and polypeptide of the present invention may be involved in modulating nerve invasion, innervation, nerve maintenance, and potentially myeline sheath maintenance and integrity.
The CAN-12v2 polynucleotides and polypeptides, including fragments and antagonists thereof, may have uses which include detecting, diagnosing, treating, ameliorating, and/or preventing diseases and disorders of the neural system, particularly Alzheimer's disease, either directly or indirectly, in addition to other neural disorders known in the art or provided in the “Neurological Diseases” section herein, such as modulating nerve invasion, innervation, nerve maintenance, potentially myelin sheath maintenance and integrity, encephalomyelitis, autoimmune encephalomyelitis, human T cell leukemia virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and neuro-inflammatory diseases.
Molecular genetic manipulation of the structure of the active site domain, particularly the predicted catalytic amino acids, and of other functional domains in the calpain family (e.g., active site domain binding pocket) enables the production of calpains with tailor-made activities. Thus, the CAN-12v2 polypeptides, and fragments thereof, as well as any homologous product resulting from genetic manipulation of the structure, are useful for NMR-based design of modulators of CAN-12v2 biological activity, and calpains, in general.
CAN-12v2 polypeptides and polynucleotides have additional uses which include diagnosing diseases related to the over and/or under expression of CAN-12v2 by identifying mutations in the CAN-12v2 gene by using CAN-12v2 sequences as probes or by determining CAN-12v2 protein or mRNA expression levels. CAN-12v2 polypeptides may be useful for screening compounds that affect the activity of the protein. CAN-12v2 peptides can also be used for the generation of specific antibodies and as bait in yeast two hybrid screens to find proteins the specifically interact with CAN-12v2 (described elsewhere herein).
The CAN-12v2 polynucleotides and polypeptides, including fragments and agonists thereof, may have uses which include detecting, diagnosing, treating, ameliorating, and/or preventing metabolic diseases and disorders, such as diabetes. Moreover, expressed human CAN-12v2 may be useful in the detection of patients susceptible to diabetes. Also paradigms that would simulate intracellular CAN-12v2 activity would be useful in treating diabetes.
The CAN-12v2 polynucleotides and polypeptides, including fragments thereof, may have uses which include identifying inhibitors of intracellular calpain inhibitors (calpastatins) leading to an effective increase in calpain activity.
Various approaches to detect alterations or allelic variants at the genomic or MRNA level of CAN-12v2, could be used as a diagnostic for identifying MS patients, or individuals susceptible to have MS. It is likely that the CAN-12v2 gene comprises polymorphic sites (i.e. SNPs), with specific alleles which may be associated with MS or other neurodegenerative disorders, or associated with an increased likelihood of developing these diseases. Therefore, the invention provides the CAN-12v2 sequence that can be used to design specific primers for the identification of polymorphisms or mutations in CAN-12v2 of patients affected with MS. The presence of a specific allele variant, such as a SNP allele or SNPs haplotype that renders the subject carrying it more susceptible to develop MS or other related diseases could be identified (e.g. a variant in the CAN-12v2 promoter region that increased transcript levels of CAN-12v2, or mutations in the coding sequence that increased the stability or half-life of the CAN-12v2 protein). Other methods such as Northern-blot analysis could be performed to measure transcript levels using a CAN-12v2 cDNA probe derived from the sequence of the invention.
Although it is believed the encoded polypeptide may share at least some biological activities with human calpains (particularly m-calpains), a number of methods of determining the exact biological function of this clone are either known in the art or are described elsewhere herein. Briefly, the function of this clone may be determined by applying microarray methodology. Nucleic acids corresponding to the CAN-12v2 polynucleotides, in addition to, other clones of the present invention, may be arrayed on microchips for expression profiling. Depending on which polynucleotide probe is used to hybridize to the slides, a change in expression of a specific gene may provide additional insight into the function of this gene based upon the conditions being studied. For example, an observed increase or decrease in expression levels when the polynucleotide probe used comes from diseased neural tissue, as compared to, normal tissue might indicate a function in modulating neural function, for example. In the case of CAN-12v2, spinal cord, lymph node, thymus, and/or spleen tissue should be used to extract RNA to prepare the probe.
In addition, the function of the protein may be assessed by applying quantitative PCR methodology, for example. Real time quantitative PCR would provide the capability of following the expression of the CAN-12v2 gene throughout development, for example. Quantitative PCR methodology requires only a nominal amount of tissue from each developmentally important step is needed to perform such experiments. Therefore, the application of quantitative PCR methodology to refining the biological function of this polypeptide is encompassed by the present invention. In the case of CAN-12v2, a disease correlation related to CAN-12v2 may be made by comparing the mRNA expression level of CAN-12v2 in normal tissue, as compared to diseased tissue (particularly diseased tissue isolated from the following: esophagus, spinal cord, lymph node, thymus, and/or spleen tissue). Significantly higher or lower levels of CAN-12v2 expression in the diseased tissue may suggest CAN-12v2 plays a role in disease progression, and antagonists against CAN-12v2 polypeptides would be useful therapeutically in treating, preventing, and/or ameliorating the disease. Alternatively, significantly higher or lower levels of CAN-12v2 expression in the diseased tissue may suggest CAN-12v2 plays a defensive role against disease progression, and agonists of CAN-12v2 polypeptides may be useful therapeutically in treating, preventing, and/or ameliorating the disease. Also encompassed by the present invention are quantitative PCR probes corresponding to the polynucleotide sequence provided as SEQ ID NO:55 (FIGS. 9A-C).
The function of the protein may also be assessed through complementation assays in yeast. For example, in the case of the CAN-12v2, transforming yeast deficient in calpain activity, particularly m-calpain activity, and assessing their ability to grow would provide convincing evidence the CAN-12v2 polypeptide has calpain activity, and possibly m-calpain activity. Additional assay conditions and methods that may be used in assessing the function of the polynucleotides and polypeptides of the present invention are known in the art, some of which are disclosed elsewhere herein.
Alternatively, the biological function of the encoded polypeptide may be determined by disrupting a homologue of this polypeptide in Mice and/or rats and observing the resulting phenotype. Such knock-out experiments are known in the art, some of which are disclosed elsewhere herein.
Moreover, the biological function of this polypeptide may be determined by the application of antisense and/or sense methodology and the resulting generation of transgenic mice and/or rats. Expressing a particular gene in either sense or antisense orientation in a transgenic mouse or rat could lead to respectively higher or lower expression levels of that particular gene. Altering the endogenous expression levels of a gene can lead to the observation of a particular phenotype that can then be used to derive indications on the function of the gene. The gene can be either over-expressed or under expressed in every cell of the organism at all times using a strong ubiquitous promoter, or it could be expressed in one or more discrete parts of the organism using a well characterized tissue-specific promoter (e.g., an esophagus, spinal cord, lymph node, thymus, or spleen specific promoter), or it can be expressed at a specified time of development using an inducible and/or a developmentally regulated promoter.
In the case of CAN-12v2 transgenic mice or rats, if no phenotype is apparent in normal growth conditions, observing the organism under diseased conditions (neural, immune, hematopoietic diseases or disorders, cancers, etc.) may lead to understanding the function of the gene. Therefore, the application of antisense and/or sense methodology to the creation of transgenic mice or rats to refine the biological function of the polypeptide is encompassed by the present invention.
In preferred embodiments, the following N-terminal CAN-12v2 deletion polypeptides are encompassed by the present invention: M1-L697, S2-L697, L3-L697, W4-L697, P5-L697, P6-L697, F7-L697, R8-L697, C9-L697, R10-L697, W11-L697, K12-L697, L13-L697, A14-L697, P15-L697, R16-L697, Y17-L697, S18-L697, R19-L697, R20-L697, A21-L697, S22-L697, P23-L697, Q24-L697, Q25-L697, P26-L697, Q27-L697, Q28-L697, D29-L697, F30-L697, E31-L697, A32-L697, L33-L697, L34-L697, A35-L697, E36-L697, C37-L697, L38-L697, R39-L697, N40-L697, G41-L697, C42-L697, L43-L697, F44-L697, E45-L697, D46-L697, T47-L697, S48-L697, F49-L697, P50-L697, A51-L697, T52-L697, L53-L697, S54-L697, S55-L697, I56-L697, G57-L697, S58-L697, G59-L697, S60-L697, L61-L697, L62-L697, Q63-L697, K64-L697, L65-L697, P66-L697, P67-L697, R68-L697, L69-L697, Q70-L697, W71-L697, K72-L697, R73-L697, P74-L697, P75-L697, E76-L697, L77-L697, H78-L697, S79-L697, N80-L697, P81-L697, Q82-L697, F83-L697, Y84-L697, F85-L697, A86-L697, K87-L697, A88-L697, K89-L697, R90-L697, L91-L697, D92-L697, L93-L697, C94-L697, Q95-L697, G96-L697, I97-L697, V98-L697, G99-L697, D100-L697, C101-L697, W102-L697, F103-L697, L104-L697, A105-L697, A106-L697, L107-L697, Q108-L697, A109-L697, L110-L697, A111-L697, L112-L697, H113-L697, Q114-L697, D115-L697, I116-L697, L117-L697, S118-L697, R119-L697, V120-L697, V121-L697, P122-L697, L123-L697, N124-L697, Q125-L697, S126-L697, F127-L697, T128-L697, E129-L697, K130-L697, Y131-L697, A132-L697, G133-L697, I134-L697, F135-L697, R136-L697, F137-L697, W138-L697, F139-L697, W140-L697, H141-L697, Y142-L697, G143-L697, N144-L697, W145-L697, V146-L697, P147-L697, V148-L697, V149-L697, I150-L697, D151-L697, D152-L697, R153-L697, L154-L697, P155-L697, V156-L697, N157-L697, E158-L697, A159-L697, G160-L697, Q161-L697, L162-L697, V163-L697, F164-L697, V165-L697, S166-L697, S167-L697, T168-L697, Y169-L697, K170-L697, N171-L697, L172-L697, F173-L697, W174-L697, G175-L697, A176-L697, L177-L697, L178-L697, E179-L697, K180-L697, A181-L697, Y182-L697, A183-L697, K184-L697, L185-L697, S186-L697, G187-L697, S188-L697, Y189-L697, E190-L697, D191-L697, L192-L697, Q193-L697, S194-L697, G195-L697, Q196-L697, V197-L697, S198-L697, E199-L697, A200-L697, L201-L697, V202-L697, D203-L697, F204-L697, T205-L697, G206-L697, G207-L697, V208-L697, T209-L697, M210-L697, T211-L697, I212-L697, N213-L697, L214-L697, A215-L697, E216-L697, A217-L697, H218-L697, G219-L697, N220-L697, L221-L697, W222-L697, D223-L697, I224-L697, L225-L697, I226-L697, E227-L697, A228-L697, T229-L697, Y230-L697, N231-L697, R232-L697, T233-L697, L234-L697, I235-L697, G236-L697, C237-L697, Q238-L697, T239-L697, H240-L697, S241-L697, G242-L697, E243-L697, K244-L697, I245-L697, L246-L697, E247-L697, N248-L697, G249-L697, L250-L697, V251-L697, E252-L697, G253-L697, H254-L697, A255-L697, Y256-L697, T257-L697, L258-L697, T259-L697, G260-L697, I261-L697, R262-L697, K263-L697, V264-L697, T265-L697, C266-L697, K267-L697, H268-L697, R269-L697, P270-L697, E271-L697, Y272-L697, L273-L697, V274-L697, K275-L697, L276-L697, R277-L697, N278-L697, P279-L697, W280-L697, G281-L697, K282-L697, V283-L697, E284-L697, W285-L697, K286-L697, G287-L697, D288-L697, W289-L697, S290-L697, D291 -L697, S292-L697, S293-L697, S294-L697, K295-L697, W296-L697, E297-L697, L298-L697, L299-L697, S300-L697, P301-L697, K302-L697, E303-L697, K304-L697, I305-L697, L306-L697, L307-L697, L308-L697, R309-L697, K310-L697, D311-L697, N312-L697, D313-L697, G314-L697, E315-L697, F316-L697, W317-L697, M318-L697, T319-L697, L320-L697, Q321-L697, D322-L697, F323-L697, K324-L697, T325-L697, H326-L697, F327-L697, V328-L697, L329-L697, L330-L697, V331-L697, I332-L697, C333-L697, K334-L697, L335-L697, T336-L697, P337-L697, G338-L697, L339-L697, L340-L697, S341-L697, Q342-L697, E343-L697, A344-L697, A345-L697, Q346-L697, K347-L697, W348-L697, T349-L697, Y350-L697, T351-L697, M352-L697, R353-L697, E354-L697, G355-L697, R356-L697, W357-L697, E358-L697, K359-L697, R360-L697, S361-L697, T362-L697, A363-L697, G364-L697, G365-L697, Q366-L697, R367-L697, Q368-L697, L369-L697, L370-L697, Q371-L697, D372-L697, T373-L697, F374-L697, W375-L697, K376-L697, N377-L697, P378-L697, Q379-L697, F380-L697, L381-L697, L382-L697, S383-L697, V384-L697, W385-L697, R386-L697, P387-L697, E388-L697, E389-L697, G390-L697, R391-L697, R392-L697, S393-L697, L394-L697, R395-L697, P396-L697, C397-L697, S398-L697, V399-L697, L400-L697, V401-L697, S402-L697, L403-L697, L404-L697, Q405-L697, K406-L697, P407-L697, R408-L697, H409-L697, R410-L697, C411-L697, R412-L697, K413-L697, R414-L697, K415-L697, P416-L697, L417-L697, L418-L697, A419-L697, I420-L697, G421-L697, F422-L697, Y423-L697, L424-L697, Y425-L697, R426-L697, M427-L697, N428-L697, K429-L697, Y430-L697, H431-L697, D432-L697, D433-L697, Q434-L697, R435-L697, R436-697, L437-L697, P438-L697, P439-L697, E440-L697, F441-L697, F442-L697, Q443-L697, R444-L697, N445-L697, T446-L697, P447-L697, L448-L697, S449-L697, Q440-L697, P451-L697, D452-L697, R453-L697, F454-L697, L455-L697, K456-L697, E457-L697, K458-L697, E459-L697, V460-L697, S461-L697, Q462-L697, E463-L697, L464-L697, C465-L697, L466-L697, E467-L697, P468-L697, G469-L697, T470-L697, Y471-L697, L472-L697, I473-L697, V474-L697, P475-L697, C476-L697, I477-L697, L478-L697, E479-L697, A480-L697, H481-L697, Q482-L697, K483-L697, S484-L697, E485-L697, F486-L697, V487-L697, L488-L697, R489-L697, V490-L697, F491-L697, S492-L697, R493-L697, K494-L697, H495-L697, I496-L697, F497-L697, Y498-L697, E499-L697, I500-L697, G501-L697, S502-L697, N503-L697, S504-L697, G505-L697, V506-L697, V507-L697, F508-L697, S509-L697, K510-L697, E511-L697, I512-L697, E513-L697, D514-L697, Q515-L697, N516-L697, E517-L697, R518-L697, Q519-L697, D520-L697, E521-L697, F522-L697, F523-L697, T524-L697, K525-L697, F526-L697, F527-L697, E528-L697, K529-L697, H530-L697, P531-L697, E532-L697, I533-L697, N534-L697, A535-L697, V536-L697, Q537-L697, L538-L697, Q539-L697, N540-L697, L541-L697, L542-L697, N543-L697, Q544-L697, and/or M545-L697 of SEQ ID NO:56. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal CAN-12v2 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal CAN-12v2 deletion polypeptides are encompassed by the present invention: M1-L697, M1-L696, M1-T695, M1-T694, M1-N693, M1-F692, M1-I691, M1-L690, M1-Y689, M1-S688, M1-T687, M1-S686, M1-H685, M1-A684, M1-V683, M1-G682, M1-G681, M1-D680, M1-I679, M1-N678, M1-A677, M1-L676, M1-I675, M1-V674, M1-P673, M1-V672, M1-I671, M1-M670, M1-F669, M1-F668, M1-T667, M1-P666, M1-T665, M1-S664, M1-Q663, M1-L662, M1-D661, M1-V660, M1-D659, M1-K658, M1-L657, M1-T656, M1-V655, M1-S654, M1-R653, M1-I652, M1-L651, M1-T650, M1-V649, M1-E648, M1-A647, M1-H646, M1-W645, M1-V644, M1-D643, M1-G642, M1-R641, M1-R640, M1-Q639, M1-R638, M1-I637, M1-L636, M1-T635, M1-C634, M1-G633, M1-A632, M1-R631, M1-T630, M1-H629, M1-G628, M1-C627, M1-S626, M1-W625, M1-S624, M1-K623, M1-R622, M1-H621, M1-R620, M1-G619, M1-A618, M1-E617, M1-R616, M1-M615, M1-A614, M1-A613, M1-H612, M1-L611, M1-Q610, M1-E609, M1-W608, M1-N607, M1-L606, M1-Y605, M1-G604, M1-S603, M1-G602, M1-R601, M1-D600, M1-Q599, M1-K598, M1-H597, M1-F596, M1-V595, M1-K594, M1-Q593, M1-S592, M1-L591, M1-K590, M1-L589, M1-Q588, M1-K587, M1-W586, M1-L585, M1-D584, M1-R583, M1-F582, M1-E581, M1-Q580, M1-I579, M1-S578, M1-M577, M1-T576, M1-G575, M1-S574, M1-A573, M1-N572, M1-L571, M1-D570, M1-L569, M1-L568, M1-A567, M1-L566, M1-I565, M1-G564, M1-Q563, M1-C562, M1-A561, M1-E560, M1-L559, M1-S558, M1-F557, M1-F556, M1-P555, M1-Q554, M1-R553, M1-S552, M1-G551, M1-L550, M1-S549, M1-S548, M1-W547, M1-T546, M1-M545, M1-Q544, M1-N543, M1-L542, M1-L541, M1-N540, M1-Q539, M1-L538, M1-Q537, M1-V536, M1-A535, M1-N534, M1-I533, M1-E532, M1-P531, M1-H530, M1-K529, M1-E528, M1-F527, M1-F526, M1-K525, M1-T524, M1-F523, M1-F522, M1-E521, M1-D520, M1-Q519, M1-R518, M1-E517, M1-N516, M1-Q515, M1-D514, M1-E513, M1-I512, M1-E511, M1-K510, M1-S509, M1-F508, M1-V507, M1-V506, M1-G505, M1-S504, M1-N503, M1-S502, M1-G501, M1-I500, M1-E499, M1-Y498, M1-F497, M1-I496, M1-H495, M1-K494, M1-R493, M1-S492, M1-F491, M1-V490, M1-R489, M1-L488, M1-V487, M1-F486, M1-E485, M1-S484, M1-K483, M1-Q482, M1-H481, M1-A480, M1-E479, M1-L478, M1-I477, M1-C476, M1-P475, M1-V474, M1-I473, M1-L472, M1-Y471, M1-T470, M1-G469, M1-P468, M1-E467, M1-L466, M1-C465, M1-L464, M1-E463, M1-Q462, M1-S461, M1-V460, M1-E459, M1-K458, M1-E457, M1-K456, M1-L455, M1-F454, M1-R453, M1-D452, M1-P451, M1-Q450, M1-S449, M1-L448, M1-P447, M1-T446, M1-N445, M1-R444, M1-Q443, M1-F442, M1-F441, M1-E440, M1-P439, M1-P438, M1-L437, M1-R436, M1-R435, M1-Q434, M1-D433, M1-D432, M1-H431, M1-Y430, M1-K429, M1-N428, and/or M1-M427 of SEQ ID NO:56. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal CAN-12v2 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
Alternatively, preferred polypeptides of the present invention may comprise polypeptide sequences corresponding to, for example, internal regions of the CAN-12v2 polypeptide (e.g., any combination of both N— and C-terminal CAN-12v2 polypeptide deletions) of SEQ ID NO:56. For example, internal regions could be defined by the equation: amino acid NX to amino acid CX, wherein NX refers to any N-terminal deletion polypeptide amino acid of CAN-12v2 (SEQ ID NO:56), and where CX refers to any C-terminal deletion polypeptide amino acid of CAN-12v2 (SEQ ID NO:56). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.
The present invention also encompasses immunogenic and/or antigenic epitopes of the CAN-12v2 polypeptide.
The CAN-12v2 polypeptides of the present invention were determined to comprise several phosphorylation sites based upon the Motif algorithm (Genetics Computer Group, Inc.). The phosphorylation of such sites may regulate some biological activity of the CAN-12v2 polypeptide. For example, phosphorylation at specific sites may be involved in regulating the proteins ability to associate or bind to other molecules (e.g., proteins, ligands, substrates, DNA, etc.). In the present case, phosphorylation may modulate the ability of the CAN-12v2 polypeptide to associate with other polypeptides, particularly the serine protease substrate for CAN-12v2, or its ability to modulate serine protease function.
The CAN-12v2 polypeptide was predicted to comprise eleven PKC phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). In vivo, protein kinase C exhibits a preference for the phosphorylation of serine or threonine residues. The PKC phosphorylation sites have the following consensus pattern: [ST]-x-[RK], where S or T represents the site of phosphorylation and ‘x’ an intervening amino acid residue. Additional information regarding PKC phosphorylation sites can be found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem. 161:177-184(1986), and Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem. 260:12492-12499(1985); which are hereby incorporated by reference herein.
In preferred embodiments, the following PKC phosphorylation site polypeptides are encompassed by the present invention: LAPRYSRRASPQQ (SEQ ID NO:76), LNQSFTEKYAGIF (SEQ ID NO:77), VFVSSTYKNLFWG (SEQ ID NO:78), GIRKVTCKHRPEY (SEQ ID NO:79), DWSDSSSKWELLS (SEQ ID NO:80), KWELLSPKEKILL (SEQ ID NO:81), QKWTYTMREGRWE (SEQ ID NO:82), EEGRRSLRPCSVL (SEQ ID NO:83), VLRVFSRKHIFYE (SEQ ID NO:84), KQLKLSQKVFHKQ (SEQ ID NO:85), and/or LIRSVTLKDVDLQ (SEQ ID NO:86). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the CAN-12v2 PKC phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The CAN-12v2 polypeptide has been shown to comprise four glycosylation site according to the Motif algorithm (Genetics Computer Group, Inc.). As discussed more specifically herein, protein glycosylation is thought to serve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.
In preferred embodiments, the following asparagine glycosylation site polypeptide is encompassed by the present invention: RVVPLNQSFTEKYA (SEQ ID NO:87), IEATYNRTLIGCQT (SEQ ID NO:102), ALLDLNASGTMSIQ (SEQ ID NO:103), and/or SYLWNTTLL (SEQ ID NO:104). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of the CAN-12v2 asparagine glycosylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The CAN-12v2 polypeptide has been shown to comprise one amidation site according to the Motif algorithm (Genetics Computer Group, Inc.). The precursor of hormones and other active peptides which are C-terminally amidated is always directly followed by a glycine residue which provides the amide group, and most often by at least two consecutive basic residues (Arg or Lys) which generally function as an active peptide precursor cleavage site. Although all amino acids can be amidated, neutral hydrophobic residues such as Val or Phe are good substrates, while charged residues such as Asp or Arg are much less reactive. A consensus pattern for amidation sites is the following: x-G-[RK]-[RK], wherein “X” represents the amidation site. Additional information relating to asparagine glycosylation may be found in reference to the following publications, which are hereby incorporated by reference herein: Kreil G., Meth. Enzymol. 106:218-223(1984); and Bradbury A. F., Smyth D. G., Biosci. Rep. 7:907-916(1987).
In preferred embodiments, the following amidation site polypeptide is encompassed by the present invention: VWRPEEGRRSLRPC (SEQ ID NO:88). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this CAN-12v2 amidation site polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The CAN-12v2 polypeptide has been shown to comprise one RGD cell attachment site domain according to the Motif algorithm (Genetics Computer Group, Inc.). The sequence Arg-Gly-Asp, found in fibronectin, is crucial for its interaction with its cell surface receptor, an integrin. What has been called the ‘RGD’ tripeptide is also found in the sequences of a number of other proteins, where it has been shown to play a role in cell adhesion. Non-limiting examples of these proteins are the following: some forms of collagens, fibrinogen, vitronectin, von Willebrand factor (VWF), snake disintegrins, and slime mold discoidins. The ‘RGD’ tripeptide is also found in other proteins where it may serve the same purpose. A consensus pattern for RGD cell attachment sites is the following: R-G-D. Additional information relating to RGD cell attachment site domains may be found in reference to the following publications, which are hereby incorporated by reference herein: Ruoslahti E., Pierschbacher M. D., Cell 44:517-518(1986); and d'Souza S. E., Ginsberg M. H., Plow E. F., Trends Biochem. Sci. 16:246-250(1991).
In preferred embodiments, the following RGD cell attachment site domain polypeptide is encompassed by the present invention: LIRQRRGDVWHAE (SEQ ID NO:89). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this RGD cell attachment site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
In confirmation of the CAN-12v2 polypeptide being a calpain, it has been shown to comprise one EF-hand calcium-binding domain according to the Motif algorithm (Genetics Computer Group, Inc.). Many calcium-binding proteins belong to the same evolutionary family and share a type of calcium-binding domain known as the EF-hand. This type of domain consists of a twelve residue loop flanked on both side by a twelve residue alpha-helical domain. In an EF-hand loop the calcium ion is coordinated in a pentagonal bipyramidal configuration. The six residues involved in the binding are in positions 1, 3, 5, 7, 9 and 12; these residues are denoted by X, Y, Z, −Y, −X and −Z. The invariant Glu or Asp at position 12 provides two oxygens for liganding Ca (bidentate ligand). Several representative proteins containing EF-hand regions are provided below: For each type of protein, the total number of EF-hand regions known or supposed to exist are provided in parenthesis: Aequorin and Renilla luciferin binding protein (LBP) (Ca=3); Alpha actinin (Ca=2); Calbindin (Ca=4); Calcineurin B subunit (protein phosphatase 2B regulatory subunit) (Ca=4); Calcium-binding protein from Streptomyces erythraeus (Ca=3?); Calcium-binding protein from Schistosoma mansoni (Ca=2?); Calcium-binding proteins TCBP-23 and TCBP-25 from Tetrahymena thermophila (Ca=4?); Calcium-dependent protein kinases (CDPK) from plants (Ca=4); Calcium vector protein from amphoxius (Ca=2); Calcyphosin (thyroid protein p24) (Ca=4?); Calmodulin (Ca=4, except in yeast where Ca=3); Calpain small and large chains (Ca=2); Calretinin (Ca=6); Calcyclin (prolactin receptor associated protein) (Ca=2); Caltractin (centrin) (Ca=2 or 4); Cell Division Control protein 31 (gene CDC31) from yeast (Ca=2?); Diacyiglycerol kinase (EC 2.7.1.107) (DGK) (Ca=2); FAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) from mammals (Ca=1); Fimbrin (plastin) (Ca-2); Flagellar calcium-binding protein (1f8) from Trypanosoma cruzi (Ca=1 or 2); Guanylate cyclase activating protein (GCAP) (Ca=3); Inositol phospholipid-specific phospholipase C isozymes gamma-1 and delta-1 (Ca=2) [10]; Intestinal calcium-binding protein (ICaBPs) (Ca=2); MIF related proteins 8 (MRP-8 or CFAG) and 14 (MRP-14) (Ca=2); Myosin regulatory light chains (Ca=1); Oncomodulin (Ca=2); Osteonectin (b)asement membrane protein BM-40) (SPARC) and proteins that contains an ‘osteonectin’ domain (QR1, matrix glycoprotein SC1) (Ca=1); Parvalbumins alpha and beta (Ca=2); Placental calcium-binding protein (18a2) (nerve growth factor induced protein 42a) (p9k) (Ca=2); Recoverins (visinin, hippocalcin, neurocalcin, S-modulin) (Ca=2 to 3); Reticulocalbin (Ca=4); S-100 protein, alpha and beta chains (Ca=2); Sarcoplasmic calcium-binding protein (SCPs) (Ca=2 to 3); Sea urchin proteins Spec 1 (Ca=4), Spec 2 (Ca=4?), Lps-1 (Ca=8); Serine/threonine protein phosphatase rdgc (EC 3.1.3.16) from Drosophila (Ca=2); Sorcin V19 from hamster (Ca=2); Spectrin alpha chain (Ca=2); Squidulin (optic lobe calcium-binding protein) from squid (Ca=4); and Troponins C; from skeletal muscle (Ca=4), from cardiac muscle (Ca=3), from arthropods and molluscs (Ca=2).

A consensus pattern for EF hand calcium binding domains is the following:

wherein X, Y, Z, −Y, −X, and −Z are as defined above, and wherein “x” represents any amino acid. Amino acid residues within the consensus at positions 1 (X), 3 (Y) and 12 (−Z) are the most conserved. The 6th residue in an EF-hand loop is in most cases a Gly.

Additional information relating to RGD cell attachment site domains may be found in reference to the following publications, which are hereby incorporated by reference herein: Kawasaki H., Kretsinger R. H., Protein Prof. 2:305490(1995); Kretsinger R. H., Cold Spring Harbor Symp. Quant. Biol. 52:499-510(1987); Moncrief N. D., Kretsinger R. H., Goodman M., J. Mol. Evol. 30:522-562(1990); Nakayama S., Moncrief N. D., Kretsinger R. H., J. Mol. Evol. 34:416448(1992); Heizmann C. W., Hunziker W., Trends Biochem. Sci. 16:98-103(1991); Kligman D., Hilt D. C., Trends Biochem. Sci. 13:437-443(1988); Strynadka N. C. J., James M. N. G., Annu. Rev. Biochem. 58:951-98(1989); Haiech J., Sallantin J., Biochimie 67:555-560(1985); Chauvaux S., Beguin P., Aubert J.-P., Bhat K. M., Gow L. A., Wood T. M., Bairoch A., Biochem. J. 265:261-265(1990); Bairoch A., Cox J. A., FEBS Lett. 269:454-456(1990).
In preferred embodiments, the following EF-hand calcium binding domain polypeptide is encompassed by the present invention: ILALLDLNASGTMSIQEFRDLWK (SEQ ID NO:90). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this EF-hand calcium binding domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
In further confirmation of the CAN-12v2 polypeptide being a calpain, it has been shown to comprise one eukaryotic thiol (cysteine) protease active site domain according to the Motif algorithm (Genetics Computer Group, Inc.). Eukaryotic thiol proteases (EC 3.4.22.-) are a family of proteolytic enzymes which contain an active site cysteine. Catalysis proceeds through a thioester intermediate and is facilitated by a nearby histidine side chain; an asparagine completes the essential catalytic triad. Non-limiting examples of proteases which are known to belong to this family are provided below: Vertebrate lysosomal cathepsins B (EC 3.4.22.1), H (EC 3.4.22.16), L (EC 3.4.22.15), and S (EC 3.4.22.27); Vertebrate lysosomal dipeptidyl peptidase I (EC 3.4.14.1) (also known as cathepsin C); Vertebrate calpains (EC 3.4.22.17) (Calpains are intracellular calcium-activated thiol protease that contain both a N-terminal catalytic domain and a C-terminal calcium-binding domain; Mammalian cathepsin K, which seems involved in osteoclastic bone resorption; Human cathepsin O; Bleomycin hydrolase (An enzyme that catalyzes the inactivation of the antitumor drug BLM (a glycopeptide); Plant enzymes: barley aleurain (EC 3.4.22.16), EP-B1/B4; kidney bean EP-C1, rice bean SH-EP; kiwi fruit actinidin (EC 3.4.22.14); papaya latex papain (EC 3.4.22.2), chymopapain (EC 3.4.22.6), caricain (EC 3.4.22.30), and proteinase IV (EC 3.4.22.25); pea turgor-responsive protein 15A; pineapple stem bromelain (EC 3.4.22.32); rape COT44; rice oryzain alpha, beta, and gamma; tomato low-temperature induced, Arabidopsis thaliana A494, RD19A and RD21 A; House-dust mites allergens DerP 1 and EurM1; Cathepsin B-like proteinases from the worms Caenorhabditis elegans (genes gcp-1, cpr-3, cpr-4, cpr-5 and cpr-6), Schistosoma mansoni (antigen SM31) and Japonica (antigen SJ31), Haemonchus contortus (genes AC-1 and AC-2), and Ostertagia ostertagi (CP-1 and CP-3); Slime mold cysteine proteinases CP1 and CP2; Cruzipain from Trypanosoma cruzi and brucei; Throphozoite cysteine proteinase (TCP) from various Plasmodium species; Proteases from Leishmania mexicana, Theileria annulata and Theileria parva; Baculoviruses cathepsin-like enzyme (v-cath); Drosophila small optic lobes protein (gene sol), a neuronal protein that contains a calpain-like domain; Yeast thiol protease BLH1/JYCP1/LAP3; and Caenorhabditis elegans hypothetical protein C06G4.2, a caipain-like protein; Two bacterial peptidases are also part of this family—Aminopeptidase C from Lactococcus lactis (gene pepC), and Thiol protease tpr from Porphyromonas gingivalis.
A consensus pattern for eukaryotic thiol (cysteine) protease active site domains is the following: Q-x(3)-[GE]-x-C-[YW]-x(2)-[STAGC]-[STAGCV], wherein C is the active site residue, and “x” represents any amino acid. The residue in position 4 of the pattern is almost always cysteine; the only exceptions are calpains (Leu), bleomycin hydrolase (Ser) and yeast YCP1 (Ser); while the residue in position 5 of the pattern is always Gly except in papaya protease IV where it is Glu.
An additional consensus pattern for eukaryotic thiol (cysteine) protease active site domains is the following: [LIVMGSTAN]-x-H-[GSACE]-[LIVM]-x-[LIVMAT](2)-G-x-[GSADNH], wherein H is the active site residue, and “x” represents any amino acid.
An additional consensus pattern for eukaryotic thiol (cysteine) protease active site domains is the following: [FYCH]-[WI]-[LIVT]-x-[KRQAG]-N-[ST]-W-x(3)-[FYW]-G-x(2)-G-[LFYW]-[LIVMFYG]-x-[LIVMF], wherein N is the active site residue, and “x” represents any amino acid.
Additional information relating to for eukaryotic thiol (cysteine) protease active site domains may be found in reference to the following publications, which are hereby incorporated by reference herein: Dufour E., Biochimie 70:1335-1342(1988); Kirschke H., Barrett A. J., Rawlings N. D., Protein Prof. 2:1587-1643(1995); Shi G.-P., Chapman H. A., Bhairi S. M., Deleeuw C., Reddy V. Y., Weiss S. J., FEBS Lett. 357:129-134(1995); Velasco G., Ferrando A. A., Puente X. S., Sanchez L. M., Lopez-Otin C., J. Biol. Chem. 269:27136-27142(1994); Chapot-Chartier M. P., Nardi M., Chopin M. C., Chopin A., Gripon J. C., Appl. Environ. Microbiol. 59:330-333(1993); Higgins D. G., McConnell D. J., Sharp P. M., Nature 340:604-604(1989); Rawlings N. D., Barrett A. J., Meth. Enzymol. 244:461-486(1994), and http://www.expasy.ch/cgi-bin/lists?peptidas.txt, which are hereby incorporated by reference in their entirety herein.
In preferred embodiments, the following for eukaryotic thiol (cysteine) protease active site domain polypeptide is encompassed by the present invention: RLDLCQGIVGDCWFLAALQALA (SEQ ID NO:91). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this for eukaryotic thiol (cysteine) protease active site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
As referenced elsewhere herein, calpains are organized in domains. As a point of reference, the larger catalytic subunit of the best characterized m-calpain is organized in four domains (I-IV)(Hosfield et al., Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation. EMBO J 18:6880-9, 1999; Strobl et al., The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium. Proc Natl Acad Sci U S A. 97:588-92, 2000). The N-terminal domain I contains an alpha helical region. Domain II contains the catalytic active domain with the active site amino acids. Domain m contains the linker between the Ca2+ binding domain in domain IV to the active site domain II.
The CAN-12v2 calpain of the present invention has the same domain I and II as the CAN-12 calpain, but differs in domains III and IV. The N-terminal domain I consists of residues Met1-Arg20. Domain II of the CAN-12v2 calpain (Ala2l-Lys333) contain the catalytic active site residue acids (Cys101, His254 and Asn 278). As can be seen in the sequence alignments (FIGS. 2A-E), there is high amino acid sequence homology in the amino acid residues bracketing the active site amino acids. Combined domains I and II of the calpains of the present invention are 42-45% homologous to m-calpain.
The CAN-12v2 calpain of the present invention, have the same domain I and II, although they differ in composition and content of domains III and IV. The CAN-12 and CAN-12v2 calpains contain both the linker (domain III) and C-terminal domain IV. The “linker” domain also contains residues Met426, Asn427 and Lys428 of SEQ ID NO56).
The present invention also provides a three-dimensional homology model of the CAN-12v2 polypeptide (see FIG. 11). The three-dimensional homology model of the CAN-12 polypeptide may also be applicable to the CAN-12v2 polypeptide. Although the CAN-12 polypeptide sequence is different than the CAN-12v2. polypeptide sequence, the fact that domain I and II are substantially the same suggests the homology model of CAN-12 may be used for designing potential ligands (including agonists and/or antagonists) for the CAN-12v2 polypeptide. A three-dimensional homology model can be constructed on the basis of the known structure of a homologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995, Yuan, et al, 1995). The homology model of the CAN-12v2 polypeptide, corresponding to amino acid residues 12 to 428 and from amino acid residues 543 to 639 of SEQ ID NO:56, was based upon the homologous structure of CAN2, a m-calpain family member (Strobl et al, 2000; hCAN2; Genbank Accession No. gi|7546423; SEQ ID NO:11) and is defined by the set of structural coordinates set forth in Table V herein. Note that amino acids 429 to 542 of SEQ ID NO:56 were omitted from the homology model. As a result, the amino acid residue numbers in Table V do not correspond to the amino acid residue numbers as provided in SEQ ID NO:56 (FIGS. 9A-C). Rather, the amino acid residue numbers in Table V for amino acid residues 12 to 428 correspond to amino acid residues 12 to 428 of SEQ ID NO:56 (FIGS. 9A-C), while amino acid residue numbers in Table V for amino acid residues 429 to 512 correspond to amino acid residues 543 to 639 of SEQ ID NO:56 (FIGS. 9A-C).
A description of the headings in Table V are as follows: “Atom No” refers to the atom number within the CAN-12v2 homology model; “Atom name” refers to the element whose coordinates are measured, the first letter in the column defines the element; “Residue” refers to the amino acid within which the atom resides, and the provided number after the amino acid refers to the amino acid number of the “residue”; “X Coord”, “Y Coord”, and “Z Coord” structurally define the atomic position of the element measured in three dimensions.
The CAN-12v2 homology model of the present invention may provide one basis for designing rational stimulators (agonists) and/or inhibitors (antagonists) of one or more of the biological functions of CAN-12v2, or of CAN-12v2 mutants having altered specificity (e.g., molecularly evolved CAN-12v2 polypeptides, engineered site-specific CAN-12v2 mutants, CAN-12v2 allelic variants, etc.).
Homology models are not only useful for designing rational agonists and/or antagonists, but are also useful in predicting the function of a particular polypeptide. The functional predictions from homology models are typically more accurate than the functional attributes derived from traditional polypeptide sequence homology alignments (e.g., CLUSTALW), particularly when the three dimensional structure of a related polypeptide is known (e.g., m-calpain family member CAN2 protein; Genbank Accession No. gi|7546423; SEQ ID NO:11). The increased prediction accuracy is based upon the fact that homology models approximate the three-dimensional structure of a protein, while homology based alignments only take into account the one dimension polypeptide sequence. Since the function of a particular polypeptide is determined not only by its primary, secondary, and tertiary structure, functional assignments derived solely upon homology alignments using the one dimensional protein sequence may be less reliable. A 3-dimensional model can be constructed on the basis of the known structure of a homologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995, Yuan, et al, 1995).
Prior to developing a homology model, those of skill in the art would appreciate that a template of a known protein, or model protein, must first be identified which will be used as a basis for constructing the homology model for the protein of unknown structure (query template). In the case of the CAN-12v2 polypeptide of the present invention, the model protein template used in constructing the CAN-12v2 homology model was the m-calpain family member CAN2 protein; Genbank Accession No. gi|7546423; SEQ ID NO:11).
Identifying a template can be accomplished using pairwise alignment of protein sequences using such programs as FASTA (Pearson, et al 1990) and BLAST (Altschul, et al, 1990). In cases where sequence similarity is high (greater than 30%), such pairwise comparison methods may be adequate for identifying an appropriate template. Likewise, multiple sequence alignments or profile-based methods can be used to align a query sequence to an alignment of multiple (structurally and biochemically) related proteins. When the sequence similarity is low, more advanced techniques may be used. Such techniques, include, for example, protein fold recognition (protein threading; Hendlich, et al, 1990), where the compatibility of a particular polypeptide sequence with the 3-dimensional fold of a potential template protein is gauged on the basis of a knowledge-based potential.
Following the initial sequence alignment, the second step would be to optimally align the query template to the model template by manual manipulation and/or by the incorporation of features specific to the polypeptides (e.g., motifs, secondary structure predictions, and allowed conservations). Preferably, the incorporated features are found within both the model and query template.
The third step would be to identify structurally conserved regions that could be used to construct secondary core structure (Sali, et al, 1995). Loops could be added using knowledge-based techniques, and by performing forcefield calculations (Sali, et al, 1995).
In order to recognize errors in a three-dimensional structure, knowledge based mean fields can be used to judge the quality of protein folds (Sippl 1993). The methods can be used to recognize misfolded structures as well as faulty parts of structural models. The technique generates an energy graph where the energy distribution for a given protein fold is displayed on the y-axis and residue position in the protein fold is displayed on the x-axis. The knowledge based mean fields compose a force field derived from a set of globular protein structures taken as a subset from the Protein Data Bank (Bernstein et. al. 1977). To analyze the quality of a model the energy distribution is plotted and compared to the energy distribution of the template from which the model was generated. FIG. 13 shows the energy graph for the CAN-12.v2 model (dotted line) and the template (1dkv, m-calpain) from which the model was generated. This graph supports the motif and sequence alignments in confirming that the three dimensional structure coordinates of CAN-12.v2 are an accurate and useful representation for the polypeptide.
The term “structure coordinates” refers to Cartesian coordinates generated from the building of a homology model. In this invention, the homology model of residues 12 to 525 of CAN-12v2 was derived from generating a sequence alignment with m-calpain (Strobl et al, 2000; hCAN2; Genbank Accession No. gi|7546423; SEQ ID NO:11) using the COMPOSER suite of software within SYBYL6.6 (Tripos Associates, St. Louis, Mo.) and then generating the backbone and side chain conformations. In the original crystal structure (pdb code 1dkv) as well as the crystal structure reported elsewhere (Hosfield et al, 1999), the active site of the enzyme comprising a cysteine, a histidine and an asparagine residue was not “formed”. The helix that contains the active site C101 was altered by moving the helix down one pitch so that the active site geometry could match that found in Papain (pdb code 1b4). This modified structure of human m-calpain was used as the template for construction of the homology model (illustrated in FIG. 11 herein).
The skilled artisan would appreciate that a set of structure coordinates for a protein represents a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates, as emanate from the generation of similar homology models using different alignment templates (i.e., other than the m-calpain (Strobl et al, 2000; hCAN2; Genbank Accession No. gi|7546423; SEQ ID NO:11), and/or using different methods in generating the homology model, will likely have minor effects on the overall shape. Variations in coordinates may also be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth in Table V could be manipulated by fractionalization of the structure coordinates; integer additions, or integer subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.
Therefore, various computational analyses are necessary to determine whether a template molecule or a portion thereof is sufficiently similar to all or part of a query template (e.g., CAN-12v2) in order to be considered the same. Such analyses may be carried out in current software applications, such as SYBYL version 6.6 or INSIGHTII (Molecular Simulations Inc., San Diego, Calif.) version 2000 and as described in the accompanying User's Guides.
Using the superimposition tool in the program SYBYL, comparisons can be made between different structures and different conformations of the same structure. The procedure used in SYBYL to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalencies in these structures; 3) perform a fitting operation; and 4) analyze the results. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); the second structure (i.e., moving structure) is identified as the source structure. The atom equivalency within SYBYL is defined by user input. For the purpose of this invention, we will define equivalent atoms as protein backbone atoms (N, Cα, C and O) for all conserved residues between the two structures being compared. We will also consider only rigid fitting operations. When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atoms is an absolute minimum. This number, given in angstroms, is reported by the SYBYL program. For the purpose of the present invention, any homology model of a CAN-12v2 that has a root mean square deviation of conserved residue backbone atoms (N, Cα, C, O) of less than 3.0 A when superimposed on the relevant backbone atoms described by structure coordinates listed in Table V are considered identical. More preferably, the root mean square deviation for the CAN-12v2 polypeptide is less than 2.0 Å.
The homology model of the present invention is useful for the structure-based design of modulators of the CAN-12v2 biological function, as well as mutants with altered biological function and/or specificity.
In accordance with the structural coordinates provided in Table V and the three dimensional homology model of CAN-12v2, the CAN-12v2 polypeptide has been shown to comprise a an active site region embodied by the following amino acids: from about amino acid R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E252 to about amino acid Y256, from about amino acid N278 to about amino acid K282, from about amino acid V328 to about amino acid V331 of SEQ ID NO:56 (FIGS. 8A-C). In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids more in either the N— or C-terminal direction of the above referenced amino acids.
Also more preferred are polypeptides comprising all or any part of the CAN-12v2 active site domain, or a mutant or homologue of said polypeptide or molecular complex. By mutant or homologue of the molecule is meant a molecule that has a root mean square deviation from the backbone atoms of said CAN-12v2 amino acids of not more than about 4.5 Angstroms, and preferably not more than about 3.5 Angstroms.
In preferred embodiments, the following CAN-12v2 active site domain polypeptide is encompassed by the present invention: RLDLCQGIVGDCWFLAALQALALHQDILSRVVPLNQSFTEKYAGIFRFWFWHYGNW VPVVIDDRLPVNEAGQLVFVSSTYKNLFWGALLEKAYAKLSGSYEDLQSGQVSEAL VDFTGGVTMTINLAEAHGNLWDILEATYNRTLIGCQTHSGEKILENGLVEGHAYTLT GIRKVTCKHRPEYLVKLRNPWGKVEWKGDWSDSSSKWELLSPKEKILLLRKDNDGE FWMTLQDFKTHFVLLV (SEQ ID NO:93). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of the CAN-12v2 active site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The present invention also encompasses polypeptides comprising at least a portion of the CAN-12v2 active site domain (SEQ ID NO: 93). Such polypeptides may correspond, for example, to the N— and/or C-terminal deletions of the active site domain.
In preferred embodiments, the following N-terminal CAN-12v2 active site domain deletion polypeptides are encompassed by the present invention: R1-V242, L2-V242, D3-V242, L4-V242, C5-V242, Q6-V242, G7-V242, I8-V242, V9-V242, G10-V242, C12-V242, W13-V242, F14-V242, L15-V242, A16-V242, A17-V242, L18-V242, Q19-V242, A20-V242, L21-V242, A22-V242, L23-V242, H24-V242, Q25-V242, D26-V242, I27-V242, L28-V242, S29-V242, R30-V242, V31-V242, V32-V242, P33-V242, L34-V242, N35-V242, Q36-V242, S37-V242, F38-V242, T39-V242, E40-V242, K41-V242, Y42-V242, A43-V242, G44-V242, I45-V242, F46-V242, R47-V242, F48-V242, W49-V242, F50-V242, W51-V242, H52-V242, Y53-V242, G54-V242, N55-V242, W56-V242, V57-V242, P58-V242, V59-V242, V60-V242, I61-V242, D62-V242, D63-V242, R64-V242, L65-V242, P66-V242, V67-V242, N68-V242, E69-V242, A70-V242, G71-V242, Q72-V242, L73-V242, V74-V242, F75-V242, V76-V242, S77-V242, S78-V242, T79-V242, Y80-V242, K81-V242, N82-V242, L83-V242, F84-V242, W85-V242, G86-V242, A87-V242, L88-V242, L89-V242, E90-V242, K91-V242, A92-V242, Y93-V242, A94-V242, K95-V242, L96-V242, S97-V242, G98-V242, S99-V242, Y100-V242, E101-V242, D102-V242, L103-V242, Q104-V242, S105-V242, G106-V242, Q107-V242, V108-V242, S109-V242, E110-V242, A111-V242, L112-V242, V113-V242, D114-V242, F115-V242, T116-V242, G117-V242, G118-V242, V119-V242, T120-V242, M121-V242, T122-V242, I123-V242, N124-V242, L125-V242, A126-V242, E127-V242, A128-V242, H129-V242, G130-V242, N131-V242, L132-V242, W133-V242, D134-V242, I135-V242, L136-V242, I137-V242, E138-V242, A139-V242, T140-V242, Y141-V242, N142-V242, R143-V242, T144-V242, L145-V242, I146-V242, G147-V242, C148-V242, Q149-V242, T150-V242, H151-V242, S152-V242, G153-V242, E154-V242, K155-V242, I156-V242, L157-V242, E158-V242, N159-V242, G160-V242, L161-V242, V162-V242, E163-V242, G164-V242, H165-V242, A166-V242, Y167-V242, T168-V242, L169-V242, T170-V242, G171-V242, I172-V242, R173-V242, K174-V242, V175-V242, T176-V242, C177-V242, K178-V242, H179-V242, R180-V242, P181-V242, E182-V242, Y183-V242, L184-V242, V185-V242, K186-V242, L187-V242, R188-V242, N189-V242, P190-V242, W191-V242, G192-V242, K193-V242, V194-V242, E195-V242, W196-V242, K197-V242, G198-V242, D199-V242, W200-V242, S201-V242, D202-V242, S203-V242, S204-V242, S205-V242, K206-V242, W207-V242, E208-V242, L209-V242, L210-V242, S211-V242, P212-V242, K213-V242, E214-V242, K215-V242, I216-V242, L217-V242, L218-V242, L219-V242, R220-V242, K221-V242, D222-V242, N223-V242, D224-V242, G225-V242, E226-V242, F227-V242, W228-V242, M229-V242, T230-V242, L231-V242, Q232-V242, D233-V242, F234-V242, K235-V242, and/or T236-V242 of SEQ ID NO:93. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal CAN-12v2 active site domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal CAN-12v2 active site domain deletion polypeptides are encompassed by the present invention: R1-V242, R1-L241, R1-L240, R1-V239, R1-F238, R1-H237, R1-T236, R1-K235, R1-F234, R1-D233, R1-Q232, R1-L231, R1-T230, R1-M229, R1-W228, R1-F227, R1-E226, R1-G225, R1-D224, R1-N223, R1-D222, R1-K221, R1-R220, R1-L219, R1-L218, R1-L217, R1-I216, R1-K215, R1-E214, R1-K213, R1-P212, R1-S211, R1-L210, R1-L209, R1-E208, R1-W207, R1-K206, R1-S205, R1-S204, R1-S203, R1-D202, R1-S201, R1-W200, R1-D199, R1-G198, R1-K197, R1-W196, R1-E195, R1-V194, R1-K193, R1-G192, R1-W191, R1-P190, R1-N189, R1-R188, R1-L187, R1-K186, R1-V185, R1-L184, R1-Y183, R1-E182, R1-P181, R1-R180, R1-H179, R1-K178, R1-C177, R1-T176, R1-V175, R1-K174, R1-R173, R1-I172, R1-G171, R1-T170, R1-L169, R1-T168, R1-Y167, R1-A166, R1-H165, R1-G164, R1-E163, R1-V162, R1-L161, R1-G160, R1-N159, R1-E158, R1-L157, R1-I156, R1-K155, R1-E154, R1-G153, R1-S152, R1-H151, R1-T150, R1-Q149, R1-C148, R1-G147, R1-I146, R1-L145, R1-T144, R1-R143, R1-N142, R1-Y141, R1-T140, R1-A139, R1-E138, R1-I137, R1-L136, R1-I135, R1-D134, R1-W133, R1-L132, R1-N131, R1-G130, R1-H129, R1-A128, R1-E127, R1-A126, R1-L125, R1-N124, R1-I123, R1-T122, R1-M121, R1-T120, R1-V119, R1-G118, R1-G117, R1-T116, R1-F115, R1-D114, R1-V113, R1-L112, R1-A111, R1-E110, R1-S109, R1-V108, R1-Q107, R1-G106, R1-S105, R1-Q104, R1-L103, R1-D102, R1-E101, R1-Y100, R1-S99, R1-G98, R1-S97, R1-L96, R1-K95, R1-A94, R1-Y93, R1-A92, R1-K91, R1-E90, R1-L89, R1-L88, R1-A87, R1-G86, R1-W85, R1-F84, R1-L83, R1-N82, R1-K81, R1-Y80, R1-T79, R1-S78, R1-S77, R1-V76, R1-F75, R1-V74, R1-L73, R1-Q72, R1-G71, R1-A70, R1-E69, R1-N68, R1-V67, R1-P66, R1-L65, R1-R64, R1-D63, R1-D62, R1-I61, R1-V60, R1-V59, R1-P58, R1-V57, R1-W56, R1-N55, R1-G54, R1-Y53, R1-H52, R1-W51, R1-F50, R1-W49, R1-F48, R1-R47, R1-F46, R1-I45, R1-G44, R1-A43, R1-Y42, R1-K41, R1-E40, R1-T39, R1-F38, R1-S37, R1-Q36, R1-N35, R1-L34, R1-P33, R1-V32, R1-V31, R1-R30, R1-S29, R1-L28, R1-I27, R1-D26, R1-Q25, R1-H24, R1-L23, R1-A22, R1-L21, R1-A20, R1-Q19, R1-L18, R1-A17, R1-A16, R1-L15, R1-F14, R1-W13, R1-C12, R1-D11, R1-G10, R1-V9, R1-I8, and/or R1-G7 of SEQ ID NO:93. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal CAN-12v2 active site domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
Alternatively, such polypeptides may comprise polypeptide sequences corresponding, for example, to internal regions of the CAN-12v2 active site domain (e.g., any combination of both N— and C-terminal CAN-12v2 active site domain deletions) of SEQ ID NO:93. For example, internal regions could be defined by the equation NX to CX, where NX refers to any N-terminal amino acid position of the CAN-12v2 active site domain (SEQ ID NO:93), and where CX refers to any C-terminal amino acid position of the CAN-12v2 active site domain (SEQ ID NO:93). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.
In preferred embodiments, the following CAN-12v2 active site domain amino acid substitutions are encompassed by the present invention: wherein R90 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein L91 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D92 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L93 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein C94 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q95 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein G96 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I97 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V98 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein G99 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D100 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C101 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W102 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein F103 is substituted with either an A, C, D, E, G. H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L104 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A105 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A106 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L107 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Q108 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein A109 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, P, S, T, V, W, or Y; wherein L110 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A111 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L112 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein H113 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q114 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein D115 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I116 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L117 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein S118 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein R119 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein V120 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein V121 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein P122 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein L123 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein N124 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein Q125 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein S126 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein F127 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T128 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein E129 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K130 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y131 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein A132 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G133 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I134 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F135 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R136 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein F137 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W138 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein F139 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W140 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein H141 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y142 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein G143 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N144 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein W145 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein V146 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein P147 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein V148 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein V149 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein I150 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D151 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D152 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R153 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein L154 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein P155 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein V156 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein N157 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein E158 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A159 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G160 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q161 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein L162 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein V163 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein F164 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V165 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein S166 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein S167 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein T168 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein Y169 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein K170 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N171 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein L172 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein F173 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, P, S, T, V, W, or Y; wherein W174 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein G175 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A176 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L177 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L178 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein E179 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K180 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A181 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y182 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein A183 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K184 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L185 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein S186 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein G187 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S188 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein Y189 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein E190 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D191 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L192 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Q193 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein S194 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein G195 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q196 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein V197 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein S198 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein E199 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A200 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, P, S, T, V, W, or Y; wherein L201 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein V202 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein D203 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F204 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T205 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein G206 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G207 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V208 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein T209 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein W110 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein T211 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein I212 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N213 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein L214 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A215 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E216 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A217 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H218 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G219 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N220 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein L221 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein W222 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein D223 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I224 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L225 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein I226 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E227 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A228 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T229 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein Y230 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein N231 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein R232 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein T233 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L234 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein I235 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G236 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, P, S, T, V, W, or Y; wherein C237 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q238 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein T239 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein H240 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S241 is substituted with either an A, C, D, E, F, G, HI I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein G242 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E243 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K244 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I245 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L246 is substituted with either an A, C, D, E F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein E247 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N248 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein G249 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L250 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein V251 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein E252 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G253 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H254 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A255 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y256 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein T257 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L258 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein T259 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein G260 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I261 is substituted with either an A, C, D, E F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R262 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein K263 is substituted with either an A, C, D, F, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V264 is substituted with either an A, C, D, E F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein T265 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein C266 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K267 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H268 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, P, S, T, V, W, or Y,; wherein R269 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein P270 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein E271 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y272 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein L273 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein V274 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein K275 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L276 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein R277 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein N278 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein P279 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein W280 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein G281 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K282 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V283 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein E284 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W285 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein K286 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G287 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D288 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W289 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein S290 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein D291 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S292 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein S293 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein S294 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein K295 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W296 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein E297 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L298 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L299 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein S300 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein P301 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein K302 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E303 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K304 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, P, S, T, V, W, or Y; wherein I305 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L306 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L307 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L308 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein R309 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein K310 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D311 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N312 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein D313 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G314 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E315 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F316 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W317 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein M318 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein T319 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L320 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Q321 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein D322 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F323 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K324 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T325 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y, wherein H326 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F327 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V328 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein L329 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L330 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; and/or wherein V331 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y of SEQ ID NO:56, in addition to any combination thereof. The present invention also encompasses the use of these CAN-12v2 active site domain amino acid substituted polypeptides as imrnunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following CAN-12v2 active site domain conservative amino acid substitutions are encompassed by the present invention: wherein R90 is substituted with either a K, or H; wherein L91 is substituted with either an A, I, or V; wherein D92 is substituted with an E; wherein L93 is substituted with either an A, I, or V; wherein C94 is a C; wherein Q95 is substituted with a N; wherein G96 is substituted with either an A, M, S, or T; wherein I97 is substituted with either an A, V, or L; wherein V98 is substituted with either an A, I, or L; wherein G99 is substituted with either an A, M, S, or T; wherein D100 is substituted with an E; wherein C101 is a C; wherein W102 is either an F, or Y; wherein F103 is substituted with either a W, or Y; wherein L104 is substituted with either an A, I, or V; wherein A105 is substituted with either a G, I, L, M, S, T, or V; wherein A106 is substituted with either a G, I, L, M, S, T, or V; wherein L107 is substituted with either an A, I, or V; wherein Q108 is substituted with a N; wherein A109 is substituted with either a G, I, L, M, S, T, or V; wherein L110 is substituted with either an A, I, or V; wherein A111 is substituted with either a G, I, L, M, S, T, or V; wherein L112 is substituted with either an A, I, or V; wherein H113 is substituted with either a K, or R; wherein Q114 is substituted with a N; wherein D115 is substituted with an E; wherein I116 is substituted with either an A, V, or L; wherein L117 is substituted with either an A, I, or V; wherein S118 is substituted with either an A, G, M, or T; wherein R119 is substituted with either a K, or H; wherein V120 is substituted with either an A, I, or L; wherein V121 is substituted with either an A, I, or L; wherein P122 is a P; wherein L123 is substituted with either an A, I, or V; wherein N124 is substituted with a Q; wherein Q125 is substituted with a N; wherein S126 is substituted with either an A, G, M, or T; wherein F127 is substituted with either a W, or Y; wherein T128 is substituted with either an A, G, M, or S; wherein E129 is substituted with a D; wherein K130 is substituted with either a R, or H; wherein Y131 is either an F, or W; wherein A132 is substituted with either a G, I, L, M, S, T, or V; wherein G133 is substituted with either an A, M, S, or T; wherein I134 is substituted with either an A, V, or L; wherein F135 is substituted with either a W, or Y; wherein R136 is substituted with either a K, or H; wherein F137 is substituted with either a W, or Y; wherein W138 is either an F, or Y; wherein F139 is substituted with either a W, or Y; wherein W140 is either an F, or Y; wherein H141 is substituted with either a K, or R; wherein Y142 is either an F, or W; wherein G143 is substituted with either an A, M, S, or T; wherein N144 is substituted with a Q; wherein W145 is either an F, or Y; wherein V146 is substituted with either an A, I, or L; wherein P147 is a P; wherein V148 is substituted with either an A, I, or L; wherein V149 is substituted with either an A, I, or L; wherein I150 is substituted with either an A, V, or L; wherein D151 is substituted with an E; wherein D152 is substituted with an E; wherein R153 is substituted with either a K, or H; wherein L154 is substituted with either an A, I, or V; wherein P155 is a P; wherein V156 is substituted with either an A, I, or L; wherein N157 is substituted with a Q; wherein E158 is substituted with a D; wherein A159 is substituted with either a G, I, L, M, S, T, or V; wherein G160 is substituted with either an A, M, S, or T; wherein Q161 is substituted with a N; wherein L162 is substituted with either an A, I, or V; wherein V163 is substituted with either an A, I, or L; wherein F164 is substituted with either a W, or Y; wherein V165 is substituted with either an A, I, or L; wherein S166 is substituted with either an A, G, M, or T; wherein S167 is substituted with either an A, G, M, or T; wherein T168 is substituted with either an A, G, M, or S; wherein Y169 is either an F, or W; wherein K170 is substituted with either a R, or H; wherein N171 is substituted with a Q; wherein L172 is substituted with either an A, I, or V; wherein F173 is substituted with either a W, or Y; wherein W174 is either an F, or Y; wherein G175 is substituted with either an A, M, S, or T; wherein A176 is substituted with either a G, I, L, M, S, T, or V; wherein L177 is substituted with either an A, I, or V; wherein L178 is substituted with either an A, I, or V; wherein E179 is substituted with a D; wherein K180 is substituted with either a R, or H; wherein A181 is substituted with either a G, I, L, M, S, T, or V; wherein Y182 is either an F, or W; wherein A183 is substituted with either a G, I, L, M, S, T, or V; wherein K184 is substituted with either a R, or H; wherein L185 is substituted with either an A, I, or V; wherein S186 is substituted with either an A, G, M, or T; wherein G187 is substituted with either an A, M, S, or T; wherein S188 is substituted with either an A, G, M, or T; wherein Y189 is either an F, or W; wherein E190 is substituted with a D; wherein D191 is substituted with an E; wherein L192 is substituted with either an A, I, or V; wherein Q193 is substituted with a N; wherein S194 is substituted with either an A, G, M, or T; wherein G195 is substituted with either an A, M, S, or T; wherein Q196 is substituted with a N; wherein V197 is substituted with either an A, I, or L; wherein S198 is substituted with either an A, G, M, or T; wherein E199 is substituted with a D; wherein A200 is substituted with either a G, I, L, M, S, T, or V; wherein L201 is substituted with either an A, I, or V; wherein V202 is substituted with either an A, I, or L; wherein D203 is substituted with an E; wherein F204 is substituted with either a W, or Y; wherein T205 is substituted with either an A, G, M, or S; wherein G206 is substituted with either an A, M, S, or T; wherein G207 is substituted with either an A, M, S, or T; wherein V208 is substituted with either an A, I, or L; wherein T209 is substituted with either an A, G, M, or S; wherein M210 is substituted with either an A, G, S, or T; wherein T211 is substituted with either an A, G, M, or S; wherein I212 is substituted with either an A, V, or L; wherein N213 is substituted with a Q; wherein L214 is substituted with either an A, I, or V; wherein A215 is substituted with either a G, I, L, M, S, T, or V; wherein E216 is substituted with a D; wherein A217 is substituted with either a G, I, L, M, S, T, or V; wherein H218 is substituted with either a K, or R; wherein G219 is substituted with either an A, M, S, or T; wherein N220 is substituted with a Q; wherein L221 is substituted with either an A, I, or V; wherein W222 is either an F, or Y; wherein D223 is substituted with an E; wherein I224 is substituted with either an A, V, or L; wherein L225 is substituted with either an A, I, or V; wherein I226 is substituted with either an A, V, or L; wherein E227 is substituted with a D; wherein A228 is substituted with either a G, I, L, M, S, T, or V; wherein T229 is substituted with either an A, G, M, or S; wherein Y230 is either an F, or W; wherein N231 is substituted with a Q; wherein R232 is substituted with either a K, or H; wherein T233 is substituted with either an A, G, M, or S; wherein L234 is substituted with either an A, I, or V; wherein I235 is substituted with either an A, V, or L; wherein G236 is substituted with either an A, M, S, or T; wherein C237 is a C; wherein Q238 is substituted with a N; wherein T239 is substituted with either an A, G, M, or S; wherein H240 is substituted with either a K, or R; wherein S241 is substituted with either an A, G, M, or T; wherein G242 is substituted with either an A, M, S, or T; wherein E243 is substituted with a D; wherein K244 is substituted with either a R, or H; wherein I245 is substituted with either an A, V, or L; wherein L246 is substituted with either an A, I, or V; wherein E247 is substituted with a D; wherein N248 is substituted with a Q; wherein G249 is substituted with either an A, M, S, or T; wherein L250 is substituted with either an A, I, or V; wherein V251 is substituted with either an A, I, or L; wherein E252 is substituted with a D; wherein G253 is substituted with either an A, M, S, or T; wherein H254 is substituted with either a K, or R; wherein A255 is substituted with either a G, I, L, M, S, T, or V; wherein Y256 is either an F, or W; wherein T257 is substituted with either an A, G, M, or S; wherein L258 is substituted with either an A, I, or V; wherein T259 is substituted with either an A, G, M, or S; wherein G260 is substituted with either an A, M, S, or T; wherein I261 is substituted with either an A, V, or L; wherein R262 is substituted with either a K, or H; wherein K263 is substituted with either a R, or H; wherein V264 is substituted with either an A, I, or L; wherein T265 is substituted with either an A, G, M, or S; wherein C266 is a C; wherein K267 is substituted with either a R, or H; wherein H268 is substituted with either a K, or R; wherein R269 is substituted with either a K, or H; wherein P270 is a P; wherein E271 is substituted with a D; wherein Y272 is either an F, or W; wherein L273 is substituted with either an A, I, or V; wherein V274 is substituted with either an A, I, or L; wherein K275 is substituted with either a R, or H; wherein L276 is substituted with either an A, I, or V; wherein R277 is substituted with either a K, or H; wherein N278 is substituted with a Q; wherein P279 is a P; wherein W280 is either an F, or Y; wherein G281 is substituted with either an A, M, S, or T; wherein K282 is substituted with either a R, or H; wherein V283 is substituted with either an A, I, or L; wherein E284 is substituted with a D; wherein W285 is either an F, or Y; wherein K286 is substituted with either a R, or H; wherein G287 is substituted with either an A, M, S, or T; wherein D288 is substituted with an E; wherein W289 is either an F, or Y; wherein S290 is substituted with either an A, G, M, or T; wherein D291 is substituted with an E; wherein S292 is substituted with either an A, G, M, or T; wherein S293 is substituted with either an A, G, M, or T; wherein S294 is substituted with either an A, G, M, or T; wherein K295 is substituted with either a R, or H; wherein W296 is either an F, or Y; wherein E297 is substituted with a D; wherein L298 is substituted with either an A, I, or V; wherein L299 is substituted with either an A, I, or V; wherein S300 is substituted with either an A, G, M, or T; wherein P301 is a P; wherein K302 is substituted with either a R, or H; wherein E303 is substituted with a D; wherein K304 is substituted with either a R, or H; wherein I305 is substituted with either an A, V, or L; wherein L306 is substituted with either an A, I, or V; wherein L307 is substituted with either an A, I, or V; wherein L308 is substituted with either an A, I, or V; wherein R309 is substituted with either a K, or H; wherein K310 is substituted with either a R, or H; wherein D311 is substituted with an E; wherein N312 is substituted with a Q; wherein D313 is substituted with an E; wherein G314 is substituted with either an A, M, S, or T; wherein E315 is substituted with a D; wherein F316 is substituted with either a W, or Y; wherein W317 is either an F, or Y; wherein M318 is substituted with either an A, G, S, or T; wherein T319 is substituted with either an A, G, M, or S; wherein L320 is substituted with either an A, I, or V; wherein Q321 is substituted with a N; wherein D322 is substituted with an E; wherein F323 is substituted with either a W, or Y; wherein K324 is substituted with either a R, or H; wherein T325 is substituted with either an A, G, M, or S; wherein H326 is substituted with either a K, or R; wherein F327 is substituted with either a W, or Y; wherein V328 is substituted with either an A, I, or L; wherein L329 is substituted with either an A, I, or V; wherein L330 is substituted with either an A, I, or V; and/or wherein V331 is substituted with either an A, I, or L of SEQ ID NO:56 in addition to any combination thereof. Other suitable substitutions within the CAN-12v2 active site domain are encompassed by the present invention and are referenced elsewhere herein. The present invention also encompasses the use of these CAN-12v2 active site domain conservative amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
For purposes of the present invention, by “at least a portion of” is meant all or any part of the CAN-12v2 active site domain defined by the structure coordinates according to Table V (e.g., fragments thereof). More preferred are molecules comprising all or any parts of the CAN-12v2 active site domain defined by the structure coordinates according to Table V, or a mutant or homologue of said molecule or molecular complex. By mutant or homologue of the molecule it is meant a molecule that has a root mean square deviation from the backbone atoms of said CAN-12v2 amino acids of not more than 4.5 Angstroms, and preferably not more than 3.5 Angstroms.
The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a term that expresses the deviation or variation from a trend or object. For the purposes of the present invention, the “root mean square deviation” defines the variation in the backbone of a protein from the relevant portion of the backbone of the AR portion of the complex as defined by the structure coordinates described herein.
A preferred embodiment is a machine-readable data storage medium that is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex that is defined by the structure coordinates of all of the amino acids in Table V±a root mean square deviation from the backbone atoms of those amino acids of not more than 4.0 ANG, preferably 3.0 ANG.
The structure coordinates of a CAN-12v2 homology model, including portions thereof, is stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery.
Accordingly, in one embodiment of this invention is provided a machine-readable data storage medium comprising a data storage material encoded with the structure coordinates set forth in Table V.
One embodiment utilizes System 10 as disclosed in WO 98/11134, the disclosure of which is incorporated herein by reference in its entirety. Briefly, one version of these embodiments comprises a computer comprising a central processing unit (“CPU”), a working memory which may be, e.g, RAM (random-access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more cathode-ray tube (“CRT”) display terminals, one or more keyboards, one or more input lines, and one or more output lines, all of which are interconnected by a conventional bidirectional system bus.
Input hardware, coupled to the computer by input lines, may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line. Alternatively or additionally, the input hardware may comprise CD-ROM drives or disk drives. In conjunction with a display terminal, keyboard may also be used as an input device.
Output hardware, coupled to the computer by output lines, may similarly be implemented by conventional devices. By way of example, output hardware may include a CRT display terminal for displaying a graphical representation of a region or domain of the present invention using a program such as QUANTA as described herein. Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use.
In operation, the CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage, and accesses to and from the working memory, and determines the sequence of data processing steps. A number of programs may be used to process the machine-readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein. Specific references to components of the hardware system are included as appropriate throughout the following description of the data storage medium.
For the purpose of the present invention, any magnetic data storage medium which can be encoded with machine-readable data would be sufficient for carrying out the storage requirements of the system. The medium could be a conventional floppy diskette or hard disk, having a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, on one or both sides, containing magnetic domains whose polarity or orientation could be altered magnetically, for example. The medium may also have an opening for receiving the spindle of a disk drive or other data storage device.
The magnetic domains of the coating of a medium may be polarized or oriented so as to encode in a manner which may be conventional, machine readable data such as that described herein, for execution by a system such as the system described herein.
Another example of a suitable storage medium which could also be encoded with such machine-readable data, or set of instructions, which could be carried out by a system such as the system described herein, could be an optically-readable data storage medium. The medium could be a conventional compact disk read only memory (CD-ROM) or a rewritable medium such as a magneto-optical disk which is optically readable and magneto-optically writable. The medium preferably has a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, usually of one side of substrate.
In the case of a CD-ROM, as is well known, the coating is reflective and is impressed with a plurality of pits to encode the machine-readable data. The arrangement of pits is read by reflecting laser light off the surface of the coating. A protective coating, which preferably is substantially transparent, is provided on top of the reflective coating.
In the case of a magneto-optical disk, as is well known, the coating has no pits, but has a plurality of magnetic domains whose polarity or orientation can be changed magnetically when heated above a certain temperature, as by a laser. The orientation of the domains can be read by measuring the polarization of laser light reflected from the coating. The arrangement of the domains encodes the data as described above.
Thus, in accordance with the present invention, data capable of displaying the three dimensional structure of the CAN-12v2 homology model, or portions thereof and their structurally similar homologues is stored in a machine-readable storage medium, which is capable of displaying a graphical three-dimensional representation of the structure. Such data may be used for a variety of purposes, such as drug discovery.
For the first time, the present invention permits the use of structure-based or rational drug design techniques to design, select, and synthesize chemical entities that are capable of modulating the biological function of CAN-12v2.
Accordingly, the present invention is also directed to the design of small molecules which imitates the structure of the CAN-12v2 active site domain (SEQ ID NO:93), or a portion thereof defined by the structure provided in Table V. Alternatively, the present invention is directed to the design of small molecules which may bind to at least part of the CAN-12v2 active site domain (SEQ ID NO:93), or some portion thereof. For purposes of this invention, by CAN-12v2 active site domain, it is also meant to include mutants or homologues thereof. In a preferred embodiment, the mutants or homologues have at least 25% identity, more preferably 50% identity, more preferably 75% identity, and most preferably 90% identity to SEQ ID NO:93. In this context, the term “small molecule” may be construed to mean any molecule described known in the art or described elsewhere herein, though may include, for example, peptides, chemicals, carbohydrates, nucleic acids, PNAs, and any derivatives thereof.
The three-dimensional model structure of CAN-12v2 will also provide methods for identifying modulators of biological function. Various methods or combination thereof can be used to identify these compounds.
For example, test compounds can be modeled that fit spatially into the active site domain in CAN-12v2 embodied by the sequence from about R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E252 to about amino acid Y256, from about amino acid N278 to about amino acid K282, from about amino acid V328 to about amino acid V331, or some portion thereof, of SEQ ID NO:56 (corresponding to SEQ ID NO:93), in accordance with the structural coordinates of Table V.
Structure coordinates of the active site domain in CAN-12v2 defined by the amino acids from about R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E252 to about amino acid Y256, from about amino acid N278 to about amino acid K282, from about amino acid V328 to about amino acid V331 of SEQ ID NO:56, can also be used to identify structural and chemical features. Identified structural or chemical features can then be employed to design or select compounds as potential CAN-12v2 modulators. By structural and chemical features it is meant to include, but is not limited to, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interaction. Alternatively, or in conjunction with, the three-dimensional structural model can be employed to design or select compounds as potential CAN-12v2 modulators. Compounds identified as potential CAN-12v2 modulators can then be synthesized and screened in an assay characterized by binding of a test compound to the CAN-12v2, or in characterizing the ability of CAN-12v2 to modulate a protease target in the presence of a small molecule. Examples of assays useful in screening of potential CAN-12v2 modulators include, but are not limited to, screening in silico, in vitro assays and high throughput assays. Finally, these methods may also involve modifying or replacing one or more amino acids at amino acid positions, C101, H254, and/or N278 of SEQ ID NO:56 in accordance with the structure coordinates of Table V.
However, as will be understood by those of skill in the art upon this disclosure, other structure based design methods can be used. Various computational structure based design methods have been disclosed in the art.
For example, a number of computer modeling systems are available in which the sequence of the CAN-12v2 and the CAN-12v2 structure (i.e., atomic coordinates of CAN-12v2 and/or the atomic coordinates of the active site domain as provided in Table V, can be input. This computer system then generates the structural details of one or more these regions in which a potential CAN-12v2 modulator binds so that complementary structural details of the potential modulators can be determined. Design in these modeling systems is generally based upon the compound being capable of physically and structurally associating with CAN-12v2. In addition, the compound must be able to assume a conformation that allows it to associate with CAN-12v2. Some modeling systems estimate the potential inhibitory or binding effect of a potential CAN-12v2 modulator prior to actual synthesis and testing.
Methods for screening chemical entities or fragments for their ability to associate with a given protein target are also well known. Often these methods begin by visual inspection of the binding site on the computer screen. Selected fragments or chemical entities are then positioned in the active site domain of CAN-12v2. Docking is accomplished using software such as INSIGHTII, QUANTA and SYBYL, following by energy minimization and molecular dynamics with standard molecular mechanic forcefields such as CHARMM and AMBER. Examples of computer programs which assist in the selection of chemical fragment or chemical entities useful in the present invention include, but are not limited to, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntz et al. 1982).
Upon selection of preferred chemical entities or fragments, their relationship to each other and CAN-12v2 can be visualized and then assembled into a single potential modulator. Programs useful in assembling the individual chemical entities include, but are not limited to CAVEAT (Bartlett et al. 1989) and 3D Database systems (Martin1992).
Alternatively, compounds may be designed de novo using either an empty active site or optionally including some portion of a known inhibitor. Methods of this type of design include, but are not limited to LUDI (Bohm 1992) and LeapFrog (Tripos Associates, St. Louis Mo.).
In addition, CAN-12v2 is overall well suited to modern methods including combinatorial chemistry.
Programs such as DOCK (Kuntz et al. 1982) can be used with the atomic coordinates from the homology model to identify potential ligands from databases or virtual databases which potentially bind CAN-12v2 active site domain, and which may therefore be suitable candidates for synthesis and testing.
Additionally, the three-dimensional homology model of CAN-12v2 will aid in the design of mutants with altered biological activity for the CAN-12v2 polypeptide.
The following are encompassed by the present invention: a machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the structure coordinates of the model CAN-12v2 according to Table V or a homologue of said model, wherein said homologue comprises backbone atoms that have a root mean square deviation from the backbone atoms of the complex of not more than 4.5 Å, preferably not more than 4.0 Å, most preferably not more than 3.5 Å, and even more preferably not more than 3.0 Å; and a machine-readable data storage medium, wherein said molecule is defined by the set of structure coordinates of the model for CAN-12v2 according to Table V, or a homologue of said molecule, said homologue having a root mean square deviation from the backbone atoms of said amino acids of not more than 4.5 Å, preferably not more than 4.0 Å, most preferably not more than 3.5 Å, and even more preferably not more than 3.0 Å; a model comprising all or any part of the model defined by structure coordinates of CAN-12v2 according to Table V, or a mutant or homologue of said molecule or molecular complex.
In a further embodiment, the following are encompassed by the present invention: a method for identifying a mutant of CAN-12v2 with altered biological properties, function, or reactivity, the method comprising any combination of steps of: use of the model or a homologue of said model according to Table V, for the design of protein mutants with altered biological function or properties which exhibit any combination of therapeutic effects provided elsewhere herein; and use of the model or a homologue of said model, for the design of a protein with mutations in the active site domain comprised of the amino acids from about R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E252 to about amino acid Y256, from about amino acid N278 to about amino acid K282, from about amino acid V328 to about amino acid V331 of SEQ ID NO:56 according to Table V with altered biological function or properties which exhibit any combination of therapeutic effects provided elsewhere herein.
In further preferred embodiments, the following are encompassed by the present invention: a method for identifying modulators of CAN-12v2 biological properties, function, or reactivity, the method comprising any combination of steps of: modeling test compounds that overlay spatially into the active site domain defined by all or any portion of residues from about R90 to about amino acid C94, from about amino acid G99 to about amino acid L104, from about amino acid Q196 to about amino acid S198, from about amino acid M210 to about amino acid I212, from about amino acid G236 to about amino acid H240, from about amino acid E252 to about amino acid Y256, from about amino acid N278 to about amino acid K282, from about amino acid V328 to about amino acid V331 of SEQ ID NO:56 according to Table V, or using a homologue or portion thereof.
The present invention encompasses using the structure coordinates as set forth herein to identify structural and chemical features of the CAN-12v2 polypeptide; employing identified structural or chemical features to design or select compounds as potential CAN-12v2 modulators; employing the three-dimensional structural model to design or select compounds as potential CAN-12v2 modulators; synthesizing the potential CAN-12v2 modulators; screening the potential CAN-12v2 modulators in an assay characterized by binding of a protein to the CAN-12v2; selecting the potential CAN-12v2 modulator from a database; designing the CAN-12v2 modulator de novo; and/or designing said CAN-12v2 modulator from a known modulator activity.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 55 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2090 of SEQ ID NO:55, b is an integer between 15 to 2104, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:55, and where b is greater than or equal to a+14.

In one embodiment, a CAN12.v2 polypeptide comprises a portion of the amino sequence depicted in FIGS. 9A-C. In another embodiment, a CAN12.v2 polypeptide comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of the amino sequence depicted in FIGS. 9A-C. In further embodiments, the CAN12.v2 polypeptide does not consist of the sequence ALLEKAYAKL (SEQ ID NO:141), and/or ALLEKAYAKLSGSYE. (SEQ ID NO:142)

TABLE I


		ATCC		NT	Total	5′ NT	3′	AA	Total
		Deposit		SEQ	NT	of Start	NT	Seq	AA
Gene	CDNA	No. Z		ID.	Seq of	Codon	of	ID	of
No.	CloneID	and Date	Vector	No. X	Clone	of ORF	ORF	No. Y	ORF

1.	CAN-12	XXXXX	pSport 1	1 and	4584	114	1397	24	428
	(protease	Xx/Xx/Xx		23
	5, clone
	70)
1.	CAN-12	XXXXX	pSport 1	1	4584	114	1995	2	581
	(protease	Xx/Xx/Xx
	5, clone
	70, CAN-
	12+) + splice
	amino
	acids

2.	CAN-	PTA-	pSport 1	53	2095	9	2090	54	694
	12v1	3434
	(protease	06/07/01
	5, clone
	1e)
3.	CAN-	PTA-	pSport 1	55	2104	9	2099	56	697
	12v2	3434
	(protease	06/07/01
	5, clone
	1e1b-1)

Table I summarizes the information corresponding to each “Gene No.” described above. The nucleotide sequence identified as “NT SEQ ID NO:X” was assembled from partially homologous (“overlapping”) sequences obtained from the “cDNA clone ID” identified in Table I and, in some cases, from additional related DNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually several overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:X.
The cDNA Clone ID was deposited on the date and given the corresponding deposit number listed in “ATCC Deposit No:Z and Date.” “Vector” refers to the type of vector contained in the cDNA Clone ID.
“Total NT Seq. Of Clone” refers to the total number of nucleotides in the clone contig identified by “Gene No.” The deposited clone may contain all or most of the sequence of SEQ ID NO:X. The nucleotide position of SEQ ID NO:X of the putative start codon (methionine) is identified as “5′ NT of Start Codon of ORF.”
The translated amino acid sequence, beginning with the methionine, is identified as “AA SEQ ID NO:Y,” although other reading frames can also be easily translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.
The total number of amino acids within the open reading frame of SEQ ID NO:Y is identified as “Total AA of ORF”.
SEQ ID NO:X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further herein. For instance, SEQ ID NO:X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the proteins encoded by the cDNA clones identified in Table I.
Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides may cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:1, 23, 53, and/or 55 and the predicted translated amino acid sequence identified as SEQ ID NO:2, 24, 54, and/or 56, but also a sample of plasmid DNA containing a cDNA of the invention deposited with the ATCC, as set forth in Table I. The nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited cDNA, collecting the protein, and determining its sequence.
The present invention also relates to the genes corresponding to SEQ ID NO:1, 23, 53, and/or 55, SEQ ID NO:2, 24, 54, and/or 56, or the deposited clone. The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
Also provided in the present invention are species homologs, allelic variants, and/or orthologs. The skilled artisan could, using procedures well-known in the art, obtain the polynucleotide sequence corresponding to full-length genes (including, but not limited to the full-length coding region), allelic variants, splice variants, orthologs, and/or species homologues of genes corresponding to SEQ ID NO:1, 23, 53, and/or 55, SEQ ID NO:2, 24, 54, and/or 56, or a deposited clone, relying on the sequence from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologues may be isolated and identified by making suitable probes or primers which correspond to the 5′, 3′, or internal regions of the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
The polypeptides may be in the form of the protein, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.
The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:3140 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using protocols described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the full-length form of the protein.
The present invention provides a polynucleotide comprising, or alternatively consisting of, the sequence identified as SEQ ID NO:1, 23, 53, and/or 55, and/or a cDNA provided in ATCC Deposit No. Z:. The present invention also provides a polypeptide comprising, or alternatively consisting of, the sequence identified as SEQ ID NO:2, 24, 54, and/or 56, and/or a polypeptide encoded by the cDNA provided in ATCC Deposit NO:Z. The present invention also provides polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:2, 24, 54, and/or 56, and/or a polypeptide sequence encoded by the cDNA contained in ATCC Deposit No:Z.
Preferably, the present invention is directed to a polynucleotide comprising, or alternatively consisting of, the sequence identified as SEQ ID NO:1, 23, 53, and/or 55, and/or a cDNA provided in ATCC Deposit No.: that is less than, or equal to, a polynucleotide sequence that is 5 mega basepairs, 1 mega basepairs, 0.5 mega basepairs, 0.1 mega basepairs, 50,000 basepairs, 20,000 basepairs, or 10,000 basepairs in length.
The present invention encompasses polynucleotides with sequences complementary to those of the polynucleotides of the present invention disclosed herein. Such sequences may be complementary to the sequence disclosed as SEQ ID NO:1, 23, 53, and/or 55, the sequence contained in a deposit, and/or the nucleic acid sequence encoding the sequence disclosed as SEQ ID NO:2, 24, 54, and/or 56.

The present invention also encompasses polynucleotides capable of hybridizing, preferably under reduced stringency conditions, more preferably under stringent conditions, and most preferably under highly stringent conditions, to polynucleotides described herein. Examples of stringency conditions are shown in Table II below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.

TABLE II


			Hybridization	Wash
Stringency	Polynucleotide	Hybrid Length	Temperature and	Temperature
Condition	Hybrid±	(bp)‡	Buffer†	and Buffer†

A	DNA:DNA	> or equal to 50	65° C.; 1xSSC -or-	65° C.; 0.3xSSC
			42° C.; 1xSSC,
			50% formamide
B	DNA:DNA	<50	Tb*; 1xSSC	Tb*; 1xSSC
C	DNA:RNA	> or equal to 50	67° C.; 1xSSC -or-	67° C.; 0.3xSSC
			45° C.; 1xSSC,
			50% formamide
D	DNA:RNA	<50	Td*; 1xSSC	Td*; 1xSSC
E	RNA:RNA	> or equal to 50	70° C.; 1xSSC -or-	70° C.; 0.3xSSC
			50° C.; 1xSSC,
			50% formamide
F	RNA:RNA	<50	Tf*; 1xSSC	Tf*; 1xSSC
G	DNA:DNA	> or equal to 50	65° C.; 4xSSC -or-	65° C.; 1xSSC
			45° C.; 4xSSC,
			50% formamide
H	DNA:DNA	<50	Th*; 4xSSC	Th*; 4xSSC
I	DNA:RNA	> or equal to 50	67° C.; 4xSSC -or-	67° C.; 1xSSC
			45° C.; 4xSSC,
			50% formamide
J	DNA:RNA	<50	Tj*; 4xSSC	Tj*; 4xSSC
K	RNA:RNA	> or equal to 50	70° C.; 4xSSC -or-	67° C.; 1xSSC
			40° C.; 6xSSC,
			50% formamide
L	RNA:RNA	<50	Tl*; 2xSSC	Tl*; 2xSSC
M	DNA:DNA	> or equal to 50	50° C.; 4xSSC -or-	50° C.; 2xSSC
			40° C. 6xSSC, 50%
			formamide
N	DNA:DNA	<50	Tn*; 6xSSC	Tn*; 6xSSC
O	DNA:RNA	> or equal to 50	55° C.; 4xSSC -or-	55° C.; 2xSSC
			42° C.; 6xSSC,
			50% formamide
P	DNA:RNA	<50	Tp*; 6xSSC	Tp*; 6xSSC
Q	RNA:RNA	> or equal to 50	60° C.; 4xSSC -or-	60° C.; 2xSSC
			45° C.; 6xSSC,
			50% formamide
R	RNA:RNA	<50	Tr*; 4xSSC	Tr*; 4xSSC

‡The “hybrid length” is the anticipated length for the hybridized region(s) of the hybridizing polynucleotides. When hybridizing a polynucleotide of unknown sequence, the hybrid is assumed to be that of the hybridizing polynucleotide of the present invention. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal
# sequence complementarity. Methods of aligning two or more polynucleotide sequences and/or determining the percent identity between two polynucleotide sequences are well known in the art (e.g., MegAlign program of the DNA*Star suite of programs, etc).
†SSPE (1xSSPE is 0.15M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1xSSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete. The hydridizations and washes may additionally include 5X Denhardt's reagent, .5-1.0% SDS, 100 ug/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, and up to 50% formamide.
*Tb-Tr: The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature Tm of the hybrids there Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(° C.) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18
# and 49 base pairs in length, Tm(° C.) = 81.5 + 16.6(log₁₀[Na+]) + 0.41(% G + C) − (600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([NA+] for 1xSSC = .165 M).
±The present invention encompasses the substitution of any one, or more DNA or RNA hybrid partners with either a PNA, or a modified polynucleotide. Such modified polynucleotides are known in the art and are more particularly described elsewhere herein.

Additional examples of stringency conditions for polynucleotide hybridization are provided, for example, in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M., Ausubel et al., eds, John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4, which are hereby incorporated by reference herein.
Preferably, such hybridizing polynucleotides have at least 70% sequence identity (more preferably, at least 80% identity; and most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which they hybridize, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps. The determination of identity is well known in the art, and discussed more specifically elsewhere herein.
The invention encompasses the application of PCR methodology to the polynucleotide sequences of the present invention, the clone deposited with the ATCC, and/or the cDNA encoding the polypeptides of the present invention. PCR techniques for the amplification of nucleic acids are described in U.S. Pat. No. 4,683,195 and Saiki et al., Science, 239:487-491 (1988). PCR, for example, may include the following steps, of denaturation of template nucleic acid (if double-stranded), annealing of primer to target, and polymerization. The nucleic acid probed or used as a template in the amplification reaction may be genomic DNA, cDNA, RNA, or a PNA. PCR may be used to amplify specific sequences from genomic DNA, specific RNA sequence, and/or cDNA transcribed from mRNA. References for the general use of PCR techniques, including specific method parameters, include Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR Technology, Stockton Press, NY, 1989; Ehrlich et al., Science, 252:1643-1650, (1991); and “PCR Protocols, A Guide to Methods and Applications”, Eds., Innis et al., Academic Press, New York, (1990).
Signal Sequences
The present invention also encompasses mature forms of the polypeptide comprising, or alternatively consisting of, the polypeptide sequence of SEQ ID NO:2, 24, 54, and/or 56, the polypeptide encoded by the polynucleotide described as SEQ ID NO:1, 23, 53, and/or 55, and/or the polypeptide sequence encoded by a cDNA in the deposited clone. The present invention also encompasses polynucleotides encoding mature forms of the present invention, such as, for example the polynucleotide sequence of SEQ ID NO:1, 23, 53, and/or 55, and/or the polynucleotide sequence provided in a cDNA of the deposited clone.
According to the signal hypothesis, proteins secreted by eukaryotic cells have a signal or secretary leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most eukaryotic cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein. Further, it has long been known that cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.
Methods for predicting whether a protein has a signal sequence, as well as the cleavage point for that sequence, are available. For instance, the method of McGeoch, Virus Res. 3:271-286 (1985), uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein. The method of von Heinje, Nucleic Acids Res. 14:4683-4690 (1986) uses the information from the residues surrounding the cleavage site, typically residues −13 to +2, where +1 indicates the amino terminus of the secreted protein. The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-80%. (von Heinje, supra.) However, the two methods do not always produce the same predicted cleavage point(s) for a given protein.
The established method for identifying the location of signal sequences, in addition, to their cleavage sites has been the SignalP program (v1.1) developed by Henrik Nielsen et al., Protein Engineering 10:1-6 (1997). The program relies upon the algorithm developed by von Heinje, though provides additional parameters to increase the prediction accuracy.
More recently, a hidden Markov model has been developed (H. Neilson, et al., Ismb 1998;6:122-30), which has been incorporated into the more recent SignalP (v2.0). This new method increases the ability to identify the cleavage site by discriminating between signal peptides and uncleaved signal anchors. The present invention encompasses the application of the method disclosed therein to the prediction of the signal peptide location, including the cleavage site, to any of the polypeptide sequences of the present invention.
As one of ordinary skill would appreciate, however, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty. Accordingly, the polypeptide of the present invention may contain a signal sequence. Polypeptides of the invention which comprise a signal sequence have an N-terminus beginning within 5 residues (i.e., + or −35 residues, or preferably at the −5, −4, −3, −2, −1, +1, +2, +3, +4, or 30 5 residue) of the predicted cleavage point. Similarly, it is also recognized that in some cases, cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.
Moreover, the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence. For example, the naturally occurring signal sequence may be further upstream from the predicted signal sequence. However, it is likely that the predicted signal sequence will be capable of directing the secreted protein to the ER. Nonetheless, the present invention provides the mature protein produced by expression of the polynucleotide sequence of SEQ ID NO:1, 23, 53, and/or 55 and/or the polynucleotide sequence contained in the cDNA of a deposited clone, in a mammalian cell (e.g., COS cells, as described below). These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.
Polynucleotide and Polypeptide Variants
The present invention also encompasses variants (e.g., allelic variants, orthologs, etc.) of the polynucleotide sequence disclosed herein in SEQ ID NO:1, 23, 53, and/or 55, the complementary strand thereto, and/or the cDNA sequence contained in the deposited clone.
The present invention also encompasses variants of the polypeptide sequence, and/or fragments therein, disclosed in SEQ ID NO:2, 24, 54, and/or 56, a polypeptide encoded by the polynucleotide sequence in SEQ ID NO:1, 23, 53, and/or 55, and/or a polypeptide encoded by a cDNA in the deposited clone.
“Variant” refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a CAN-12 related polypeptide having an amino acid sequence as shown in the sequence listing and described in SEQ ID NO:1, 23, 53, and/or 55 or the cDNA contained in ATCC deposit No:PTA-3434 ; (b) a nucleotide sequence encoding a mature CAN-12 related polypeptide having the amino acid sequence as shown in the sequence listing and described in SEQ ID NO:1, 23, 53, and/or 55 or the cDNA contained in ATCC deposit No:PTA-3434 ; (c) a nucleotide sequence encoding a biologically active fragment of a CAN-12 related polypeptide having an amino acid sequence shown in the sequence listing and described in SEQ ID NO:1, 23, 53, and/or 55 or the cDNA contained in ATCC deposit No:PTA-3434; (d) a nucleotide sequence encoding an antigenic fragment of a CAN-12 related polypeptide having an amino acid sequence sown in the sequence listing and described in SEQ ID NO:1, 23, 53, and/or 55 or the cDNA contained in ATCC deposit No:PTA-3434; (e) a nucleotide sequence encoding a CAN-12 related polypeptide comprising the complete amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1, 23, 53, and/or 55 or the cDNA contained in ATCC deposit No:PTA-3434; (f) a nucleotide sequence encoding a mature CAN-12 related polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1, 23, 53, and/or 55 or the cDNA contained in ATCC deposit No:PTA-3434; (g) a nucleotide sequence encoding a biologically active fragment of a CAN-12 related polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1, 23, 53, and/or 55 or the cDNA contained in ATCC deposit No:PTA-3434; (h) a nucleotide sequence encoding an antigenic fragment of a CAN-12 related polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1, 23, 53, and/or 55 or the cDNA contained in ATCC deposit No:PTA-3434; (I) a nucleotide sequence complimentary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above.
The present invention is also directed to polynucleotide sequences which comprise, or alternatively consist of, a polynucleotide sequence which is at least about 80%, 85%, 90%, 91%, 92%, 92.4%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides encoded by these nucleic acid molecules are also encompassed by the invention. In another embodiment, the invention encompasses nucleic acid molecules which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polypeptides.
Another aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively, consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a CAN-12 related polypeptide having an amino acid sequence as shown in the sequence listing and descried in Table I; (b) a nucleotide sequence encoding a mature CAN-12 related polypeptide having the amino acid sequence as shown in the sequence listing and descried in Table I; (c) a nucleotide sequence encoding a biologically active fragment of a CAN-12 related polypeptide having an amino acid sequence as shown in the sequence listing and descried in Table I; (d) a nucleotide sequence encoding an antigenic fragment of a CAN-12 related polypeptide having an amino acid sequence as shown in the sequence listing and descried in Table I; (e) a nucleotide sequence encoding a CAN-12 related polypeptide comprising the complete amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC Deposit and described in Table I; (f) a nucleotide sequence encoding a mature CAN-12 related polypeptide having an amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC Deposit and described in Table I: (g) a nucleotide sequence encoding a biologically active fragment of a CAN-12 related polypeptide having an amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC Deposit and described in Table I; (h) a nucleotide sequence encoding an antigenic fragment of a CAN-12 related polypeptide having an amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC deposit and described in Table I; (i) a nucleotide sequence complimentary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h) above.
The present invention is also directed to nucleic acid molecules which comprise, or alternatively, consist of, a nucleotide sequence which is at least about 80%, 85%, 90%, 91%, 92%, 92.4%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above.
The present invention encompasses polypeptide sequences which comprise, or alternatively consist of, an amino acid sequence which is at least about 60.3%, 61.4%, 80%, 81.8%, 82.2%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, the following non-limited examples, the polypeptide sequence identified as SEQ ID NO:2, 24, 54, and/or 56, the polypeptide sequence encoded by a cDNA provided in the deposited clone, and/or polypeptide fragments of any of the polypeptides provided herein. Polynucleotides encoded by these nucleic acid molecules are also encompassed by the invention. In another embodiment, the invention encompasses nucleic acid molecules which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polypeptides.
The present invention is also directed to polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least about 60.3%, 61.4%, 80%, 81.8%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for example, the polypeptide sequence shown in SEQ ID NO:2, 24, 54, and/or 56, a polypeptide sequence encoded by the nucleotide sequence in SEQ ID NO:1, 23, 53, and/or 55, a polypeptide sequence encoded by the cDNA in cDNA plasmid:Z, and/or polypeptide fragments of any of these polypeptides (e.g., those fragments described herein). Polynucleotides which hybridize to the complement of the nucleic acid molecules encoding these polypeptides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompasses by the present invention, as are the polypeptides encoded by these polynucleotides.
By a nucleic acid having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence referenced in Table I, the ORF (open reading frame), or any fragment specified as described herein.
As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least about 60.3%, 61.4%, 80%, 81.8%, 80%, 85%, 90%, 91%, 92%, 92.4%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the CLUSTALW computer program (Thompson, J. D., et al., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based on the algorithm of Higgins, D. G., et al., Computer Applications in the Biosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. However, the CLUSTALW algorithm automatically converts U's to T's when comparing RNA sequences to DNA sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a CLUSTALW alignment of DNA sequences to calculate percent identity via pairwise alignments are: Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent, Window Size=5 or the length of the subject nucleotide sequence, whichever is shorter. For multiple alignments, the following CLUSTALW parameters are preferred: Gap Opening Penalty=10; Gap Extension Parameter=0.05; Gap Separation Penalty Range=8; End Gap Separation Penalty=Off; % Identity for Alignment Delay=40%; Residue Specific Gaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. The pairwise and multple alignment parameters provided for CLUSTALW above represent the default parameters as provided with the AlignX software program (Vector NTI suite of programs, version 6.0).
The present invention encompasses the application of a manual correction to the percent identity results, in the instance where the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions. If only the local pairwise percent identity is required, no manual correction is needed. However, a manual correction may be applied to determine the global percent identity from a global polynucleotide alignment. Percent identity calculations based upon global polynucleotide alignments are often preferred since they reflect the percent identity between the polynucleotide molecules as a whole (i.e., including any polynucleotide overhangs, not just overlapping regions), as opposed to, only local matching polynucleotides. Manual corrections for global percent identity determinations are required since the CLUSTALW program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the CLUSTALW sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above CLUSTALW program using the specified parameters, to arrive at a final percent identity score. This corrected score may be used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the CLUSTALW alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the CLUSTALW alignment does not show a matched/alignment of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the CLUSTALW program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by CLUSTALW is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are required for the purposes of the present invention.
By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least about 60.3%, 61.4%, 80%, 81.8%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for instance, an amino acid sequence referenced in Table 1 (SEQ ID NO:2) or to the amino acid sequence encoded by cDNA contained in a deposited clone, can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the CLUSTALW computer program (Thompson, J. D., et al., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based on the algorithm of Higgins, D. G., et al., Computer Applications in the Biosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment the query and subject sequences are both amino acid sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a CLUSTALW alignment of DNA sequences to calculate percent identity via pairwise alignments are: Matrix=BLOSUM, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent, Window Size=5 or the length of the subject nucleotide sequence, whichever is shorter. For multiple alignments, the following CLUSTALW parameters are preferred: Gap Opening Penalty=10; Gap Extension Parameter=0.05; Gap Separation Penalty Range=8; End Gap Separation Penalty=Off; % Identity for Alignment Delay=40%; Residue Specific Gaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. The pairwise and multple alignment parameters provided for CLUSTALW above represent the default parameters as provided with the AlignX software program (Vector NTI suite of programs, version 6.0).
The present invention encompasses the application of a manual correction to the percent identity results, in the instance where the subject sequence is shorter than the query sequence because of N— or C-terminal deletions, not because of internal deletions. If only the local pairwise percent identity is required, no manual correction is needed. However, a manual correction may be applied to determine the global percent identity from a global polypeptide alignment. Percent identity calculations based upon global polypeptide alignments are often preferred since they reflect the percent identity between the polypeptide molecules as a whole (i.e., including any polypeptide overhangs, not just overlapping regions), as opposed to, only local matching polypeptides. Manual corrections for global percent identity determinations are required since the CLUSTALW program does not account for N— and C-terminal truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the N— and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N— and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the CLUSTALW sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above CLUSTALW program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what may be used for the purposes of the present invention. Only residues to the N— and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N— and C-terminal residues of the subject sequence.
For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the CLUSTALW alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N— and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the CLUSTALW program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N— or C-termini of the subject sequence, which are not matched/aligned with the query. In this case the percent identity calculated by CLUSTALW is not manually corrected. Once again, only residue positions outside the N— and C-terminal ends of the subject sequence, as displayed in the CLUSTALW alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are required for the purposes of the present invention.
In addition to the above method of aligning two or more polynucleotide or polypeptide sequences to arrive at a percent identity value for the aligned sequences, it may be desirable in some circumstances to use a modified version of the CLUSTALW algorithm which takes into account known structural features of the sequences to be aligned, such as for example, the SWISS-PROT designations for each sequence. The result of such a modifed CLUSTALW algorithm may provide a more accurate value of the percent identity for two polynucleotide or polypeptide sequences. Support for such a modified version of CLUSTALW is provided within the CLUSTALW algorithm and would be readily appreciated to one of skill in the art of bioinformatics.
The variants may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the MRNA to those preferred by a bacterial host such as E. coli).
Naturally occurring variants are called “allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.
Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the protein without substantial loss of biological function. The authors of Ron et al., J. Biol. Chem. 268: 2984-2988 (1993), reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J. Biotechnology 7:199-216 (1988)).
Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that “[m]ost of the molecule could be altered with little effect on either [binding or biological activity].” In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.
Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the protein will likely be retained when less than the majority of the residues of the protein are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N— or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.
Alternatively, such N-terminus or C-terminus deletions of a polypeptide of the present invention may, in fact, result in a significant increase in one or more of the biological activities of the polypeptide(s). For example, biological activity of many polypeptides are governed by the presence of regulatory domains at either one or both termini. Such regulatory domains effectively inhibit the biological activity of such polypeptides in lieu of an activation event (e.g., binding to a cognate ligand or receptor, phosphorylation, proteolytic processing, etc.). Thus, by eliminating the regulatory domain of a polypeptide, the polypeptide may effectively be rendered biologically active in the absence of an activation event.
Thus, the invention further includes polypeptide variants that show substantial biological activity. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant molecules can then be tested for biological activity.
As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved.
The invention encompasses polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by the polypeptide of the present invention. Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics (e.g., chemical properties). According to Cunningham et al above, such conservative substitutions are likely to be phenotypically silent. Additional guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).
Tolerated conservative amino acid substitutions of the present invention involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

In addition, the present invention also encompasses the conservative substitutions provided in Table III below.

TABLE III


For Amino Acid	Code	Replace with any of:

Alanine	A	D-Ala, Gly, beta-Ala, L-Cys, D-Cys
Arginine	R	D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-Met, D-
		Ile, Orn, D-Orn
Asparagine	N	D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln
Aspartic Acid	D	D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln
Cysteine	C	D-Cys, S—Me-Cys, Met, D-Met, Thr, D-Thr
Glutamine	Q	D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp
Glutamic Acid	E	D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln
Glycine	G	Ala, D-Ala, Pro, D-Pro, β-Ala, Acp
Isoleucine	I	D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met
Leucine	L	D-Leu, Val, D-Val, Met, D-Met
Lysine	K	D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-
		Ile, Orn, D-Orn
Methionine	M	D-Met, S—Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val
Phenylalanine	F	D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4,
		or 5-phenylproline, cis-3,4, or 5-phenylproline
Proline	P	D-Pro, L-1-thioazolidine-4-carboxylic acid, D- or L-1-
		oxazolidine-4-carboxylic acid
Serine	S	D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D-Met(O), L-
		Cys, D-Cys
Threonine	T	D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O),
		Val, D-Val
Tyrosine	Y	D-Tyr, Phe, D-Phe, L-Dopa, His, D-His
Valine	V	D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

Aside from the uses described above, such amino acid substitutions may also increase protein or peptide stability. The invention encompasses amino acid substitutions that contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the protein or peptide sequence. Also included are substitutions that include amino acid residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., B or y amino acids.
Both identity and similarity can be readily calculated by reference to the following publications: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Informatics Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press,New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.
In addition, the present invention also encompasses substitution of amino acids based upon the probability of an amino acid substitution resulting in conservation of function. Such probabilities are determined by aligning multiple genes with related function and assessing the relative penalty of each substitution to proper gene function. Such probabilities are often described in a matrix and are used by some algorithms (e.g., BLAST, CLUSTALW, GAP, etc.) in calculating percent similarity wherein similarity refers to the degree by which one amino acid may substitute for another amino acid without lose of function. An example of such a matrix is the PAM250 or BLOSUM62 matrix.
Aside from the canonical chemically conservative substitutions referenced above, the invention also encompasses substitutions which are typically not classified as conservative, but that may be chemically conservative under certain circumstances. Analysis of enzymatic catalysis for proteases, for example, has shown that certain amino acids within the active site of some enzymes may have highly perturbed pKa's due to the unique microenvironment of the active site. Such perturbed pKa's could enable some amino acids to substitute for other amino acids while conserving enzymatic structure and function. Examples of amino acids that are known to have amino acids with perturbed pKa's are the Glu-35 residue of Lysozyme, the Ile-16 residue of Chymotrypsin, the His-159 residue of Papain, etc. The conservation of function relates to either anomalous protonation or anomalous deprotonation of such amino acids, relative to their canonical, non-perturbed pKa. The pKa perturbation may enable these amino acids to actively participate in general acid-base catalysis due to the unique ionization environment within the enzyme active site. Thus, substituting an amino acid capable of serving as either a general acid or general base within the microenvironment of an enzyme active site or cavity, as may be the case, in the same or similar capacity as the wild-type amino acid, would effectively serve as a conservative amino substitution.
Besides conservative amino acid substitution, variants of the present invention include, but are not limited to, the following: (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification. Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.
For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).)
Moreover, the invention further includes polypeptide variants created through the application of molecular evolution (“DNA Shuffling”) methodology to the polynucleotide disclosed as SEQ ID NO:1, 23, 53, and/or 55, the sequence of the clone submitted in a deposit, and/or the cDNA encoding the polypeptide disclosed as SEQ ID NO:2, 24, 54, and/or 56. Such DNA Shuffling technology is known in the art and more particularly described elsewhere herein (e.g., WPC, Stemmer, PNAS, 91:10747, (1994)), and in the Examples provided herein).
A further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of the present invention having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions. Of course, in order of ever-increasing preference, it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of the present invention, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. In specific embodiments, the number of additions, substitutions, and/or deletions in the amino acid sequence of the present invention or fragments thereof (e.g., the mature form and/or other fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions are preferable.
Polynucleotide and Polypeptide Fragments
The present invention is directed to polynucleotide fragments of the polynucleotides of the invention, in addition to polypeptides encoded therein by said polynucleotides and/or fragments.
In the present invention, a “polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence which: is a portion of that contained in a deposited clone, or encoding the polypeptide encoded by the cDNA in a deposited clone; is a portion of that shown in SEQ ID NO:1, 23, 53, and/or 55 or the complementary strand thereto, or is a portion of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2, 24, 54, and/or 56. The nucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length. A fragment “at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in a deposited clone or the nucleotide sequence shown in SEQ ID NO:1, 23, 53, and/or 55. In this context “about” includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus, or at both termini. These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600, 2000 nucleotides) are preferred.
Moreover, representative examples of polynucleotide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:1, 23, 53, and/or 55, or the complementary strand thereto, or the cDNA contained in a deposited clone. In this context “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein. Also encompassed by the present invention are polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions, as are the polypeptides encoded by these polynucleotides.
In the present invention, a “polypeptide fragment” refers to an amino acid sequence which is a portion of that contained in SEQ ID NO:2, 24, 54, and/or 56 or encoded by the cDNA contained in a deposited clone. Protein (polypeptide) fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81 - 100, 102-120, 121-140, 141-160, or 161 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context “about” includes the particularly recited ranges or values, and ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Preferred polypeptide fragments include the full-length protein. Further preferred polypeptide fragments include the full-length protein having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of the full-length polypeptide. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the full-length protein. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.
Also preferred are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Polypeptide fragments of SEQ ID NO:2, 24, 54, and/or 56 falling within conserved domains are specifically contemplated by the present invention. Moreover, polynucleotides encoding these domains are also contemplated.
Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
In a preferred embodiment, the functional activity displayed by a polypeptide encoded by a polynucleotide fragment of the invention may be one or more biological activities typically associated with the full-length polypeptide of the invention. Illustrative of these biological activities includes the fragments ability to bind to at least one of the same antibodies which bind to the full-length protein, the fragments ability to interact with at lease one of the same proteins which bind to the full-length, the fragments ability to elicit at least one of the same immune responses as the full-length protein (i.e., to cause the immune system to create antibodies specific to the same epitope, etc.), the fragments ability to bind to at least one of the same polynucleotides as the full-length protein, the fragments ability to bind to a receptor of the full-length protein, the fragments ability to bind to a ligand of the full-length protein, and the fragments ability to multimerize with the full-length protein. However, the skilled artisan would appreciate that some fragments may have biological activities which are desirable and directly inapposite to the biological activity of the full-length protein. The functional activity of polypeptides of the invention, including fragments, variants, derivatives, and analogs thereof can be determined by numerous methods available to the skilled artisan, some of which are described elsewhere herein.
The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:2, 24, 54, and/or 56, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. Z or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO:1, 23, 53, and/or 55 or contained in ATCC deposit No. Z under stringent hybridization conditions or lower stringency hybridization conditions as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:1), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.
The term “epitopes,” as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An “immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998- 4002 (1983)). The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211).
In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length, or longer. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μg of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences. For example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides. Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion disulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (“HA”) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972- 897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO:1, 23, 53, and/or 55 and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
Antibodies
Further polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of SEQ ID NO:2, 24, 54, and/or 56, and/or an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding). Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Moreover, the term “antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules, as well as, antibody fragments (such as, for example, Fab and F(ab′)2 fragments) which are capable of specifically binding to protein. Fab and F(ab′)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation of the animal or plant, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and humanized antibodies.
Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homologue of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologues of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10-2 M, 10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5 ×10-5 M, 10-5 M, 5×10-6 M, 10-6M, 5×10-7 M, 107 M,5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M, 10-10 M, 5 ×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M, 5×10-14 M, 10-14 M,5×10-15 M, or 10-15 M.
The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Preferably, antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.
The invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).
Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).
As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionucleotides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.
The antibodies of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
The antibodies of the present invention may be generated by any suitable method known in the art.
The antibodies of the present invention may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan (Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2^nded. (1988); and Current Protocols, Chapter 2; which are hereby incorporated herein by reference in its entirety). In a preferred method, a preparation of the XXXXX protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. The administration of the polypeptides of the present invention may entail one or more injections of an immunizing agent and, if desired, an adjuvant. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art. For the purposes of the invention, “immunizing agent” may be defined as a polypeptide of the invention, including fragments, variants, and/or derivatives thereof, in addition to fusions with heterologous polypeptides and other forms of the polypeptides described herein.
Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections, though they may also be given intramuscularly, and/or through IV). The immunizing agent may include polypeptides of the present invention or a fusion protein or variants thereof. Depending upon the nature of the polypeptides (i.e., percent hydrophobicity, percent hydrophilicity, stability, net charge, isoelectric point etc.), it may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Such conjugation includes either chemical conjugation by derivitizing active chemical functional groups to both the polypeptide of the present invention and the immunogenic protein such that a covalent bond is formed, or through fusion-protein based methodology, or other methods known to the skilled artisan. Examples of such immunogenic proteins include, but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Additional examples of adjuvants which may be employed includes the MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
The antibodies of the present invention may comprise monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975) and U.S. Pat. No. 4,376,110, by Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2^nded. (1988), by Hammerling, et al., Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, N.Y., pp. 563-681 (1981); K6hler et al., Eur. J. Immunol. 6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976), or other methods known to the artisan. Other examples of methods which may be employed for producing monoclonal antibodies includes, but are not limited to, the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.
In a hybridoma method, a mouse, a humanized mouse, a mouse with a human immune system, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.
The immunizing agent will typically include polypeptides of the present invention or a fusion protein thereof. Preferably, the immunizing agent consists of an XXXXX polypeptide or, more preferably, with a XXXXX polypeptide-expressing cell. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56 degrees C), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin. Generally, either peripheral blood lymphocytes (“PBLs”) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986), pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. More preferred are the parent myeloma cell line (SP20) as provided by the ATCC. As inferred throughout the specification, human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptides of the present invention. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbant assay (ELISA). Such techniques are known in the art and within the skill of the artisan. The binding affmity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollart, Anal. Biochem., 107:220 (1980).
After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra, and/or according to Wands et al. (Gastroenterology 80:225-232 (1981)). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-sepharose, hydroxyapatite chromatography, gel exclusion chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The skilled artisan would acknowledge that a variety of methods exist in the art for the production of monoclonal antibodies and thus, the invention is not limited to their sole production in hydridomas. For example, the monoclonal antibodies may be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. In this context, the term “monoclonal antibody” refers to an antibody derived from a single eukaryotic, phage, or prokaryotic clone. The DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies, or such chains from human, humanized, or other sources). The hydridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transformed into host cells such as Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for hurman heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et al, supra) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art and are discussed in detail in the Examples described herein. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain.
For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.
As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988).
For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; Cabilly et al., Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entirety. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the methods of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possible some FR residues are substituted from analogous sites in rodent antibodies.
In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988)1 and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).
Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety. The techniques of cole et al., and Boerder et al., are also available for the preparation of human monoclonal antibodies (cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol., 147(1):86-95, (1991)).
Human antibodies can also be produced using transgenic mice which are incapable of expressing fimctional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-fimctional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc. (Princeton, N.J.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and creation of an antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,106, and in the following scientific publications: Marks et al., Biotechnol., 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Fishwild et al., Nature Biotechnol., 14:845-51 (1996); Neuberger, Nature Biotechnol., 14:826 (1996); Lonberg and Huszer, Intern. Rev. Immunol., 13:65-93 (1995).
Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).
Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.
Such anti-idiotypic antibodies capable of binding to the XXXXX polypeptide can be produced in a two-step procedure. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody that binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones that produce an antibody whose ability to bind to the protein-specific antibody can be blocked by the polypeptide. Such antibodies comprise anti-idiotypic antibodies to the protein-specific antibody and can be used to immunize an animal to induce formation of fuirther protein-specific antibodies.
The antibodies of the present invention may be bispecific antibodies. Bispecific antibodies are monoclonal, Preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present invention, one of the binding specificities may be directed towards a polypeptide of the present invention, the other may be for any other antigen, and preferably for a cell-surface protein, receptor, receptor subunit, tissue-specific antigen, virally derived protein, virally encoded envelope protein, bacterially derived protein, or bacterial surface protein, etc.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transformed into a suitable host organism. For further details of generating bispecific antibodies see, for example Suresh et al., Meth. In Enzym., 121:210 (1986).
Heteroconjugate antibodies are also contemplated by the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4, 676, 980), and for the treatment of HIV infection (WO 91/00360; WO 92/20373; and EP03089). It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioester bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.
Polynucleotides Encoding Antibodies
The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:2, 24, 54, and/or 56.
The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.
Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties ), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.
In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423- 42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038- 1041 (1988)).
More preferably, a clone encoding an antibody of the present invention may be obtained according to the method described in the Example section herein.
Methods of Producing Antibodies
The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.
The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.
The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. lrmunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341(1992) (said references incorporated by reference in their entireties).
As discussed, supra, the polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:2, 24, 54, and/or 56 may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NO:2, 24, 54, and/or 56 may be fused or conjugated to the above antibody portions to facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide-linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP A 232,262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.
The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 111In or 99Tc.
Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologues thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).
Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.
An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
The present invention also encompasses the creation of synthetic antibodies directed against the polypeptides of the present invention. One example of synthetic antibodies is described in Radrizzani, M., et al., Medicina, (Aires), 59(6):753-8, (1999)). Recently, a new class of synthetic antibodies has been described and are referred to as molecularly imprinted polymers (MIPs) (Semorex, Inc.). Antibodies, peptides, and enzymes are often used as molecular recognition elements in chemical and biological sensors. However, their lack of stability and signal transduction mechanisms limits their use as sensing devices. Molecularly imprinted polymers (MIPs) are capable of mimicking the function of biological receptors but with less stability constraints. Such polymers provide high sensitivity and selectivity while maintaining excellent thermal and mechanical stability. MIPs have the ability to bind to small molecules and to target molecules such as organics and proteins' with equal or greater potency than that of natural antibodies. These “super” MIPs have higher affinities for their target and thus require lower concentrations for efficacious binding.
During synthesis, the MIPs are imprinted so as to have complementary size, shape, charge and functional groups of the selected target by using the target molecule itself (such as a polypeptide, antibody, etc.), or a substance having a very similar structure, as its “print” or “template.” MIPs can be derivatized with the same reagents afforded to antibodies. For example, fluorescent ‘super’ MIPs can be coated onto beads or wells for use in highly sensitive separations or assays, or for use in high throughput screening of proteins.
Moreover, MIPs based upon the structure of the polypeptide(s) of the present invention may be useful in screening for compounds that bind to the polypeptide(s) of the invention. Such a MIP would serve the role of a synthetic “receptor” by minimicking the native architecture of the polypeptide. In fact, the ability of a MIP to serve the role of a synthetic receptor has already been demonstrated for the estrogen receptor (Ye, L., Yu, Y., Mosbach, K, Analyst., 126(6):760-5, (2001); Dickert, F, L., Hayden, O., Halilias, K, P, Analyst., 126(6):766-71, (2001)). A synthetic receptor may either be mimicked in its entirety (e.g., as the entire protein), or mimicked as a series of short peptides corresponding to the protein (Rachkov, A., Minoura, N, Biochim, Biophys, Acta., 1544(1-2):255-66, (2001)). Such a synthetic receptor MIPs may be employed in any one or more of the screening methods described elsewhere herein.
MIPs have also been shown to be useful in “sensing” the presence of its mimicked molecule (Cheng, Z., Wang, E., Yang, X, Biosens, Bioelectron., 16(3):179-85, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst., 126(6):798-802, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst., 126(6):798-802, (2001)). For example, a MIP designed using a polypeptide of the present invention may be used in assays designed to identify, and potentially quantitate, the level of said polypeptide in a sample. Such a MIP may be used as a substitute for any component described in the assays, or kits, provided herein (e.g., ELISA, etc.).
A number of methods may be employed to create MIPs to a specific receptor, ligand, polypeptide, peptide, organic molecule. Several preferred methods are described by Esteban et al in J. Anal, Chem., 370(7):795-802, (2001), which is hereby incorporated herein by reference in its entirety in addition to any references cited therein. Additional methods are known in the art and are encompassed by the present invention, such as for example, Hart, B, R., Shea, K, J. J. Am. Chem, Soc., 123(9):2072-3, (2001); and Quaglia, M., Chenon, K., Hall, A, J., De, Lorenzi, E., Sellergren, B, J. Am. Chem, Soc., 123(10):2146-54, (2001); which are hereby incorporated by reference in their entirety herein.
Uses for Antibodies Directed Against Polypeptides of the Invention
The antibodies of the present invention have various utilities. For example, such antibodies may be used in diagnostic assays to detect the presence or quantification of the polypeptides of the invention in a sample. Such a diagnostic assay may be comprised of at least two steps. The first, subjecting a sample with the antibody, wherein the sample is a tissue (e.g., human, animal, etc.), biological fluid (e.g., blood, urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract (e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g., See Arenkov P, et al., Anal Biochem., 278(2):123-131 (2000)), or a chromatography column, etc. And a second step involving the quantification of antibody bound to the substrate. Alternatively, the method may additionally involve a first step of attaching the antibody, either covalently, electrostatically, or reversibly, to a solid support, and a second step of subjecting the bound antibody to the sample, as defmed above and elsewhere herein.
Various diagnostic assay techniques are known in the art, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogenous phases (Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., (1987), ppl47-158). The antibodies used in the diagnostic assays can be labeled with a detectable moiety. The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as 2H, 14C, 32P, or 125I, a florescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase, green fluorescent protein, or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); Dafvid et al., Biochem., 13:1014 (1974); Pain et al., J. Immunol. Metho., 40:219(1981); and Nygren, J. Histochem. And Cytochem., 30:407 (1982).
Antibodies directed against the polypeptides of the present invention are useful for the affinity purification of such polypeptides from recombinant cell culture or natural sources. In this process, the antibodies against a particular polypeptide are immobilized on a suitable support, such as a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody then is contacted with a sample containing the polypeptides to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except for the desired polypeptides, which are bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the desired polypeptide from the antibody.
Immunophenotyping
The antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples. The translation product of the gene of the present invention may be usefuil as a cell specific marker, or more specifically as a cellular marker that is differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-oated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).
These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and “non-self” cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.
Assays For Antibody Binding
The antibodies of the invention may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-ompetitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 14 hours) at 4° C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.
ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.
The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.
Therapeutic Uses of Antibodies
The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.
The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.
It is preferred to use high affmity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affmity for polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10-2 M, 10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M, 10-6 M, 5×10-7 M, 10-7 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M, 10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M, 5×10-14 M, 10-14 M, 5×10-15 M, and 10-15 M.
Antibodies directed against polypeptides of the present invention are useful for inhibiting allergic reactions in animals. For example, by administering a therapeutically acceptable dose of an antibody, or antibodies, of the present invention, or a cocktail of the present antibodies, or in combination with other antibodies of varying sources, the animal may not elicit an allergic response to antigens.
Likewise, one could envision cloning the gene encoding an antibody directed against a polypeptide of the present invention, said polypeptide having the potential to elicit an allergic and/or immune response in an organism, and transforming the organism with said antibody gene such that it is expressed (e.g., constitutively, inducibly, etc.) in the organism. Thus, the organism would effectively become resistant to an allergic response resulting from the ingestion or presence of such an immune/allergic reactive polypeptide. Moreover, such a use of the antibodies of the present invention may have particular utility in preventing and/or ameliorating autoimmune diseases and/or disorders, as such conditions are typically a result of antibodies being directed against endogenous proteins. For example, in the instance where the polypeptide of the present invention is responsible for modulating the immune response to auto-antigens, transforming the organism and/or individual with a construct comprising any of the promoters disclosed herein or otherwise known in the art, in addition, to a polynucleotide encoding the antibody directed against the polypeptide of the present invention could effective inhibit the organisms immune system from eliciting an immune response to the auto-antigen(s). Detailed descriptions of therapeutic and/or gene therapy applications of the present invention are provided elsewhere herein.
Alternatively, antibodies of the present invention could be produced in a plant (e.g., cloning the gene of the antibody directed against a polypeptide of the present invention, and transforming a plant with a suitable vector comprising said gene for constitutive expression of the antibody within the plant), and the plant subsequently ingested by an animal, thereby conferring temporary immunity to the animal for the specific antigen the antibody is directed towards (See, for example, U.S. Pat. Nos. 5,914,123 and 6,034,298).
In another embodiment, antibodies of the present invention, preferably polyclonal antibodies, more preferably monoclonal antibodies, and most preferably single-chain antibodies, can be used as a means of inhibiting gene expression of a particular gene, or genes, in a human, mammal, and/or other organism. See, for example, International Publication Number WO 00/05391, published Feb. 3, 2000, to Dow Agrosciences LLC. The application of such methods for the antibodies of the present invention are known in the art, and are more particularly described elsewhere herein.
In yet another embodiment, antibodies of the present invention may be useful for multimerizing the polypeptides of the present invention. For example, certain proteins may confer enhanced biological activity when present in a multimeric state (i.e., such enhanced activity may be due to the increased effective concentration of such proteins whereby more protein is available in a localized location).
Antibody-Based Gene Therapy
In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.
Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).
In a preferred aspect, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435438 (1989). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.
Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid- carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by adrninistering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO93114188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).
In a specific embodiment, viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).
Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In a preferred embodiment, adenovirus vectors are used.
Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146).
Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
In a preferred embodiment, the cell used for gene therapy is autologous to the patient.
In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).
In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription. Demonstration of Therapeutic or Prophylactic Activity The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
Therapeutic/Prophylactic Administration and Compositions
The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred aspect, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.
Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.
In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y. (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with - cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
Diagnosis and Imaging with Antibodies
Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.
The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell . Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
One aspect of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “lrmunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).
Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
Kits
The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).
In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.
In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.
In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.
In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, Mo.).
The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface- bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.
Fusion Proteins
Any polypeptide of the present invention can be used to generate fusion proteins. For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide. Moreover, because certain proteins target cellular locations based on trafficking signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins.
Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.
Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. Similarly, peptide cleavage sites can be introduced in-between such peptide moieties, which could additionally be subjected to protease activity to remove said peptide(s) from the protein of the present invention. The addition of peptide moieties, including peptide cleavage sites, to facilitate handling of polypeptides are familiar and routine techniques in the art.
Moreover, polypeptides of the present invention, including fragments, and specifically epitopes, can be combined with parts of the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86 (1988).) Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).) Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of the constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).)
Moreover, the polypeptides of the present invention can be fused to marker sequences (also referred to as “tags”). Due to the availability of antibodies specific to such “tags”, purification of the fused polypeptide of the invention, and/or its identification is significantly facilitated since antibodies specific to the polypeptides of the invention are not required. Such purification may be in the form of an affinity purification whereby an anti-tag antibody or another type of affinity matrix (e.g., anti-tag antibody attached to the matrix of a flow-thru column) that binds to the epitope tag is present. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767 (1984)).
The skilled artisan would acknowledge the existence of other “tags” which could be readily substituted for the tags referred to supra for purification and/or identification of polypeptides of the present invention (Jones C., et al., J Chromatogr A. 707(1):3-22 (1995)). For example, the c-myc tag and the 8F9, 3C7, 6E10, G4m B7 and 9E10 antibodies thereto (Evan et al., Molecular and Cellular Biology 5:3610-3616 (1985)); the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering, 3(6):547-553 (1990), the Flag-peptide—i.e., the octapeptide sequence DYKDDDDK (SEQ ID NO:47), (Hopp et al., Biotech. 6:1204-1210 (1988); the KT3 epitope peptide (Martin et al., Science, 255:192-194 (1992)); a-tubulin epitope peptide (Skinner et al., J. Biol. Chem., 266:15136-15166, (1991)); the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Sci. USA, 87:6363-6397 (1990)), the FITC epitope (Zymed, Inc.), the GFP epitope (Zymed, Inc.), and the Rhodamine epitope (Zymed, Inc.).
The present invention also encompasses the attachment of up to nine codons encoding a repeating series of up to nine arginine amino acids to the coding region of a polynucleotide of the present invention. The invention also encompasses chemically derivitizing a polypeptide of the present invention with a repeating series of up to nine arginine amino acids. Such a tag, when attached to a polypeptide, has recently been shown to serve as a universal pass, allowing compounds access to the interior of cells without additional derivitization or manipulation (Wender, P., et al., unpublished data).
Protein fusions involving polypeptides of the present invention, including fragrnents and/or variants thereof, can be used for the following, non-limiting examples, subcellular localization of proteins, determination of protein-protein interactions via immunoprecipitation, purification of proteins via affinity chromatography, functional and/or structural characterization of protein. The present invention also encompasses the application of hapten specific antibodies for any of the uses referenced above for epitope fusion proteins. For example, the polypeptides of the present invention could be chemically derivatized to attach hapten molecules (e.g., DNP, (Zymed, Inc.)). Due to the availability of monoclonal antibodies specific to such haptens, the protein could be readily purified using immunoprecipation, for example.
Polypeptides of the present invention, including fragments and/or variants thereof, in addition to, antibodies directed against such polypeptides, fragments, and/or variants, may be fused to any of a number of known, and yet to be determined, toxins, such as ricin, saporin (Mashiba H. et al., Ann. N. Y. Acad. Sci. 1999;886:233-5), or HC toxin (Tonukari N J, et al., Plant Cell. 2000 February;12(2):237-248), for example. Such fusions could be used to deliver the toxins to desired tissues for which a ligand or a protein capable of binding to the polypeptides of the invention exists.
The invention encompasses the fusion of antibodies directed against polypeptides of the present invention, including variants and fragments thereof, to said toxins for delivering the toxin to specific locations in a cell, to specific tissues, and/or to specific species. Such bifunctional antibodies are known in the art, though a review describing additional advantageous fusions, including citations for methods of production, can be found in P. J. Hudson, Curr. Opp. In. Imm. 11:548-557, (1999); this publication, in addition to the references cited therein, are hereby incorporated by reference in their entirety herein. In this context, the term “toxin” may be expanded to include any heterologous protein, a small molecule, radionucleotides, cytotoxic drugs, liposomes, adhesion molecules, glycoproteins, ligands, cell or tissue-specific ligands, enzymes, of bioactive agents, biological response modifiers, anti-fingal agents, hormones, steroids, vitamins, peptides, peptide analogs, anti-allergenic agents, anti-tubercular agents, anti-viral agents, antibiotics, anti-protozoan agents, chelates, radioactive particles, radioactive ions, X-ray contrast agents, monoclonal antibodies, polyclonal antibodies and genetic material. In view of the present disclosure, one skilled in the art could determine whether any particular “toxin” could be used in the compounds of the present invention. Examples of suitable “toxins” listed above are exemplary only and are not intended to limit the “toxins” that may be used in the present invention.
Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.
Vectors, Host Cells, and Protein Production
The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlsbad, Calif.). Other suitable vectors will be readily apparent to the skilled artisan.
Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.
A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
Polypeptides of the present invention, and preferably the secreted form, can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
In one embodiment, the yeast Pichia pastoris is used to express the polypeptide of the present invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O2. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O2. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See, Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, et al., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
In one example, the plasmid vector pPIC9K is used to express DNA encoding a polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in “Pichia Protocols: Methods in Molecular Biology,” D.R. Higgins and J. Cregg, eds. The Humana Press, Totowa, N.J., 1998. This expression vector allows expression and secretion of a protein of the invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.
Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG, as required.
In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.
In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with the polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination, resulting in the formation of a new transcription unit (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No. 5,733,761, issued Mar. 31, 1998; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide sequence of the invention can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).
The invention encompasses polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protectingiblocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein, the addition of epitope tagged peptide fragments (e.g., FLAG, HA, GST, thioredoxin, maltose binding protein, etc.), attachment of affinity tags such as biotin and/or streptavidin, the covalent attachment of chemical moieties to the amino acid backbone, N- or C-terminal processing of the polypeptides ends (e.g., proteolytic processing), deletion of the N-terminal methionine residue, etc.
Also provided by the invention are chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycolipropylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
The invention further encompasses chemical derivitization of the polypeptides of the present invention, preferably where the chemical is a hydrophilic polymer residue. Exemplary hydrophilic polymers, including derivatives, may be those that include polymers in which the repeating units contain one or more hydroxy groups (polyhydroxy polymers), including, for example, poly(vinyl alcohol); polymers in which the repeating units contain one or more amino groups (polyamine polymers), including, for example, peptides, polypeptides, proteins and lipoproteins, such as albumin and natural lipoproteins; polymers in which the repeating units contain one or more carboxy groups (polycarboxy polymers), including, for example, carboxymethylcellulose, alginic acid and salts thereof, such as sodium and calcium alginate, glycosaminoglycans and salts thereof, including salts of hyaluronic acid, phosphorylated and sulfonated derivatives of carbohydrates, genetic material, such as interleukin-2 and interferon, and phosphorothioate oligomers; and polymers in which the repeating units contain one or more saccharide moieties (polysaccharide polymers), including, for example, carbohydrates.
The molecular weight of the hydrophilic polymers may vary, and is generally about 50 to about 5,000,000, with polymers having a molecular weight of about 100 to about 50,000 being preferred. The polymers may be branched or unbranched. More preferred polymers have a molecular weight of about 150 to about 10,000, with molecular weights of 200 to about 8,000 being even more preferred.
For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).
Additional preferred polymers which may be used to derivatize polypeptides of the invention, include, for example, poly(ethylene glycol) (PEG), poly(vinylpyrrolidine), polyoxomers, polysorbate and poly(vinyl alcohol), with PEG polymers being particularly preferred. Preferred among the PEG polymers are PEG polymers having a molecular weight of from about 100 to about 10,000. More preferably, the PEG polymers have a molecular weight of from about 200 to about 8,000, with PEG 2,000, PEG 5,000 and PEG 8,000, which have molecular weights of 2,000, 5,000 and 8,000, respectively, being even more preferred. Other suitable hydrophilic polymers, in addition to those exemplified above, will be readily apparent to one skilled in the art based on the present disclosure. Generally, the polymers used may include polymers that can be attached to the polypeptides of the invention via alkylation or acylation reactions.
The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminus) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
As with the various polymers exemplified above, it is contemplated that the polymeric residues may contain functional groups in addition, for example, to those typically involved in linking the polymeric residues to the polypeptides of the present invention. Such functionalities include, for example, carboxyl, amine, hydroxy and thiol groups. These functional groups on the polymeric residues can be further reacted, if desired, with materials that are generally reactive with such functional groups and which can assist in targeting specific tissues in the body including, for example, diseased tissue. Exemplary materials which can be reacted with the additional functional groups include, for example, proteins, including antibodies, carbohydrates, peptides, glycopeptides, glycolipids, lectins, and nucleosides.
In addition to residues of hydrophilic polymers, the chemical used to derivatize the polypeptides of the present invention can be a saccharide residue. Exemplary saccharides which can be derived include, for example, monosaccharides or sugar alcohols, such as erythrose, threose, ribose, arabinose, xylose, lyxose, fructose, sorbitol, mannitol and sedoheptulose, with preferred monosaccharides being fructose, mannose, xylose, arabinose, mannitol and sorbitol; and disaccharides, such as lactose, sucrose, maltose and cellobiose. Other saccharides include, for example, inositol and ganglioside head groups. Other suitable saccharides, in addition to those exemplified above, will be readily apparent to one skilled in the art based on the present disclosure. Generally, saccharides which may be used for derivitization include saccharides that can be attached to the polypeptides of the invention via alkylation or acylation reactions.
Moreover, the invention also encompasses derivitization of the polypeptides of the present invention, for example, with lipids (including cationic, anionic, polymerized, charged, synthetic, saturated, unsaturated, and any combination of the above, etc.). stabilizing agents.
The invention encompasses derivitization of the polypeptides of the present invention, for example, with compounds that may serve a stabilizing function (e.g., to increase the polypeptides half-life in solution, to make the polypeptides more water soluble, to increase the polypeptides hydrophilic or hydrophobic character, etc.). Polymers useful as stabilizing materials may be of natural, semi-synthetic (modified natural) or synthetic origin. Exemplary natural polymers include naturally occurring polysaccharides, such as, for example, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans (such as, for example, inulin), levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectins, including amylose, pullulan, glycogen, amylopectin, cellulose, dextran, dextrin, dextrose, glucose, polyglucose, polydextrose, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum, starch and various other natural homopolymer or heteropolymers, such as those containing one or more of the following aldoses, ketoses, acids or amines: erythose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid, and naturally occurring derivatives thereof Accordingly, suitable polymers include, for example, proteins, such as albumin, polyalginates, and polylactide-coglycolide polymers. Exemplary semi-synthetic polymers include carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose. Exemplary synthetic polymers include polyphosphazenes, hydroxyapatites, fluoroapatite polymers, polyethylenes (such as, for example, polyethylene glycol (including for example, the class of compounds referred to as Pluronics.RTM., commercially available from BASF, Parsippany, N.J.), polyoxyethylene, and polyethylene terephthlate), polypropylenes (such as, for example, polypropylene glycol), polyurethanes (such as, for example, polyvinyl alcohol (PVA), polyvinyl chloride and polyvinylpyrrolidone), polyamides including nylon, polystyrene, polylactic acids, fluorinated hydrocarbon polymers, fluorinated carbon polymers (such as, for example, polytetrafluoroethylene), acrylate, methacrylate, and polymethylmethacrylate, and derivatives thereof Methods for the preparation of derivatized polypeptides of the invention which employ polymers as stabilizing compounds will be readily apparent to one skilled in the art, in view of the present disclosure, when coupled with information known in the art, such as that described and referred to in Unger, U.S. Pat. No. 5,205,290, the disclosure of which is hereby incorporated by reference herein in its entirety.
Moreover, the invention encompasses additional modifications of the polypeptides of the present invention. Such additional modifications are known in the art, and are specifically provided, in addition to methods of derivitization, etc., in U.S. Pat. No. 6,028,066, which is hereby incorporated in its entirety herein.
The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.
Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term homomer, refers to a multimer containing only polypeptides corresponding to the amino acid sequence of SEQ ID NO:2, 24, 54, and/or 56 or encoded by the cDNA contained in a deposited clone (including fragments, variants, splice variants, and fusion proteins, corresponding to these polypeptides as described herein). These homomers may contain polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing polypeptides having identical and/or different amino acid sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in the sequence listing, or contained in the polypeptide encoded by a deposited clone). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein of the invention.
In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in an Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated mulfimers, such as for example, osteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.
Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.
Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference. Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trmeric polypeptides of the invention.
In another example, proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide sequence. In a further embodiment, associations proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti-Flag® antibody.
The multimers of the invention may be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).
Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hydrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).
In addition, the polynucleotide insert of the present invention could be operatively linked to “artificial” or chimeric promoters and transcription factors. Specifically, the artificial promoter could comprise, or alternatively consist, of any combination of cis-acting DNA sequence elements that are recognized by trans-acting transcription factors. Preferably, the cis acting DNA sequence elements and trans-acting transcription factors are operable in mammals. Further, the trans-acting transcription factors of such “artificial” promoters could also be “artificial” or chimeric in design themselves and could act as activators or repressors to said “artificial” promoter.
Uses of the Polynucleotides
Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.
The polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. Each polynucleotide of the present invention can be used as a chromosome marker.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NO:1, 23, 53, and/or 55. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the SEQ ID NO: 1, 23, 53, and/or 55 will yield an amplified fragment.
Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome specific-cDNA libraries.
Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread. This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. For a review of this technique, see Verma et al., “Human Chromosomes: a Manual of Basic Techniques,” Pergamon Press, New York (1988).
For chromosome mapping, the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes). Preferred polynucleotides correspond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross hybridization during chromosomal mapping.
Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. Disease mapping data are known in the art. Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.
Thus, once coinheritance is established, differences in the polynucleotide and the corresponding gene between affected and unaffected organisms can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected organisms, but not in normal organisms, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the corresponding gene from several normal organisms is required to distinguish the mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.
Furthermore, increased or decreased expression of the gene in affected organisms as compared to unaffected organisms can be assessed using polynucleotides of the present invention. Any of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker.
Thus, the invention also provides a diagnostic method useful during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an organism and comparing the measured gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder.
By “measuring the expression level of a polynucleotide of the present invention” is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the present invention or the level of the mRNA encoding the polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of organisms not having a disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
By “biological sample” is intended any biological sample obtained from an organism, body fluids, cell line, tissue culture, or other source which contains the polypeptide of the present invention or mRNA. As indicated, biological samples include body fluids (such as the following non-limiting examples, sputum, amniotic fluid, urine, saliva, breast milk, secretions, interstitial fluid, blood, serum, spinal fluid, etc.) which contain the polypeptide of the present invention, and other tissue sources found to express the polypeptide of the present invention. Methods for obtaining tissue biopsies and body fluids from organisms are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
The method(s) provided above may Preferably be applied in a diagnostic method and/or kits in which polynucleotides and/or polypeptides are attached to a solid support. In one exemplary method, the support may be a “gene chip” or a “biological chip” as described in U.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip with polynucleotides of the present invention attached may be used to identify polymorphisms between the polynucleotide sequences, with polynucleotides isolated from a test subject. The knowledge of such polymorphisms (i.e. their location, as well as, their existence) would be beneficial in identifying disease loci for many disorders, including proliferative diseases and conditions. Such a method is described in U.S. Pat. Nos. 5,858,659 and 5,856,104. The US Patents referenced supra are hereby incorporated by reference in their entirety herein.
The present invention encompasses polynucleotides of the present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art. The use of PNAs would serve as the preferred form if the polynucleotides are incorporated onto a solid support, or gene chip. For the purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M. Egholm, R. H. Berg and 0. Buchardt, Science 254, 1497 (1991); and M. Egholm, O. Buchardt, L.Christensen, C. Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365, 666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the stronger binding characteristics of PNA:DNA hybrids. In addition, it is more likely that single base mismatches can be determined with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by 8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis.
In addition to the foregoing, a polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA. Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56: 560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991). Both methods rely on binding of the polynucleotide to a complementary DNA or RNA. For these techniques, preferred polynucleotides are usually oligonucleotides 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991) ) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an niRNA molecule into polypeptide. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat or prevent disease.
The present invention encompasses the addition of a nuclear localization signal, operably linked to the 5′ end, 3′ end, or any location therein, to any of the oligonucleotides, antisense oligonucleotides, triple helix oligonucleotides, ribozymes, PNA oligonucleotides, and/or polynucleotides, of the present invention. See, for example, G. Cutrona, et al., Nat. Biotech., 18:300-303, (2000); which is hereby incorporated herein by reference.
Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell. In one example, polynucleotide sequences of the present invention may be used to construct chimeric RNA/DNA oligonucleotides corresponding to said sequences, specifically designed to induce host cell mismatch repair mechanisms in an organism upon systemic injection, for example (Bartlett, R. J., et al., Nat. Biotech, 18:615-622 (2000), which is hereby incorporated by reference herein in its entirety). Such RNA/DNA oligonucleotides could be designed to correct genetic defects in certain host strains, and/or to introduce desired phenotypes in the host (e.g., introduction of a specific polymorphism within an endogenous gene corresponding to a polynucleotide of the present invention that may ameliorate and/or prevent a disease symptom and/or disorder, etc.). Alternatively, the polynucleotide sequence of the present invention may be used to construct duplex oligonucleotides corresponding to said sequence, specifically designed to correct genetic defects in certain host strains, and/or to introduce desired phenotypes into the host (e.g., introduction of a specific polymorphism within an endogenous gene corresponding to a polynucleotide of the present invention that may ameliorate and/or prevent a disease symptom and/or disorder, etc). Such methods of using duplex oligonucleotides are known in the art and are encompassed by the present invention (see EP1007712, which is hereby incorporated by reference herein in its entirety).
The polynucleotides are also useful for identifying organisms from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel. This method does not suffer from the current limitations of “Dog Tags” which can be lost, switched, or stolen, making positive identification difficult. The polynucleotides of the present invention can be used as additional DNA markers for RFLP.
The polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an organisms genome. These sequences can be used to prepare PCR primers for amplifiing and isolating such selected DNA, which can then be sequenced. Using this technique, organisms can be identified because each organism will have a unique set of DNA sequences. Once an unique ID database is established for an organism, positive identification of that organism, living or dead, can be made from extremely small tissue samples. Similarly, polynucleotides of the present invention can be used as polymorphic markers, in addition to, the identification of transformed or non-transformed cells and/or tissues.
There is also a need for reagents capable of identifying the source of a particular tissue. Such need arises, for example, when presented with tissue of unknown origin. Appropriate reagents can comprise, for example, DNA probes or primers specific to particular tissue prepared from the sequences of the present invention. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination. Moreover, as mentioned above, such reagents can be used to screen and/or identify transformed and non-transformed cells and/or tissues.
In the very least, the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to “subtract-out” known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a “gene chip” or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.
Uses of the Polypeptides
Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.
A polypeptide of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques. For example, protein expression in tissues can be studied with classical immunohistological methods. (Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987).) Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (1 12In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
In addition to assaying protein levels in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NoM or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 131I, 112In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or intraperitoneally) into the mammal. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)
Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Moreover, polypeptides of the present invention can be used to treat, prevent, and/or diagnose disease. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor suppressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).
Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat, prevent, and/or diagnose disease. For example, administration of an antibody directed to a polypeptide of the present invention can bind and reduce overproduction of the polypeptide. Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).
At the very least, the polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the following biological activities.
Gene Therapy Methods
Another aspect of the present invention is to gene therapy methods for treating or preventing disorders, diseases and conditions. The gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of a polypeptide of the present invention. This method requires a polynucleotide which codes for a polypeptide of the invention that operatively linked to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques are known in the art, see, for example, WO90/11092, which is herein incorporated by reference.
Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide of the invention ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, see Belldegrun et al., J. Natl. Cancer Inst., 85:207-216 (1993); Ferrantini et al., Cancer Research, 53:107-1112 (1993); Ferrantini et al., J. Immunology 153: 46044615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura et al., Cancer Research 50: 5102-5106 (1990); Santodonato, et al., Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy 4:1246-1255 (1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38 (1996)), which are herein incorporated by reference. In one embodiment, the cells which are engineered are arterial cells. The arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.
As discussed in more detail below, the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like). The polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
In one embodiment, the polynucleotide of the invention is delivered as a naked polynucleotide. The term “naked” polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.
The polynucleotide vector constructs of the invention used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.
Any strong promoter known to those skilled in the art can be used for driving the expression of polynucleotide sequence of the invention. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAl promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; the b-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter for the polynucleotides of the invention.
Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.
The polynucleotide construct of the invention can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
For the naked nucleic acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.
The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.
The naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called “gene guns”. These delivery methods are known in the art.
The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc. Such methods of delivery are known in the art.
In certain embodiments, the polynucleotide constructs of the invention are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA , 84:7413-7416 (1987), which is herein incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA, 86:6077-6081 (1989), which is herein incorporated by reference); and purified transcription factors (Debs et al., J. Biol. Chem., 265:10189-10192 (1990), which is herein incorporated by reference), in functional form.
Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA , 84:7413-7416 (1987), which is herein incorporated by reference). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).
Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. PCT Publication NO: WO 90/11092 (which is herein incorporated by reference) for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA liposomes is explained in the literature, see, e.g., Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417, which is herein incorporated by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials.
Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.
For example, commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water. The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size. Other methods are known and available to those of skill in the art.
The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art. See, e.g., Straubinger et al., Methods of Immunology, 101:512-527 (1983), which is herein incorporated by reference. For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be entrapped is added to a suspension of preformed MLVs and then sonicated. When using liposomes containing cationic lipids, the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are mixed directly with the DNA. The liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art. Commonly used methods include Ca2+-EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilson et al., Cell, 17:77 (1979)); ether injection (Deamer et al., Biochim. Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res. Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl. Acad. Sci. USA , 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem., 255:10431 (1980); Szoka et al., Proc. Natl. Acad. Sci. USA, 75:145 (1978); Schaefer-Ridder et al., Science, 215:166 (1982)), which are herein incorporated by reference.
Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10. Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.
U.S. Pat. No.: 5,676,954 (which is herein incorporated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein incorporated by reference) provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein incorporated by reference) provide methods for delivering DNA-cationic lipid complexes to mammals.
In certain embodiments, cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding polypeptides of the invention. Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy, 1:5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
The producer cell line generates infectious retroviral vector particles which include polynucleotide encoding polypeptides of the invention. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express polypeptides of the invention.
In certain other embodiments, cells are engineered, ex vivo or in vivo, with polynucleotides of the invention contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses polypeptides of the invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartzet al., Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-I-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld et al., Science, 252:431-434 (1991); Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA, 76:6606 (1979)).
Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel., 3:499-503 (1993); Rosenfeld et al., Cell, 68:143-155 (1992); Engelhardt et al., Human Genet. Ther., 4:759-769 (1993); Yang et al., Nature Genet., 7:362-369 (1994); Wilson et al., Nature, 365:691-692 (1993); and U.S. Pat. No.: 5,652,224, which are herein incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be grown in human 293 cells. These cells contain the E1 region of adenovirus and constitutively express E1a and E1b, which complement the defective adenoviruses by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.
Preferably, the adenoviruses used in the present invention are replication deficient. Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, but cannot replicate in most cells. Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: E1a, E1b, E3, E4, E2a, or L1 through L5.
In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol., 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.
For example, an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration. The polynucleotide construct containing polynucleotides of the invention is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc. Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses. Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct of the invention. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the polynucleotide construct integrated into its genome, and will express the desired gene product.
Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. encoding the polypeptide sequence of interest) via homologous recombination (see, e.g., U.S. Pat. No.: 5,641,670, issued Jun. 24, 1997; International Publication NO: WO 96/29411, published Sep. 26, 1996; International Publication NO: WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not normally expressed in the cells, or is expressed at a lower level than desired.
Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the 5′ end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination.
The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter. The amplified promoter and targeting sequences are digested and ligated together.
The promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc., described in more detail above. The P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.
The promoter-targeting sequence construct is taken up by cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence.
The polynucleotides encoding polypeptides of the present invention may be administered along with other polynucleotides encoding angiogenic proteins. Angiogenic proteins include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.
Preferably, the polynucleotide encoding a polypeptide of the invention contains a secretory signal sequence that facilitates secretion of the protein. Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5′ end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art.
Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter infusion, biolistic injectors, particle accelerators (i.e., “gene guns”), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery. For example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasmid into the portal vein has resulted in gene expression of the foreign gene in the rat livers. (Kaneda et al., Science, 243:375 (1989)).
A preferred method of local administration is by direct injection. Preferably, a recombinant molecule of the present invention complexed with a delivery vehicle is administered by direct injection into or locally within the area of arteries. Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.
Another method of local administration is to contact a polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound.
Therapeutic compositions useful in systemic administration, include recombinant molecules of the present invention complexed to a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site.
Preferred methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA , 189:11277-11281 (1992), which is incorporated herein by reference). Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.
Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian. Therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly preferred.
Biological Activities
The polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists could be used to treat the associated disease.
Immune Activity
The polynucleotides or polypeptides, or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing diseases, disorders, and/or conditions of the immune system, by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune diseases, disorders, and/or conditions may be genetic, somatic, such as cancer or some autoimmune diseases, disorders, and/or conditions, acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, a polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used as a marker or detector of a particular immune system disease or disorder.
A polynucleotides or polypeptides, or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing diseases, disorders, and/or conditions of hematopoietic cells. A polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types hematopoietic cells. Examples of immunologic deficiency syndromes include, but are not limited to: blood protein diseases, disorders, and/or conditions (e.g. agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.
Moreover, a polynucleotides or polypeptides, or agonists or antagonists of the present invention could also be used to modulate hemostatic (the stopping of bleeding) or thrombolytic activity (clot formation). For example, by increasing hemostatic or thrombolytic activity, a polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to treat or prevent blood coagulation diseases, disorders, and/or conditions (e.g., afibrinogenemia, factor deficiencies, arterial thrombosis, venous thrombosis, etc.), blood platelet diseases, disorders, and/or conditions (e.g. thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, a polynucleotides or polypeptides, or agonists or antagonists of the present invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. Polynucleotides or polypeptides, or agonists or antagonists of the present invention are may also be useful for the detection, prognosis, treatment, and/or prevention of heart attacks (infarction), strokes, scarring, fibrinolysis, uncontrolled bleeding, uncontrolled coagulation, uncontrolled complement fixation, and/or inflammation.
A polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be useful in treating, preventing, and/or diagnosing autoimmune diseases, disorders, and/or conditions. Many autoimmune diseases, disorders, and/or conditions result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of a polynucleotides or polypeptides, or agonists or antagonists of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune diseases, disorders, and/or conditions.
Examples of autoimmune diseases, disorders, and/or conditions that can be treated, prevented, and/or diagnosed or detected by the present invention include, but are not limited to: Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.
Similarly, allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated, prevented, and/or diagnosed by polynucleotides or polypeptides, or agonists or antagonists of the present invention. Moreover, these molecules can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
A polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be used to treat, prevent, and/or diagnose organ rejection or graft-versus-host disease (GVHD). Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. The administration of a polynucleotides or polypeptides, or agonists or antagonists of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD.
Similarly, a polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be used to modulate inflammation. For example, the polypeptide or polynucleotide or agonists or antagonist may inhibit the proliferation and differentiation of cells involved in an inflammatory response. These molecules can be used to treat, prevent, and/or diagnose inflammatory conditions, both chronic and acute conditions, including chronic prostatitis, granulomatous prostatitis and malacoplakia, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-1.)
Hyperproliferative Disorders
A polynucleotides or polypeptides, or agonists or antagonists of the invention can be used to treat, prevent, and/or diagnose hyperproliferative diseases, disorders, and/or conditions, including neoplasms. A polynucleotides or polypeptides, or agonists or antagonists of the present invention may inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, a polynucleotides or polypeptides, or agonists or antagonists of the present invention may proliferate other cells which can inhibit the hyperproliferative disorder.
For example, by increasing an immune response, particularly increasing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing T-cells, hyperproliferative diseases, disorders, and/or conditions can be treated, prevented, and/or diagnosed. This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, decreasing an immune response may also be a method of treating, preventing, and/or diagnosing hyperproliferative diseases, disorders, and/or conditions, such as a chemotherapeutic agent.
Examples of hyperproliferative diseases, disorders, and/or conditions that can be treated, prevented, and/or diagnosed by polynucleotides or polypeptides, or agonists or antagonists of the present invention include, but are not limited to neoplasms located in the: colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
Similarly, other hyperproliferative diseases, disorders, and/or conditions can also be treated, prevented, and/or diagnosed by a polynucleotides or polypeptides, or agonists or antagonists of the present invention. Examples of such hyperproliferative diseases, disorders, and/or conditions include, but are not limited to: hypergammaglobulinemia, lymphoproliferative diseases, disorders, and/or conditions, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
One preferred embodiment utilizes polynucleotides of the present invention to inhibit aberrant cellular division, by gene therapy using the present invention, and/or protein fusions or fragments thereof.
Thus, the present invention provides a method for treating or preventing cell proliferative diseases, disorders, and/or conditions by inserting into an abnormally proliferating cell a polynucleotide of the present invention, wherein'said polynucleotide represses said expression.
Another embodiment of the present invention provides a method of treating or preventing cell-proliferative diseases, disorders, and/or conditions in individuals comprising administration of one or more active gene copies of the present invention to an abnormally proliferating cell or cells. In a preferred embodiment, polynucleotides of the present invention is a DNA construct comprising a recombinant expression vector effective in expressing a DNA sequence encoding said polynucleotides. In another preferred embodiment of the present invention, the DNA construct encoding the polynucleotides of the present invention is inserted into cells to be treated utilizing a retrovirus, or more Preferably an adenoviral vector (See G J. Nabel, et. al., PNAS 1999 96: 324-326, which is hereby incorporated by reference). In a most preferred embodiment, the viral vector is defective and will not transform non-proliferating cells, only proliferating cells. Moreover, in a preferred embodiment, the polynucleotides of the present invention inserted into proliferating cells either alone, or in combination with or fused to other polynucleotides, can then be modulated via an external stimulus (i.e. magnetic, specific small molecule, chemical, or drug administration, etc.), which acts upon the promoter upstream of said polynucleotides to induce expression of the encoded protein product. As such the beneficial therapeutic affect of the present invention may be expressly modulated (i.e. to increase, decrease, or inhibit expression of the present invention) based upon said external stimulus.
Polynucleotides of the present invention may be useful in repressing expression of oncogenic genes or antigens. By “repressing expression of the oncogenic genes ” is intended the suppression of the transcription of the gene, the degradation of the gene transcript (pre-message RNA), the inhibition of splicing, the destruction of the messenger RNA, the prevention of the post-translational modifications of the protein, the destruction of the protein, or the inhibition of the normal function of the protein.
For local administration to abnormally proliferating cells, polynucleotides of the present invention may be administered by any method known to those of skill in the art including, but not limited to transfection, electroporation, microinjection of cells, or in vehicles such as liposomes, lipofectin, or as naked polynucleotides, or any other method described throughout the specification. The polynucleotide of the present invention may be delivered by known gene delivery systems such as, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol. Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yates et al., Nature 313:812 (1985)) known to those skilled in the art. These references are exemplary only and are hereby incorporated by reference. In order to specifically deliver or transfect cells which are abnormally proliferating and spare non-dividing cells, it is preferable to utilize a retrovirus, or adenoviral (as described in the art and elsewhere herein) delivery system known to those of skill in the art. Since host DNA replication is required for retroviral DNA to integrate and the retrovirus will be unable to self replicate due to the lack of the retrovirus genes needed for its life cycle. Utilizing such a retroviral delivery system for polynucleotides of the present invention will target said gene and constructs to abnormally proliferating cells and will spare the non-dividing normal cells.
The polynucleotides of the present invention may be delivered directly to cell proliferative disorder/disease sites in internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the disease site. The polynucleotides of the present invention may also be administered to disease sites at the time of surgical intervention.
By “cell proliferative disease” is meant any human or animal disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which is characterized by single or multiple local abnormal proliferations of cells, groups of cells, or tissues, whether benign or malignant.
Any amount of the polynucleotides of the present invention may be administered as long as it has a biologically inhibiting effect on the proliferation of the treated cells. Moreover, it is possible to administer more than one of the polynucleotide of the present invention simultaneously to the same site. By “biologically inhibiting” is meant partial or total growth inhibition as well as decreases in the rate of proliferation or growth of the cells. The biologically inhibitory dose may be determined by assessing the effects of the polynucleotides of the present invention on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and cell cultures, or any other method known to one of ordinary skill in the art.
The present invention is further directed to antibody-based therapies which involve administering of anti-polypeptides and anti-polynucleotide antibodies to a mammalian, preferably human, patient for treating, preventing, and/or diagnosing one or more of the described diseases, disorders, and/or conditions. Methods for producing anti-polypeptides and anti-polynucleotide antibodies polyclonal and monoclonal antibodies are described in detail elsewhere herein. Such antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.
In particular, the antibodies, fragments and derivatives of the present invention are useful for treating, preventing, and/or diagnosing a subject having or developing cell proliferative and/or differentiation diseases, disorders, and/or conditions as described herein. Such treatment comprises administering a single or multiple doses of the antibody, or a fragment, derivative, or a conjugate thereof.
The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors, for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of diseases, disorders, and/or conditions related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10-6M, 10-6M, 5×10-7M, 10-7M, 5×10-8M, 10-8M, 5×10-9M, 10-9M, 5×10-10M, 10-10M, 5×10-11M, 10-11M, 5×10-12M, 10-12M, 5×10-13M, 10-13M, 5×10-14M, 10-14M, 5×10-15M, 10-15M.
Moreover, polypeptides of the present invention may be useful in inhibiting the angiogenesis of proliferative cells or tissues, either alone, as a protein fusion, or in combination with other polypeptides directly or indirectly, as described elsewhere herein. In a most preferred embodiment, said anti-angiogenesis effect may be achieved indirectly, for example, through the inhibition of hematopoietic, tumor-specific cells, such as tumor-associated macrophages (See Joseph I B, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is hereby incorporated by reference). Antibodies directed to polypeptides or polynucleotides of the present invention may also result in inhibition of angiogenesis directly, or indirectly (See Witte L, et al., Cancer Metastasis Rev. 17(2):155-61 (1998), which is hereby incorporated by reference)).
Polypeptides, including protein fusions, of the present invention, or fragments thereof may be useful in inhibiting proliferative cells or tissues through the induction of apoptosis. Said polypeptides may act either directly, or indirectly to induce apoptosis of proliferative cells and tissues, for example in the activation of a death-domain receptor, such as tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K, et al., Eur J Biochem 254(3):439-59 (1998), which is hereby incorporated by reference). Moreover, in another preferred embodiment of the present invention, said polypeptides may induce apoptosis through other mechanisms, such as in the activation of other proteins which will activate apoptosis, or through stimulating the expression of said proteins, either alone or in combination with small molecule drugs or adjuvants, such as apoptonin, galectins, thioredoxins, antiinflammatory proteins (See for example, Mutat. Res. 400(1-2):447-55 (1998), Med Hypotheses.50(5):423-33 (1998), Chem. Biol. Interact Apr 24;1 1-112:23-34 (1998), J Mol Med.76(6):402-12 (1998), Int. J. Tissue React. 20(1):3-15 (1998), which are all hereby incorporated by reference).
Polypeptides, including protein fusions to, or fragments thereof, of the present invention are useful in inhibiting the metastasis of proliferative cells or tissues. Inhibition may occur as a direct result of administering polypeptides, or antibodies directed to said polypeptides as described elsewhere herein, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol 1998;231:12541, which is hereby incorporated by reference). Such therapeutic affects of the present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.
In another embodiment, the invention provides a method of delivering compositions containing the polypeptides of the invention (e.g., compositions containing polypeptides or polypeptide antibodies associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells expressing the polypeptide of the present invention. Polypeptides or polypeptide antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.
Polypeptides, protein fusions to, or fragments thereof, of the present invention are useful in enhancing the immunogenicity and/or antigenicity of proliferating cells or tissues, either directly, such as would occur if the polypeptides of the present invention ‘vaccinated’ the immune response to respond to proliferative antigens and immunogens, or indirectly, such as in activating the expression of proteins known to enhance the immune response (e.g. chemokines), to said antigens and immunogens.
Cardiovascular Disorders
Polynucleotides or polypeptides, or agonists or antagonists of the invention may be used to treat, prevent, and/or diagnose cardiovascular diseases, disorders, and/or conditions, including peripheral artery disease, such as limb ischemia.
Cardiovascular diseases, disorders, and/or conditions include cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.
Cardiovascular diseases, disorders, and/or conditions also include heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.
Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.
Heart valve disease include aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.
Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.
Myocardial ischemias include coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.
Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular diseases, disorders, and/or conditions, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.
Aneurysms include dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.
Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.
Cerebrovascular diseases, disorders, and/or conditions include carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.
Embolisms include air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms. Thrombosis include coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.
Ischemia includes cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitis includes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.
Polynucleotides or polypeptides, or agonists or antagonists of the invention, are especially effective for the treatment of critical limb ischemia and coronary disease.
Polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Polypeptides of the invention may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides of the invention are described in more detail herein.
Diseases at the Cellular Level
Diseases associated with increased cell survival or the inhibition of apoptosis that could be treated, prevented, and/or diagnosed by the polynucleotides or polypeptides and/or antagonists or agonists of the invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune diseases, disorders, and/or conditions (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection. In preferred embodiments, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention are used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.
Additional diseases or conditions associated with increased cell survival that could be treated, prevented or diagnosed by the polynucleotides or poly eptides, or agonists or antagonists of the invention, include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Diseases associated with increased apoptosis that could be treated, prevented, and/or diagnosed by the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, include AIDS; neurodegenerative diseases, disorders, and/or conditions (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune diseases, disorders, and/or conditions (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer);: toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.
Wound Healing and Epithelial Cell Proliferation
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, for therapeutic purposes, for example, to stimulate epithelial cell proliferation and basal keratinocytes for the purpose of wound healing, and to stimulate hair follicle production and healing of dermal wounds. Polynucleotides or polypeptides, as well as agonists or antagonists of the invention, may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resulting from heat exposure or chemicals, and other abnormal wound healing conditions such as uremia, malnutrition, vitamin deficiencies and complications associated with systemic treatment with steroids, radiation therapy and antineoplastic drugs and antimetabolites. Polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to promote dermal reestablishment subsequent to dermal loss
The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. The following are a non-exhaustive list of grafts that polynucleotides or polypeptides, agonists or antagonists of the invention, could be used to increase adherence to a wound bed: autografts, artificial skin, allografts, autodermic graft, autoepidermic grafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, split skin graft, thick split graft. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, can be used to promote skin strength and to improve the appearance of aged skin.
It is believed that the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intestine, and large intestine. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.
The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may have a cytoprotective effect on the small intestine mucosa. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may also stimulate healing of mucositis (mouth ulcers) that result from chemotherapy and viral infections.
The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could further be used in full regeneration of skin in full and partial thickness skin defects, including burns, (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful blisters by accelerating reepithelialization of these lesions. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly. Inflamamatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases which result in destruction of the mucosal surface of the small or large intestine, respectively. Thus, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to promote the resurfacing of the mucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease. Treatment with the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to treat diseases associate with the under expression of the polynucleotides of the invention.
Moreover, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to prevent and heal damage to the lungs due to various pathological states. A growth factor such as the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, which could stimulate proliferation and differentiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of aveoli, and inhalation injuries, i.e., resulting from smoke inhalation and burns, that cause necrosis of the bronchiolar epithelium and alveoli could be effectively treated, prevented, and/or diagnosed using the polynucleotides or polypeptides, and/or agonists or antagonists of the invention. Also, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary displasia, in premature infants.
The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).
In addition, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used treat or prevent the onset of diabetes mellitus. In patients with newly diagnosed Types I and H diabetes, where some islet cell function remains, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease. Also, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.
Neurological Diseases
Nervous system diseases, disorders, and/or conditions, which can be treated, prevented, and/or diagnosed with the compositions of the invention (e.g., polypeptides, polynucleotides, and/or agonists or antagonists), include, but are not limited to, nervous system injuries, and diseases, disorders, and/or conditions which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated, prevented, and/or diagnosed in a patient (including human and non-human mammalian patients) according to the invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis; (5) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associated with nutritional diseases, disorders, and/or conditions, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (7) neurological lesions associated with systemic diseases including, but not limited to, diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (9) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including, but not limited to, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.
In a preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to protect neural cells from the damaging effects of cerebral hypoxia. According to this embodiment, the compositions of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with cerebral hypoxia. In one aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with cerebral ischemia. In another aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with cerebral infarction. In another aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose or prevent neural cell injury associated with a stroke. In a further aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with a heart attack.
The compositions of the invention which are useful for treating or preventing a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, compositions of the invention which elicit any of the following effects may be useful according to the invention: (1) increased survival time of neurons in culture; (2) increased sprouting of neurons in culture or in vivo; (3) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms of neuron dysfunction in vivo. Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may routinely be measured using a method set forth herein or otherwise known in the art, such as, for example, the method set forth in Arakawa et al. (J. Neurosci. 10:3507-3515 (1990)); increased sprouting of neurons may be detected by methods known in the art, such as, for example, the methods set forth in Pestronk et al. (Exp. Neurol. 70:65-82 (1980)) or Brown et al. (Ann. Rev. Neurosci. 4:1742 (1981)); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., using techniques known in the art and depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.
In specific embodiments, motor neuron diseases, disorders, and/or conditions that may be treated, prevented, and/or diagnosed according to the invention include, but are not limited to, diseases, disorders, and/or conditions such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as diseases, disorders, and/or conditions that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
Infectious Disease
A polypeptide or polynucleotide and/or agonist or antagonist of the present invention can be used to treat, prevent, and/or diagnose infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated, prevented, and/or diagnosed. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, polypeptide or polynucleotide and/or agonist or antagonist of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.
Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention. Examples of viruses, include, but are not limited to Examples of viruses, include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivinis, Bimaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae, Picomaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additional specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines. In a further specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose AIDS.
Similarly, bacterial or fungal agents that can cause disease or symptoms and that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, include, but not limited to, the following Gram-Negative and Gram-positive bacteria and bacterial families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, and Salmonella paratyphi), Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis, Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g., Heamophilus influenza type B), Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal, Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcus pneumoniae and Group B Streptococcus). These bacterial or fingal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A and B), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections. Polynucleotides or polypeptides, agonists or antagonists of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat, prevent, and/or diagnose: tetanus, Diptheria, botulism, and/or meningitis type B.
Moreover, parasitic agents causing disease or symptoms that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, the following families or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), malaria, pregnancy complications, and toxoplasmosis. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used totreat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose malaria.
Preferably, treatment or prevention using a polypeptide or polynucleotide and/or agonist or antagonist of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.
Regeneration
A polynucleotide or polypeptide and/or agonist or antagonist of the present invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues. (See, Science 276:59-87 (1997).) The regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.
Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably, regeneration occurs without or decreased scarring. Regeneration also may include angiogenesis.
Moreover, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage. A polynucleotide or polypeptide and/or agonist or antagonist of the present invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated, prevented, and/or diagnosed include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.
Similarly, nerve and brain tissue could also be regenerated by using a polynucleotide or polypeptide and/or agonist or antagonist of the present invention to proliferate and differentiate nerve cells. Diseases that could be treated, prevented, and/or diagnosed using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic diseases, disorders, and/or conditions (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stoke). Specifically, diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated, prevented, and/or diagnosed using the polynucleotide or polypeptide and/or agonist or antagonist of the present invention.
Binding Activity
A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors),or small molecules.
Preferably, the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991).) Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds, or at least, a fragment of the receptor capable of being bound by the polypeptide (e.g., active site). In either case, the molecule can be rationally designed using known techniques.
Preferably, the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.
The assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.
Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.
Preferably, an ELISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody. The antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.
Additionally, the receptor to which a polypeptide of the invention binds can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in lmuun., 1(2), Chapter 5, (1991)). For example, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the polypeptides, for example, NIH3T3 cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptides. Transfected cells which are grown on glass slides are exposed to the polypeptide of the present invention, after they have been labeled. The polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase.
Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clones that encodes the putative receptor.
As an alternative approach for receptor identification, the labeled polypeptides can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptors of the polypeptides can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.
Moreover, the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”) may be employed to modulate the activities of polypeptides of the invention thereby effectively generating agonists and antagonists of polypeptides of the invention. See generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference). In one embodiment, alteration of polynucleotides and corresponding polypeptides of the invention may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into a desired polynucleotide sequence of the invention molecule by homologous, or site-specific, recombination. In another embodiment, polynucleotides and corresponding polypeptides of the invention may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of the polypeptides of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are family members. In further preferred embodiments, the heterologous molecule is a growth factor such as, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and glial-derived neurotrophic factor (GDNF).
Other preferred fragments are biologically active fragments of the polypeptides of the invention. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
Additionally, this invention provides a method of screening compounds to identify those which modulate the action of the polypeptide of the present invention. An example of such an assay comprises combining a mammalian fibroblast cell, a the polypeptide of the present invention, the compound to be screened and 3[H] thymidine under cell culture conditions where the fibroblast cell would normally proliferate. A control assay may be performed in the absence of the compound to be screened and compared to the amount of fibroblast proliferation in the presence of the compound to determine if the compound stimulates proliferation by determining the uptake of 3[H] thymidine in each case. The amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incorporation of 3[H] thymidine. Both agonist and antagonist compounds may be identified by this procedure.
In another method, a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the present invention is incubated with a labeled polypeptide of the present invention in the presence of the compound. The ability of the compound to enhance or block this interaction could then be measured. Alternatively, the response of a known second messenger system following interaction of a compound to be screened and the receptor is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to determine if the compound is a potential agonist or antagonist. Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.
All of these above assays can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat, prevent, and/or diagnose disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule. Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptides of the invention from suitably manipulated cells or tissues. Therefore, the invention includes a method of identifying compounds which bind to the polypeptides of the invention comprising the steps of: (a) incubating a candidate binding compound with the polypeptide; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with the polypeptide, (b) assaying a biological activity, and (b) determining if a biological activity of the polypeptide has been altered.
Also, one could identify molecules bind a polypeptide of the invention experimentally by using the beta-pleated sheet regions contained in the polypeptide sequence of the protein. Accordingly, specific embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, the amino acid sequence of each beta pleated sheet regions in a disclosed polypeptide sequence. Additional embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, any combination or all of contained in the polypeptide sequences of the invention. Additional preferred embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, the amino acid sequence of each of the beta pleated sheet regions in one of the polypeptide sequences of the invention. Additional embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, any combination or all of the beta pleated sheet regions in one of the polypeptide sequences of the invention.
Targeted Delivery
In another embodiment, the invention provides a method of delivering compositions to targeted cells expressing a receptor for a polypeptide of the invention, or cells expressing a cell bound form of a polypeptide of the invention.
As discussed herein, polypeptides or antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions. In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (including antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention (e.g., polypeptides of the invention or antibodies of the invention) in association with toxins or cytotoxic prodrugs.
By “toxin” is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the methods of the invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.
Drug Screening
Further contemplated is the use of the polypeptides of the present invention, or the polynucleotides encoding these polypeptides, to screen for molecules which modify the activities of the polypeptides of the present invention. Such a method would include contacting the polypeptide of the present invention with a selected compound(s) suspected of having antagonist or agonist activity, and assaying the activity of these polypeptides following binding.
This invention is particularly useful for screening therapeutic compounds by using the polypeptides of the present invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. One may measure, for example, the formulation of complexes between the agent being tested and a polypeptide of the present invention.
Thus, the present invention provides methods of screening for drugs or any other agents which affect activities mediated by the polypeptides of the present invention. These methods comprise contacting such an agent with a polypeptide of the present invention or a fragment thereof and assaying for the presence of a complex between the agent and the polypeptide or a fragment thereof, by methods well known in the art. In such a competitive binding assay, the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular agent to bind to the polypeptides of the present invention.
Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the polypeptides of the present invention, and is described in great detail in European Patent Application 84/03564, published on Sep. 13, 1984, which is incorporated herein by reference herein. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with polypeptides of the present invention and washed. Bound polypeptides are then detected by methods well known in the art. Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support.
This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding polypeptides of the present invention specifically compete with a test compound for binding to the polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic epitopes with a polypeptide of the invention.
The human CAN-12 polypeptides and/or peptides of the present invention, or immunogenic fragments or oligopeptides thereof, can be used for screening therapeutic drugs or compounds in a variety of drug screening techniques. The fragment employed in such a screening assay may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The reduction or abolition of activity of the formation of binding complexes between the ion channel protein and the agent being tested can be measured. Thus, the present invention provides a method for screening or assessing a plurality of compounds for their specific binding affinity with a CAN-12 polypeptide, or a bindable peptide fragment, of this invention, comprising providing a plurality of compounds, combining the CAN-12 polypeptide, or a bindable peptide fragment, with each of a plurality of compounds for a time sufficient to allow binding under suitable conditions and detecting binding of the CAN-12 polypeptide or peptide to each of the plurality of test compounds, thereby identifying the compounds that specifically bind to the CAN-12 polypeptide or peptide.
Methods of identifying compounds that modulate the activity of the novel human CAN-12 polypeptides and/or peptides are provided by the present invention and comprise combining a potential or candidate compound or drug modulator of calpain biological activity with an CAN-12 polypeptide or peptide, for example, the CAN-12 amino acid sequence as set forth in SEQ ID NOS:2, and measuring an effect of the candidate compound or drug modulator on the biological activity of the CAN-12 polypeptide or peptide. Such measurable effects include, for example, physical binding interaction; the ability to cleave a suitable calpain substrate; effects on native and cloned CAN-12-expressing cell line; and effects of modulators or other calpain-mediated physiological measures.
Another method of identifying compounds that modulate the biological activity of the novel CAN-12 polypeptides of the present invention comprises combining a potential or candidate compound or drug modulator of a calpain biological activity with a host cell that expresses the CAN-12 polypeptide and measuring an effect of the candidate compound or drug modulator on the biological activity of the CAN-12 polypeptide. The host cell can also be capable of being induced to express the CAN-12 polypeptide, e.g., via inducible expression. Physiological effects of a given modulator candidate on the CAN-12 polypeptide can also be measured. Thus, cellular assays for particular calpain modulators may be either direct measurement or quantification of the physical biological activity of the CAN-1-2 polypeptide, or they may be measurement or quantification of a physiological effect. Such methods preferably employ a CAN-12 polypeptide as described herein, or an overexpressed recombinant CAN-12 polypeptide in suitable host cells containing an expression vector as described herein, wherein the CAN-12 polypeptide is expressed, overexpressed, or undergoes upregulated expression.
Another aspect of the present invention embraces a method of screening for a compound that is capable of modulating the biological activity of a CAN-12 polypeptide, comprising providing a host cell containing an expression vector harboring a nucleic acid sequence encoding a CAN-12 polypeptide, or a functional peptide or portion thereof (e.g., SEQ ID NOS:2); determining the biological activity of the expressed CAN-12 polypeptide in the absence of a modulator compound; contacting the cell with the modulator compound and determining the biological activity of the expressed CAN-12 polypeptide in the presence of the modulator compound. In such a method, a difference between the activity of the CAN-12 polypeptide in the presence of the modulator compound and in the absence of the modulator compound indicates a modulating effect of the compound.
Essentially any chemical compound can be employed as a potential modulator or ligand in the assays according to the present invention. Compounds tested as calpain modulators can be any small chemical compound, or biological entity (e.g., protein, sugar, nucleic acid, lipid). Test compounds will typically be small chemical molecules and peptides. Generally, the compounds used as potential modulators can be dissolved in aqueous or organic (e.g., DMSO-based) solutions. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source. Assays are typically run in parallel, for example, in microtiter formats on microtiter plates in robotic assays. There are many suppliers of chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland), for example. Also, compounds may be synthesized by methods known in the art.
High throughput screening methodologies are particularly envisioned for the detection of modulators of the novel CAN-12 polynucleotides and polypeptides described herein. Such high throughput screening methods typically involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (e.g., ligand or modulator compounds). Such combinatorial chemical libraries or ligand libraries are then screened in one or more assays to identify those library members (e.g., particular chemical species or subclasses) that display a desired characteristic activity. The compounds so identified can serve as conventional lead compounds, or can themselves be used as potential or actual therapeutics.
A combinatorial chemical library is a collection of diverse chemical compounds generated either by chemical synthesis or biological synthesis, by combining a number of chemical building blocks (i.e., reagents such as amino acids). As an example, a linear combinatorial library, e.g., a polypeptide or peptide library, is formed by combining a set of chemical building blocks in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide or peptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
The preparation and screening of combinatorial chemical libraries is well known to those having skill in the pertinent art. Combinatorial libraries include, without limitation, peptide libraries (e.g. U.S. Pat. No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; and Houghton et al., 1991, Nature, 354:84-88). Other chemistries for generating chemical diversity libraries can also be used. Nonlimiting examples of chemical diversity library chemistries include, peptides (PCT Publication No. WO 91/019735), encoded peptides (PCT Publication No. WO 93/20242), random bio-oligomers (PCT Publication No. WO 92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc. Natl. Acad. Sci. USA, 90:6909-6913), vinylogous polypeptides (Hagihara et al., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J. Amer. Chem. Soc., 114:9217-9218), analogous organic synthesis of small compound libraries (Chen et al., 1994, J. Amer. Chem. Soc., 116:2661), oligocarbamates (Cho et al., 1993, Science, 261:1303), and/or peptidyl phosphonates (Campbell et al., 1994, J. Org. Chem., 59:658), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries (e.g., Vaughn et al., 1996, Nature Biotechnology, 14(3):309-314) and PCT/US96/10287), carbohydrate libraries (e.g., Liang et al., 1996, Science, 274-1520-1522) and U.S. Pat. No. 5,593,853), small organic molecule libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993, page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; and the like).
Devices for the preparation of combinatorial libraries are commercially available (e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition, a large number of combinatorial libraries are commercially available (e.g., ComGenex, Princeton, N.J.; Asinex, Moscow, Russia; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd., Moscow, Russia; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md., and the like).
In one embodiment, the invention provides solid phase based in vitro assays in a high throughput format, where the cell or tissue expressing an ion channel is attached to a solid phase substrate. In such high throughput assays, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to perform a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 96 modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay several different plates per day; thus, for example, assay screens for up to about 6,000-20,000 different compounds are possible using the described integrated systems.
In another of its aspects, the present invention encompasses screening and small molecule (e.g., drug) detection assays which involve the detection or identification of small molecules that can bind to a given protein, i.e., a CAN-12 polypeptide or peptide. Particularly preferred are assays suitable for high throughput screening methodologies.
In such binding-based detection, identification, or screening assays, a functional assay is not typically required. All that is needed is a target protein, preferably substantially purified, and a library or panel of compounds (e.g., ligands, drugs, small molecules) or biological entities to be screened or assayed for binding to the protein target. Preferably, most small molecules that bind to the target protein will modulate activity in some manner, due to preferential, higher affinity binding to functional areas or sites on the protein.
An example of such an assay is the fluorescence based thermal shift assay (3-Dimensional Pharmaceuticals, Inc., 3DP, Exton, Pa.) as described in U.S. Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano et al.; see also, J. Zimmerman, 2000, Gen. Eng. News, 20(8)). The assay allows the detection of small molecules (e.g., drugs, ligands) that bind to expressed, and preferably purified, ion channel polypeptide based on affinity of binding determinations by analyzing thermal unfolding curves of protein-drug or ligand complexes. The drugs or binding molecules determined by this technique can be further assayed, if desired, by methods, such as those described herein, to determine if the molecules affect or modulate function or activity of the target protein.
To purify a CAN-12 polypeptide or peptide to measure a. biological binding or ligand binding activity, the source may be a whole cell lysate that can be prepared by successive freeze-thaw cycles (e.g., one to three) in the presence of standard protease inhibitors. The CAN-12 polypeptide may be partially or completely purified by standard protein purification methods, e.g., affinity chromatography using specific antibody described infra, or by ligands specific for an epitope tag engineered into the recombinant CAN-12 polypeptide molecule, also as described herein. Binding activity can then be measured as described.
Compounds which are identified according to the methods provided herein, and which modulate or regulate the biological activity or physiology of the CAN-12 polypeptides according to the present invention are a preferred embodiment of this invention. It is contemplated that such modulatory compounds may be employed in treatment and therapeutic methods for treating a condition that is mediated by the novel CAN-12 polypeptides by administering to an individual in need of such treatment a therapeutically effective amount of the compound identified by the methods described herein.
In addition, the present invention provides methods for treating an individual in need of such treatment for a disease, disorder, or condition that is mediated by the CAN-12 polypeptides of the invention, comprising administering to the individual a therapeutically effective amount of the CAN-12-modulating compound identified by a method provided herein.
Antisense And Ribozyme (Antagonists)
In specific embodiments, antagonists according to the present invention are nucleic acids corresponding to the sequences contained in SEQ ID NO: 1, 23, 53, and/or 55, or the complementary strand thereof, and/or to nucleotide sequences contained a deposited clone. In one embodiment, antisense sequence is generated internally by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation. Antisense techniques are discussed for example, in Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research, 6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan et al., Science, 251:1300 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA.
For example, the use of c-myc and c-myb antisense RNA constructs to inhibit the growth of the non-lymphocytic leukemia cell line HL-60 and other cell lines was previously described. (Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments were performed in vitro by incubating cells with the oligoribonucleotide. A similar procedure for in vivo use is described in WO 91/15580. Briefly, a pair of oligonucleotides for a given antisense RNA is produced as follows: A sequence complimentary to the first 15 bases of the open reading frame is flanked by an EcoRl site on the 5 end and a HindIII site on the 3 end. Next, the pair of oligonucleotides is heated at 90° C. for one minute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5, 10 mM MgCl2, 100 MM dithiotlireitol (DTT) and 0.2 mM ATP) and then ligated to the EcoRl/Hind Im site of the retroviral vector PMV7 (WO 91/15580).
For example, the 5′ coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into receptor polypeptide.
In one embodiment, the antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding the antisense nucleic acid of the invention. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding a polypeptide of the invention, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature, 29:304-310 (1981), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell, 22:787-797 (1980), the herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445 (1981), the regulatory sequences of the metallothionein gene (Brinster et al., Nature, 296:39-42 (1982)), etc.
The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a gene of interest. However, absolute complementarity, although preferred, is not required. A sequence “complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded antisense nucleic acids of the invention, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA sequence of the invention it may contain and still form a stable duplex (or triplex as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
Oligonucleotides that are complementary to the 5′ end of the message, e.g., the 5′ untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3′ untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., Nature, 372:333-335 (1994). Thus, oligonucleotides complementary to either the 5′- or 3′-non-translated, non-coding regions of a polynucleotide sequence of the invention could be used in an antisense approach to inhibit translation of endogenous mRNA. Oligonucleotides complementary to the 5′ untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5′-, 3′- or coding region of mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
The polynucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci., 84:648-652 (1987); PCT Publication NO: WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication NO: WO89/10134, published Apr. 25, 1988), hybridization-triggered cleavage agents. (See, e.g., Krol et al., BioTechniques, 6:958-976 (1988)) or intercalating agents. (See, e.g., Zon, Pharm. Res., 5:539-549 (1988)). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacefic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
In yet another embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al., Nucl. Acids Res., 15:6625-6641 (1987)). The oligonucleotide is a 2-0-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215:327-330 (1987)).
Polynucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (Nucl. Acids Res., 16:3209 (1988)), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A., 85:7448-7451 (1988)), etc.
While antisense nucleotides complementary to the coding region sequence of the invention could be used, those complementary to the transcribed untranslated region are most preferred.
Potential antagonists according to the invention also include catalytic RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364, published Oct. 4, 1990; Sarver et al, Scienice, 247:1222-1225 (1990). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy muRNAs corresponding to the polynucleotides of the invention, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5′-UG-3′. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature, 334:585-591 (1988). There are numerous potential hammerhead ribozyme cleavage sites within each nucleotide sequence disclosed in the sequence listing. Preferably, the ribozyme is engineered so that the cleavage recognition site is located near the 5′ end of the mRNA corresponding to the polynucleotides of the invention; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
As in the antisense approach, the ribozymes of the invention can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express the polynucleotides of the invention in vivo. DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA. A preferred method of delivery involves using a DNA construct “encoding” the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous messages and inhibit translation. Since ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
Antagonist/agonist compounds may be employed to inhibit the cell growth and proliferation effects of the polypeptides of the present invention on neoplastic cells and tissues, i.e. stimulation of angiogenesis of tumors, and, therefore, retard or prevent abnormal cellular growth and proliferation, for example, in tumor formation or growth.
The antagonist/agonist may also be employed to prevent hyper-vascular diseases, and prevent the proliferation of epithelial lens cells after extracapsular cataract surgery. Prevention of the mitogenic activity of the polypeptides of the present invention may also be desirous in cases such as restenosis after balloon angioplasty.
The antagonist/agonist may also be employed to prevent the growth of scar tissue during wound healing.
The antagonist/agonist may also be employed to treat, prevent, and/or diagnose the diseases described herein.
Thus, the invention provides a method of treating or preventing diseases, disorders, and/or conditions, including but not limited to the diseases, disorders, and/or conditions listed throughout this application, associated with overexpression of a polynucleotide of the present invention by administering to a patient (a) an antisense molecule directed to the polynucleotide of the present invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention.
invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention.
Biotic Associations
A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase the organisms ability, either directly or indirectly, to initiate and/or maintain biotic associations with other organisms. Such associations may be symbiotic, nonsymbiotic, endosymbiotic, macrosymbiotic, and/or microsymbiotic in nature. In general, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase the organisms ability to form biotic associations with any member of the fungal, bacterial, lichen, mycorrhizal, cyanobacterial, dinoflaggellate, and/or algal, kingdom, phylums, families, classes, genuses, and/or species.
The mechanism by which a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase the host organisms ability, either directly or indirectly, to initiate and/or maintain biotic associations is variable, though may include, modulating osmolarity to desirable levels for the symbiont, modulating pH to desirable levels for the symbiont, modulating secretions of organic acids, modulating the secretion of specific proteins, phenolic compounds, nutrients, or the increased expression of a protein required for host-biotic organisms interactions (e.g., a receptor, ligand, etc.). Additional mechanisms are known in the art and are encompassed by the invention (see, for example, “Microbial Signalling and Communication”, eds., R. England, G. Hobbs, N. Bainton, and D. McL. Roberts, Cambridge University Press, Cambridge, (1999); which is hereby incorporated herein by reference).
In an alternative embodiment, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may decrease the host organisms ability to form biotic associations with another organism, either directly or indirectly. The mechanism by which a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may decrease the host organisms ability, either directly or indirectly, to initiate and/or maintain biotic associations with another organism is variable, though may include, modulating osmolarity to undesirable levels, modulating pH to undesirable levels, modulating secretions of organic acids, modulating the secretion of specific proteins, phenolic compounds, nutrients, or the decreased expression of a protein required for host-biotic organisms interactions (e.g., a receptor, ligand, etc.). Additional mechanisms are known in the art and are encompassed by the invention (see, for example, “Microbial Signalling and Communication”, eds., R. England, G. Hobbs, N. Bainton, and D. McL. Roberts, Cambridge University Press, Cambridge, (1999); which is hereby incorporated herein by reference).
The hosts ability to maintain biotic associations with a particular pathogen has significant implications for the overall health and fitness of the host. For example, human hosts have symbiosis with enteric bacteria in their gastrointestinal tracts, particularly in the small and large intestine. In fact, bacteria counts in feces of the distal colon often approach 1012 per milliliter of feces. Examples of bowel flora in the gastrointestinal tract are members of the Enterobacteriaceae, Bacteriodes, in addition to a-hemolytic streptococci, E. coli, Bifobacteria, Anaerobic cocci, Eubacteria, Costridia, lactobacilli, and yeasts. Such bacteria, among other things, assist the host in the assimilation of nutrients by breaking down food stuffs not typically broken down by the hosts digestive system, particularly in the hosts bowel. Therefore, increasing the hosts ability to maintain such a biotic association would help assure proper nutrition for the host.
Aberrations in the enteric bacterial population of mammals, particularly humans, has been associated with the following disorders: diarrhea, ileus, chronic inflammatory disease, bowel obstruction, duodenal diverticula, biliary calculous disease, and malnutrition. A polynucleotide or polypeptide and/or agonist or antagonist of the present invention are useful for treating, detecting, diagnosing, prognosing, and/or ameliorating, either directly or indirectly, and of the above mentioned diseases and/or disorders associated with aberrant enteric flora population.
The composition of the intestinal flora, for example, is based upon a variety of factors, which include, but are not limited to, the age, race, diet, malnutrition, gastric acidity, bile salt excretion, gut motility, and immune mechanisms. As a result, the polynucleotides and polypeptides, including agonists, antagonists, and fragments thereof, may modulate the ability of a host to form biotic associations by affecting, directly or indirectly, at least one or more of these factors.
Although the predominate intestinal flora comprises anaerobic organisms, an underlying percentage represents aerobes (e.g., E. coli). This is significant as such aerobes rapidly become the predominate organisms in intraabdominal infections—effectively becoming opportunistic early in infection pathogenesis. As a result, there is an intrinsic need to control aerobe populations, particularly for immune compromised individuals.
In a preferred embodiment, a polynucleotides and polypeptides, including agonists, antagonists, and fragments thereof, are useful for inhibiting biotic associations with specific enteric symbiont organisms in an effort to control the population of such organisms.
Biotic associations occur not only in the gastrointestinal tract, but also on an in the integument. As opposed to the gastrointestinal flora, the cutaneous flora is comprised almost equally with aerobic and anaerobic organisms. Examples of cutaneous flora are members of the gram-positive cocci (e.g., S. aureus, coagulase-negative staphylococci, micrococcus, M. sedentarius), gram-positive bacilli (e.g., Corynebacterium species, C. minutissimum, Brevibacterium species, Propoionibacterium species, P. acnes), gram-negative bacilli (e.g., Acinebacter species), and fuingi (Pityrosporum orbiculare). The relatively low number of flora associated with the integument is based upon the inability of many organisms to adhere to the skin. The organisms referenced above have acquired this unique ability. Therefore, the polynucleotides and polypeptides of the present invention may have uses which include modulating the population of the cutaneous flora, either directly or indirectly.
Aberrations in the cutaneous flora are associated with a number of significant diseases and/or disorders, which include, but are not limited to the following: impetigo, ecthyma, blistering distal dactulitis, pustules, folliculitis, cutaneous abscesses, pitted keratolysis, trichomycosis axcillaris, dermatophytosis complex, axillary odor, erthyrasma, cheesy foot odor, acne, tinea versicolor, seborrheic dermititis, and Pityrosporum folliculitis, to name a few. A polynucleotide or polypeptide and/or agonist or antagonist of the present invention are useful for treating, detecting, diagnosing, prognosing, and/or ameliorating, either directly or indirectly, and of the above mentioned diseases and/or disorders associated with aberrant cutaneous flora population.
Additional biotic associations, including diseases and disorders associated with the aberrant growth of such associations, are known in the art and are encompassed by the invention. See, for example, “Infectious Disease”, Second Edition, Eds., S. L., Gorbach, J. G., Bartlett, and N. R., Blacklow, W. B. Saunders Company, Philadelphia, (1998); which is hereby incorporated herein by reference).
Pheromones
In another embodiment, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase the organisms ability to synthesize and/or release a pheromone. Such a pheromone may, for example, alter the organisms behavior and/or metabolism.
A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may modulate the biosynthesis and/or release of pheromones, the organisms ability to respond to pheromones (e.g., behaviorally, and/or metabolically), and/or the organisms ability to detect pheromones. Preferably, any of the pheromones, and/or volatiles released from the organism, or induced, by a polynucleotide or polypeptide and/or agonist or antagonist of the invention have behavioral effects the organism.
Other Activities
The polypeptide of the present invention, as a result of the ability to stimulate vascular endothelial cell growth, may be employed in treatment for stimulating re-vascularization of ischemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and other cardiovascular conditions. These polypeptide may also be employed to stimulate angiogenesis and limb regeneration, as discussed above.
The polypeptide may also be employed for treating wounds due to injuries, burns, post-operative tissue repair, and ulcers since they are mitogenic to various cells of different origins, such as fibroblast cells and skeletal muscle cells, and therefore, facilitate the repair or replacement of damaged or diseased tissue.
The polypeptide of the present invention may also be employed stimulate neuronal growth and to treat, prevent, and/or diagnose neuronal damage which occurs in certain neuronal disorders or neuro-degenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complex. The polypeptide of the invention may have the ability to stimulate chondrocyte growth, therefore, they may be employed to enhance bone and periodontal regeneration and aid in tissue transplants or bone grafts.
The polypeptide of the present invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth.
The polypeptide of the invention may also be employed for preventing hair loss, since FGF family members activate hair-forming cells and promotes melano-cyte growth. Along the same lines, the polypeptides of the present invention may be employed to stimulate growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines.
The polypeptide of the invention may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues.
The polypeptide of the present invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos.
The polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.
The polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery). Similarly, polypeptides or polynucleotides and/or agonist or antagonists of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.
Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, caricadic rhythms, depression (including depressive diseases, disorders, and/or conditions), tendency for violence, tolerance for pain, reproductive capabilities (preferably by Activin or Inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.
Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used to increase the efficacy of a pharmaceutical composition, either directly or indirectly. Such a use may be administered in simultaneous conjunction with said pharmaceutical, or separately through either the same or different route of administration (e.g., intravenous for the polynucleotide or polypeptide of the present invention, and orally for the pharmaceutical, among others described herein.).
Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used to prepare individuals for extraterrestrial travel, low gravity environments, prolonged exposure to extraterrestrial radiation levels, low oxygen levels, reduction of metabolic activity, exposure to extraterrestrial pathogens, etc. Such a use may be administered either prior to an extraterrestrial event, during an extraterrestrial event, or both. Moreover, such a use may result in a number of beneficial changes in the recipient, such as, for example, any one of the following, non-limiting, effects: an increased level of hematopoietic cells, particularly red blood cells which would aid the recipient in coping with low oxygen levels; an increased level of B-cells, T-cells, antigen presenting cells, and/or macrophages, which would aid the recipient in coping with exposure to extraterrestrial pathogens, for example; a temporary (i.e., reversible) inhibition of hematopoietic cell production which would aid the recipient in coping with exposure to extraterrestrial radiation levels; increase and/or stability of bone mass which would aid the recipient in coping with low gravity environments; and/or decreased metabolism which would effectively facilitate the recipients ability to prolong their extraterrestrial travel by any one of the following, non-limiting means: (i) aid the recipient by decreasing their basal daily energy requirements; (ii) effectively lower the level of oxidative and/or metabolic stress in recipient (i.e., to enable recipient to cope with increased extraterrestial radiation levels by decreasing the level of internal oxidative/metabolic damage acquired during normal basal energy requirements; and/or (iii) enabling recipient to subsist at a lower metabolic temperature (i.e., cryogenic, and/or sub-cryogenic environment).
Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.

Other Preferred Embodiments

Further preferred is a method of making a recombinant vector comprising inserting any of the above isolated nucleic acid molecule(s) into a vector. Also preferred is the recombinant vector produced by this method. Also preferred is a method of making a recombinant host cell comprising introducing the vector into a host cell, as well as the recombinant host cell produced by this method.
Also preferred is a method of making an isolated polypeptide comprising culturing this recombinant host cell under conditions such that said polypeptide is expressed and recovering said polypeptide. Also preferred is this method of making an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a protein comprising an amino acid sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:2, 24, 54, and/or 56 wherein Y is an integer set forth in Table I and said position of the “Total AA of ORF” of SEQ ID NO:2, 24, 54, and/or 56 is defined in Table I; and an amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I. The isolated polypeptide produced by this method is also preferred.
Also preferred is a method of treatment of an individual in need of an increased level of a protein activity, which method comprises administering to such an individual a pharmaceutical composition comprising an amount of an isolated polypeptide, polynucleotide, or antibody of the claimed invention effective to increase the level of said protein activity in said individual.
Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

REFERENCES

Altschul S F; Gish W; Miller W; Myers E W; Lipman D J Basic local alignment search tool. J. Mol. Biol. 215:403-10 (1990).
Bartlett et al. Molecular Recognition in Chemical and Biological Problems Special Publication, Royal Chem. Soc. 78:182-196 (1989).
Bohm H-J, LUDI: rule-based automatic design of new substituents for enzyme inhibitor leads. J. Comp. Aid. Molec. Design 6:61-78 (1992)
Cardozo T; Totrov M; Abagyan R Homology modeling by the ICM method. Proteins 23:403-14 (1995).
Goodford, P. J. A computational procedure for determining energetically favorable binding sites on biologically important macromolecules. J. Med. Chem. 28:849-857 (1985)
Goodsell, D. S. and Olsen, A. J. Automated docking of substrates to proteins by simulated annealing. Proteins 8:195-202 (1990)
Greer J Comparative modeling of homologous proteins. Methods Enzymol 202:239-52 (1991).
Hendlich M; Lackner P; Weitckus S; Floeckner H; Froschauer R; Gottsbacher K; Casari G; Sippl M J Identification of native protein folds amongst a large number of incorrect models. The calculation of low energy conformations from potentials of mean force. J. Mol. Biol. 216:167-80 (1990).
Hosfield, C., Elce, J. S., Davies, P., Jia, Z., Crystal structure of calpain reveals the structural basis for Ca²⁺-dependent protease activity and a novel mode of enzyme activation., EMBO J., 18, 6880-6889 (1999).
Kuntz I D, Blaney J M, Oatley S J, Langridge R, Ferrin T E. A geometric approach to macromolecule-ligand interactions. J. Mol. Biol. 161:269-288 (1982)
Lesk, A. M., Boswell, D. R., Homology Modeling: Inferences from Tables of Aligned Sequences. Curr. Op. Struc. Biol. 2:242-247 (1992)
Martin, Y. C. 3D database searching in drug design. J. Med. Chem. 35:2145-2154 (1992)
Pearson W R Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol 183:63-98 (1990).
Sali A; Potterton L; Yuan F; van Vlijmen H; Karplus M Evaluation of comparative protein modeling by MODELLER. PROTEINS 23:318-26 (1995).
Strobl, S., Femandez-Catalan, C., Braun, M., Huber, R, Masumoto, H., Nakagawa., K, Irie, A., Sorimachi, S., Bourenkow, G., Bartunik, H., Suzuki, K, Bode, W., The crystal structure of calcium-free human mcalpain suggests an electrostatic switch mechanism for activation by calcium. Proc. Natl. Acad. Sci., 2000, 97(2), 588-592.

EXAMPLES

Description of the Preferred Embodiments

Example 1

Bioinformatics Analysis

To search for novel protease inhibitors, a Hidden-Markov Model (HMM) of cysteine proteases (obtained from the Pfam database in Sanger center) was used to search against the human genomic sequence database using a computer program called GENEWISEDB. Genomic sequences that were found to have a GENEWISEDB matching score of more than 15 were selected for further analysis. The genomic sequence contained in BAC (bacteria artificial chromosome) AC015980 (Genbank Accession No. AC015980) was found to contain a putative exon sequence. The portion of the sequence from AC015980 that matched was extracted and back-searched against the Genbank non-redundant protein database using the BLASTX program (SEQ ID NO:26). The most similar protein sequence, the human CAN5 protein (SEQ ID NO:4), was used as a template to predict more exons from AC015980 using the GENEWISEDB program (see FIG. 7). The final predicted exons were assembled and a full length clone of the gene was obtained using the predicted exon sequences. The protein sequence was found to have significant sequence homology with a family of known proteases. Based on the sequence, structure and known calpain signature sequences, the novel gene was determined to represent a novel human calpain protease and have provisionally named the gene CAN-12 (calcium activated neutral protease 12) and the protease itself as calpain 12.

Example 2

Method for Constructing a Size Fractionated Brain and Testis cDNA Library

Brain and testis poly A+ RNA was purchased from Clontech and converted into double stranded cDNA using the SuperScript™ Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies) except that no radioisotope was incorporated in either of the cDNA synthesis steps and that the cDNA was fractionated by HPLC. This was accomplished on a TransGenomics HPLC system equipped with a size exclusion column (TosoHass) with dimensions of 7.8 mm×30 cm and a particle size of 10 um. Tris buffered saline was used as the mobile phase and the column was run at a flow rate of 0.5 mL/min.
The resulting chromatograms were analyzed to determine which fractions should be pooled to obtain the largest cDNA's; generally fractions that eluted in the range of 12 to 15 minutes were pooled. The cDNA was precipitated prior to ligation into the Sal I/Not I sites in the pSport vector supplied with the kit. Using a combination of PCR with primers to the ends of the vector and Sal I/Not I restriction enzyme digestion of mini-prep DNA, it was determined that the average insert size of the library was greater the 3.5 Kb. The overall complexity of the library was greater that 10⁷independent clones. The library was amplified in semi-solid agar for 2 days at 30° C. An aliquot (200 microliters) of the amplified library was inoculated into a 200 ml culture for single-stranded DNA isolation by super-infection with a f1 helper phage. After overnight growth, the released phage particles with precipitated with PEG and the DNA isolated with proteinase K, SDS and phenol extractions. The single stranded circular DNA was concentrated by ethanol precipitation and used for the cDNA capture experiments.

Example 3

Method of Cloning the Novel Human CAN-12 Calpain

Using the predicted exon genomic sequence from bac AC015980 (FIG. 7; SEQ ID NO:13, 14, 15, 16, 17, and 26) an antisense 80 bp oligonucleotide with biotin on the 5′ end was designed with the following sequence;

(SEQ ID NO:18)

5′bAGGGAGCCACTGCCGATGGAGCTCAGGGTGGCCGGGAAGCTGGTGTC

TTCAAAGAGGCAGCCATTCCTCAGGCACTC-3′
One microliter (one hundred and fifty nanograms) of the biotinylated oligonucleotide was added to six microliters (six micrograms) of a mixture of single-stranded covalently closed circular liver, brain, testis, and spleen cDNA libraries and seven microliters of 100% formamide in a 0.5 ml PCR tube. The library was a mixture of the brain and testis cDNA library referenced in Example 2, in addition to, commercially available liver and spleen cDNA libraries from Life Technologies (Rockville, Md.). The mixture was heated in a thermal cycler to 95° C. for 2 mins. Fourteen microliters of 2× hybridization buffer (50% formamide, 1.5 M NaCl, 0.04 M NaPO₄, pH 7.2, 5 mM EDTA, 0.2% SDS) was added to the heated probe/cDNA library mixture and incubated at 42° C. for 26 hours. Hybrids between the biotinylated oligonucleotide and the circular cDNA were isolated by diluting the hybridization mixture to 220 microliters in a solution containing 1 M NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, pH 8.0 and adding 125 microliters of streptavidin magnetic beads. This solution was incubated at 42° C. for 60 mins, mixing every 5 mins to resuspend the beads. The beads were separated from the solution with a magnet and the beads washed three times in 200 microliters of 0.1×SSPE, 0.1% SDS at 45° C.
The single stranded cDNAs were released from the biotinlyated oligonucleotide/streptavidin magnetic bead complex by adding 50 microliters of 0.1 N NaOH and incubating at room temperature for 10 mins. Six microliters of 3 M Sodium Acetate was added along with 15 micrograms of glycogen and the solution ethanol precipitated with 120 microliters of 100% ethanol. The DNA was resuspended in 12 microliters of TE (10 mM Tris-HCl, pH 8.0), 1 mM EDTA, pH 8.0). The single stranded cDNA was converted into double strands in a thermal cycler by mixing 5 microliters of the captured DNA with 1.5 microliters 10 micromolar standard SP6 primer (homologous to a sequence on the cDNA cloning vector) and 1.5 microliters of 10×PCR buffer. The mixture was heated to 95° C. for 20 seconds then ramped down to 59° C. At this time 15 microliters of a repair mix, that was preheated to 70° C. (Repair mix contains 4 microliters of 5 mM dNTPs (1.25 mM each), 1.5 microliters of 10×PCR buffer, 9.25 microliters of water, and 0.25 microliters of Taq polymerase). The solution was ramped back to 73° C. and incubated for 23 mins. The repaired DNA was ethanol precipitate and resuspended in 10 microliters of TE. Two microliters were electroporated in E. coli DH12S cells and resulting colonies were screen by PCR, using a primer pair designed from the genomic exonic sequence to identify the proper cDNAs.
Oligonucleotides used to identity the cDNA by PCR.

AC015980-L1 GACTTTGAGGCCCTGCTG (SEQ ID NO: 19)
AC015980-R1 ACAGGAACCCAGTTCCCATA (SEQ ID NO:20)

A single cDNA clone was positive by PCR. The insert was sized and the clone was sequenced. The sequence of the cDNA clone revealed an unspliced intron, in addition to a three base-pair deletion (SEQ ID NO: 1). Further attempts to obtain more clones with this method were unsuccessful, so an RT-PCR cloning approach was applied.
The nucleotide sequence and the encoded polypeptide for CAN-12 containing the unspliced intron and three base-pair deletion is shown in FIGS. 1A-E (SEQ ID NO: 1).

Example 4

Method of Cloning the Novel Human CAN-12 Calpain via RT-PCR

The cDNA was amplified from testis stranded cDNA and spinal cord first strand cDNA using the following RT-PCR primer pair in a standard PCR reaction (25 ng of DNA template were added to the reaction mixture along with each oligonucleotide primer at a final concentration of 0.2 μM each. The total volume of the reactions was 50 μL).

RT-PCR CACCTGCCATGTCTCTGTG SEQ ID NO:21

Sense

PRIMER

RT-PCR GATTATAACAAGGTGGTGTTGAAGA (SEQ ID NO:22)

Antisense

PRIMER
The thermal cycling conditions for the PCR were as follows:

96° C. 2 minutes

Then 45 cycles of:

94° C. 30 seconds

55° C. 30 seconds

72° C. 3 minutes

Then one cycle of:

72° C. 10 minutes

4° C. hold
The PCR was then subjected to electrophoresis on a 1% agarose gel. Bands on the gel were visualized of the predicted size. The bands were excised from the gel with a razor blade.
The PCR products were then extracted from the agarose gel slice using the Qiagen QIAquick™ Gel Extraction kit. Briefly, 3 volumes of buffer QG are added to the gel slice. The mixture is incubated at 50° C. until the agarose is melted. Then one volume of isopropanol is added. The sample is applied to a QIAquick spin column and centrifuged for 1 minute at high speed. The DNA binds to the column. The column is washed by applying 750 μL of buffer PE to and centrifuging for 1 minute. The column is then dried by spinning for an additional minute at high speed. The DNA is eluted from the column by applying 30 μL of elution buffer (buffer EB), letting the column stand for 1 minute, then centrifuging the column at high speed for 1 minute. The eluate is collected in a microcentrifuge tube.
Next, a ‘TA’ cloning procedure was used to insert the amplified fragment into a plasmid vector. In order to use the ‘TA’ cloning strategy, the PCR amplicon must have a 3+ ‘A’ overhang which is generated by Taq polymerase. Since a high fidelity, proofreading enzyme was used for the PCR amplification, the proofreading properties of the enzyme mix cause the ‘A’ overhang to be removed. Therefore, before the ‘TA’ cloning could be done, ‘A’ overhangs had to be added to the PCR product. To do this, the PCR product was incubated for 15 minutes at 72° C. in a mixture containing 5 units of Taq polymerase, 1×PCR buffer and 0.2 mM dATP (all from Roche). The Taq polymerase is from Thermus aquaticus BM, recombinant E. coli. The 10× PCR buffer contains 100 mM Tris-HCl, 15 mM MgCl₂, 500 mM KCl, pH 8.3.
The PCR products with added 3′ ‘A’ overhangs was then immediately used for ‘TA’ cloning. To do this, the TOPO TA Cloning® Kit for Sequencing from Invitrogen was used.
The following reaction mixture was set up:

- −4 μL PCR product
- −1 μL Salt Solution
- −1 μL pCR®4-TOPO® vector
  This was incubated at room temperature for 5 minutes.

Then 2 μl of this reaction were added to a vial of TOP10 One Shot® chemically competent E. Coli. This was incubated on ice for 5 minutes. The cells were then heat shocked at 42° C. for 30 seconds. Cells were transferred to ice for another 5 minutes. 250 μl of room temperature S.O.C. medium was added to the cells. The cells were then incubated at 37° C. for 1 hour with shaking for aeration. 50 μL of cells were spread on selective plates containing 50 82 g/μL carbenicillin and incubated at 37° C. overnight. A more detailed protocol for this kit from Invitrogen is available from their website (http://www.invitrogen.com).
The next step was to screen colonies that grew on the selective plates for positive clones. This was done by growing 7 colonies from the testis PCR and 7 from the spinal cord PCR overnight in 4 mL of LB broth containing 50 μg/μL carbenicillin. The plasmid DNA was then isolated from the bacteria using the Qiagen QIAquick Spin Miniprep Kit. Protocols for this are available from the Qiagen company web site (http:H/www.qiagen.com).
Once the plasmid DNA was purified, a restriction digest analysis was performed to determine if the clones were correct. A restriction enzyme digestion was performed with NotI and SpeI restriction endonucleases, using the suggested buffer and 5 μl of the purified plasmid, 5 units of enzyme in a total volume of 20 μl. The mixture was incubated at 37° C. for 2 hours. The digest was visualized by electrophoretic separation on a 1% agarose gel stained with ethidium bromide. From this analysis it was apparent the plasmids contained one size insert corresponding to the predicted insert size of the transcript. Two clones from each PCR reaction were sequenced using Applied Biosystems BigDye™ dideoxy terminator cycle sequencing on an Applied Biosystems 3700 capillary array DNA sequencer.
The above method resulted in several positive clones. After sequencing, it was determined that all of them had an unspliced intron that introduced a stop codon, with the exception of clone 1 e (CAN-12v1; FIGS. 8A-C; SEQ ID NO:53). Clone 1 e was correct, except for it was missing 3 amino acids in the middle of the protein (See FIGS. 10A-B). It is thought that these three amino acids may represent an exon of the CAN-12v1 polynucleotide.
Although the other clones contained the unspliced intron, one of the clones, clone 1 b, had the missing 3 amino acids. Thus, clone 1 e and clone 1 b were recombinately combined together effectively cutting out the region of clone 1 e that was missing the terminal three amino acids, and replacing it with the same region from clone 1 b which did have the three amino acids. The resulting recombinant clone is named clone 1 e 1 b (CAN-12v2), and is provided in FIGS. 9A-C (SEQ ID NO:55).
The method of recombinately combining clone 1 e and clone 1 b are provided below. The part of clone 1 e that had the missing exon was excised by restriction digestion with unique cutting enzymes BamHI and Aval. The corresponding fragment with the small exon (three amino acids) from clone 1 b was excised with the same enzymes and inserted into clone 1 e. This yielded the complete correct coding sequence that was predicted. The clone was called 1 e 1 b (CAN-12v2; FIGS. 9A-C; SEQ ID NO:55). CAN-12v2 is believed to represent the true physioligical form of CAN-12.

Example 5

Expression Profiling of the Novel Human CAN-12 Calpain

The same PCR primer pair that was used to identify the novel CAN-12 cDNA clones via RT-PCR (SEQ ID NO: 21 and 22) was used to measure the steady state levels of mRNA by quantitative PCR. Briefly, first strand cDNA was made from commercially available mRNA. The relative amount of cDNA used in each assay was determined by performing a parallel experiment using a primer pair for a gene expressed in equal amounts in all tissues, cyclophilin. The cyclophilin primer pair detected small variations in the amount of cDNA in each sample and these data were used for normalization of the data obtained with the primer pair for the novel CAN-12. The PCR data was converted into a relative assessment of the difference in transcript abundance amongst the tissues tested and the data is presented in FIG. 4. Transcripts corresponding to CAN-12 were expressed highly in the spinal cord; significantly in lymph node, thymus, and to a lesser extent, in spleen.

Example 6

Method of Assessing the Expression Profile of the Novel CAN-12 Polypeptides of the Present Invention using Expanded mRNA Tissue and Cell Sources

Total RNA from tissues was isolated using the TriZol protocol (Invitrogen) and quantified by determining its absorbance at 260 nM. An assessment of the 18s and 28s ribosomal RNA bands was made by denaturing gel electrophoresis to determine RNA integrity.
The specific sequence to be measured was aligned with related genes found in GenBank to identity regions of significant sequence divergence to maximize primer and probe specificity. Gene-specific primers and probes were designed using the ABI primer express software to amplify small amplicons (150 base pairs or less) to maximize the likelihood that the primers function at 100% efficiency. All primer/probe sequences were searched against Public Genbank databases to ensure target specificity. Primers and probes were obtained from ABI.

For CAN-12, the primer probe sequences were as follows


Forward Primer	5′-TCGTGCCCTGCATATTGGA-3′
	(SEQ ID NO:143)

Reverse Primer	5′-AAAAGATGTGCTTCCTGGAGAAGA-3′
	(SEQ ID NO:144)

TaqMan Probe	5′-CCCACCAGAAGTCAGAGTTCGTCCTCAG-3′
	(SEQ ID NO:145)

DNA Contamination

To access the level of contaminating genomic DNA in the RNA, the RNA was divided into 2 aliquots and one half was treated with Rnase-free Dnase (Invitrogen). Samples from both the Dnase-treated and non-treated were then subjected to reverse transcription reactions with (RT+) and without (RT−) the presence of reverse transcriptase. TaqMan assays were carried out with gene-specific primers (see above) and the contribution of genomic DNA to the signal detected was evaluated by comparing the threshold cycles obtained with the RT+/RT− non-Dnase treated RNA to that on the RT+/RT− Dnase treated RNA. The amount of signal contributed by genomic DNA in the Dnased RT− RNA must be less that 10% of that obtained with Dnased RT+ RNA. If not the RNA was not used in actual experiments.
Reverse Transcription reaction and Sequence Detection
100 ng of Dnase-treated total RNA was annealed to 2.5 μM of the respective gene-specific reverse primer in the presence of 5.5 mM Magnesium Chloride by heating the sample to 72° C. for 2 min and then cooling to 55° C. for 30 min. 1.25 U/μl of MuLv reverse transcriptase and 500 μM of each DNTP was added to the reaction and the tube was incubated at 37° C. for 30 min. The sample was then heated to 90° C. for 5 min to denature enzyme.
Quantitative sequence detection was carried out on an ABI PRISM 7700 by adding to the reverse transcribed reaction 2.5 μM forward and reverse primers, 500 μM of each dNTP, buffer and 5U AmpliTaq Gold™. The PCR reaction was then held at 94° C. for 12 min, followed by 40 cycles of 94° C. for 15 sec and 60° C. for 30 sec.
Data Handling
The threshold cycle (Ct) of the lowest expressing tissue (the highest Ct value) was used as the baseline of expression and all other tissues were expressed as the relative abundance to that tissue by calculating the difference in Ct value between the baseline and the other tissues and using it as the exponent in 2^(ΔCt)
The expanded expression profile of the CAN-12 polypeptide, is provided in FIG. 12 and described elsewhere herein.

Example 7

Method of Measuring the Protease Activity of CAN-12 Polypeptides

Protease activity of the CAN-12 polypeptide are measured by following the inhibition of proteolytic activity in cells, tissues, and/or in in vitro assays. Cysteine proteases of the calpain family (of which the present invention is a member) catalyze the hydrolysis of peptide, amide, ester, thiol ester and thiono ester bonds. Any assay that measures cleavage of these bonds can be used to quantitate enzymatic activity. In vitro assays for measuring protease activity using synthetic peptide fluorescent, spectrophotometric either through the use of single substrates (see below for examples), and fluorescence resonance transfer assays are well described in the art, as single substrates or as part of substrate libraries (Backes et al., 2000; Knight, C. G. Fluorimetric Assays of Proteolytic Enzymes. Meth. Enzymol. 248: 18-34 (1995)). In addition proteolytic activity is measured by following production of peptide products. Such approaches are well known to those familiar with the art (reviewed in McGeehan, G. M., Bickett, D. M., Wiseman, J. S., Green, M., Berman, Meth. Enzymol. 248: 35-46 (1995))
A complete set of protocols that have been used to evaluate calpain activity and are provided in Calpain Methods and Protocols John Elce ed. In Meth. Mol. Biol. Volume 144, 2000 (Humana Press, Totowa, N.J.).
Inhibitor Identification
Early work on calpain inhibitors produced nonselective enzyme inhibitors. Peptidyl aldehydes such as leupeptin and antipain inhibit calpain but also other proteases including serine proteases. Irreversible inhibitors such as the E64 family have also been studied, and peptidyl halomethanes and diazomethanes have long been used as protease inhibitors (Hayes et al., Drug News Perspect 11:215-222, 1998). Given the multiple therapeutic indications for the inhibition of calpain it appears that the achievement of selective modulators including specific inhibitors of this enzyme is an important goal.
The CAN-12 may be incubated with potential inhibitors (preferably small molecule inhibitors or antibodies provided elsewhere herein) for different times and with varying concentrations. Residual protease activity could then be measured according to any appropriate means known in the art. Enzyme activity in the presence of control may be expressed as fraction of control and curve fit to pre-incubation time and serpin concentration to determine inhibitory parameters including concentration that half maximally inhibits the enzyme activity.
Non-limiting examples of in vitro protease assays are well described in the art. Non-limiting examples of a spectrophotometric protease assays are the thrombin and tryptase assays measuring time-dependent optical density change followed at 405 nm using a kinetic microplate reader (Molecular Devices UVmax)(Balasubramanian, et al., Active site-directed synthetic thrombin inhibitors: synthesis, in vitro and in vivo activity profile of BMY 44621 and analogs an examination of the role of the amino group in the D-Phe-Pro-Arg-H series. J. Med. Chem. 36:300-303 (1993); and Combrink et al., Novel 1,2-Benzisothiazol-3-one-1,1-dioxide Inhibitors of Human Mast Cell Tryptase. J. Med. Chem. 41:4854-4860 (1998)).
An example of a fluorescence assay which m-ay be used for the present invention is the Factor VIIa assay. Briefly, the Factor VIIa assay is measured in the presence of human recombinant tissue factor (INNOVIN from Dade Behring Cat.# B4212-100). Human Factor VIIa may be obtained from Enzyme Research Labs (Cat.# HFVIIA 1640). Enzymatic activity could be measured in a buffer containing 150 mM NaCl, 5 mM CaCl₂, 1 mM CHAPS and 1 mg/ml PEG 6000 (pH 7.4) with 1 nM FVIIa and 100 μM D-Ile-Pro-Arg-AFC (Enzyme Systems Products, Km>200 ) 0.66% DMSO. The assay (302 μl total volume) may be incubated at room temperature for 2 hr prior to reading fluorometric signal (Ex 405/Em 535) using a Molecular Devices or Victor 2 (Wallac) fluorescent plate reader.
In addition to the methods described above, protease activity (and therefore serpin activity) can be measured using fluorescent resonance energy transfer (FRET with Quencher -P_n-P₃-P₂-P₁- -P₁′-P₂′-Fluorophore), fluorescent peptide bound to beads (Fluorophore -P_n-P₃- P₂-P₁- -P₁′-P₂′-Bead), dye-protein substrates and serpin-protease gel shifts. All of which are well known to those skilled in the art (see a non-limiting review in Knight, C. G. Fluorimetric Assays of Proteolytic Enzymes. Meth. Enzymol. 248: 18-34 (1995)).
Additional assay methods are known in the art and are encompassed by the present invention. See, for example, Backes B J, Harris J L, Leonetti F, Craik C S, Ellman J A. Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin. Nat Biotechnol. 18:187-93 (2000); Balasubramanian, et al., Active site-directed synthetic thrombin inhibitors: synthesis, in vitro and in vivo activity profile of BMY 44621 and analogs an examination of the role of the amino group in the D-Phe-Pro-Arg-H series. J. Med. Chem. 36:300-303 (1993); and Combrink et al., Novel 1,2-Benzisothiazol-3-one-1,1-dioxide Inhibitors of Human Mast Cell Tryptase. J. Med. Chem. 41:4854-4860 (1998) and those methods described in: Calpain Methods and Protocols (ed J. S. Elce) Meth. Mol. Biol. 144, 2000 and Calpain: Pharmacology and Toxicology of a calcium-dependent protease (K. Wang & P.-W. Yuen editors) Taylor & Francis Philadelphia, Pa., 1999; which are hereby incorporated herein by reference in their entirety.

Example 8

Determination of the Preferred Substrate Sequence of the CAN-12 Protease

The preferred substrate sequence specificity of the CAN-12 metalliprotease of the present invention may be determined using using two redundant peptide libraries and Edman peptide sequencing (1-2) as shown below.
The first peptide library is random, can vary in length and incorporates a modification at the N-terminus to block Edman sequencing. In the example provided, biotin is used as the blocking group. Proteolytic cleavage of the library is allowed to proceed long enough to turn over approximately 5-10% of the library. Edman sequencing of the peptide mixture provides the preferred substrate residues for the P′ sites on the protease. The second peptide library has fixed P′ residues to restrict the proteolytic cleavage site and an affinity tag for removing the C-terminal product of the proteolysis, leaving the N-terminal peptide product pool behind for Edman sequencing to determine the amino acid residues preferred in the P1, P2, P3 etc. sites of the protease.
Reagents.
The endoproteases Factor Xa (New England BioLabs, Inc., Beverly, Mass.) and human kidney Renin (Calbiochem, San Diego, Calif.) were purchased for validation experiments. A hexapeptide library containing 4.7×10⁷peptide species was synthesized by the Molecular Redesign group (Natarajan & Riexinger) at Bristol-Myers Squibb Company (Princeton, N.J.). The library contained equivalent representation of 19 amino acid residues at each of the six degenerate positions and incorporated an N-terminal biotin group and a C-terminal amide. Cysteine residues were excluded from the peptide pool and Methionine residues were replaced with Norleucine.
Endoprotease Cleavage of the Peptide Library.
The following method may be used to determine the preferred substrate sequence downstream of the cleavage site. A 1.88 mM peptide library solution is prepared in phosphate buffer (10 mM Sodium Phosphate (pH 7.6), 0.1 M NaCl, and 10% DMSO) and is incubated with 2-30 μg endoprotease at 37° C. Using a fluorescamine assay to estimate the extent of peptide cleavage, the reaction is stopped at 5-10% completion with incubation at 100° C. for 2.0 minutes. Peptide pools are subjected to Edman sequencing. The data obtained is normalized and corrected for differences in efficiency of cleavage and recovery in the sequencer.
Fluorescamine Assay to Monitor Peptide Cleavage.
Primary amines generated during peptide cleavage is measured by reaction with fluorescamine (Aldrich, St. Louis, Mo.), as described in reference 3. The relative fluorescence is determined by measuring signals at □^ex=355 nm and □^em=460 nm on a PerkinElmer Wallac 1420 Spectrofluorometer. Reactions are sampled at multiple time points and assayed in triplicate. The amount of cleavage product formed is determined using the relative fluorescence produced by varying concentrations of a peptide standard of known concentration.

References

(1) “Substrate Specificity of Cathepsins D and E Determined by N-Terminal and C-Terminal Sequencing of Peptide Pools” D. Arnold et al. (1997) Eur. J. Biochem. 249, 171.
(2) “Determination of Protease Cleavage Site Motifs Using Mixture-Based Oriented Peptide Libraries” B. E. Turk et al. (2001) Nature Biotech. 19, 661.
(3) “Fluorescamine: a Reagent for Assay of Amino Acids, Peptides, Proteins, and Primary Amines in the Picomole Range” S. Udenfirend, S. Stein, P. Bohlen, W. Dairman, W. Leimgruber, and M. Weigele (1972) Science 178, 87.

Example 9

Chromosomal Mapping of Calpain 12 and Linkage to Neurodegenerative Disorders

The calpain 12 polynucleotides of the present invention were used to determine the chromosomal localization of the calpain 12 gene. The comparison of the chromosomal location of the calpain 12 gene with the location of chromosomal regions which have been shown to be associated with specific diseases or conditions, e.g. by linkage analysis, can be indicative of diseases in which calpain 12 may play a role.
A chromosomal localization of the calpain 12 gene was performed by using the nucleic acid sequence (SEQ ID NO:1) of the invention in a database search of the recently completed draft of human genome sequence. Using the Basic Local Alignment Search Tool 2 (BLAST2), the first 200 bp of calpain 12 cDNA sequence showed a perfect alignment of nucleotides 62 to 200 with the minus strand of Homo sapiens chromosome 2 clone RP11-541A15 nucleotide 177952 to 177814, and a perfect alignment of nucleotide 1 to 62 of calpain 12 cDNA (SEQ ID NO:1) with the nucleotide 94118 to 94179 of the same clone, suggesting the likelihood of an intron intervening this 200 cDNA fragment. To confirm the map of calpain 12 gene, another BLAST2 search was done with a 300 pb fragment of calpain 12 cDNA sequence containing nucleotides 4251 up to poly A signal sequence at position 4550 (SEQ ID NO:1). An alignment with 97% identities (292/300) of this calpain 3′ sequence was found with nucleotide 125724 to 126023 of the same Homo sapiens chromosome 2 clone RP11-541A15. In order to get a refined map of this clone containing the locus of calpain 12, a map search of NCBI genome database was performed. RP11-541A15 (Acc# AC015980.2) was found within a BAC contig adjacent to clone 852C13 (AL133246.2) mapping in 2p21-p22.
A whole-genome linkage scan in multiple sclerosis families (Ebers et al. A full genome search in multiple sclerosis. Nature Genet. 13: 472-476, 1996.) identified 5 susceptibility loci on chromosomes 2, 3, 5, 11, and X. In particular, an association was identified with marker D2S119 on chromosome 2 and MS. We further delineated the localization of this marker, D2S119, on 2p16-p21 based on a radiation hybrid linkage map retrieved from an online query at NCBI web site: http:/www.ncbi.nlm.nih.gov/genome/sts/. Since the map of calpain 12 and the susceptibility marker D2S119 overlaps, it is reasonable to postulate that calpain 12 may contribute to MS. Furthermore, the transcription profile of calpain 12 indicated a prominent expression in spinal cord, and implication of calpains in MS has been suggested (Shields D C et al. A putative mechanism of demyelination in multiple sclerosis by a proteolytic enzyme, calpain. Proc Natl Acad Sci USA. 96:11486-91.1999).

Example 10

Method of Screening for Compounds that Interact with the CAN-12 Polypeptide

The following assays are designed to identify compounds that bind to the CAN-12 polypeptide, bind to other cellular proteins that interact with the CAN-12 polypeptide, and to compounds that interfere with the interaction of the CAN-12 polypeptide with other cellular proteins.
Such compounds can include, but are not limited to, other cellular proteins. Specifically, such compounds can include, but are not limited to, peptides, such as, for example, soluble peptides, including, but not limited to Ig-tailed fusion peptides, comprising extracellular portions of CAN-12 polypeptide transmembrane receptors, and members of random peptide libraries (see, e.g., Lam, K. S. et al., 1991, Nature 354:82-84; Houghton, R. et al., 1991, Nature 354:84-86), made of D- and/or L-configuration amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate phosphopeptide libraries; see, e.g., Songyang, Z., et al., 1993, Cell 72:767-778), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab′).sub.2 and FAb. expression libary fragments, and epitope-binding fragments thereof), and small organic or inorganic molecules.
Compounds identified via assays such as those described herein can be useful, for example, in elaborating the biological function of the CAN-12 polypeptide, and for ameliorating symptoms of tumor progression, for example. In instances, for example, whereby a tumor progression state or disorder results from a lower overall level of CAN-12 expression, CAN-12 polypeptide, and/or CAN-12 polypeptide activity in a cell involved in the tumor progression state or disorder, compounds that interact with the CAN-12 polypeptide can include ones which accentuate or amplify the activity of the bound CAN-12 polypeptide. Such compounds would bring about an effective increase in the level of CAN-12 polypeptide activity, thus ameliorating symptoms of the tumor progression disorder or state. In instances whereby mutations within the CAN-12 polypeptide cause aberrant CAN-12 polypeptides to be made which have a deleterious effect that leads to tumor progression, compounds that bind CAN-12 polypeptide can be identified that inhibit the activity of the bound CAN-12 polypeptide. Assays for testing the effectiveness of such compounds are known in the art and discussed, elsewhere herein.

Example 11

Method of Screening, In Vitro, Compounds that Bind to the CAN-12 Polypeptide

In vitro systems can be designed to identify compounds capable of binding the CAN-12 polypeptide of the invention. Compounds identified can be useful, for example, in modulating the activity of wild type and/or mutant CAN-12 polypeptide, preferably mutant CAN-12 polypeptide, can be useful in elaborating the biological function of the CAN-12 polypeptide, can be utilized in screens for identifying compounds that disrupt normal CAN-12 polypeptide interactions, or can in themselves disrupt such interactions.
The principle of the assays used to identify compounds that bind to the CAN-12 polypeptide involves preparing a reaction mixture of the CAN-12 polypeptide and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring CAN-12 polypeptide or the test substance onto a solid phase and detecting CAN-12 polypeptide /test compound complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the CAN-12 polypeptide can be anchored onto a solid surface, and the test compound, which is not anchored, can be labeled, either directly or indirectly.
In practice, microtitre plates can conveniently be utilized as the solid phase. The anchored component can be immobilized by non-covalent or covalent attachments. Non-covalent attachment can be accomplished by simply coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized can be used to anchor the protein to the solid surface. The surfaces can be prepared in advance and stored.
In order to conduct the assay, the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nonimmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody).
Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for CAN-12 polypeptide or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.

Example 12

Method of Identifying Compounds that Interfere with CAN-12 Polypeptide/Cellular Product Interaction

The CAN-12 polypeptide of the invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. Such macromolecules include, but are not limited to, nucleic acid molecules and those products identified via methods such as those described, elsewhere herein. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as “binding partner(s)”. For the purpose of the present invention, “binding partner” may also encompass small molecule compounds, polysaccarides, lipids, and any other molecule or molecule type referenced herein. Compounds that disrupt such interactions can be useful in regulating the activity of the CAN-12 polypeptide, especially mutant CAN-12 polypeptide. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and the like described in elsewhere herein.
The basic principle of the assay systems used to identify compounds that interfere with the interaction between the CAN-12 polypeptide and its cellular or extracellular binding partner or partners involves preparing a reaction mixture containing the CAN-12 polypeptide, and the binding partner under conditions and for a time sufficient to allow the two products to interact and bind, thus forming a complex. In order to test a compound for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of CAN-12 polypeptide and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the CAN-12 polypeptide and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the CAN-12 polypeptide and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal CAN-12 polypeptide can also be compared to complex formation within reaction mixtures containing the test compound-and mutant CAN-12 polypeptide. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal CAN-12 polypeptide.
The assay for compounds that interfere with the interaction of the CAN-12 polypeptide and binding partners can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the CAN-12 polypeptide or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the CAN-12 polypeptide and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the CAN-12 polypeptide and interactive cellular or extracellular binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g. compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below.
In a heterogeneous assay system, either the CAN-12 polypeptide or the interactive cellular or extracellular binding partner, is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly. In practice, microtitre plates are conveniently utilized. The anchored species can be immobilized by non-covalent or covalent attachments. Non-covalent attachment can be accomplished simply by coating the solid surface with a solution of the CAN-12 polypeptide or binding partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface. The surfaces can be prepared in advance and stored.
In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.
Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds which inhibit complex or which disrupt preformed complexes can be identified.
In an alternate embodiment of the invention, a homogeneous assay can be used. In this approach, a preformed complex of the CAN-12 polypeptide and the interactive cellular or extracellular binding partner product is prepared in which either the CAN-12 polypeptide or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein which utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances which disrupt CAN-12 polypeptide -cellular or extracellular binding partner interaction can be identified.
In a particular embodiment, the CAN-12 polypeptide can be prepared for immobilization using recombinant DNA techniques known in the art. For example, the CAN-12 polypeptide coding region can be fused to a glutathione-S-transferase (GST) gene using a fusion vector such as pGEX-5×-1, in such a manner that its binding activity is maintained in the resulting fusion product. The interactive cellular or extracellular product can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art and described above. This antibody can be labeled with the radioactive isotope sup. 125 I, for example, by methods routinely practiced in the art. In a heterogeneous assay, e.g., the GST-CAN-12 polypeptide fusion product can be anchored to glutathione-agarose beads. The interactive cellular or extracellular binding partner product can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur. At the end of the reaction period, unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components. The interaction between the CAN-12 polypeptide and the interactive cellular or extracellular binding partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.
Alternatively, the GST-CAN-12 polypeptide fusion product and the interactive cellular or extracellular binding partner product can be mixed together in liquid in the absence of the solid glutathione-agarose beads. The test compound can be added either during or after the binding partners are allowed to interact. This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the binding partner interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads.
In another embodiment of the invention, these same techniques can be employed using peptide fragments that correspond to the binding domains of the CAN-12 polypeptide product and the interactive cellular or extracellular binding partner (in case where the binding partner is a product), in place of one or both of the full length products.
Any number of methods routinely practiced in the art can be used to identify and isolate the protein's binding site. These methods include, but are not limited to, mutagenesis of one of the genes encoding one of the products and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can be selected. Sequence analysis of the genes encoding the respective products will reveal the mutations that correspond to the region of the product involved in interactive binding. Alternatively, one product can be anchored to a solid surface using methods described in this Section above, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the binding domain can remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene coding for the cellular or extracellular binding partner product is obtained, short gene segments can be engineered to express peptide fragments of the product, which can then be tested for binding activity and purified or synthesized.

Example 13

Isolation of a Specific Clone from the Deposited Sample

The deposited material in the sample assigned the ATCC Deposit Number cited in Table I for any given cDNA clone also may contain one or more additional plasmids, each comprising a cDNA clone different from that given clone. Thus, deposits sharing the same ATCC Deposit Number contain at least a plasmid for each cDNA clone identified in Table I. Typically, each ATCC deposit sample cited in Table I comprises a mixture of approximately equal amounts (by weight) of about 1-10 plasmid DNAs, each containing a different cDNA clone and/or partial cDNA clone; but such a deposit sample may include plasmids for more or less than 2 cDNA clones.
Two approaches can be used to isolate a particular clone from the deposited sample of plasmid DNA(s) cited for that clone in Table I. First, a plasmid is directly isolated by screening the clones using a polynucleotide probe corresponding to SEQ ID NO: 1.
Particularly, a specific polynucleotide with 3040 nucleotides is synthesized using an Applied Biosystems DNA synthesizer according to the sequence reported. The oligonucleotide is labeled, for instance, with 32P-ATP using T4 polynucleotide kinase and purified according to routine methods. (E.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmid mixture is transformed into a suitable host, as indicated above (such as XL-1 Blue (Stratagene)) using techniques known to those of skill in the art, such as those provided by the vector supplier or in related publications or patents cited above. The transformants are plated on 1.5% agar plates (containing the appropriate selection agent, e.g., ampicillin) to a density of about 150 transformants (colonies) per plate. These plates are screened using Nylon membranes according to routine methods for bacterial colony screening (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press, pages 1.93 to 1.104), or other techniques known to those of skill in the art.
Alternatively, two primers of 17-20 nucleotides derived from both ends of the SEQ ID NO: 1 (i.e., within the region of SEQ ID NO: 1 bounded by the 5′ NT and the 3 ′ NT of the clone defined in Table I) are synthesized and used to amplify the desired cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is carried out under routine conditions, for instance, in 25 ul of reaction mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl2, 0.01% (w/v) gelatin, 20 uM each of DATP, dCTP, dGTP, dTTP, 25 μmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94 degree C. for 1 min; annealing at 55 degree C. for 1 min; elongation at 72 degree C. for 1 min) are performed with a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product.
The polynucleotide(s) of the present invention, the polynucleotide encoding the polypeptide of the present invention, or the polypeptide encoded by the deposited clone may represent partial, or incomplete versions of the complete coding region (i.e., full-length gene). Several methods are known in the art for the identification of the 5′ or 3, non-coding and/or coding portions of a gene which may not be present in the deposited clone. The methods that follow are exemplary and should not be construed as limiting the scope of the invention. These methods include but are not limited to, filter probing, clone enrichment using specific probes, and protocols similar or identical to 5′ and 3, “RACE” protocols that are well known in the art. For instance, a method similar to 5′ RACE is available for generating the missing 5′ end of a desired full-length transcript. (Fromont-Racine et al., Nucleic Acids Res. 21(7):1683-1684 (1993)).
Briefly, a specific RNA oligonucleotide is ligated to the 5, ends of a population of RNA presumably containing full-length gene RNA transcripts. A primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest is used to PCR amplify the 5′ portion of the desired full-length gene. This amplified product may then be sequenced and used to generate the full-length gene.
This above method starts with total RNA isolated from the desired source, although poly-A+ RNA can be used. The RNA preparation can then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA that may interfere with the later RNA ligase step. The phosphatase should then be inactivated and the RNA treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase.
This modified RNA preparation is used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction is used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the desired gene. Moreover, it may be advantageous to optimize the RACE protocol to increase the probability of isolating additional 5′ or 3′ coding or non-coding sequences. Various methods of optimizing a RACE protocol are known in the art, though a detailed description summarizing these methods can be found in B. C. Schaefer, Anal. Biochem., 227:255-273, (1995).
An alternative method for carrying out 5′ or 3′ RACE for the identification of coding or non-coding sequences is provided by Frohman, M. A., et al., Proc. Nat'l. Acad. Sci. USA, 85:8998-9002 (1988). Briefly, a cDNA clone missing either the 5′ or 3′ end can be reconstructed to include the absent base pairs extending to the translational start or stop codon, respectively. In some cases, cDNAs are missing the start of translation, therefor. The following briefly describes a modification of this original 5′ RACE procedure. Poly A+or total RNAs reverse transcribed with Superscript II (Gibco/BRL) and an antisense or I complementary primer specific to the cDNA sequence. The primer is removed from the reaction with a Microcon Concentrator (Amicon). The first-strand cDNA is then tailed with dATP and terminal deoxynucleotide transferase (Gibco/BRL). Thus, an anchor sequence is produced which is needed for PCR amplification. The second strand is synthesized from the da-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), an oligo-dT primer containing three adjacent restriction sites (XhoIJ Sail and ClaI) at the 5′ end and a primer containing just these restriction sites. This double-stranded cDNA is PCR amplified for 40 cycles with the same primers as well as a nested cDNA-specific antisense primer. The PCR products are size-separated on an ethidium bromide-agarose gel and the region of gel containing cDNA products the predicted size of missing protein-coding DNA is removed. cDNA is purified from the agarose with the Magic PCR Prep kit (Promega), restriction digested with XhoI or SalI, and ligated to a plasmid such as pBluescript SKII (Stratagene) at XhoI and EcoRV sites. This DNA is transformed into bacteria and the plasmid clones sequenced to identify the correct protein-coding inserts. Correct 5′ ends are confirmed by comparing this sequence with the putatively identified homologue and overlap with the partial cDNA clone. Similar methods known in the art and/or commercial kits are used to amplify and recover 3′ ends.
Several quality-controlled kits are commercially available for purchase. Similar reagents and methods to those above are supplied in kit form from Gibco/BRL for both 5′ and 3′ RACE for recovery of full length genes. A second kit is available from Clontech which is a modification of a related technique, SLIC (single-stranded ligation to single-stranded cDNA), developed by Dumas et al., Nucleic Acids Res., 19:5227-32(1991). The major differences in procedure are that the RNA is alkaline hydrolyzed after reverse transcription and RNA ligase is used to join a restriction site-containing anchor primer to the first-strand cDNA. This obviates the necessity for the dA-tailing reaction which results in a polyT stretch that is difficult to sequence past.
An alternative to generating 5′ or 3′ cDNA from RNA is to use cDNA library double-stranded DNA. An asymmetric PCR-amplified antisense cDNA strand is synthesized with an antisense cDNA-specific primer and a plasmid-anchored primer. These primers are removed and a symmetric PCR reaction is performed with a nested cDNA-specific antisense primer and the plasmid-anchored primer.
RNA Ligase Protocol for Generating the 5′ or 3′ End Sequences to Obtain Full Length Genes
Once a gene of interest is identified, several methods are available for the identification of the 5′ or 3′ portions of the gene which may not be present in the original cDNA plasmid. These methods include, but are not limited to, filter probing, clone enrichment using specific probes and protocols similar and identical to 5′ and 3′ RACE. While the full-length gene may be present in the library and can be identified by probing, a useful method for generating the 5′ or 3′ end is to use the existing sequence information from the original cDNA to generate the missing information. A method similar to 5′ RACE is available for generating the missing 5′ end of a desired full-length gene. (This method was published by Fromont-Racine et al., Nucleic Acids Res., 21(7): 1683-1684 (1993)). Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of a population of RNA presumably 30 containing full-length gene RNA transcript and a primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest, is used to PCR amplify the 5′ portion of the desired full length gene which may then be sequenced and used to generate the full length gene. This method starts with total RNA isolated from the desired source, poly A RNA may be used but is not a prerequisite for this procedure. The RNA preparation may then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase if used is then inactivated and the RNA is treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase. This modified RNA preparation can then be used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction can then be used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the apoptosis related of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the relevant apoptosis related.

Example 14

Bacterial Expression of a Polypeptide

A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in Example 13, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites, such as BamHI and XbaI, at the 5′ end of the primers in order to clone the amplified product into the expression vector. For example, BamHI and XbaI correspond to the restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.
The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture is then used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4, that expresses the lacI repressor and also confers kanamycin resistance (Kanr). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.
Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.600) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expression.
Cells are grown for an extra 3 to 4 hours. Cells are then harvested by centrifugation (20 mins at 600×g). The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at 4 degree C. The cell debris is removed by centrifugation, and the supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind to the Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist (1995) QIAGEN, Inc., supra).
Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.
The purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on the Ni-NTA column. The recommended conditions are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM imidazole. Imidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein is stored at 4 degree C. or frozen at −80 degree C.

Example 15

Purification of a Polypeptide from an Inclusion Body

The following alternative method can be used to purify a polypeptide expressed in E coli when it is present in the form of inclusion bodies. Unless otherwise specified, all of the following steps are conducted at 4-10 degree C.
Upon completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4-10 degree C. and the cells harvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste, by weight, is suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneous suspension using a high shear mixer.
The cells are then lysed by passing the solution through a microfluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000×g for 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
The resulting washed inclusion bodies are solubilized with 1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×g centrifugation for 15 min., the pellet is discarded and the polypeptide containing supernatant is incubated at 4 degree C. overnight to allow further GuHCl extraction.
Following high speed centrifugation (30,000×g) to remove insoluble particles, the GuHCl solubilized protein is refolded by quickly mixing the GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded diluted protein solution is kept at 4 degree C. without mixing for 12 hours prior to further purification steps.
To clarify the refolded polypeptide solution, a previously prepared tangential filtration unit equipped with 0.16 um membrane filter with appropriate surface area (e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loaded onto a cation exchange resin (e.g., Poros HS-50, Perceptive Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. The absorbance at 280 nm of the effluent is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.
Fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample is then loaded onto a previously prepared set of tandem columns of strong anion (Poros HQ-50, Perceptive Biosystems) and weak anion (Poros CM-20, Perceptive Biosystems) exchange resins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10 column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected under constant A280 monitoring of the effluent. Fractions containing the polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.
The resultant polypeptide should exhibit greater than 95% purity after the above refolding and purification steps. No major contaminant bands should be observed from Coomassie blue stained 16% SDS-PAGE gel when 5 ug of purified protein is loaded. The purified protein can also be tested for endotoxin/LPS contamination, and typically the LPS content is less than 0.1 ng/ml according to LAL assays.

Example 16

Cloning and Expression of a Polypeptide in a Baculovirus Expression System

In this example, the plasmid shuttle vector pAc373 is used to insert a polynucleotide into a baculovirus to express a polypeptide. A typical baculovirus expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites, which may include, for example BamHI, Xba I and Asp718. The polyadenylation site of the simian virus 40 (“SV40”) is often used for efficient polyadenylation. For easy selection of recombinant virus, the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate a viable virus that express the cloned polynucleotide.
Many other baculovirus vectors can be used in place of the vector above, such as pVL941 and pAcIMN1, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required. Such vectors are described, for instance, in Luckow et al., Virology 170:31-39 (1989).
A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in Example 13, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites at the 5′ end of the primers in order to clone the amplified product into the expression vector. Specifically, the cDNA sequence contained in the deposited clone, including the AUG initiation codon and the naturally associated leader sequence identified elsewhere herein (if applicable), is amplified using the PCR protocol described in Example 13. If the naturally occurring signal sequence is used to produce the protein, the vector used does not need a second signal peptide. Alternatively, the vector can be modified to include a baculovirus leader sequence, using the standard methods described in Summers et al., “A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures,” Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.
The plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).
The fragment and the dephosphorylated plasmid are ligated together with T4 DNA ligase. E. coli HB 101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells are transformed with the ligation mixture and spread on culture plates. Bacteria containing the plasmid are identified by digesting DNA from individual colonies and analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.
Five ug of a plasmid containing the polynucleotide is co-transformed with 1.0 ug of a commercially available linearized baculovirus DNA (“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofection method described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One ug of BaculoGoldtm virus DNA and 5 ug of the plasmid are mixed in a sterile well of a microtiter plate containing 50 ul of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated for 5 hours at 27 degrees C. The transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. Cultivation is then continued at 27 degrees C. for four days.
After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra. An agarose gel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a “plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10.) After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 ul of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4 degree C.
To verify the expression of the polypeptide, Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus containing the polynucleotide at a multiplicity of infection (“MOI”) of about 2. If radiolabeled proteins are desired, 6 hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of 35S-methionine and 5 uCi 35S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then are harvested by centrifugation. The proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).
Microsequencing of the amino acid sequence of the amino terminus of purified protein may be used to determine the amino terminal sequence of the produced protein.

Example 17

Expression of a Polypeptide in Mammalian Cells

The polypeptide of the present invention can be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription is achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).
Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
Alternatively, the polypeptide can be expressed in stable cell lines containing the polynucleotide integrated into a chromosome. The co-transformation with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transformed cells.
The transformed gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the gene of interest. (See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A., Biotechnology 9:64-68 (1991).) Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.
A polynucleotide of the present invention is amplified according to the protocol outlined in herein. If the naturally occurring signal sequence is used to produce the protein, the vector does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/34891.) The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.
The amplified fragment is then digested with the same restriction enzyme and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 using, for instance, restriction enzyme analysis.
Chinese hamster ovary cells lacking an active DHFR gene is used for transformation. Five pg of an expression plasmid is cotransformed with 0.5 ug of the plasmid pSVneo using lipofectin (Felgner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G4 18. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100 -200 uM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.

Example 18

Protein Fusions

The polypeptides of the present invention are preferably fused to other proteins. These fusion proteins can be used for a variety of applications. For example, fusion of the present polypeptides to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification. (See Example described herein; see also EP A 394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusion to IgG-1, IgG-3, and albumin increases the half-life time in vivo. Nuclear localization signals fused to the polypeptides of the present invention can target the protein to a specific subcellular localization, while covalent heterodimer or homodimers can increase or decrease the activity of a fusion protein. Fusion proteins can also create chimeric molecules having more than one function. Finally, fusion proteins can increase solubility and/or stability of the fused protein compared to the non-fused protein. All of the types of fusion proteins described above can be made by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG molecule.
Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using primers that span the 5′ and 3′ ends of the sequence described below. These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian expression vector. Note that the polynucleotide is cloned without a stop codon, otherwise a fusion protein will not be produced.
The naturally occurring signal sequence may be used to produce the protein (if applicable). Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/34891 and/or U.S. Pat. No. 6,066,781, supra.)

Human IgG Fc region:


(SEQ ID NO:48)

GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGC
CCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGG
TGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG
GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT
GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA
ACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC
ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTG
GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG
TAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 19

Method of Creating N- and C-terminal Deletion Mutants Corresponding to the CAN-12, CAN-12v1, and CAN-12v2 Polypeptides of the Present Invention.

As described elsewhere herein, the present invention encompasses the creation of N- and C-terminal deletion mutants, in addition to any combination of N- and C-terminal deletions thereof, corresponding to the CAN-12, CAN-12v1, and CAN-12v2 polypeptides of the present invention. A number of methods are available to one skilled in the art for creating such mutants. Such methods may include a combination of PCR amplification and gene cloning methodology. Although one of skill in the art of molecular biology, through the use of the teachings provided or referenced herein, and/or otherwise known in the art as standard methods, could readily create each deletion mutant of the present invention, exemplary methods are described below.
Briefly, using the isolated cDNA clone encoding the full-length CAN-12, CAN-12v1, or CAN-12v2 polypeptide sequence (as described in Example 13, for example), appropriate primers of about 15-25 nucleotides derived from the desired 5′ and 3′ positions of SEQ ID NO: 1, 53, or 55 may be designed to PCR amplify, and subsequently clone, the intended N- and/or C-terminal deletion mutant. Such primers could comprise, for example, an inititation and stop codon for the 5′ and 3′ primer, respectively. Such primers may also comprise restriction sites to facilitate cloning of the deletion mutant post amplification. Moreover, the primers may comprise additional sequences, such as, for example, flag-tag sequences, kozac sequences, or other sequences discussed and/or referenced herein.

For example, in the case of the P23 to L581 CAN-12 N-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant:


5′ Primer	5′-GCAGCA GCGGCCGC CCACAGCAACCCCAACAGGACTTTG-3′
	(SEQ ID NO:49)
	NotI

3′ Primer	5′-GCAGCA GTCGAC TAACAAGGTGGTGTTGAAGATTAAA-3′
	(SEQ ID NO:50)
	SalI

For example, in the case of the M1 to L423 CAN-12 C-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant:


5′ Primer	5′-GCAGCA GCGGCCGC ATGTCTCTGTGGCCACCTTTCCG-3′
	(SEQ ID NO:51)
	NotI

3′ Primer	5′-GCAGCA GTCGAC GAGGTAGAAGCCAATGGCGAGGAG-3′
	(SEQ ID NO:52)
	SalI

For example, in the case of the R90 to L694 CAN-12v1 N-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant:


5′ Primer	5′-GCAGCA GCGGCCGC AGGCTGGATCTGTGCCAGGGGATAG-3′
	(SEQ ID NO:94)
	NotI

3′ Primer	5′-GCAGCA GTCGAC TAACAAGGTGGTGTTGAAG-3′
	(SEQ ID NO: 95)
	SalI

For example, in the case of the M1 to G561 CAN-12v1 C-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant:


5′ Primer	5′- GCAGCA GCGGCCGC ATGTCTCTGTGGCCACCTTTCCG -3′ (SEQ ID NO:96)
	NotI

3′ Primer	5′- GCAGCA GTCGAC CCCCTGGCAGGCTTCCAGGCTAAAG -3′ (SEQ ID NO:97)
	SalI

For example, in the case of the R90 to L697 CAN-12v2 N-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant:


5′ Primer	5′- GCAGCA GCGGCCGC AGGCTGGATCTGTGCCAGGGGATAG -3′ (SEQ ID NO:98)
	NotI

3′ Primer	5′- GCAGCA GTCGAC TAACAAGGTGGTGTTGAAG -3′ (SEQ ID NO:99)
	SalI

For example, in the case of the MI to G564 CAN-12v2 C-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant:


5′ Primer	5′- GCAGCA GCGGCCGC ATGTCTCTGTGGCCACCTTTCCG -3′ (SEQ ID NO:100)
	NotI

3′ Primer	5′- GCAGCA GTCGAC CCCCTGGCAGGCTTCCAGGCTAAAG -3′ (SEQ ID NO:101)
	SalI

Representative PCR amplification conditions are provided below, although the skilled artisan would appreciate that other conditions may be required for efficient amplification. A 100 ul PCR reaction mixture may be prepared using 10 ng of the template DNA (cDNA clone of CAN-12), 200 uM 4dNTPs, 1 uM primers, 0.25U Taq DNA polymerase (PE), and standard Taq DNA polymerase buffer. Typical PCR cycling condition are as follows:

20-25 cycles: 45 sec, 93 degrees

2 min, 50 degrees

2 min, 72 degrees

1 cycle: 10 min, 72 degrees
After the final extension step of PCR, 5U Klenow Fragment may be added and incubated for 15 min at 30 degrees.
Upon digestion of the fragment with the NotI and SalI restriction enzyres, the fragment could be cloned into an appropriate expression and/or cloning vector which has been similarly digested (e.g., pSport1, among others). The skilled artisan would appreciate that other plasmids could be equally substituted, and may be desirable in certain circumstances. The digested fragment and vector are then ligated using a DNA ligase, and then used to transform competent E. coli cells using methods provided herein and/or otherwise known in the art.
The 5′ primer sequence for amplifying any additional N-terminal deletion mutants may be determined by reference to the following formula:
(S+(X*3)) to ((S+(X*3))+25), wherein ‘S’ is equal to the nucleotide position of the initiating start codon of the CAN-12, CAN-12v1, or CAN12-v2 gene (SEQ ID NO: 1, 53, or 55), and ‘X’ is equal to the most N-terminal amino acid of the intended N-terminal deletion mutant. The first term will provide the start 5′ nucleotide position of the 5′ primer, while the second term will provide the end 3′ nucleotide position of the 5′ primer corresponding to sense strand of SEQ ID NO: 1, 53, or 55. Once the corresponding nucleotide positions of the primer are determined, the final nucleotide sequence may be created by the addition of applicable restriction site sequences to the 5′ end of the sequence, for example. As referenced herein, the addition of other sequences to the 5′ primer may be desired in certain circumstances (e.g., kozac sequences, etc.).
The 3′ primer sequence for amplifying any additional N-terminal deletion mutants may be determined by reference to the following formula:
(S+(X*3)) to ((S+(X*3))−25), wherein ‘S’ is equal to the nucleotide position of the initiating start codon of the CAN-12, CAN-12v1, or CAN-12-v2 gene (SEQ ID NO:1, 53, or 55), and ‘X’ is equal to the most C-terminal amino acid of the intended N-terminal deletion mutant. The first term will provide the start 5′ nucleotide position of the 3′ primer, while the second term will provide the end 3′ nucleotide position of the 3′ primer corresponding to the anti-sense strand of SEQ ID NO: 1, 53, or 55. Once the corresponding nucleotide positions of the primer are determined, the final nucleotide sequence may be created by the addition of applicable restriction site sequences to the 5′ end of the sequence, for example. As referenced herein, the addition of other sequences to the 3′ primer may be desired in certain circumstances (e.g., stop codon sequences, etc.). The skilled artisan would appreciate that modifications of the above nucleotide positions may be necessary for optimizing PCR amplification.
The same general formulas provided above may be used in identifying the 5′ and 3′ primer sequences for amplifying any C-terminal deletion mutant of the present invention. Moreover, the same general formulas provided above may be used in identifying the 5′ and 3′ primer sequences for amplifying any combination of N-terminal and C-terminal deletion mutant of the present invention. The skilled artisan would appreciate that modifications of the above nucleotide positions may be necessary for optimizing PCR amplification.

Example 20

Site Directed/Site Specific Mutagenesis

In vitro site-directed mutagenesis is an invaluable technique for studying protein structure-function relationships and gene expression, for example, as well as for vector modification. Site-directed mutagenesis can also be used for creating any of one or more of the mutants of the present invention, particularly the conservative and/or non-conservative amino acid substitution mutants of the present invention. Approaches utilizing single stranded DNA (ssDNA) as the template have been reported (e.g., T. A. Kunkel et al., 1985, Proc. Natl. Acad. Sci. USA), 82:488-492; M. A. Vandeyar et al., 1988, Gene, 65(1):129-133; M. Sugimoto et al., 1989, Anal. Biochem., 179(2):309-31 1; and J. W. Taylor et al., 1985, Nuc. Acids. Res., 13(24):8765-8785).
The use of PCR in site-directed mutagenesis accomplishes strand separation by using a denaturing step to separate the complementary strands and to allow efficient polymerization of the PCR primers. PCR site-directed mutagenesis methods thus permit site specific mutations to be incorporated in virtually any double stranded plasmid, thus eliminating the need for re-subcloning into M13-based bacteriophage vectors or single-stranded rescue. (M. P. Weiner et al., 1995, Molecular Biology: Current Innovations and Future Trends, Eds. A. M. Griffin and H. G. Griffin, Horizon Scientific Press, Norfolk, UK; and C. Papworth et al., 1996, Strategies, 9(3):3-4).
A protocol for performing site-directed mutagenesis, particularly employing the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, Calif.; U.S. Pat. Nos. 5,789,166 and 5,923,419) is provided for making point mutations, to switch or substitute amino acids, and to delete or insert single or multiple amino acids in the RATL1d6 amino acid sequence of this invention.
Primer Design
For primer design using this protocol, the mutagenic oligonucleotide primers are designed individually according to the desired mutation. The following considerations should be made for designing mutagenic primers: 1) Both of the mutagenic primers must contain the desired mutation and anneal to the same sequence on opposite strands of the plasmid; 2) Primers should be between 25 and 45 bases in length, and the melting temperature (T_m) of the primers should be greater than, or equal to, 78° C. The following formula is commonly used for estimating the T_mof primers: T=81.5+0.41 (%GC)−675/N− %mismatch. For calculating T_m, N is the primer length in bases; and values for %GC and % mismatch are whole numbers. For calculating T_mfor primers intended to introduce insertions or deletions, a modified version of the above formula is employed: T=81.5+0.41 (%GC)−675/N, where N does not include the bases which are being inserted or deleted; 3) The desired mutation (deletion or insertion) should be in the middle of the primer with approximately 10-15 bases of correct sequence on both sides; 4) The primers optimally should have a minimum GC content of 40%, and should terminate in one or more C or G bases; 5) Primers need not be 5′-phosphorylated, but must be purified either by fast polynucleotide liquid chromatography (FPLC) or by polyacrylamide gel electrophoresis (PAGE). Failure to purify the primers results in a significant decrease in mutation efficiency; and 6). It is important that primer concentration is in excess. It is suggested to vary the amount of template while keeping the concentration of the primers constantly in excess (QuikChange™ Site-Directed Mutagenesis Kit, Stratagene, La Jolla, Calif.).
Protocol for Setting UP the Reactions
Using the above-described primer design, two complimentary oligonucleotides containing the desired mutation, flanked by unmodified nucleic acid sequence, are synthesized. The resulting oligonucleotide primers are purified.
A control reaction is prepared using 5 μl 10× reaction buffer (100 mM KCl; 100 mM (NH₄)₂SO₄; 200 mM Tris-HCl, pH 8.8; 20 mM MgSO₄; 1% Triton® X-100; 1 mg/ml nuclease-free bovine serum albumin, BSA); 2 μl (10 ng) of pWhitescript™, 4.5-kb control plasmid (5 ng/μl); 1.25 μl (125 ng) of oligonucleotide control primer #1 (34-mer, 100 ng/l); 1.25 μl (125 ng) of oligonucleotide control primer #2 (34-mer, 100 ng/μl); 1 μl of dNTP mix; double distilled H₂O; to a final volume of 50 μl. Thereafter, 1 μl of DNA polymerase (PfuTurbo® DNA Polymerase, Stratagene), (2.5 U/gl) is added. PfuTurbo® DNA Polymerase is stated to have 6-fold higher fidelity in DNA synthesis than does Taq polymerase. To maximize temperature cycling performance, use of thin-walled test tubes is suggested to ensure optimum contact with the heating blocks of the temperature cycler.
The sample reaction is prepared by combining 5 μl of 10× reaction buffer; x μl (5-50 ng) of dsDNA template; x μl (125 ng) of oligonucleotide primer #1; x μl (5-50 ng) of dsDNA template; x μg (125 ng) of oligonucleotide primer #2; 1 μl of dNTP mix; and ddH₂O to a final volume of 50 μl. Thereafter, 1 μl of DNA polymerase (PfuTurbo DNA Polymerase, Stratagene), (2.5 U/gl) is added.
It is suggested that if the thermal cycler does not have a hot-top assembly, each reaction should be overlaid with approximately 30 μl of mineral oil.
Cycling the Reactions
Each reaction is cycled using the following cycling parameters:

Segment Cycles Temperature Time

1 1 95° C. 30 seconds

2 12-18 95° C. 30 seconds

55° C. 1 minute

68° C. 2 minutes/kb of

plasmid length
For the control reaction, a 12-minute extension time is used and the reaction is run for 12 cycles. Segment 2 of the above cycling parameters is adjusted in accordance with the type of mutation desired. For example, for point mutations, 12 cycles are used; for single amino acid changes, 16 cycles are used; and for multiple amino acid deletions or insertions, 18 cycles are used. Following the temperature cycling, the reaction is placed on ice for 2 minutes to cool the reaction to ≦37° C.
Digesting the Products and Transforming Competent Cells
One μl of the DpnI restriction enzyme (10 U/gl) is added directly (below mineral oil overlay) to each amplification reaction using a small, pointed pipette tip. The reaction mixture is gently and thoroughly mixed by pipetting the solution up and down several times. The reaction mixture is then centrifuged for 1 minute in a microcentrifuge. Immediately thereafter, each reaction is incubated at 37° C. for 1 hour to digest the parental (i.e., the non-mutated) supercoiled dsDNA.
Competent cells (i.e., XL1-Blue supercompetent cells, Stratagene) are thawed gently on ice. For each control and sample reaction to be transformed, 50 μl of the supercompetent cells are aliquotted to a prechilled test tube (Falcon 2059 polypropylene). Next, 1 μl of the DpnI-digested DNA is transferred from the control and the sample reactions to separate aliquots of the supercompetent cells. The transformation reactions are gently swirled to mix and incubated for 30 minutes on ice. Thereafter, the transformation reactions are heat-pulsed for 45 seconds at 42° C. for 2 minutes.
0.5 ml of NZY+ broth, preheated to 42° C. is added to the transformation reactions which are then incubated at 37° C. for 1 hour with shaking at 225-250 rpm. An aliquot of each transformation reaction is plated on agar plates containing the appropriate antibiotic for the vector. For the mutagenesis and transformation controls, cells are spread on LB-ampicillin agar plates containing 80 μg/ml of X-gal and 20 mM MIPTG. Transformation plates are incubated for >6 hours at 37° C.

Example 21

Regulation of Protein Expression Via Controlled Aggregation in the Endoplasmic Reticulum

As described more particularly herein, proteins regulate diverse cellular processes in higher organisms, ranging from rapid metabolic changes to growth and differentiation. Increased production of specific proteins could be used to prevent certain diseases and/or disease states. Thus, the ability to modulate the expression of specific proteins in an organism would provide significant benefits.
Numerous methods have been developed to date for introducing foreign genes, either under the control of an inducible, constitutively active, or endogenous promoter, into organisms. Of particular interest are the inducible promoters (see, M. Gossen, et al., Proc. Natl. Acad. Sci. USA., 89:5547 (1992); Y. Wang, et al., Proc. Natl. Acad. Sci. USA, 91:8180 (1994), D. No., et al., Proc. Natl. Acad. Sci. USA, 93:3346 (1996); and V. M. Rivera, et al., Nature Med, 2:1028 (1996); in addition to additional examples disclosed elsewhere herein). In one example, the gene for erthropoietin (Epo) was transferred into mice and primates under the control of a small molecule inducer for expression (e.g., tetracycline or rapamycin) (see, D. Bohl, et al., Blood, 92:1512, (1998); K. G. Rendahl, et al., Nat. Biotech, 16:757, (1998); V. M. Rivera, et al., Proc. Natl. Acad. Sci. USA, 96:8657 (1999); and X. Ye et al., Science, 283:88 (1999). Although such systems enable efficient induction of the gene of interest in the organism upon addition of the inducing agent (i.e., tetracycline, rapamycin, etc,.), the levels of expression tend to peak at 24 hours and trail off to background levels after 4 to 14 days. Thus, controlled transient expression is virtually impossible using these systems, though such control would be desirable.
A new alternative method of controlling gene expression levels of a protein from a transgene (i.e., includes stable and transient transformants) has recently been elucidated (V. M. Rivera., et al., Science, 287:826-830, (2000)). This method does not control gene expression at the level of the mRNA like the aforementioned systems. Rather, the system controls the level of protein in an active secreted form. In the absence of the inducing agent, the protein aggregates in the ER and is not secreted. However, addition of the inducing agent results in dis-aggregation of the protein and the subsequent secretion from the ER. Such a system affords low basal secretion, rapid, high level secretion in the presence of the inducing agent, and rapid cessation of secretion upon removal of the inducing agent. In fact, protein secretion reached a maximum level within 30 minutes of induction, and a rapid cessation of secretion within 1 hour of removing the inducing agent. The method is also applicable for controlling the level of production for membrane proteins.
Detailed methods are presented in V. M. Rivera., et al., Science, 287:826-830, (2000)), briefly:
Fusion protein constructs are created using polynucleotide sequences of the present invention with one or more copies (preferably at least 2, 3, 4, or more) of a conditional aggregation domain (CAD) a domain that interacts with itself in a ligand-reversible manner (i.e., in the presence of an inducing agent) using molecular biology methods known in the art and discussed elsewhere herein. The CAD domain may be the mutant domain isolated from the human FKBP12 (Phe³⁶to Met) protein (as disclosed in V. M. Rivera., et al., Science, 287:826-830, (2000), or alternatively other proteins having domains with similar ligand-reversible, self-aggregation properties. As a principle of design the fusion protein vector would contain a furin cleavage sequence operably linked between the polynucleotides of the present invention and the CAD domains. Such a cleavage site would enable the proteolytic cleavage of the CAD domains from the polypeptide of the present invention subsequent to secretion from the ER and upon entry into the trans-Golgi (J. B. Denault, et al., FEBS Lett., 379:113, (1996)). Alternatively, the skilled artisan would recognize that any proteolytic cleavage sequence could be substituted for the furin sequence provided the substituted sequence is cleavable either endogenously (e.g., the furin sequence) or exogenously (e.g., post secretion, post purification, post production, etc.). The preferred sequence of each feature of the fusion protein construct, from the 5′ to 3′ direction with each feature being operably linked to the other, would be a promoter, signal sequence, “X” number of (CAD)x domains, the furin sequence (or other proteolytic sequence), and the coding sequence of the polypeptide of the present invention. The artisan would appreciate that the promotor and signal sequence, independent from the other, could be either the endogenous promotor or signal sequence of a polypeptide of the present invention, or alternatively, could be a heterologous signal sequence and promotor.
The specific methods described herein for controlling protein secretion levels through controlled ER aggregation are not meant to be limiting are would be generally applicable to any of the polynucleotides and polypeptides of the present invention, including variants, homologues, orthologs, and fragments therein.

Example 22

Alteration of Protein Glycosylation Sites to Enhance Characteristics of Polypeptides of the Invention

Many eukaryotic cell surface and proteins are post-translationally processed to incorporate N-linked and O-linked carbohydrates (Kornfeld and Kornfeld (1985) Annu. Rev. Biochem. 54:631-64; Rademacher et al., (1988) Annu. Rev. Biochem. 57:785-838). Protein glycosylation is thought to serve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion (Fieldler and Simons (1995) Cell, 81:309-312; Helenius (1994) Mol. Biol. Of the Cell 5:253-265; Olden et al., (1978) Cell, 13:461-473; Caton et al., (1982) Cell, 37:417-427; Alexander and Elder (1984), Science, 226:1328-1330; and Flack et al., (1994), J. Biol. Chem., 269:14015-14020). In higher organisms, the nature and extent of glycosylation can markedly affect the circulating half-life and bio-availability of proteins by mechanisms involving receptor mediated uptake and clearance (Ashwell and Morrell, (1974), Adv. Enzymol., 41:99-128; Ashwell and Harford (1982), Ann. Rev. Biochem., 51:531-54). Receptor systems have been identified that are thought to play a major role in the clearance of serum proteins through recognition of various carbohydrate structures on the glycoproteins (Stockert (1995), Physiol. Rev., 75:591-609; Kery et al., (1992), Arch. Biochem. Biophys., 298:49-55). Thus, production strategies resulting in incomplete attachment of terminal sialic acid residues might provide a means of shortening the bioavailability and half-life of glycoproteins. Conversely, expression strategies resulting in saturation of terminal sialic acid attachment sites might lengthen protein bioavailability and half-life.
In the development of recombinant glycoproteins for use as pharmaceutical products, for example, it has been speculated that the pharmacodynamics of recombinant proteins can be modulated by the addition or deletion of glycosylation sites from a glycoproteins primary structure (Berman and Lasky (1985a) Trends in Biotechnol., 3:51-53). However, studies have reported that the deletion of N-linked glycosylation sites often impairs intracellular transport and results in the intracellular accumulation of glycosylation site variants (Machamer and Rose (1988), J. Biol Chem., 263:5955-5960; Gallagher et al., (1992), J. Virology., 66:7136-7145; Collier et al., (1993), Biochem., 32:7818-7823; Claffey et al., (1995) Biochemica et Biophysica Acta, 1246:1-9; Dube et al., (1988), J. Biol. Chem. 263:17516-17521). While glycosylation site variants of proteins can be expressed intracellularly, it has proved difficult to recover useful quantities from growth conditioned cell culture medium.
Moreover, it is unclear to what extent a glycosylation site in one species will be recognized by another species glycosylation machinery. Due to the importance of glycosylation in protein metabolism, particularly the secretion and/or expression of the protein, whether a glycosylation signal is recognized may profoundly determine a proteins ability to be expressed, either endogenously or recombinately, in another organism (i.e., expressing a human protein in E. coli, yeast, or viral organisms; or an E. coli, yeast, or viral protein in human, etc.). Thus, it may be desirable to add, delete, or modify a glycosylation site, and possibly add a glycosylation site of one species to a protein of another species to improve the proteins functional, bioprocess purification, and/or structural characteristics (e.g., a polypeptide of the present invention).
A number of methods may be employed to identify the location of glycosylation sites within a protein. One preferred method is to run the translated protein sequence through the PROSITE computer program (Swiss Institute of Bioinformatics). Once identified, the sites could be systematically deleted, or impaired, at the level of the DNA using mutagenesis methodology known in the art and available to the skilled artisan, Preferably using PCR-directed mutagenesis (See Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982)). Similarly, glycosylation sites could be added, or modified at the level of the DNA using similar methods, preferably PCR methods (See, Maniatis, supra). The results of modifying the glycosylation sites for a particular protein (e.g., solubility, secretion potential, activity, aggregation, proteolytic resistance, etc.) could then be analyzed using methods know in the art.
The skilled artisan would acknowledge the existence of other computer algorithms capable of predicting the location of glycosylation sites within a protein. For example, the Motif computer program (Genetics Computer Group suite of programs) provides this function, as well.

Example 23

Method of Enhancing the Biological Activity/Functional Characteristics of Invention through Molecular Evolution

Although many of the most biologically active proteins known are highly effective for their specified function in an organism, they often possess characteristics that make them undesirable for transgenic, therapeutic, and/or industrial applications. Among these traits, a short physiological half-life is the most prominent problem, and is present either at the level of the protein, or the level of the proteins mRNA. The ability to extend the half-life, for example, would be particularly important for a proteins use in gene therapy, transgenic animal production, the bioprocess production and purification of the protein, and use of the protein as a chemical modulator among others. Therefore, there is a need to identify novel variants of isolated proteins possessing characteristics which enhance their application as a therapeutic for treating diseases of animal origin, in addition to the proteins applicability to common industrial and pharmaceutical applications.
Thus, one aspect of the present invention relates to the ability to enhance specific characteristics of invention through directed molecular evolution. Such an enhancement may, in a non-limiting example, benefit the inventions utility as an essential component in a kit, the inventions physical attributes such as its solubility, structure, or codon optimization, the inventions specific biological activity, including any associated enzymatic activity, the proteins enzyme kinetics, the proteins Ki, Kcat, Km, Vmax, Kd, protein-protein activity, protein-DNA binding activity, antagonist/inhibitory activity (including direct or indirect interaction), agonist activity (including direct or indirect interaction), the proteins antigenicity (e.g., where it would be desirable to either increase or decrease the antigenic potential of the protein), the immunogenicity of the protein, the ability of the protein to form dimers, trimers, or multimers with either itself or other proteins, the antigenic efficacy of the invention, including its subsequent use a preventative treatment for disease or disease states, or as an effector for targeting diseased genes. Moreover, the ability to enhance specific characteristics of a protein may also be applicable to changing the characterized activity of an enzyme to an activity completely unrelated to its initially characterized activity. Other desirable enhancements of the invention would be specific to each individual protein, and would thus be well known in the art and contemplated by the present invention.
For example, an engineered calpain may be constitutively active upon binding of its cognate substrate. Alternatively, an engineered calpain may be constitutively active in the absence of substrate binding, and/or may exhibit increased efficacy in inhibiting cysteine proteases. In yet another example, an engineered calpain may be capable of being activated with less than all of the regulatory factors and/or conditions typically required for calpain activation (e.g., substrate binding, phosphorylation, cofactor binding, Ca+ binding, Ca+ activation, conformational changes, etc.). Such calpain would be useful in screens to identify calpain modulators, among other uses described herein.
Directed evolution is comprised of several steps. The first step is to establish a library of variants for the gene or protein of interest. The most important step is to then select for those variants that entail the activity you wish to identify. The design of the screen is essential since your screen should be selective enough to eliminate non-useful variants, but not so stringent as to eliminate all variants. The last step is then to repeat the above steps using the best variant from the previous screen. Each successive cycle, can then be tailored as necessary, such as increasing the stringency of the screen, for example.
Over the years, there have been a number of methods developed to introduce mutations into macromolecules. Some of these methods include, random mutagenesis, “error-prone” PCR, chemical mutagenesis, site-directed mutagenesis, and other methods well known in the art (for a comprehensive listing of current mutagenesis methods, see Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982)). Typically, such methods have been used, for example, as tools for identifying the core functional region(s) of a protein or the function of specific domains of a protein (if a multi-domain protein). However, such methods have more recently been applied to the identification of macromolecule variants with specific or enhanced characteristics.
Random mutagenesis has been the most widely recognized method to date. Typically, this has been carried out either through the use of “error-prone” PCR (as described in Moore, J., et al, Nature Biotechnology 14:458, (1996), or through the application of randomized synthetic oligonucleotides corresponding to specific regions of interest (as described by Derbyshire, K. M. et al, Gene, 46:145-152, (1986), and Hill, D E, et al, Methods Enzymol., 55:559-568, (1987). Both approaches have limits to the level of mutagenesis that can be obtained. However, either approach enables the investigator to effectively control the rate of mutagenesis. This is particularly important considering the fact that mutations beneficial to the activity of the enzyme are fairly rare. In fact, using too high a level of mutagenesis may counter or inhibit the desired benefit of a useful mutation.
While both of the aforementioned methods are effective for creating randomized pools of macromolecule variants, a third method, termed “DNA Shuffling”, or “sexual PCR” (WPC, Stemmer, PNAS, 91:10747, (1994)) has recently been elucidated. DNA shuffling has also been referred to as “directed molecular evolution”, “exon-shuffling”, “directed enzyme evolution”, in vitro evolution”, and “artificial evolution”. Such reference terms are known in the art and are encompassed by the invention. This new, preferred, method apparently overcomes the limitations of the previous methods in that it not only propagates positive traits, but simultaneously eliminates negative traits in the resulting progeny.
DNA shuffling accomplishes this task by combining the principal of in vitro recombination, along with the method of “error-prone” PCR. In effect, you begin with a randomly digested pool of small fragments of your gene, created by Dnase I digestion, and then introduce said random fragments into an “error-prone” PCR assembly reaction. During the PCR reaction, the randomly sized DNA fragments not only hybridize to their cognate strand, but also may hybridize to other DNA fragments corresponding to different regions of the polynucleotide of interest—regions not typically accessible via hybridization of the entire polynucleotide. Moreover, since the PCR assembly reaction utilizes “error-prone” PCR reaction conditions, random mutations are introduced during the DNA synthesis step of the PCR reaction for all of the fragments -further diversifying the potential hybridization sites during the annealing step of the reaction.
A variety of reaction conditions could be utilized to carry-out the DNA shuffling reaction. However, specific reaction conditions for DNA shuffling are provided, for example, in PNAS, 91:10747, (1994). Briefly:
Prepare the DNA substrate to be subjected to the DNA shuffling reaction. Preparation may be in the form of simply purifying the DNA from contaminating cellular material, chemicals, buffers, oligonucleotide primers, deoxynucleotides, RNAs, etc., and may entail the use of DNA purification kits as those provided by Qiagen, Inc., or by the Promega, Corp., for example.
Once the DNA substrate has been purified, it would be subjected to Dnase I digestion. About 2-4 ug of the DNA substrate(s) would be digested with 0.0015 units of Dnase I (Sigma) per ul in 100 ul of 50 mM Tris-HCl, pH 7.4/1 mM MgCl2 for 10-20 min. at room temperature. The resulting fragments of 10-50 bp could then be purified by running them through a 2% low-melting point agarose gel by electrophoresis onto DE81 ion-exchange paper (Whatmann) or could be purified using Microcon concentrators (Amicon) of the appropriate molecular weight cutoff, or could use oligonucleotide purification columns (Qiagen), in addition to other methods known in the art. If using DE81 ion-exchange paper, the 10-50 bp fragments could be eluted from said paper using 1M NaCl, followed by ethanol precipitation.
The resulting purified fragments would then be subjected to a PCR assembly reaction by re-suspension in a PCR mixture containing: 2 mM of each dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM TriseHCl, pH 9.0, and 0.1% Triton X-100, at a final fragment concentration of 10-30 ng/ul. No primers are added at this point. Taq DNA polymerase (Promega) would be used at 2.5 units per 100 ul of reaction mixture. A PCR program of 94 C for 60s; 94 C for 30s, 50-55 C for 30s, and 72 C for 30s using 3045 cycles, followed by 72 C for 5 min using an MJ Research (Cambridge, Mass.) PTC-150 thermocycler. After the assembly reaction is completed, a 1:40 dilution of the resulting primerless product would then be introduced into a PCR mixture (using the same buffer mixture used for the assembly reaction) containing 0.8 um of each primer and subjecting this mixture to 15 cycles of PCR (using 94 C for 30s, 5° C. for 30s, and 72 C for 30s). The referred primers would be primers corresponding to the nucleic acid sequences of the polynucleotide(s) utilized in the shuffling reaction. Said primers could consist of modified nucleic acid base pairs using methods known in the art and referred to else where herein, or could contain additional sequences (i.e., for adding restriction sites, mutating specific base-pairs, etc.).
The resulting shuffled, assembled, and amplified product can be purified using methods well known in the art (e.g., Qiagen PCR purification kits) and then subsequently cloned using appropriate restriction enzymes.
Although a number of variations of DNA shuffling have been published to date, such variations would be obvious to the skilled artisan and are encompassed by the invention. The DNA shuffling method can also be tailored to the desired level of mutagenesis using the methods described by Zhao, et al. (Nucl Acid Res., 25(6):1307-1308, (1997).
As described above, once the randomized pool has been created, it can then be subjected to a specific screen to identify the variant possessing the desired characteristic(s). Once the variant has been identified, DNA corresponding to the variant could then be used as the DNA substrate for initiating another round of DNA shuffling. This cycle of shuffling, selecting the optimized variant of interest, and then re-shuffling, can be repeated until the ultimate variant is obtained. Examples of model screens applied to identify variants created using DNA shuffling technology may be found in the following publications: J. C., Moore, et al., J. Mol. Biol., 272:336-347, (1997), F. R., Cross, et al., Mol. Cell. Biol., 18:2923-2931, (1998), and A. Crameri., et al., Nat. Biotech., 15:436-438, (1997).
DNA shuffling has several advantages. First, it makes use of beneficial mutations. When combined with screening, DNA shuffling allows the discovery of the best mutational combinations and does not assume that the best combination contains all the mutations in a population. Secondly, recombination occurs simultaneously with point mutagenesis. An effect of forcing DNA polymerase to synthesize full-length genes from the small fragment DNA pool is a background mutagenesis rate. In combination with a stringent selection method, enzymatic activity has been evolved up to 16000 fold increase over the wild-type form of the enzyme. In essence, the background mutagenesis yielded the genetic variability on which recombination acted to enhance the activity.
A third feature of recombination is that it can be used to remove deleterious mutations. As discussed above, during the process of the randomization, for every one beneficial mutation, there may be at least one or more neutral or inhibitory mutations. Such mutations can be removed by including in the assembly reaction an excess of the wild-type random-size fragments, in addition to the random-size fragments of the selected mutant from the previous selection. During the next selection, some of the most active variants of the polynucleotide/polypeptide/enzyme, should have lost the inhibitory mutations.
Finally, recombination enables parallel processing. This represents a significant advantage since there are likely multiple characteristics that would make a protein more desirable (e.g. solubility, activity, etc.). Since it is increasingly difficult to screen for more than one desirable trait at a time, other methods of molecular evolution tend to be inhibitory. However, using recombination, it would be possible to combine the randomized fragments of the best representative variants for the various traits, and then select for multiple properties at once.
DNA shuffling can also be applied to the polynucleotides and polypeptides of the present invention to decrease their immunogenicity in a specified host. For example, a particular variant of the present invention may be created and isolated using DNA shuffling technology. Such a variant may have all of the desired characteristics, though may be highly immunogenic in a host due to its novel intrinsic structure. Specifically, the desired characteristic may cause the polypeptide to have a non-native structure which could no longer be recognized as a “self” molecule, but rather as a “foreign”, and thus activate a host immune response directed against the novel variant. Such a limitation can be overcome, for example, by including a copy of the gene sequence for a xenobiotic ortholog of the native protein in with the gene sequence of the novel variant gene in one or more cycles of DNA shuffling. The molar ratio of the ortholog and novel variant DNAs could be varied accordingly. Ideally, the resulting hybrid variant identified would contain at least some of the coding sequence which enabled the xenobiotic protein to evade the host immune system, and additionally, the coding sequence of the original novel variant that provided the desired characteristics.
Likewise, the invention encompasses the application of DNA shuffling technology to the evolution of polynucleotides and polypeptides of the invention, wherein one or more cycles of DNA shuffling include, in addition to the gene template DNA, oligonucleotides coding for known allelic sequences, optimized codon sequences, known variant sequences, known polynucleotide polymorphism sequences, known ortholog sequences, known homologue sequences, additional homologous sequences, additional non-homologous sequences, sequences from another species, and any number and combination of the above.
In addition to the described methods above, there are a number of related methods that may also be applicable, or desirable in certain cases. Representative among these are the methods discussed in PCT applications WO 98/31700, and WO 98/32845, which are hereby incorporated by reference. Furthermore, related methods can also be applied to the polynucleotide sequences of the present invention in order to evolve invention for creating ideal variants for use in gene therapy, protein engineering, evolution of whole cells containing the variant, or in the evolution of entire enzyme pathways containing polynucleotides of the invention as described in PCT applications WO 98/13485, WO 98/13487, WO 98/27230, WO 98/31837, and Crameri, A., et al., Nat. Biotech., 15:436-438, (1997), respectively.
Additional methods of applying “DNA Shuffling” technology to the polynucleotides and polypeptides of the present invention, including their proposed applications, may be found in U.S. Pat. No. 5,605,793; PCT Application No. WO 95/22625; PCT Application No. WO 97/20078; PCT Application No. WO 97/35966; and PCT Application No. WO 98/42832; PCT Application No. WO 00/09727 specifically provides methods for applying DNA shuffling to the identification of herbicide selective crops which could be applied to the polynucleotides and polypeptides of the present invention; additionally, PCT Application No. WO 00/12680 provides methods and compositions for generating, modifying, adapting, and optimizing polynucleotide sequences that confer detectable phenotypic properties on plant species; each of the above are hereby incorporated in their entirety herein for all purposes.

Example 24

Method of Determining Alterations in a Gene Corresponding to a Polynucleotide

RNA isolated from entire families or individual patients presenting with a phenotype of interest (such as a disease) is be isolated. cDNA is then generated from these RNA samples using protocols known in the art. (See, Sambrook.) The cDNA is then used as a template for PCR, employing primers surrounding regions of interest in SEQ ID NO:1. Suggested PCR conditions consist of 35 cycles at 95 degrees C. for 30 seconds; 60-120 seconds at 52-58 degrees C.; and 60-120 seconds at 70 degrees C., using buffer solutions described in Sidransky et al., Science 252:706 (1991).
PCR products are then sequenced using primers labeled at their 5′ end with T4 polynucleotide kinase, employing SequiTherm Polymerase. (Epicentre Technologies). The intron-exon borders of selected exons is also determined and genomic PCR products analyzed to confirm the results. PCR products harboring suspected mutations is then cloned and sequenced to validate the results of the direct sequencing.
PCR products is cloned into T-tailed vectors as described in Holton et al., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations not present in unaffected individuals.
Genomic rearrangements are also observed as a method of determining alterations in a gene corresponding to a polynucleotide. Genomic clones isolated according to Example 2 are nick-translated with digoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISH performed as described in Johnson et al., Methods Cell Biol. 35:73-99 (1991). Hybridization with the labeled probe is carried out using a vast excess of human cot-i DNA for specific hybridization to the corresponding genomic locus.
Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C- and R-bands. Aligned images for precise mapping are obtained using a triple-band filter set (Chroma Technology, Brattleboro, Vt.) in combination with a cooled charge-coupled device camera (Photometrics, Tucson, Ariz.) and variable excitation wavelength filters. (Johnson et al., Genet. Anal. Tech. Appl., 8:75 (1991).) Image collection, analysis and chromosomal fractional length measurements are performed using the ISee Graphical Program System. (Inovision Corporation, Durham, N.C.) Chromosome alterations of the genomic region hybridized by the probe are identified as insertions, deletions, and translocations. These alterations are used as a diagnostic marker for an associated disease.

Example 25

Method of Detecting Abnormal Levels of a Polypeptide in a Biological Sample

A polypeptide of the present invention can be detected in a biological sample, and if an increased or decreased level of the polypeptide is detected, this polypeptide is a marker for a particular phenotype. Methods of detection are numerous, and thus, it is understood that one skilled in the art can modify the following assay to fit their particular needs.
For example, antibody-sandwich ELISAs are used to detect polypeptides in a sample, preferably a biological sample. Wells of a microtiter plate are coated with specific antibodies, at a final concentration of 0.2 to 10 ug/ml. The antibodies are either monoclonal or polyclonal and are produced by the method described elsewhere herein. The wells are blocked so that non-specific binding of the polypeptide to the well is reduced.
The coated wells are then incubated for >2 hours at RT with a sample containing the polypeptide. Preferably, serial dilutions of the sample should be used to validate results. The plates are then washed three times with deionized or distilled water to remove unbounded polypeptide.
Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at a concentration of 25-400 ng, is added and incubated for 2 hours at room temperature. The plates are again washed three times with deionized or distilled water to remove unbounded conjugate.
Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution to each well and incubate I hour at room temperature. Measure the reaction by a microtiter plate reader. Prepare a standard curve, using serial dilutions of a control sample, and plot polypeptide concentration on the X-axis (log.scale) and fluorescence or absorbance of the Y-axis (linear scale). Interpolate the concentration of the polypeptide in the sample using the standard curve.

Example 26

Formulation

The invention also provides methods of treatment and/or prevention diseases, disorders, and/or conditions (such as, for example, any one or more of the diseases or disorders disclosed herein) by administration to a subject of an effective amount of a Therapeutic. By therapeutic is meant a polynucleotides or polypeptides of the invention (including fragments and variants), agonists or antagonists thereof, and/or antibodies thereto, in combination with a pharmaceutically acceptable carrier type (e.g., a sterile carrier).
The Therapeutic will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the Therapeutic alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” for purposes herein is thus determined by such considerations.
As a general proposition, the total pharmaceutically effective amount of the Therapeutic administered parenterally per dose will be in the range of about 1 ug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, the Therapeutic is typically administered at a dose rate of about 1 ug/kg/hour to about 50 ug/kg/hour, either by 14 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.
Therapeutics can be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
In yet an additional embodiment, the Therapeutics of the invention are delivered orally using the drug delivery technology described in U.S. Pat. No. 6,258,789, which is hereby incorporated by reference herein.
Therapeutics of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics are administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
Therapeutics of the invention may also be suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).
Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).
Sustained-release Therapeutics also include liposomally entrapped Therapeutics of the invention (see, generally, Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 317 -327 and 353-365 (1989)). Liposomes containing the Therapeutic are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal Therapeutic.
In yet an additional embodiment, the Therapeutics of the invention are delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).
Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
For parenteral administration, in one embodiment, the Therapeutic is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to the Therapeutic.
Generally, the formulations are prepared by contacting the Therapeutic uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.
The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.
The Therapeutic will typically be formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts.
Any pharmaceutical used for therapeutic administration can be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutics generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
Therapeutics ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous Therapeutic solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized Therapeutic using bacteriostatic Water-for-Injection.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the Therapeutics of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the Therapeutics may be employed in conjunction with other therapeutic compounds.
The Therapeutics of the invention may be administered alone or in combination with adjuvants. Adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG, and MPL. In a specific embodiment, Therapeutics of the invention are administered in combination with alum. In another specific embodiment, Therapeutics of the invention are administered in combination with QS-21. Further adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the Therapeutics of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.
The Therapeutics of the invention may be administered alone or in combination with other therapeutic agents. Therapeutic agents that may be administered in combination with the Therapeutics of the invention, include but not limited to, other members of the TNF family, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, cytokines and/or growth factors. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.
In one embodiment, the Therapeutics of the invention are administered in combination with members of the TNF family. TNF, TNF-related or TNF-like molecules that may be administered with the Therapeutics of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), endokine-alpha (International Publication No. WO 98/07880), TR6 (International Publication No. WO 98/30694), OPG, and neutrokine-alpha (International Publication No. WO 98/18921, OX40, and nerve growth factor (NOF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9 (International Publication No. WO 98/56892),TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms CD154, CD70, and CD153.
In certain embodiments, Therapeutics of the invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors. Nucleoside reverse transcriptase inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, RETROVIR (zidovudine/AZT), VIDEX (didanosine/ddI), HIVID (zalcitabine/ddC), ZERIT (stavudine/d4T), EPIVIR (lamivudine/3TC), and COMBIVIR (zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, VIRAMUE (nevirapine), RESCRIPTOR (delavirdine), and SUSTIVA (efavirenz). Protease inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, CRIXIVAN (indinavir), NORVIR (ritonavir), INVIRASE (saquinavir), and VIRACEPT (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with Therapeutics of the invention to treat AIDS and/or to prevent or treat HIV infection.
In other embodiments, Therapeutics of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE, DAPSONE, PENTAMIDINE, ATOVAQUONE, ISONIAZID, RIFAMPIN, PYRAZIAMIDE, ETHAMBUTOL, RIFABUTIN, CLARITHROMYCIN, AZITHROMYCIN, GANCICLOVK, FOSCARNET, CIDOFOVK, FLUCONAZOLE, ITRACONAZOLE, KETOCONAZOLE, ACYCLOVIR, FAMCICOLVIR, PYRIMETHAMMNE, LEUCOVORIN, NEUPOGEN (filgrastim/G-CSF), and LEUKINE (sargramostim/GM-CSF). In a specific embodiment, Therapeutics of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLE, DAPSONE, PENTAMIDINE, and/or ATOVAQUONE to prophylactically treat or prevent an opportunistic Pneumocystis carinii pneumonia infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ISONLAZID, RIFAMPIN, PYRAZINAMIDE, and/or ETHAMBUTOL to prophylactically treat or prevent an opportunistic Mycobacterium avium complex infection. In another specific embodiment, Therapeutics of the invention are used in any combination with RIFABUTIN, CLARITHROMYCIN, and/or AZITHROMYCIN to prophylactically treat or prevent an opportunistic Mycobacterium tuberculosis infection. In another specific embodiment, Therapeutics of the invention are used in any combination with GANCICLOVIR, FOSCARNET, and/or CIDOFOVIR to prophylactically treat or prevent an opportunistic cytomegalovirus infection. In another specific embodiment, Therapeutics of the invention are used in any combination with FLUCONAZOLE, ITRACONAZOLE, and/or KETOCONAZOLE to prophylactically treat or prevent an opportunistic fungal infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ACYCLOVIR and/or FAMCICOLVIR to prophylactically treat or prevent an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment, Therapeutics of the invention are used in any combination with PYRIMETHAMINE and/or LEUCOVORIN to prophylactically treat or prevent an opportunistic Toxoplasma -gondii infection. In another specific embodiment, Therapeutics of the invention are used in any combination with LEUCOVORIN and/or NEUPOGEN to prophylactically treat or prevent an opportunistic bacterial infection.
In a further embodiment, the Therapeutics of the invention are administered in combination with an antiviral agent. Antiviral agents that may be administered with the Therapeutics of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.
In a further embodiment, the Therapeutics of the invention are administered in combination with an antibiotic agent. Antibiotic agents that may be administered with the Therapeutics of the invention include, but are not limited to, amoxicillin, beta-lactamases, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.
Conventional nonspecific immunosuppressive agents, that may be administered in combination with the Therapeutics of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells.
In specific embodiments, Therapeutics of the invention are administered in combination with immunosuppressants. Immunosuppressants preparations that may be administered with the Therapeutics of the invention include, but are not limited to, ORTHOCLONE (OKT3), SANDIMMUNE/NEORAL/SANGDYA (cyclosporin), PROGRAF (tacrolimus), CELLCEPT (mycophenolate), Azathioprine, glucorticosteroids, and RAPAMUNE (sirolimus). In a specific embodiment, immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.
In an additional embodiment, Therapeutics of the invention are administered alone or in combination with one or more intravenous immune globulin preparations. Intravenous immune globulin preparations that may be administered with the Therapeutics of the invention include, but not limited to, GAMMAR, IVEEGAM, SANDOGLOBULIN, GAMMAGARD S/D, and GAMIMUNE. In a specific embodiment, Therapeutics of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).
In an additional embodiment, the Therapeutics of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered with the Therapeutics of the invention include, but are not limited to, glucocorticoids and the nonsteroidal anti-inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.
In another embodiment, compositions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the Therapeutics of the invention include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogen mustard) and thiotepa); steroids and combinations (e.g., bethamethasone sodium phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).
In a specific embodiment, Therapeutics of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or any combination of the components of CHOP. In another embodiment, Therapeutics of the invention are administered in combination with Rituximab. In a further embodiment, Therapeutics of the invention are administered with Rituxmab and CHOP, or Rituxmab and any combination of the components of CHOP.
In an additional embodiment, the Therapeutics of the invention are administered in combination with cytokines. Cytokines that may be administered with the Therapeutics of the invention include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7, EL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment, Therapeutics of the invention may be administered with any interleukin, including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-S, IL-6, IL-7, L-8, IL-9 IL-10, IL-11, IL-12, IL-13, IL,14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.
In an additional embodiment, the Therapeutics of the invention are administered in combination with angiogenic proteins. Angiogenic proteins that may be administered with the Therapeutics of the invention include, but are not limited to, Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-682110; Platelet Derived Growth Factor-B (P)DGF-B), as disclosed in European Patent Number EP-2823 17; Placental Growth Factor (PlGF), as disclosed in International Publication Number WO 92/06194; Placental Growth Factor-2 (PlGF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in International Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in International Publication Number WO 96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-1 86 (VEGF-B186), as disclosed in International Publication Number WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number DE19639601. The above mentioned references are incorporated herein by reference herein.
In an additional embodiment, the Therapeutics of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered with the Therapeutics of the invention include, but are not limited to, LEUKINE (SARGRAMOSTIM) and NEUPOGEN (FILGRASTIM).
In an additional embodiment, the Therapeutics of the invention are administered in combination with Fibroblast Growth Factors. Fibroblast Growth Factors that may be administered with the Therapeutics of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
In a specific embodiment, formulations of the present invention may further comprise antagonists of P-glycoprotein (also referred to as the multiresistance protein, or PGP), including antagonists of its encoding polynucleotides (e.g., antisense oligonucleotides, ribozymes, zinc-finger proteins, etc.). P-glycoprotein is well known for decreasing the efficacy of various drug administrations due to its ability to export intracellular levels of absorbed drug to the cell exterior. While this activity has been particularly pronounced in cancer cells in response to the administration of chemotherapy regimens, a variety of other cell types and the administration of other drug classes have been noted (e.g., T-cells and anti-HIV drugs). In fact, certain mutations in the PGP gene significantly reduces PGP function, making it less able to force drugs out of cells. People who have two versions of the mutated gene—one inherited from each parent—have more than four times less PGP than those with two normal versions of the gene. People may also have one normal gene and one mutated one. Certain ethnic populations have increased incidence of such PGP mutations. Among individuals from Ghana, Kenya, the Sudan, as well as African Americans, frequency of the normal gene ranged from 73% to 84%. In contrast, the frequency was 34% to 59% among British whites, Portuguese, Southwest Asian, Chinese, Filipino and Saudi populations. As a result, certain ethnic populations may require increased administration of PGP antagonist in the formulation of the present invention to arrive at the an efficacious dose of the therapeutic (e.g., those from African descent). Conversely, certain ethnic populations, particularly those having increased frequency of the mutated PGP (e.g., of Caucasian descent, or non-African descent) may require less pharmaceutical compositions in the formulation due to an effective increase in efficacy of such compositions as a result of the increased effective absorption (e.g., less PGP activity) of said composition.
Moreover, in another specific embodiment, formulations of the present invention may further comprise antagonists of OATP2 (also referred to as the multiresistance protein, or MRP2), including antagonists of its encoding polynucleotides (e.g., antisense oligonucleotides, ribozymes, zinc-finger proteins, etc.). The invention also further comprises any additional antagonists known to inhibit proteins thought to be attributable to a multidrug resistant phenotype in proliferating cells.
Preferred antagonists that formulations of the present may comprise include the potent P-glycoprotein inhibitor elacridar, and/or LY-335979. Other P-glycoprotein known in the art are also encompassed by the present invention.
In additional embodiments, the Therapeutics of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

Example 27

Method of Treating Decreased Levels of the Polypeptide

The present invention relates to a method for treating an individual in need of an increased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an agonist of the invention (including polypeptides of the invention). Moreover, it will be appreciated that conditions caused by a decrease in the standard or normal expression level of a secreted protein in an individual can be treated by administering the polypeptide of the present invention, preferably in the secreted form. Thus, the invention also provides a method of treatment of an individual in need of an increased level of the polypeptide comprising administering to such an individual a Therapeutic comprising an amount of the polypeptide to increase the activity level of the polypeptide in such an individual.
For example, a patient with decreased levels of a polypeptide receives a daily dose 0.1-100 ug/kg of the polypeptide for six consecutive days. Preferably, the polypeptide is in the secreted form. The exact details of the dosing scheme, based on administration and formulation, are provided herein.

Example 28

Method of Treating Increased Levels of the Polypeptide

The present invention also relates to a method of treating an individual in need of a decreased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an antagonist of the invention (including polypeptides and antibodies of the invention).
In one example, antisense technology is used to inhibit production of a polypeptide of the present invention. This technology is one example of a method of decreasing levels of a polypeptide, preferably a secreted form, due to a variety of etiologies, such as cancer. For example, a patient diagnosed with abnormally increased levels of a polypeptide is administered intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day rest period if the treatment was well tolerated. The formulation of the antisense polynucleotide is provided herein.

Example 29

Method of Treatment Using Gene Therapy-Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable of expressing a polypeptide, onto a patient. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37 degree C. for approximately one week.
At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.
pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention can be amplified using PCR primers which correspond to the 5′ and 3′ end sequences respectively as set forth in Example 13 using primers and having appropriate restriction sites and initiation/stop codons, if necessary. Preferably, the 5′ primer contains an EcoRI site and the 3′ primer includes a HindHI site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is then used to transform bacteria HB101, which are then plated onto agar containing kanamycin for the purpose of confirming that the vector has the gene of interest properly inserted.
The amphotropic pA317 or GP+am12 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether protein is produced.
The engineered fibroblasts are then transplanted onto the host, either alone or after having been-grown to confluence on cytodex 3 microcarrier-beads.

Example 30

Gene Therapy using Endogenous Genes Corresponding to Polynucleotides of the Invention

Another method of gene therapy according to the present invention involves operably associating the endogenous polynucleotide sequence of the invention with a promoter via homologous recombination as described, for example, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication NO: WO 96/29411, published Sep. 26, 1996; International Publication NO: WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not expressed in the cells, or is expressed at a lower level than desired.
Polynucleotide constructs are made which contain a promoter and targeting sequences, which are homologous to the 5′ non-coding sequence of endogenous polynucleotide sequence, flanking the promoter. The targeting sequence will be sufficiently near the 5′ end of the polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination. The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter.
The amplified promoter and the amplified targeting sequences are digested with the appropriate restriction enzymes and subsequently treated with calf intestinal phosphatase. The digested promoter and digested targeting sequences are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The construct is size fractionated on an agarose gel then purified by phenol extraction and ethanol precipitation.
In this Example, the polynucleotide constructs are administered as naked polynucleotides via electroporation. However, the polynucleotide constructs may also be administered with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, precipitating agents, etc. Such methods of delivery are known in the art.
Once the cells are transfected, homologous recombination will take place which results in the promoter being operably linked to the endogenous polynucleotide sequence. This results in the expression of polynucleotide corresponding to the polynucleotide in the cell. Expression may be detected by immunological staining, or any other method known in the art.
Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in DMEM +10% fetal calf serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the plastic surface with nutrient medium. An aliquot of the cell suspension is removed for counting, and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2 HPO4, 6 mM dextrose). The cells are recentrifuged, the supernatant aspirated, and the cells resuspended in electroporation buffer containing 1 mg/ml acetylated bovine serum alburnin. The final cell suspension contains approximately 3×106 cells/ml. Electroporation should be performed immediately following resuspension.
Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to the locus corresponding to the polynucleotide of the invention, plasmid pUC18 (MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The CMV promoter is amplified by PCR with an XbaI site on the 5′ end and a BamHI site on the 3′end. Two non-coding sequences are amplified via PCR: one non-coding sequence (fragment 1) is amplified with a HindHI site at the 5′ end and an Xba site at the 3′end; the other non-coding sequence (fragment 2) is amplified with a BamHI site at the 5′end and a HindIII site at the 3′end. The CMV promoter and the fragments (1 and 2) are digested with the appropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1—XbaI; fragment 2-BamHI) and ligated together. The resulting ligation product is digested with HindIII, and ligated with the HindIII-digested pUC18 plasmid.
Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap (Bio-Rad). The final DNA concentration is generally at least 120 μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5.×106 cells) is then added to the cuvette, and the cell suspension and DNA solutions are gently mixed. Electroporation is performed with a Gene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960 PF and 250-300 V, respectively. As voltage increases, cell survival decreases, but the percentage of surviving cells that stably incorporate the introduced DNA into their genome increases dramatically. Given these parameters, a pulse time of approximately 14-20 mSec should be observed.
Electroporated cells are maintained at room temperature for approximately 5 min, and the contents of the cuvette are then gently removed with a sterile transfer pipette. The cells are added directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cm dish and incubated at 37 degree C. The following day, the media is aspirated and replaced with 10 ml of fresh media and incubated for a further 16-24 hours.
The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product. The fibroblasts can then be introduced into a patient as described above.

Example 31

Method of Treatment Using Gene Therapy—In Vivo

Another aspect of the present invention is using in vivo gene therapy methods to treat disorders, diseases and conditions. The gene therapy method relates to the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to increase or decrease the expression of the polypeptide. The polynucleotide of the present invention may be operatively linked to a promoter or any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. No. 5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res. 35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997); Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., Gene Ther. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290 (1996) (incorporated herein by reference).
The polynucleotide constructs may be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and the like). The polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
The term “naked” polynucleotide, DNA or RNA, refers to sequences that are free from any delivery vehicle that acts to assist, promote, or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the present invention may also be delivered in liposome formulations (such as those taught in Felgner P. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods well known to those skilled in the art.
The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Any strong promoter known to those skilled in the art can be used for driving the expression of DNA. Unlike other gene therapies techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.
The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
For the naked polynucleotide injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 g/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.
The dose response effects of injected polynucleotide in muscle in vivo is determined as follows. Suitable template DNA for production of mRNA coding for polypeptide of the present invention is prepared in accordance with a standard recombinant DNA methodology. The template DNA, which may be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps muscles of mice are then injected with various amounts of the template DNA.
Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. The template DNA is injected in 0.1 ml of carrier in a I cc syringe through a 27 gauge needle over one minute, approximately 0.5 cm from the distal insertion site of the muscle into the knee and about 0.2 cm deep. A suture is placed over the injection site for future localization, and the skin is closed with stainless steel clips.
After an appropriate incubation time (e.g., 7 days) muscle extracts are prepared by excising the entire quadriceps. Every fifth 15 urn cross-section of the individual quadriceps muscles is histochemically stained for protein expression. A time course for protein expression may be done in a similar fashion except that quadriceps from different mice are harvested at different times. Persistence of DNA in muscle following injection may be determined by Southern blot analysis after preparing total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experimentation in mice can be use to extrapolate proper dosages and other treatment parameters in humans and other animals using naked DNA.

Example 32

Transgenic Animals

The polypeptides of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.
Any technique known in the art may be used to introduce the transgene (i.e., polynucleotides of the invention) into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al., Science 259:1745 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For a review of such techniques, see Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by reference herein in its entirety.
Any technique known in the art may be used to produce transgenic clones containing polynucleotides of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).
The present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR(RT-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.
Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.
Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying diseases, disorders, and/or conditions associated with aberrant expression, and in screening for compounds effective in ameliorating such diseases, disorders, and/or conditions.

Example 33

Knock-Out Animals

Endogenous gene expression can also be reduced by inactivating or “knocking out” the gene and/or its promoter using targeted homologous recombination. (E.g., see Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989); each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo. In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra). However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that will be apparent to those of skill in the art.
In further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally.
Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated by reference herein in its entirety).
When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
Transgenic and “knock-out” animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying diseases, disorders, and/or conditions associated with aberrant expression, and in screening for compounds effective in ameliorating such diseases, disorders, and/or conditions.

Example 34

Method of Isolating Antibody Fragments Directed Against CAN-12 From a Library of scFvs

Naturally occurring V-genes isolated from human PBLs are constructed into a library of antibody fragments which contain reactivities against CAN-12 to which the donor may or may not have been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein by reference in its entirety).
Rescue of the Library. A library of scFvs is constructed from the RNA of human PBLs as described in PCT publication WO 92/01047. To rescue phage displaying antibody fragments, approximately 109 E. coli harboring the phagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and 100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of this culture is used to inoculate 50 ml of 2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, see PCT publication WO 92/01047) are added and the culture incubated at 37° C. for 45 minutes without shaking and then at 37° C. for 45 minutes with shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillin and 50 ug/ml kanamycin and grown overnight. Phage are prepared as described in PCT publication WO 92/01047.
M13 delta gene III is prepared as follows: M13 delta gene El[ helper phage does not encode gene Ell protein, hence the phage(mid) displaying antibody fragments have a greater avidity of binding to antigen. Infectious M13 delta gene III particles are made by growing the helper phage in cells harboring a pUC19 derivative supplying the wild type gene III protein during phage morphogenesis. The culture is incubated for 1 hour at 37° C. without shaking and then for a further hour at 37° C. with shaking. Cells are spun down (EEC-Centra 8,400 r.p.m. for 10 min), resuspended in 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25 μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C. Phage particles are purified and concentrated from the culture medium by two PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS and passed through a 0.45 μm filter (Minisart NML; Sartorius) to give a final concentration of approximately 1013 transducing units/ml (ampicillin-resistant clones).
Panning of the Library. Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of the present invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage is applied to the tube and incubated for 30 minutes at room temperature tumbling on an over and under turntable and then left to stand for another 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes on an under and over turntable after which the solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG1 by incubating eluted phage with bacteria for 30 minutes at 37° C. The E. coli are then plated on TYE plates containing 1% glucose and 100 μg/ml ampicillin. The resulting bacterial library is then rescued with delta gene 3 helper phage as described above to prepare phage for a subsequent round of selection. This process is then repeated for a total of 4 rounds of affinity purification with tube-washing increased to 20 times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.
Characterization of Binders. Eluted phage from the 3rd and 4th rounds of selection are used to infect E. coli HB 2151 and soluble scFv is produced (Marks, et al., 1991) from single colonies for assay. ELISAs are performed with microtitre plates coated with either 10 pg/ml of the polypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clones positive in ELISA are further characterized by PCR fingerprinting (see, e.g., PCT publication WO 92/01047) and then by sequencing. These ELISA positive clones may also be further characterized by techniques known in the art, such as, for example, epitope mapping, binding affinity, receptor signal transduction, ability to block or competitively inhibit antibody/antigen binding, and competitive agonistic or antagonistic activity.
Moreover, in another preferred method, the antibodies directed against the polypeptides of the present invention may be produced in plants. Specific methods are disclosed in U.S. Pat. Nos. 5,959,177, and 6,080,560, which are hereby incorporated in their entirety herein. The methods not only describe methods of expressing antibodies, but also the means of assembling foreign multimeric proteins in plants (i.e., antibodies, etc,), and the subsequent secretion of such antibodies from the plant.

Example 35

Identification and Cloning of VH and VL domains of Antibodies Directed Against the CAN-12 Polypeptide

VH and VL domains may be identified and cloned from cell lines expressing an antibody directed against a CAN-12 epitope by performing PCR with VH and VL specific primers on cDNA made from the antibody expressing cell lines. Briefly, RNA is isolated from the cell lines and used as a template for RT-PCR designed to amplify the VH and VL domains of the antibodies expressed by the EBV cell lines. Cells may be lysed using the TRIzol reagent (Life Technologies, Rockville, Md.) and extracted with one fifth volume of chloroform. After addition of chloroform, the solution is allowed to incubate at room temperature for 10 minutes, and then centrifuged at 14,000 rpm for 15 minutes at 4 C in a tabletop centrifuge. The supernatant is collected and RNA is precipitated using an equal volume of isopropanol. Precipitated RNA is pelleted by centrifuging at 14,000 rpm for 15 minutes at 4 C in a tabletop centrifuge.
Following centrifugation, the supernatant is discarded and washed with 75% ethanol. Following the wash step, the RNA is centrifuged again at 800 rpm for 5 minutes at 4 C. The supernatant is discarded and the pellet allowed to air dry. RNA is the dissolved in DEPC water and heated to 6° C. for 10 minutes. Quantities of RNA can be determined using optical density measurements. cDNA may be synthesized, according to methods well-known in the art and/or described herein, from 1.5-2.5 micrograms of RNA using reverse transciptase and random hexamer primers. cDNA is then used as a template for PCR amplification of VH and VL domains.

Primers used to amplify VH and VL genes are shown below. Typically a PCR reaction makes use of a single 5′primer and a single 3′primer. Sometimes, when the amount of available RNA template is limiting, or for greater efficiency, groups of 5′ and/or 3′primers may be used. For example, sometimes all five VH-5′primers and all JH3′primers are used in a single PCR reaction. The PCR reaction is carried out in a 50 microliter volume containing 1× PCR buffer, 2 mM of each DNTP, 0.7 units of High Fidelity Taq polymerse, 5′primer mix, 3′primer mix and 7.5 microliters of cDNA. The 5′and 3′primer mix of both VH and VL can be made by pooling together 22 pmole and 28 pmole, respectively, of each of the individual primers. PCR conditions are: 96 C for 5 minutes; followed by 25 cycles of 94 C for 1 minute, 5° C. for 1 minute, and 72 C for 1 minute; followed by an extension cycle of 72 C for 10 minutes. After the reaction has been completed, sample tubes may be stored at 4 C.

Primer Sequences Used to Amplify VH domains.

		SEQ ID
Primer name	Primer Sequence	NO:

Hu VH1 -5′	CAGGTGCAGCTGGTGCAGTCTGG	105

Hu VH2 -5′	CAGGTCAACTTAAGGGAGTCTGG	106

Hu VH3 -5′	GAGGTGCAGCTGGTGGAGTCTGG	107

Hu VH4 -5′	CAGGTGCAGCTGCAGGAGTCGGG	108

Hu VH5 -5′	GAGGTGCAGCTGTTGCAGTCTGC	109

Hu VH6 -5′	CAGGTACAGCTGCAGCAGTCAGG	110

Hu JH1 -5′	TGAGGAGACGGTGACCAGGGTGCC	111

Hu JH3 -5′	TGAAGAGACGGTGACCATTGTCCC	112

Hu JH4 -5′	TGAGGAGACGGTGACCAGGGTTCC	113

Hu JH6 -5′	TGAGGAGACGGTGACCGTGGTCCC	114

Hu Vkappa1 -5′	GACATCCAGATGACCCAGTCTCC	115

Hu Vkappa2a -5′	GATGTTGTGATGACTCAGTCTCC	116

Hu Vkappa2b -5′	GATATTGTGATGACTCAGTCTCC	117

Hu Vkappa3 -5′	GAAATTGTGTTGACGCAGTCTCC	118

Hu Vkappa4 -5′	GACATCGTGATGACCCAGTCTCC	119

Hu Vkappa5 -5′	GAAACGACACTCACGCAGTCTCC	120

Hu Vkappa6 -5′	GAAATTGTGCTGACTCAGTCTCC	121

Hu Vlambda1 -5′	CAGTCTGTGTTGACGCAGCCGCC	122

Hu Vlambda2 -5′	CAGTCTGCCCTGACTCAGCCTGC	123

Hu Vlambda3 -5′	TCCTATGTGCTGACTCAGCCACC	124

Hu Vlambda3b -5′	TCTTCTGAGCTGACTCAGGACCC	125

Hu Vlambda4 -5′	CACGTTATACTGACTCAACCGCC	126

Hu Vlambda5 -5′	CAGGCTGTGCTCACTCAGCCGTC	127

Hu Vlambda6 -5′	AATTTTATGCTGACTCAGCCCCA	128

Hu Jkappa1 -3′	ACGTTTGATTTCCACCTTGGTCCC	129

Hu Jkappa2 -3′	ACGTTTGATCTCCAGCTTGGTCCC	130

Hu Jkappa3 -3′	ACGTTTGATATCCACTTTGGTCCC	131

Hu Jkappa4 -3′	ACGTTTGATCTCCACCTTGGTCCC	132

Hu Jkappa5 -3′	ACGTTTAATCTCCAGTCGTGTCCC	133

Hu Vlambda1 -3′	CAGTCTGTGTTGACGCAGCCGCC	134

Hu Vlambda2 -3′	CAGTCTGCCCTGACTCAGCCTGC	135

Hu Vlambda3 -3′	TCCTATGTGCTGACTCAGCCACC	136

Hu Vlambda3b -3′	TCTTCTGAGCTGACTCAGGACCC	137

Hu Vlambda4 -3′	CACGTTATACTGACTCAACCGCC	138

Hu Vlambda5 -3′	CAGGCTGTGCTCACTCAGCCGTC	139

Hu Vlambda6 -3′	AATTTTATGCTGACTCAGCCCCA	140

PCR samples are then electrophoresed on a 1.3% agarose gel. DNA bands of the expected sizes (−506 base pairs for VH domains, and 344 base pairs for VL domains) can be cut out of the gel and purified using methods well known in the art and/or described herein.
Purified PCR products can be ligated into a PCR cloning vector (TA vector from Invitrogen Inc., Carlsbad, Calif.). Individual cloned PCR products can be isolated after transfection of E. coli and blue/white color selection. Cloned PCR products may then be sequenced using methods commonly known in the art and/or described herein.
The PCR bands containing the VH domain and the VL domains can also be used to create full-length Ig expression vectors. VH and VL domains can be cloned into vectors containing the nucleotide sequences of a heavy (e.g., human IgG1 or human IgG4) or light chain (human kappa or human ambda) constant regions such that a complete heavy or light chain molecule could be expressed from these vectors when transfected into an appropriate host cell. Further, when cloned heavy and light chains are both expressed in one cell line (from either one or two vectors), they can assemble into a complete functional antibody molecule that is secreted into the cell culture medium. Methods using polynucleotides encoding VH and VL antibody domain to generate expression vectors that encode complete antibody molecules are well known within the art.

Example 36

Assays Detecting Stimulation or Inhibition of B cell Proliferation and Differentiation

Generation of functional humoral immune responses requires both soluble and cognate signaling between B-lineage cells and their microenvironment. Signals may impart a positive stimulus that allows a B-lineage cell to continue its programmed development, or a negative stimulus that instructs the cell to arrest its current developmental pathway. To date, numerous stimulatory and inhibitory signals have been found to influence B cell responsiveness including IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-13, IL-14 and IL-15. Interestingly, these signals are by themselves weak effectors but can, in combination with various co-stimulatory proteins, induce activation, proliferation, differentiation, homing, tolerance and death among B cell populations.
One of the best studied classes of B-cell co-stimulatory proteins is the TNF-superfamily. Within this family CD40, CD27, and CD30 along with their respective higands CD154, CD70, and CD153 have been found to regulate a variety of immune responses. Assays which allow for the detection and/or observation of the proliferation and differentiation of these B-cell populations and their precursors are valuable tools in determining the effects various proteins may have on these B-cell populations in terms of proliferation and differentiation. Listed below are two assays designed to allow for the detection of the differentiation, proliferation, or inhibition of B-cell populations and their precursors.
In Vitro Assay—Purified polypeptides of the invention, or truncated forms thereof, is assessed for its ability to induce activation, proliferation, differentiation or inhibition and/or death in B-cell populations and their precursors. The activity of the polypeptides of the invention on purified human tonsillar B cells, measured qualitatively over the dose range from 0.1 to 10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulation assay in which purified tonsillar B cells are cultured in the presence of either formalin-fixed Staphylococcus aureus Cowan I (SAC) or immobilized anti-human IgM antibody as the priming agent. Second signals such as IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicit B cell proliferation as measured by tritiated-thymidine incorporation. Novel synergizing agents can be readily identified using this assay. The assay involves isolating human tonsillar B cells by magnetic bead (MACS) depletion of CD3-positive cells. The resulting cell population is greater than 95% B cells as assessed by expression of CD45R(B220).
Various dilutions of each sample are placed into individual wells of a 96-well plate to which are added 105 B-cells suspended in culture medium (RPMI 1640 containing 10% FBS, 5×10-5M 2ME, 100 U/ml penicillin, 10 ug/ml streptomycin, and 10-5 dilution of SAC) in a total volume of 150 ul. Proliferation or inhibition is quantitated by a 20 h pulse (1 uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factor addition. The positive and negative controls are IL2 and medium respectively.
In Vivo Assay—BALB/c mice are injected (i.p.) twice per day with buffer only, or 2 mg/Kg of a polypeptide of the invention, or truncated forms thereof. Mice receive this treatment for 4 consecutive days, at which time they are sacrificed and various tissues and serum collected for analyses. Comparison of H&E sections from normal spleens and spleens treated with polypeptides of the invention identify the results of the activity of the polypeptides on spleen cells, such as the diffusion of peri-arterial lymphatic sheaths, and/or significant increases in the nucleated cellularity of the red pulp regions, which may indicate the activation of the differentiation and proliferation of B-cell populations. Immunohistochemical studies using a B cell marker, anti-CD45R(B220), are used to determine whether any physiological changes to splenic cells, such as splenic disorganization, are due to increased B-cell representation within loosely defined B-cell zones that infiltrate established T-cell regions.
Flow cytometric analyses of the spleens from mice treated with polypeptide is used to indicate whether the polypeptide specifically increases the proportion of ThB+, CD45R(B220)dull B cells over that which is observed in control mice.
Likewise, a predicted consequence of increased mature B-cell representation in vivo is a relative increase in serum Ig titers. Accordingly, serum IgM and IgA levels are compared between buffer and polypeptide-treated mice.
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 37

T Cell Proliferation Assay

A CD3-induced proliferation assay is performed on PBMCs and is measured by the uptake of 3H-thymidine. The assay is performed as follows. Ninety-six well plates are coated with 100 (l/well of mAb to CD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4 degrees C. (1 (g/ml in 0.05M bicarbonate buffer, pH 9.5), then washed three times with PBS. PBMC are isolated by F/H gradient centrifugation from human peripheral blood and added to quadruplicate wells (5×104/well) of mAb coated plates in RPMI containing 10% FCS and P/S in the presence of varying concentrations of polypeptides of the invention (total volume 200 ul). Relevant protein buffer and medium alone are controls. After 48 hr. culture at 37 degrees C., plates are spun for 2 min. at 1000 rpm and 100 (1 of supernatant is removed and stored −20 degrees C. for measurement of IL-2 (or other cytokines) if effect on proliferation is observed. Wells are supplemented with 100 ul of medium containing 0.5 uCi of 3H-thymidine and cultured at 37 degrees C. for 18-24 hr. Wells are harvested and incorporation of 3H-thymidine used as a measure of proliferation. Anti-CD3 alone is the positive control for proliferation. IL-2 (100 U/ml) is also used as a control which enhances proliferation. Control antibody which does not induce proliferation of T cells is used as the negative controls for the effects of polypeptides of the invention.
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 38

Effect of Polypeptides of the Invention on the Expression of MHC Class II, Costimulatory and Adhesion Molecules and Cell Differentiation of Monocytes and Monocyte-Derived Human Dendritic Cells

Dendritic cells are generated by the expansion of proliferating precursors found in the peripheral blood: adherent PBMC or elutriated monocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml) and IL4 (20 ng/ml). These dendritic cells have the characteristic phenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with activating factors, such as TNF-, causes a rapid change in surface phenotype (increased expression of MHC class I and II, costimulatory and adhesion molecules, downregulation of FC(RII, upregulation of CD83). These changes correlate with increased antigen-presenting capacity and with functional maturation of the dendritic cells.
FACS analysis of surface antigens is performed as follows. Cells are treated 1-3 days with increasing concentrations of polypeptides of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).
Effect on the production of cytokines. Cytokines generated by dendritic cells, in particular IL-12, are important in the initiation of T-cell dependent immune responses. IL-12 strongly influences the development of Th1 helper T-cell immune response, and induces cytotoxic T and NK cell function. An ELISA is used to measure the IL-12 release as follows. Dendritic cells (106/ml) are treated with increasing concentrations of polypeptides of the invention for 24 hours. LPS (100 ng/ml) is added to the cell culture as positive control. Supernatants from the cell cultures are then collected and analyzed for IL-12 content using commercial ELISA kit(e.g., R & D Systems (Minneapolis, Minn.)). The standard protocols provided with the kits are used.
Effect on the expression of MHC Class II, costimulatory and adhesion molecules. Three major families of cell surface antigens can be identified on monocytes: adhesion molecules, molecules involved in antigen presentation, and Fc receptor. Modulation of the expression of MHC class II antigens and other costimulatory molecules, such as B7 and ICAM-1, may result in changes in the antigen presenting capacity of monocytes and ability to induce T cell activation. Increase expression of Fc receptors may correlate with improved monocyte cytotoxic activity, cytokine release and phagocytosis.
FACS analysis is used to examine the surface antigens as follows. Monocytes are treated 1-5 days with increasing concentrations of polypeptides of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).
Monocyte activation and/or increased survival. Assays for molecules that activate (or alternatively, inactivate) monocytes and/or increase monocyte survival (or alternatively, decrease monocyte survival) are known in the art and may routinely be applied to determine whether a molecule of the invention functions as an inhibitor or activator of monocytes. Polypeptides, agonists, or antagonists of the invention can be screened using the three assays described below. For each of these assays, Peripheral blood mononuclear cells (PBMC) are purified from single donor leukopacks (American Red Cross, Baltimore, Md.) by centrifugation through a Histopaque gradient (Sigma). Monocytes are isolated from PBMC by counterflow centrifugal elutriation.
Monocyte Survival Assay. Human peripheral blood monocytes progressively lose viability when cultured in absence of serum or other stimuli. Their death results from internally regulated process (apoptosis). Addition to the culture of activating factors, such as TNF-alpha dramatically improves cell survival and prevents DNA fragmentation. Propidium iodide (PI) staining is used to measure apoptosis as follows. Monocytes are cultured for 48 hours in polypropylene tubes in serum-free medium (positive control), in the presence of 100 ng/ml TNF-alpha (negative control), and in the presence of varying concentrations of the compound to be tested. Cells are suspended at a concentration of 2×106/ml in PBS containing PI at a final concentration of 5 (g/ml, and then incubated at room temperature for 5 minutes before FACScan analysis. PI uptake has been demonstrated to correlate with DNA fragmentation in this experimental paradigm.
Effect on cytokine release. An important function of monocytes/macrophages is their regulatory activity on other cellular populations of the immune system through the release of cytokines after stimulation. An ELISA to measure cytokine release is performed as follows. Human monocytes are incubated at a density of 5×105 cells/ml with increasing concentrations of the a polypeptide of the invention and under the same conditions, but in the absence of the polypeptide. For IL-12 production, the cells are primed overnight with IFN (100 U/ml) in presence of a polypeptide of the invention. LPS (10 ng/ml) is then added. Conditioned media are collected after 24h and kept frozen until use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then performed using a commercially available ELISA kit(e.g., R & D Systems (Minneapolis, Minn.)) and applying the standard protocols provided with the kit.
Oxidative burst. Purified monocytes are plated in 96-w plate at 2-1×105 cell/well. Increasing concentrations of polypeptides of the invention are added to the wells in a total volume of 0.2 ml culture medium (RPMI 1640+10% FCS, glutamine and antibiotics). After 3 days incubation, the plates are centrifuged and the medium is removed from the wells. To the macrophage monolayers, 0.2 ml per well of phenol red solution (140 mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is added, together with the stimulant (200 nM PMA). The plates are incubated at 37(C for 2 hours and the reaction is stopped by adding 20 μl 1N NaOH per well. The absorbance is read at 610 nm. To calculate the amount of H2O2 produced by the macrophages, a standard curve of a H2O2 solution of known molarity is performed for each experiment.
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 39

Biological Effects of Polypeptides of the Invention

Astrocyte and Neuronal Assays.
Recombinant polypeptides of the invention, expressed in Escherichia coli and purified as described above, can be tested for activity in promoting the survival, neurite outgrowth, or phenotypic differentiation of cortical neuronal cells and for inducing the proliferation of glial fibrillary acidic protein immunopositive cells, astrocytes. The selection of cortical cells for the bioassay is based on the prevalent expression of FGF-1 and FGF-2 in cortical structures and on the previously reported enhancement of cortical neuronal survival resulting from FGF-2 treatment. A thymidine incorporation assay, for example, can be used to elucidate a polypeptide of the invention's activity on these cells.
Moreover, previous reports describing the biological effects of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro have demonstrated increases in both neuron survival and neurite outgrowth (Walicke et al., “Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension.” Proc. Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated by reference in its entirety). However, reports from experiments done on PC-12 cells suggest that these two responses are not necessarily synonymous and may depend on not only which FGF is being tested but also on which receptor(s) are expressed on the target cells. Using the primary cortical neuronal culture paradigm, the ability of a polypeptide of the invention to induce neurite outgrowth can be compared to the response achieved with FGF-2 using, for example, a thymidine incorporation assay.
Fibroblast and Endothelial Cell Assays.
Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.) and maintained in growth media from Clonetics. Dermal microvascular endothelial cells are obtained from Cell Applications (San Diego, Calif.). For proliferation assays, the human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 cells/well in a 96-well plate for one day in growth medium. The cells are then incubated for one day in 0.1% BSA basal medium. After replacing the medium with fresh 0.1% BSA medium, the cells are incubated with the test proteins for 3 days. Alamar Blue (Alamar Biosciences, Sacramento, Calif.) is added to each well to a final concentration of 10%. The cells are incubated for 4 hr. Cell viability is measured by reading in a CytoFluor fluorescence reader. For the PGE2 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or polypeptides of the invention with or without IL-1( for 24 hours. The supernatants are collected and assayed for PGE2 by EIA kit (Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or with or without polypeptides of the invention IL-1 (for 24 hours. The supernatants are collected and assayed for IL-6 by ELISA kit (Endogen, Cambridge, Mass.).
Human lung fibroblasts are cultured with FGF-2 or polypeptides of the invention for 3 days in basal medium before the addition of Alamar Blue to assess effects on growth of the fibroblasts. FGF-2 should show a stimulation at 10 -2500 ng/ml which can be used to compare stimulation with polypeptides of the invention.
Parkinson Models.
The loss of motor function in Parkinson's disease is attributed to a deficiency of striatal dopamine resulting from the degeneration of the nigrostriatal dopaminergic projection neurons. An animal model for Parkinson's that has been extensively characterized involves the systemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized by monoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP+) and released. Subsequently, MPP+ is actively accumulated in dopaminergic neurons by the high-affinity reuptake transporter for dopamine. MPP+ is then concentrated in mitochondria by the electrochemical gradient and selectively inhibits nicotidamide adenine disphosphate: ubiquinone oxidoreductionase (complex I), thereby interfering with electron transport and eventually generating oxygen radicals.
It has been demonstrated in tissue culture paradigms that FGF-2 (basic FGF) has trophic activity towards nigral dopaminergic neurons (Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's group has demonstrated that administering FGF-2 in gel foam implants in the striatum results in the near complete protection of nigral dopaminergic neurons from the toxicity associated with MPTP exposure (Otto and Unsicker, J. Neuroscience, 1990).
Based on the data with FGF-2, polypeptides of the invention can be evaluated to determine whether it has an action similar to that of FGF-2 in enhancing dopaminergic neuronal survival in vitro and it can also be tested in vivo for protection of dopaminergic neurons in the striatum from the damage associated with MPTP treatment. The potential effect of a polypeptide of the invention is first examined in vitro in a dopaminergic neuronal cell culture paradigm. The cultures are prepared by dissecting the midbrain floor plate from gestation day 14 Wistar rat embryos. The tissue is dissociated with trypsin and seeded at a density of 200,000 cells/ctn2 on polyorthinine-laminin coated glass coverslips. The cells are maintained in Dulbecco's Modified Eagle's medium and F12 medium containing hormonal supplements (N1). The cultures are fixed with paraformaldehyde after 8 days in vitro and are processed for tyrosine hydroxylase, a specific marker for dopaminergic neurons, immunohistochemical staining. Dissociated cell cultures are prepared from embryonic rats. The culture medium is changed every third day and the factors are also added at that time.
Since the dopaminergic neurons are isolated from animals at gestation day 14, a developmental time which is past the stage when the dopaminergic precursor cells are proliferating, an increase in the number of tyrosine hydroxylase immunopositive neurons would represent an increase in the number of dopaminergic neurons surviving in vitro. Therefore, if a polypeptide of the invention acts to prolong the survival of dopaminergic neurons, it would suggest that the polypeptide may be involved in Parkinson's Disease.
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 40

The Effect of Polypeptides of the Invention on the Growth of Vascular Endothelial Cells

On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at 2-5×104 cells/35 mm dish density in M199 medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the medium is replaced with M199 containing 10% FBS, 8 units/ml heparin. A polypeptide having the amino acid sequence of SEQ ID NO:2, and positive controls, such as VEGF and basic FGF (bFGF) are added, at varying concentrations. On days 4 and 6, the medium is replaced. On day 8, cell number is determined with a Coulter Counter.
An increase in the number of HUVEC cells indicates that the polypeptide of the invention may proliferate vascular endothelial cells.
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 41

Stimulatory Effect of Polypeptides of the Invention on the Proliferation of Vascular Endothelial Cells

For evaluation of mitogenic activity of growth factors, the colorimetric MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) assay with the electron coupling reagent PMS (phenazine methosulfate) was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-well plate (5,000 cells/well) in 0.1 mL serum-supplemented medium and are allowed to attach overnight. After serum-starvation for 12 hours in 0.5% FBS, conditions (bFGF, VEGF165 or a polypeptide of the invention in 0.5% FBS) with or without Heparin (8 U/ml) are added to wells for 48 hours. 20 mg of MTS/PMS mixture (1:0.05) are added per well and allowed to incubate for 1 hour at 37° C. before measuring the absorbance at 490 nm in an ELISA plate reader. Background absorbance from control wells (some media, no cells) is subtracted, and seven wells are performed in parallel for each condition. See, Leak et al. In Vitro Cell. Dev. Biol. 30A:512-518 (1994).
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 42

Inhibition of PDGF-induced Vascular Smooth Muscle Cell Proliferation Stimulatory Effect

HAoSMC proliferation can be measured, for example, by BrdUrd incorporation. Briefly, subconfluent, quiescent cells grown on the 4-chamber slides are transfected with CRP or FITC-labeled AT2-3-LP. Then, the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24 h, immunocytochemistry is performed by using BrdUrd Staining Kit (Zymed Laboratories). In brief, the cells are incubated with the biotinylated mouse anti-BrdUrd antibody at 4 degrees C. for 2 h after being exposed to denaturing solution and then incubated with the streptavidin-peroxidase and diaminobenzidine. After counterstaining with hematoxylin, the cells are mounted for microscopic examination, and the BrdUrd-positive cells are counted. The BrdUrd index is calculated as a percent of the BrdUrd-positive cells to the total cell number. In addition, the simultaneous detection of the BrdUrd staining (nucleus) and the FITC uptake (cytoplasm) is performed for individual cells by the concomitant use of bright field illumination and dark field-UV fluorescent illumination. See, Hayashida et al., J. Biol. Chem. 6:271(36):21985-21992 (1996).
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 43

Stimulation of Nitric Oxide Production by Endothelial Cells

Nitric oxide released by the vascular endothelium is believed to be a mediator of vascular endothelium relaxation. Thus, activity of a polypeptide of the invention can be assayed by determining nitric oxide production by endothelial cells in response to the polypeptide.
Nitric oxide is measured in 96-well plates of confluent microvascular endothelial cells after 24 hours starvation and a subsequent 4 hr exposure to various levels of a positive control (such as VEGF-1) and the polypeptide of the invention. Nitric oxide in the medium is determined by use of the Griess reagent to measure total nitrite after reduction of nitric oxide-derived nitrate by nitrate reductase. The effect of the polypeptide of the invention on nitric oxide release is examined on HUVEC.
Briefly, NO release from cultured HUVEC monolayer is measured with a NO-specific polarographic electrode connected to a NO meter (Iso-NO, World Precision Instruments Inc.) (1049). Calibration of the NO elements is performed according to the following equation:
2 KNO2+2 KI+2 H2SO4 6 2 NO+I2+2 H2O+2 K2SO4
The standard calibration curve is obtained by adding graded concentrations of KNO2 (0, 5, 10, 25, 50, 100, 250, and 500 mmol) into the calibration solution containing KI and H2S04. The specificity of the Iso-NO electrode to NO is previously determined by measurement of NO from authentic NO gas (1050). The culture medium is removed and HUVECs are washed twice with Dulbecco's phosphate buffered saline. The cells are then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-well plates, and the cell plates are kept on a slide warmer (Lab Line Instruments Inc.) To maintain the temperature at 37° C. The NO sensor probe is inserted vertically into the wells, keeping the tip of the electrode 2 mm under the surface of the solution, before addition of the different conditions. S-nitroso acetyl penicillamin (SNAP) is used as a positive control. The amount of released NO is expressed as picomoles per 1×106 endothelial cells. All values reported are means of four to six measurements in each group (number of cell culture wells). See, Leak et al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 44

Rescue of Ischemia in Rabbit Lower Limb Model

To study the in vivo effects of polynucleotides and polypeptides of the invention on ischemia, a rabbit hindlimb ischemia model is created by surgical removal of one femoral arteries as described previously (Takeshita et al., Am J. Pathol 147:1649-1660 (1995)). The excision of the femoral artery results in retrograde propagation of thrombus and occlusion of the external iliac artery. Consequently, blood flow to the ischemic limb is dependent upon collateral vessels originating from the internal iliac artery (Takeshitaet al. Am J. Pathol 147:1649-1660 (1995)). An interval of 10 days is allowed for post-operative recovery of rabbits and development of endogenous collateral vessels. At 10 day post-operatively (day 0), after performing a baseline angiogram, the internal iliac artery of the ischemic limb is transfected with 500 mg naked expression plasmid containing a polynucleotide of the invention by arterial gene transfer technology using a hydrogel-coated balloon catheter as described (Riessen et al. Hum Gene Ther. 4:749-758 (1993); Leclerc et al. J. Clin. Invest. 90: 936-944 (1992)). When a polypeptide of the invention is used in the treatment, a single bolus of 500 mg polypeptide of the invention or control is delivered into the internal iliac artery of the ischemic limb over a period of 1 min. through an infusion catheter. On day 30, various parameters are measured in these rabbits: (a) BP ratio—The blood pressure ratio of systolic pressure of the ischemic limb to that of normal limb; (b) Blood Flow and Flow Reserve—Resting FL: the blood flow during undilated condition and Max FL: the blood flow during fully dilated condition (also an indirect measure of the blood vessel amount) and Flow Reserve is reflected by the ratio of max FL: resting FL; (c) Angiographic Score—This is measured by the angiogram of collateral vessels. A score is determined by the percentage of circles in an overlaying grid that with crossing opacified arteries divided by the total number m the rabbit thigh; (d) Capillary density—The number of collateral capillaries determined in light microscopic sections taken from hindlimbs.
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polyp eptides of the invention.

Example 45

Effect of Polypeptides of the Invention on Vasodilation

Since dilation of vascular endothelium is important in reducing blood pressure, the ability of polypeptides of the invention to affect the blood pressure in spontaneously hypertensive rats (SHR) is examined. Increasing doses (0, 10, 30, 100, 300, and 900 mg/kg) of the polypeptides of the invention are administered to 13-14 week old spontaneously hypertensive rats (SHR). Data are expressed as the mean±SEM. Statistical analysis are performed with a paired t-test and statistical significance is defined as p<0.05 vs. the response to buffer alone.
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 46

Rat Ischemic Skin Flap Model

The evaluation parameters include skin blood flow, skin temperature, and factor VII immunohistochemistry or endothelial alkaline phosphatase reaction. Expression of polypeptides of the invention, during the skin ischemia, is studied using in situ hybridization. The study in this model is divided into three parts as follows:
a) Ischemic skin
b) Ischemic skin wounds
c) Normal wounds
The experimental protocol includes:
a) Raising a 3×4 cm, single pedicle full-thickness random skin flap (myocutaneous flap over the lower back of the animal).
b) An excisional wounding (4-6 mm in diameter) in the ischemic skin (skin-flap).
c) Topical treatment with a polypeptide of the invention of the excisional wounds ( day 0, 1, 2, 3, 4 post-wounding) at the following various dosage ranges: 1 mg to 100 mg.
d) Harvesting the wound tissues at day 3, 5, 7, 0, 14 and 21 post-wounding for histological, immunohistochemical, and in situ studies.
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 47

Peripheral Arterial Disease Model

Angiogenic therapy using a polypeptide of the invention is a novel therapeutic strategy to obtain restoration of blood flow around the ischemia in case of peripheral arterial diseases. The experimental protocol includes:
a) One side of the femoral artery is ligated to create ischemic muscle of the hindlimb, the other side of hindlimb serves as a control.
b) a polypeptide of the invention, in a dosage range of 20 mg-500 mg, is delivered intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-3 weeks.
c) The ischemic muscle tissue is collected after ligation of the femoral
artery at 1, 2, and 3 weeks for the analysis of expression of a polypeptide of the invention and histology. Biopsy is also performed on the other side of normal muscle of the contralateral hindlimb.
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 48

Ischemic Myocardial Disease Model

A polypeptide of the invention is evaluated as a potent mitogen capable of stimulating the development of collateral vessels, and restructuring new vessels after coronary artery occlusion. Alteration of expression of the polypeptide is investigated in situ. The experimental protocol includes:
a) The heart is exposed through a left-side thoracotomy in the rat. Immediately, the left coronary artery is occluded with a thin suture (6-0) and the thorax is closed.
b) a polypeptide of the invention, in a dosage range of 20 mg-500 mg, is delivered intravenously and/or intramuscularly 3 times (perhaps more) per week for 24 weeks.
c) Thirty days after the surgery, the heart is removed and cross-sectioned for morphometric and in situ analyzes.
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 49

Rat Corneal Wound Healing Model

This animal model shows the effect of a polypeptide of the invention on neovascularization. The experimental protocol includes:
a) Making a 1-1.5 mm long incision from the center of cornea into the stromal layer.
b) Inserting a spatula below the lip of the incision facing the outer corner of the eye.
c) Making a pocket (its base is 1-1.5 mm form the edge of the eye).
d) Positioning a pellet, containing 50 ng-5 ug of a polypeptide of the invention, within the pocket.
e) Treatment with a polypeptide of the invention can also be applied topically to the corneal wounds in a dosage range of 20 mg-500 mg (daily treatment for five days).
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 50

Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models

A. Diabetic db+/db+ Mouse Model.
To demonstrate that a polypeptide of the invention accelerates the healing process, the genetically diabetic mouse model of wound healing is used. The full thickness wound healing model in the db+/db+ mouse is a well characterized, clinically relevant and reproducible model of impaired wound healing. Healing of the diabetic wound is dependent on formation of granulation tissue and re-epithelialization rather than contraction (Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)).
The diabetic animals have many of the characteristic features observed in Type II diabetes mellitus. Homozygous (db+/db+) mice are obese in comparison to their normal heterozygous (db+/+m) littermates. Mutant diabetic (db+/db+) mice have a single autosomal recessive mutation on chromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293 (1982)). Animals show polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose, increased or normal insulin levels, and suppressed cell-mediated immunity (Mandel et al., J. Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol. 51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)). Peripheral neuropathy, myocardial complications, and microvascular lesions, basement membrane thickening and glomerular filtration abnormalities have been described in these animals (Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl): 1-6 (1982)). These homozygous diabetic mice develop hyperglycemia that is resistant to insulin analogous to human type II diabetes (Mandel et al., J. Immunol. 120:1375-1377 (1978)).
The characteristics observed in these animals suggests that healing in this model may be similar to the healing observed in human diabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).
Genetically diabetic female C57BL/KsJ (db+/db+) mice and their non-diabetic (db+/+m) heterozygous littermates are used in this study (Jackson Laboratories). The animals are purchased at 6 weeks of age and are 8 weeks old at the beginning of the study. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. The experiments are conducted according to the rules and guidelines of Bristol-Myers Squibb Company's Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.
Wounding protocol is performed according to previously reported methods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)). Briefly, on the day of wounding, animals are anesthetized with an intraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. The dorsal region of the animal is shaved and the skin washed with 70% ethanol solution and iodine. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is then created using a Keyes tissue punch. Immediately following wounding, the surrounding skin is gently stretched to eliminate wound expansion. The wounds are left open for the duration of the experiment. Application of the treatment is given topically for 5 consecutive days commencing on the day of wounding. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.
Wounds are visually examined and photographed at a fixed distance at the day of surgery and at two day intervals thereafter. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.
A polypeptide of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.
Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.
Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3) treated group.
Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total square area of the wound. Contraction is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64mm2, the corresponding size of the dermal punch. Calculations are made using the following formula:
[Open area on day 8]−[Open area on day 1]/ [Open area on day 1]
Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5mm) and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds are used to assess whether the healing process and the morphologic appearance of the repaired skin is altered by treatment with a polypeptide of the invention. This assessment included verification of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometer is used by a blinded observer.
Tissue sections are also stained immunohistochemically with a polyclonal rabbit anti-human keratin antibody using ABC Elite detection system. Human skin is used as a positive tissue control while non-immune IgG is used as a negative control. Keratinocyte growth is determined by evaluating the extent of reepithelialization of the wound using a calibrated lens micrometer.
Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens is demonstrated by using anti-PCNA antibody (1:50) with an ABC Elite detection system. Human colon cancer can serve as a positive tissue control and human brain tissue can be used as a negative tissue control. Each specimen includes a section with omission of the primary antibody and substitution with non-immune mouse IgG. Ranking of these sections is based on the extent of proliferation on a scale of 0-8, the lower side of the scale reflecting slight proliferation to the higher side reflecting intense proliferation.
Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.
B. Steroid Impaired Rat Model
The inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid Action: Basic and Clinical Aspects. 280-302 (1989); Wahiet al., J. Immunol. 115: 476-481 (1975); Werb et al., J. Exp. Med. 147:1684-1694 (1978)). Glucocorticoids retard wound healing by inhibiting angiogenesis, decreasing vascular permeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)), fibroblast proliferation, and collagen synthesis (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978)) and producing a transient reduction of circulating monocytes (Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989)). The systemic administration of steroids to impaired wound healing is a well establish phenomenon in rats (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad. Sci. USA 86: 2229-2233 (1989)).
To demonstrate that a polypeptide of the invention can accelerate the healing process, the effects of multiple topical applications of the polypeptide on full thickness excisional skin wounds in rats in which healing has been impaired by the systemic administration of methylprednisolone is assessed.
Young adult male Sprague Dawley rats weighing 250-300 g (Charles River Laboratories) are used in this example. The animals are purchased at 8 weeks of age and are 9 weeks old at the beginning of the study. The healing response of rats is impaired by the systemic administration of methylprednisolone (17 mg/kg/rat intramuscularly) at the time of wounding. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. This study would be conducted according to the rules and guidelines of Bristol-Myers Squibb Corporations Guidelines for the Care and Use of Laboratory Animals.
The wounding protocol is followed according to section A, above. On the day of wounding, animals are anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsal region of the animal is shaved and the skin washed with 70% ethanol and iodine solutions. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is created using a Keyes tissue punch. The wounds are left open for the duration of the experiment. Applications of the testing materials are given topically once a day for 7 consecutive days commencing on the day of wounding and subsequent to methylprednisolone administration. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.
Wounds are visually examined and photographed at a fixed distance at the day of wounding and at the end of treatment. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.
The polypeptide of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.
Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.
Four groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebo control 3) treated groups.
Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total area of the wound. Closure is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm2, the corresponding size of the dermal punch. Calculations are made using the following formula:
[Open area on day 8]−[Open area on day 1]/[Open area on day 1]
Specimens are fixed in 10% buffered formalin and paraffm embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds allows assessment of whether the healing process and the morphologic appearance of the repaired skin is improved by treatment with a polypeptide of the invention. A calibrated lens micrometer is used by a blinded observer to determine the distance of the wound gap.
Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.
It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background of the Invention, Detailed Description, and Examples is hereby incorporated herein by reference. Further, the hard copy of the sequence listing submitted herewith and the corresponding computer readable form are both incorporated herein by reference in their entireties.

TABLE IV


Residue	Atom name	x coord	y coord	z coord

1	LYS12	N	−27.2159	19.7201	−6.2824
2	LYS12	CA	−26.6971	18.4591	−5.7744
3	LYS12	C	−27.7349	17.3482	−5.7385
4	LYS12	O	−27.4109	16.2063	−6.0352
5	LYS12	CB	−26.1266	18.6653	−4.3594
6	LYS12	CG	−24.7332	18.0137	−4.2714
7	LYS12	CD	−24.8483	16.4855	−4.433
8	LYS12	CE	−23.4439	15.8647	−4.5476
9	LYS12	NZ	−22.7011	16.0707	−3.2959
10	LEU13	N	−28.9809	17.682	−5.4127
11	LEU13	CA	−30.064	16.6951	−5.4047
12	LEU13	C	−30.2537	16.2158	−6.8405
13	LEU13	O	−30.3861	15.0234	−7.0985
14	LEU13	CB	−31.3563	17.3552	−4.8907
15	LEU13	CG	−31.1721	17.7804	−3.421
16	LEU13	CD1	−32.4543	18.4737	−2.9249
17	LEU13	CD2	−30.8835	16.5465	−2.5441
18	ALA14	N	−30.2416	17.1744	−7.7676
19	ALA14	CA	−30.3825	16.9101	−9.195
20	ALA14	C	−29.2866	15.9587	−9.6613
21	ALA14	O	−29.5553	14.9807	−10.3628
22	ALA14	CB	−30.3313	18.232	−9.9835
23	PRO15	N	−28.0569	16.2571	−9.2419
24	PRO15	CA	−26.8772	15.4556	−9.563
25	PRO15	C	−27.029	14.0783	−8.9231
26	PRO15	O	−26.6765	13.0642	−9.5273
27	PRO15	CB	−25.7231	16.2219	−8.8922
28	PRO15	CG	−26.2623	17.6337	−8.5936
29	PRO15	CD	−27.7987	17.5229	−8.5821
30	ARG16	N	−27.58	14.0482	−7.7102
31	ARG16	CA	−27.7908	12.7957	−6.9958
32	ARG16	C	−28.7852	11.9184	−7.7336
33	ARG16	O	−28.7192	10.6897	−7.6578
34	ARG16	CB	−28.4328	13.1445	−5.6398
35	ARG16	CG	−27.3904	13.7645	−4.6917
36	ARG16	CD	−28.1224	14.4083	−3.4996
37	ARG16	NE	−27.4893	15.6684	−3.1552
38	ARG16	CZ	−26.8721	15.823	−2.0196
39	ARG16	NH1	−26.3196	16.9665	−1.7426
40	ARG16	NH2	−26.797	14.8537	−1.1553
41	TYR17	N	−29.9822	12.8509	−8.5832
42	TYR17	CA	−30.4267	11.5765	−9.1118
43	TYR17	C	−29.9697	11.2849	−10.5385
44	TYR17	O	−30.2553	10.2132	−11.0727
45	TYR17	CB	−31.971	11.5383	−9.0666
46	TYR17	CG	−32.529	12.9543	−8.9375
47	TYR17	CD1	−32.5301	13.8102	−10.0419
48	TYR17	CD2	−33.0158	13.3997	−7.706
49	TYR17	CE1	−32.8944	15.1493	−9.8813
50	TYR17	CE2	−33.3803	14.7389	−7.5456
51	TYR17	CZ	−33.287	15.6204	−8.6256
52	TYR17	OH	−33.5844	16.9663	−8.4507
53	GLN28	N	−17.3167	19.6634	−20.9577
54	GLN28	CA	−16.6717	19.1545	−22.1761
55	GLN28	C	−17.6399	18.3959	−23.1004
56	GLN28	O	−17.691	18.6208	−24.3087
57	GLN28	CB	−15.9024	20.2732	−22.9059
58	GLN28	CG	−16.8571	21.4214	−23.2838
59	GLN28	CD	−16.0631	22.5632	−23.8481
60	GLN28	OE1	−16.2528	22.9259	−24.9971
61	GLN28	NE2	−15.1606	23.145	−23.0375
62	ASP29	N	−17.812	18.2585	−24.2329
63	ASP29	CA	−18.2937	17.1126	−24.9955
64	ASP29	C	−17.3081	16.7493	−26.0905
65	ASP29	O	−17.1225	17.4942	−27.052
66	ASP29	CB	−19.7095	17.3082	−25.5761
67	ASP29	CG	−20.2664	15.967	−25.9577
68	ASP29	OD1	−21.0819	15.42	−25.1689
69	ASP29	OD2	−19.8983	15.4607	−27.0508
70	PHE30	N	−16.6686	15.5986	−25.92
71	PHE30	CA	−15.6883	15.1073	−26.876
72	PHE30	C	−16.2374	15.0541	−28.2917
73	PHE30	O	−15.5848	15.4889	−29.2215
74	PHE30	CB	−15.0913	13.7425	−26.4865
75	PHE30	CG	−13.9312	13.4564	−27.4363
76	PHE30	CD1	−12.8189	14.302	−27.4553
77	PHE30	CD2	−13.9836	12.3569	−28.2963
78	PHE30	CE1	−11.7856	14.0734	−28.367
79	PHE30	CE2	−12.9192	12.0914	−29.1615
80	PHE30	CZ	−11.8222	12.9557	−29.2041
81	GLU31	N	−17.4227	14.4765	−28.4391
82	GLU31	CA	−18.0695	14.3384	−29.74
83	GLU31	C	−18.4961	15.6958	−30.2978
84	GLU31	O	−18.1746	16.0281	−31.4366
85	GLU31	CB	−19.3248	13.4596	−29.5707
86	GLU31	CG	−18.9969	12.1952	−28.7512
87	GLU31	CD	−19.1929	12.4724	−27.2876
88	GLU31	OE1	−20.3728	12.5627	−26.8547
89	GLU31	OE2	−18.1684	12.5927	−26.5636
90	ALA32	N	−19.1724	16.4914	−29.4728
91	ALA32	CA	−19.6256	17.817	−29.8767
92	ALA32	C	−18.4792	18.7175	−30.3487
93	ALA32	O	−18.5771	19.3496	−31.4051
94	ALA32	CB	−20.3076	18.4749	−28.6631
95	LEU33	N	−17.3954	18.7607	−29.5751
96	LEU33	CA	−16.2231	19.5772	−29.9031
97	LEU33	C	−15.4331	19.0228	−31.0884
98	LEU33	O	−14.8897	19.7818	−31.891
99	LEU33	CB	−15.3011	19.5611	−28.6684
100	LEU33	CG	−16.0154	20.2097	−27.4667
101	LEU33	CD1	−15.182	19.9944	−26.1896
102	LEU33	CD2	−16.1953	21.7178	−27.7216
103	LEU34	N	−15.3571	17.6969	−31.1802
104	LEU34	CA	−14.6678	17.0334	−32.2855
105	LEU34	C	−15.4723	17.3381	−33.5408
106	LEU34	O	−14.9271	17.7085	−34.5669
107	LEU34	CB	−14.7381	15.5166	−32.0247
108	LEU34	CG	−13.4308	14.8282	−32.4594
109	LEU34	CD1	−13.4802	13.3423	−32.0608
110	LEU34	CD2	−13.2453	14.9435	−33.9839
111	ALA35	N	−16.7854	17.2046	−33.4234
112	ALA35	CA	−17.7074	17.4638	−34.5157
113	ALA35	C	−17.5894	18.9123	−34.9803
114	ALA35	O	−17.5165	19.1899	−36.1781
115	ALA35	CB	−19.1407	17.2174	−34.0109
116	GLU36	N	−17.5819	19.8209	−34.0104
117	GLU36	CA	−17.4616	21.2591	−34.2319
118	GLU36	C	−16.2171	21.5709	−35.0692
119	GLU36	O	−16.2857	22.2732	−36.0777
120	GLU36	CB	−17.3366	21.903	−32.8349
121	GLU36	CG	−16.8291	23.3574	−32.9143
122	GLU36	CD	−15.3265	23.3815	−32.8896
123	GLU36	OE1	−14.7352	22.8224	−31.927
124	GLU36	OE2	−14.7316	23.9552	−33.8408
125	CYS37	N	−15.0851	21.0145	−34.6518
126	CYS37	CA	−13.8228	21.2122	−35.3467
127	CYS37	C	−13.8265	20.4262	−36.6526
128	CYS37	O	−13.2082	20.8268	−37.6325
129	CYS37	CB	−12.7031	20.6447	−34.4549
130	CYS37	SG	−11.7988	22.0477	−33.7396
131	LEU38	N	−14.5137	19.2872	−36.6354
132	LEU38	CA	−14.6365	18.4176	−37.7991
133	LEU38	C	−15.2427	19.2608	−38.8935
134	LEU38	O	−14.7294	19.3257	−40.0108
135	LEU38	CB	−15.5583	17.2359	−37.4444
136	LEU38	CG	−14.8353	15.9028	−37.7188
137	LEU38	CD1	−13.5226	15.8275	−36.9155
138	LEU38	CD2	−15.7503	14.7352	−37.3062
139	ARG39	N	−16.3056	19.9654	−38.5243
140	ARG39	CA	−17.0297	20.8292	−39.4381
141	ARG39	C	−16.244	22.0579	−39.8902
142	ARG39	O	−15.9049	22.1629	−41.0629
143	ARG39	CB	−18.3186	21.316	−38.7483
144	ARG39	CG	−19.1868	20.1166	−38.3268
145	ARG39	CD	−20.103	20.5459	−37.1664
146	ARG39	NE	−20.3618	19.4053	−36.3068
147	ARG39	CZ	−21.5716	18.9628	−36.1218
148	ARG39	NH1	−21.7652	17.9388	−35.3449
149	ARG39	NH2	−22.5913	19.5263	−36.7005
150	ASN40	N	−15.8973	22.9463	−38.9598
151	ASN40	CA	−15.158	24.169	−39.2938
152	ASN40	C	−13.7759	23.9349	−39.8974
153	ASN40	O	−13.082	24.8845	−40.2596
154	ASN40	CB	−14.9873	25.0138	−38.0155
155	ASN40	CG	−16.3169	25.3188	−37.3873
156	ASN40	OD1	−16.5252	25.0099	−36.2256
157	ASN40	ND2	−17.2341	25.9335	−38.1556
158	GLY41	N	−13.3893	22.667	−40.0176
159	GLY41	CA	−12.0967	22.3184	−40.5824
160	GLY41	C	−10.9452	22.9275	−39.8082
161	GLY41	O	−10.0967	23.6122	−40.3791
162	CYS42	N	−10.9167	22.6693	−38.5057
163	CYS42	CA	−9.8803	23.2064	−37.6375
164	CYS42	C	−9.4616	22.2276	−36.5439
165	CYS42	O	−9.9692	21.1126	−36.4619
166	CYS42	CB	−10.4265	24.4707	−36.9437
167	CYS42	SG	−10.753	25.7585	−38.1837
168	LEU43	N	−8.5193	22.6577	−35.7118
169	LEU43	CA	−8.0132	21.8337	−34.6213
170	LEU43	C	−8.4703	22.3473	−33.2651
171	LEU43	O	−8.5778	23.5611	−33.0479
172	LEU43	CB	−6.4773	21.7117	−34.6571
173	LEU43	CG	−5.9576	21.4349	−36.0819
174	LEU43	CD1	−4.4199	21.3664	−36.0489
175	LEU43	CD2	−6.5092	20.0994	−36.615
176	PHE44	N	−8.6955	21.4162	−32.3455
177	PHE44	CA	−9.1464	21.7627	−31.0043
178	PHE44	C	−8.0883	22.4337	−30.1214
179	PHE44	O	−6.9495	21.9754	−29.9899
180	PHE44	CB	−9.8202	20.5294	−30.3676
181	PHE44	CG	−9.882	20.6174	−28.8472
182	PHE44	CD1	−10.7478	21.5258	−28.233
183	PHE44	CD2	−9.0725	19.7821	−28.073
184	PHE44	CE1	−10.8371	21.5679	−26.8396
185	PHE44	CE2	−9.1608	19.8264	−26.6796
186	PHE44	CZ	−10.0598	20.7022	−26.0661
187	GLU45	N	−8.4811	23.5795	−29.5902
188	GLU45	CA	−7.6634	24.3474	−28.6926
189	GLU45	C	−8.5174	24.3954	−27.4403
190	GLU45	O	−9.5537	25.0596	−27.3985
191	GLU45	CB	−7.4462	25.7576	−29.2734
192	GLU45	CG	−6.7926	25.6599	−30.6663
193	GLU45	CD	−5.4031	25.1005	−30.5591
194	GLU45	OE1	−4.5005	25.842	−30.0872
195	GLU45	OE2	−5.2083	23.9213	−30.9566
196	ASP46	N	−8.1279	23.5759	−26.4713
197	ASP46	CA	−8.8271	23.4565	−25.2064
198	ASP46	C	−8.9745	24.79	−24.4679
199	ASP46	O	−7.9792	25.4257	−24.1015
200	ASP46	CB	−7.9973	22.5011	−24.3257
201	ASP46	CG	−8.8587	21.8017	−23.3145
202	ASP46	OD1	−9.4405	22.4985	−22.4406
203	ASP46	OD2	−8.9499	20.5471	−23.39
204	THR47	N	−10.2319	25.236	−24.2636
205	THR47	CA	−10.5788	26.4827	−23.5731
206	THR47	C	−10.5448	26.3291	−22.0414
207	THR47	O	−10.6702	27.3036	−21.297
208	THR47	CB	−11.991	26.9051	−24.0256
209	THR47	OG1	−12.0685	26.8763	−25.4538
210	THR47	CG2	−12.2993	28.3333	−23.5373
211	SER48	N	−10.3562	25.0947	−21.5841
212	SER48	CA	−10.3202	24.7963	−20.1625
213	SER48	C	−8.9327	24.3765	−19.7007
214	SER48	O	−8.7044	24.1661	−18.5061
215	SER48	CB	−11.3054	23.6448	−19.8871
216	SER48	OG	−12.6213	24.0291	−20.2945
217	PHE49	N	−8.0123	24.2454	−20.6548
218	PHE49	CA	−6.6435	23.8505	−20.3588
219	PHE49	C	−5.7089	24.3915	−21.4418
220	PHE49	O	−5.1154	23.6305	−22.2142
221	PHE49	CB	−6.5801	22.3116	−20.2651
222	PHE49	CG	−5.3298	21.8633	−19.5126
223	PHE49	CD1	−5.3312	21.8194	−18.1156
224	PHE49	CD2	−4.1833	21.4888	−20.2187
225	PHE49	CE1	−4.1956	21.3837	−17.4269
226	PHE49	CE2	−3.047	21.0549	−19.5305
227	PHE49	CZ	−3.0541	20.9974	−18.1343
228	PRO50	N	−5.5643	25.7267	−21.5041
229	PRO50	CA	−4.7293	26.4558	−22.4561
230	PRO50	C	−3.2687	26.0165	−22.478
231	PRO50	O	−2.7439	25.4808	−21.4869
232	PRO50	CB	−4.8313	27.9279	−22.0172
233	PRO50	CG	−5.8165	27.9803	−20.831
234	PRO50	CD	−6.2821	26.5397	−20.5405
235	ALA51	N	−2.6121	26.2739	−23.6058
236	ALA51	CA	−1.2098	25.9377	−23.7851
237	ALA51	C	−0.3497	26.9614	−23.0391
238	ALA51	O	0.7223	27.3612	−23.5021
239	ALA51	CB	−0.8481	25.8622	−25.281
240	THR52	N	−0.8405	27.3848	−21.8767
241	THR52	CA	−0.1559	28.3625	−21.0525
242	THR52	C	0.6958	27.7065	−19.9669
243	THR52	O	0.709	26.4813	−19.8398
244	THR52	CB	−1.2136	29.2515	−20.369
245	THR52	OG1	−2.0784	28.4487	−19.5588
246	THR52	CG2	−2.0467	29.9875	−21.4353
247	LEU53	N	1.5163	28.5131	−19.2586
248	LEU53	CA	2.3863	28.037	−18.1786
249	LEU53	C	1.5758	27.5585	−16.9718
250	LEU53	O	2.0681	26.7947	−16.1515
251	LEU53	CB	3.3336	29.1723	−17.7501
252	LEU53	CG	4.6099	29.1306	−18.6121
253	LEU53	CD1	5.5137	30.3224	−18.247
254	LEU53	CD2	5.3734	27.8177	−18.3543
255	SER54	N	0.3319	28.0127	−16.8819
256	SER54	CA	−0.569	27.6427	−15.7938
257	SER54	C	−0.9496	26.1564	−15.8412
258	SER54	O	−1.2905	25.5583	−14.8192
259	SER54	CB	−1.8575	28.4696	−15.9596
260	SER54	OG	−1.5491	29.864	−15.8839
261	SER55	N	−0.9164	25.584	−17.0444
262	SER55	CA	−1.2204	24.1714	−17.2571
263	SER55	C	−0.0414	23.3364	−16.7588
264	SER55	O	−0.2092	22.2061	−16.2832
265	SER55	CB	−1.469	23.9085	−18.7566
266	SER55	OG	−0.2429	23.9254	−19.4941
267	ILE56	N	1.1563	23.8991	−16.9025
268	ILE56	CA	2.3905	23.2585	−16.4678
269	ILE56	C	2.5322	23.3656	−14.9373
270	ILE56	O	2.886	22.3901	−14.2855
271	ILE56	CB	3.5575	23.9946	−17.1549
272	ILE56	CG1	3.3514	23.9704	−18.6829
273	ILE56	CG2	4.8919	23.3115	−16.798
274	ILE56	CD1	4.3716	24.8964	−19.3715
275	GLY57	N	2.2641	24.5079	−14.3713
276	GLY57	CA	2.3598	24.7298	−12.9266
277	GLY57	C	3.7996	24.4235	−12.4989
278	GLY57	O	4.2274	23.2821	−12.5859
279	LEU65	N	12.3115	25.9569	−15.7051
280	LEU65	CA	12.1314	24.6761	−16.4042
281	LEU65	C	11.0916	24.8704	−17.538
282	LEU65	O	11.2794	24.3854	−18.6495
283	LEU65	CB	11.5248	23.6404	−15.4356
284	LEU65	CG	12.5547	23.1647	−14.3945
285	LEU65	CD1	11.8521	22.277	−13.351
286	LEU65	CD2	13.6664	22.3566	−15.0896
287	PRO66	N	10.0696	25.653	−17.3149
288	PRO66	CA	9.0282	25.8331	−18.322
289	PRO66	C	9.2286	27.0866	−19.1893
290	PRO66	O	8.2609	27.7518	−19.5641
291	PRO66	CB	7.7834	26.0207	−17.4333
292	PRO66	CG	8.2941	26.5192	−16.0641
293	PRO66	CD	9.8033	26.2118	−16.0068
294	PRO67	N	10.4842	27.3788	−19.5276
295	PRO67	CA	10.8245	28.5473	−20.3404
296	PRO67	C	11.2929	28.1703	−21.7447
297	PRO67	O	12.161	27.3217	−21.9075
298	PRO67	CB	12.0302	29.1474	−19.5952
299	PRO67	CG	12.6726	27.9643	−18.8471
300	PRO67	CD	11.6812	26.7941	−18.9858
301	ARG68	N	10.701	28.7991	−22.754
302	ARG68	CA	11.0793	28.5286	−24.135
303	ARG68	C	10.7253	27.1529	−24.6599
304	ARG68	O	11.4609	26.5524	−25.4496
305	ARG68	CB	12.5578	28.8764	−24.4017
306	ARG68	CG	12.8181	30.3504	−24.0425
307	ARG68	CD	14.3237	30.6448	−24.1658
308	ARG68	NE	14.5854	31.9972	−23.709
309	ARG68	CZ	15.264	32.2199	−22.6207
310	ARG68	NH1	15.4798	33.445	−22.2438
311	ARG68	NH2	15.7293	31.2393	−21.9035
312	LEU69	N	9.5924	26.6432	−24.2168
313	LEU69	CA	9.1505	25.3393	−24.6592
314	LEU69	C	7.8526	25.4596	−25.456
315	LEU69	O	6.9058	26.1202	−25.0331
316	LEU69	CB	8.9084	24.3992	−23.457
317	LEU69	CG	8.7632	25.1657	−22.1248
318	LEU69	CD1	7.4743	26.0088	−22.1242
319	LEU69	CD2	8.7105	24.1644	−20.9568
320	GLN70	N	7.8405	24.8246	−26.6228
321	GLN70	CA	6.7115	24.8565	−27.5489
322	GLN70	C	5.7536	23.6717	−27.4633
323	GLN70	O	6.1667	22.5133	−27.4887
324	GLN70	CB	7.2115	24.9668	−29.0024
325	GLN70	CG	8.2553	26.0927	−29.1291
326	GLN70	CD	9.6245	25.5437	−28.8497
327	GLN70	OE1	10.2487	25.9357	−27.8779
328	GLN70	NE2	10.105	24.6265	−29.7079
329	TRP71	N	4.465	23.9829	−27.3854
330	TRP71	CA	3.4258	22.9642	−27.3331
331	TRP71	C	3.1746	22.4423	−28.7393
332	TRP71	O	2.4693	23.0698	−29.5419
333	TRP71	CB	2.1377	23.5984	−26.7739
334	TRP71	CG	2.312	23.8643	−25.3078
335	TRP71	CD1	2.8263	24.9684	−24.7439
336	TRP71	CD2	1.94	22.9265	−24.192
337	TRP71	NE1	2.8321	24.8419	−23.4397
338	TRP71	CE2	2.3239	23.6463	−23.0764
339	TRP71	CE3	1.3639	21.6594	−24.1194
340	TRP71	CZ2	2.1738	23.1341	−21.788
341	TRP71	CZ3	1.2152	21.1334	−22.8307
342	TRP71	CH2	1.6176	21.8522	−21.6955
343	LYS72	N	3.8006	21.3124	−29.0428
344	LYS72	CA	3.6586	20.6789	−30.3357
345	LYS72	C	2.6951	19.49	−30.1769
346	LYS72	O	2.6561	18.7499	−29.2878
347	LYS72	CB	5.0384	20.2965	−30.9014
348	LYS72	CG	5.9482	21.5389	−30.8451
349	LYS72	CD	7.3908	21.1728	−31.2361
350	LYS72	CE	8.3114	22.3529	−30.8736
351	LYS72	NZ	9.384	22.4688	−31.8714
352	ARG73	N	1.887	19.3481	−31.3241
353	ARG73	CA	0.9543	18.2391	−31.4196
354	ARG73	C	1.7324	17.134	−32.1228
355	ARG73	O	2.7467	17.4047	−32.7786
356	ARG73	CB	−0.2282	18.6367	−32.3243
357	ARG73	CG	−1.0143	19.8161	−31.7239
358	ARG73	CD	−2.4537	19.344	−31.4564
359	ARG73	NE	−3.4166	20.2295	−32.081
360	ARG73	CZ	−4.5688	20.4563	−31.5206
361	ARG73	NH1	−4.888	19.8999	−30.3895
362	ARG73	NH2	−5.4157	21.2509	−32.1024
363	PRO74	N	1.299	15.8673	−31.9654
364	PRO74	CA	1.9489	14.7052	−32.5823
365	PRO74	C	2.1161	14.8143	−34.1023
366	PRO74	O	2.9741	14.1518	−34.6932
367	PRO74	CB	0.9333	13.5833	−32.2893
368	PRO74	CG	−0.0003	14.0863	−31.1698
369	PRO74	CD	0.1523	15.6169	−31.1164
370	PRO75	N	1.2926	15.6575	−34.7186
371	PRO75	CA	1.316	15.8916	−36.1551
372	PRO75	C	2.6997	16.3246	−36.5983
373	PRO75	O	3.2238	15.8412	−37.6031
374	PRO75	CB	0.2915	17.0171	−36.3851
375	PRO75	CG	−0.6163	17.027	−35.1413
376	PRO75	CD	0.1047	16.187	−34.0712
377	GLU76	N	3.2625	17.3134	−35.8124
378	GLU76	CA	4.4344	18.0945	−36.1982
379	GLU76	C	5.7322	17.2973	−35.9839
380	GLU76	O	6.7912	17.6086	−36.527
381	GLU76	CB	4.5004	19.329	−35.2796
382	GLU76	CG	3.3103	20.2616	−35.5694
383	GLU76	CD	2.2824	20.1052	−34.4879
384	GLU76	OE1	2.3162	20.9111	−33.5203
385	GLU76	OE2	1.4303	19.1867	−34.6128
386	LEU77	N	5.6511	16.3624	−34.9705
387	LEU77	CA	6.8391	15.8446	−34.2955
388	LEU77	C	7.4537	14.6814	−35.0966
389	LEU77	O	8.6507	14.4037	−34.9876
390	LEU77	CB	6.3855	15.2602	−32.9414
391	LEU77	CG	5.3161	16.1448	−32.2704
392	LEU77	CD1	4.7594	15.4336	−31.0233
393	LEU77	CD2	5.9284	17.4984	−31.8652
394	HIS78	N	6.5733	13.885	−35.8006
395	HIS78	CA	7.0516	12.7057	−36.5296
396	HIS78	C	6.221	12.4993	−37.8079
397	HIS78	O	5.1972	13.1305	−38.0482
398	HIS78	CB	6.9335	11.4315	−35.6679
399	HIS78	CG	7.2747	11.6999	−34.231
400	HIS78	ND1	6.394	12.1963	−33.3935
401	HIS78	CD2	8.4737	11.4727	−33.6617
402	HIS78	CE1	6.9793	12.313	−32.244
403	HIS78	NE2	8.1679	11.9184	−32.3293
404	SER79	N	6.744	11.5449	−38.6654
405	SER79	CA	6.2259	11.3421	−40.0167
406	SER79	C	4.9636	10.4715	−40.0374
407	SER79	O	4.1412	10.5656	−40.9488
408	SER79	CB	7.3217	10.6505	−40.8505
409	SER79	OG	7.6769	9.3977	−40.2577
410	ASN80	N	4.9115	9.4953	−39.0614
411	ASN80	CA	3.7097	8.6832	−38.8418
412	ASN80	C	3.3877	8.7954	−37.3409
413	ASN80	O	3.9182	8.0475	−36.5135
414	ASN80	CB	4.0308	7.2212	−39.216
415	ASN80	CG	2.8922	6.2781	−38.9316
416	ASN80	OD1	1.8258	6.6843	−38.4983
417	ASN80	ND2	3.1233	4.9769	−39.1805
418	PRO81	N	2.5675	9.8295	−36.9434
419	PRO81	CA	2.0432	9.8923	−35.5806
420	PRO81	C	0.9728	8.7992	−35.4421
421	PRO81	O	−0.0481	8.7744	−36.1332
422	PRO81	CB	1.3513	11.2709	−35.5758
423	PRO81	CG	1.275	11.7676	−37.0367
424	PRO81	CD	2.1119	10.8087	−37.9067
425	GLN82	N	1.2765	7.8106	−34.525
426	GLN82	CA	0.2983	6.7865	−34.207
427	GLN82	C	−0.3517	7.1645	−32.8705
428	GLN82	O	0.2082	7.817	−31.9873
429	GLN82	CB	1.0091	5.4307	−34.0417
430	GLN82	CG	1.6042	5.0014	−35.3964
431	GLN82	CD	0.583	4.3049	−36.2521
432	GLN82	OE1	−0.5485	4.1029	−35.8418
433	GLN82	NE2	0.9893	3.9225	−37.4754
434	PHE83	N	−1.6335	6.6481	−32.7332
435	PHE83	CA	−2.2914	6.7368	−31.4406
436	PHE83	C	−1.7335	5.594	−30.5892
437	PHE83	O	−1.0996	5.8522	−29.5679
438	PHE83	CB	−3.8224	6.6601	−31.5886
439	PHE83	CG	−4.4571	7.7012	−30.672
440	PHE83	CD1	−4.1099	9.0492	−30.7975
441	PHE83	CD2	−5.3827	7.3067	−29.7024
442	PHE83	CE1	−4.6423	9.9939	−29.9166
443	PHE83	CE2	−5.929	8.2541	−28.8324
444	PHE83	CZ	−5.5502	9.5954	−28.9318
445	ASP92	N	8.7138	1.3421	−24.7856
446	ASP92	CA	9.9333	2.1258	−24.6143
447	ASP92	C	11.0305	1.4805	−25.5119
448	ASP92	O	10.7546	0.699	−26.4246
449	ASP92	CB	10.407	2.1775	−23.1449
450	ASP92	CG	9.7248	3.2612	−22.3582
451	ASP92	OD1	9.2374	4.2466	−22.9756
452	ASP92	OD2	9.6647	3.1203	−21.1075
453	LEU93	N	12.3299	1.9183	−25.302
454	LEU93	CA	13.4513	1.2276	−25.9597
455	LEU93	C	13.9838	0.2162	−24.9175
456	LEU93	O	13.6738	−0.9711	−24.9345
457	LEU93	CB	14.548	2.2237	−26.3839
458	LEU93	CG	13.9455	3.3288	−27.2731
459	LEU93	CD1	15.0357	4.3611	−27.6145
460	LEU93	CD2	13.3912	2.7184	−28.5745
461	CYS94	N	14.7737	0.7976	−23.9419
462	CYS94	CA	15.3553	0.0678	−22.8027
463	CYS94	C	15.5177	1.1607	−21.7285
464	CYS94	O	15.0841	1.0768	−20.5819
465	CYS94	CB	16.7457	−0.4768	−23.1815
466	CYS94	SG	16.5289	−1.9599	−24.2076
467	GLN95	N	16.1911	2.2917	−22.1814
468	GLN95	CA	16.3642	3.4819	−21.3572
469	GLN95	C	15.0323	4.2548	−21.3495
470	GLN95	O	14.8373	5.2658	−22.0181
471	GLN95	CB	17.487	4.3453	−21.962
472	GLN95	CG	18.8381	3.6285	−21.7829
473	GLN95	CD	19.9443	4.4988	−22.304
474	GLN95	OE1	20.6087	4.1307	−23.2586
475	GLN95	NE2	20.1536	5.6691	−21.6745
476	GLY96	N	14.0855	3.7102	−20.5023
477	GLY96	CA	12.7456	4.2719	−20.3779
478	GLY96	C	12.6237	5.1896	−19.1601
479	GLY96	O	11.5433	5.522	−18.6759
480	ILE97	N	13.8142	5.7522	−18.7335
481	ILE97	CA	13.9329	6.3569	−17.4039
482	ILE97	C	13.1112	7.6447	−17.1971
483	ILE97	O	12.9717	8.1418	−16.0811
484	ILE97	CB	15.428	6.6321	−17.1406
485	ILE97	CG1	16.2594	5.3785	−17.4804
486	ILE97	CG2	15.6462	7.0035	−15.6609
487	ILE97	CD1	17.7616	5.7174	−17.456
488	VAL98	N	12.6429	8.2331	−18.3549
489	VAL98	CA	11.7495	9.3858	−18.3377
490	VAL98	C	10.252	8.9878	−18.3753
491	VAL98	O	9.3646	9.8424	−18.3641
492	VAL98	CB	12.0704	10.3188	−19.5223
493	VAL98	CG1	13.5084	10.8545	−19.3896
494	VAL98	CG2	11.9206	9.5627	−20.8569
495	GLY99	N	9.9718	7.6392	−18.4239
496	GLY99	CA	8.6184	7.1278	−18.3169
497	GLY99	C	8.2272	6.8629	−16.8609
498	GLY99	O	9.0457	6.6416	−15.9726
499	ASP100	N	6.8553	6.8641	−16.6641
500	ASP100	CA	6.2623	6.5337	−15.3641
501	ASP100	C	5.831	5.0446	−15.3993
502	ASP100	O	5.6976	4.4108	−16.4423
503	ASP100	CB	5.0095	7.4013	−15.1404
504	ASP100	CG	4.7068	7.4901	−13.6731
505	ASP100	OD1	5.1626	8.4764	−13.0357
506	ASP100	OD2	4.0011	6.5834	−13.1557
507	CYS101	N	5.6063	4.4998	−14.1533
508	CYS101	CA	5.5167	3.0637	−13.8994
509	CYS101	C	4.1114	2.4834	−14.084
510	CYS101	O	3.9375	1.2843	−14.319
511	CYS101	CB	6.0579	2.7259	−12.4996
512	CYS101	SG	6.8261	1.0845	−12.6355
513	TRP102	N	3.0511	3.3388	−13.8879
514	TRP102	CA	1.6744	2.8555	−14.0637
515	TRP102	C	1.3911	2.5082	−15.5374
516	TRP102	O	0.521	1.7013	−15.8714
517	TRP102	CB	0.7186	3.986	−13.6263
518	TRP102	CG	0.7217	5.1136	−14.6207
519	TRP102	CD1	1.4421	6.2439	−14.5577
520	TRP102	CD2	−0.0869	5.1729	−15.889
521	TRP102	NE1	1.1984	6.9888	−15.6076
522	TRP102	CE2	0.3029	6.3884	−16.4185
523	TRP102	CE3	−1.0105	4.334	−16.5113
524	TRP102	CZ2	−0.2065	6.8568	−17.6295
525	TRP102	CZ3	−1.5256	4.7953	−17.7289
526	TRP102	CH2	−1.1332	6.0272	−18.2731
527	PHE103	N	2.1799	3.1817	−16.4499
528	PHE103	CA	1.97	3.0654	−17.8948
529	PHE103	C	2.26	1.6029	−18.2845
530	PHE103	O	1.6619	1.0124	−19.1813
531	PHE103	CB	2.9686	4.0053	−18.5934
532	PHE103	CG	2.3833	4.5108	−19.9084
533	PHE103	CD1	0.9999	4.5431	−20.1068
534	PHE103	CD2	3.2434	4.9464	−20.9194
535	PHE103	CE1	0.4757	4.9946	−21.3197
536	PHE103	CE2	2.7199	5.416	−22.1263
537	PHE103	CZ	1.3397	5.3971	−22.3407
538	LEU104	N	3.3014	1.0417	−17.5789
539	LEU104	CA	3.8472	−0.2778	−17.8623
540	LEU104	C	2.8276	−1.3489	−17.424
541	LEU104	O	2.6573	−2.3938	−18.0553
542	LEU104	CB	5.1372	−0.4506	−17.0364
543	LEU104	CG	6.3276	0.2252	−17.7462
544	LEU104	CD1	6.1177	1.7499	−17.8154
545	LEU104	CD2	7.6261	−0.0697	−16.973
546	ALA105	N	2.1932	−1.0749	−16.2233
547	ALA105	CA	1.1276	−1.9355	−15.7244
548	ALA105	C	−0.1106	−1.8301	−16.6245
549	ALA105	O	−0.8567	−2.7933	−16.8042
550	ALA105	CB	0.7518	−1.4414	−14.3154
551	ALA106	N	−0.3581	−0.5791	−17.1656
552	ALA106	CA	−1.487	−0.3921	−18.0657
553	ALA106	C	−1.22	−1.1369	−19.392
554	ALA106	O	−2.1309	−1.6826	−20.017
555	ALA106	CB	−1.5879	1.1155	−18.3643
556	LEU107	N	0.0829	−1.0988	−19.8597
557	LEU107	CA	0.4617	−1.7496	−21.1241
558	LEU107	C	0.418	−3.2763	−20.9538
559	LEU107	O	0.1156	−4.0209	−21.8892
560	LEU107	CB	1.8699	−1.2468	−21.4836
561	LEU107	CG	1.7736	0.2519	−21.8284
562	LEU107	CD1	3.1721	0.8904	−21.8436
563	LEU107	CD2	1.0896	0.4491	−23.1921
564	GLN108	N	0.7506	−3.7767	−19.7065
565	GLN108	CA	0.7222	−5.2282	−19.4859
566	GLN108	C	−0.7099	−5.7747	−19.7101
567	GLN108	O	−0.933	−6.9481	−20.0032
568	GLN108	CB	1.1255	−5.4721	−18.0208
569	GLN108	CG	2.6571	−5.3768	−17.8879
570	GLN108	CD	3.0337	−5.1535	−16.4519
571	GLN108	OE1	3.7327	−4.2021	−16.1437
572	GLN108	NE2	2.5673	−6.0397	−15.5542
573	ALA109	N	−1.6921	−4.8226	−19.5244
574	ALA109	CA	−3.1166	−5.0242	−19.7711
575	ALA109	C	−3.5236	−4.8583	−21.2595
576	ALA109	O	−4.7013	−4.7503	−21.6008
577	ALA109	CB	−3.9504	−4.0865	−18.8771
578	LEU110	N	−2.5076	−5.0135	−22.1825
579	LEU110	CA	−2.7165	−5.3006	−23.6053
580	LEU110	C	−2.2962	−6.7648	−23.8919
581	LEU110	O	−1.8484	−7.1455	−24.9707
582	LEU110	CB	−1.9913	−4.2966	−24.5207
583	LEU110	CG	−2.7136	−2.9386	−24.4503
584	LEU110	CD1	−2.133	−2.0943	−23.2995
585	LEU110	CD2	−2.5442	−2.1967	−25.7885
586	ALA111	N	−2.6269	−7.6359	−22.8693
587	ALA111	CA	−2.5563	−9.0971	−23.007
588	ALA111	C	−3.8628	−9.5213	−23.7149
589	ALA111	O	−4.7955	−8.7411	−23.9273
590	ALA111	CB	−2.5057	−9.7403	−21.6089
591	LEU112	N	−3.9772	−10.8455	−24.0885
592	LEU112	CA	−5.1466	−11.3563	−24.8259
593	LEU112	C	−6.3949	−11.5635	−23.9092
594	LEU112	O	−7.2128	−12.4664	−24.0847
595	LEU112	CB	−4.7856	−12.6517	−25.5768
596	LEU112	CG	−4.4419	−12.3208	−27.0409
597	LEU112	CD1	−3.8983	−13.5827	−27.7356
598	LEU112	CD2	−5.7085	−11.8411	−27.7752
599	ILE116	N	−8.104	−3.5179	−19.3071
600	ILE116	CA	−8.2759	−2.3901	−18.3846
601	ILE116	C	−7.8945	−1.0951	−19.1202
602	ILE116	O	−8.4238	−0.0119	−18.8715
603	ILE116	CB	−7.2846	−2.5885	−17.2211
604	ILE116	CG1	−7.3947	−4.0199	−16.6603
605	ILE116	CG2	−7.5752	−1.5673	−16.1043
606	ILE116	CD1	−6.1486	−4.3382	−15.8139
607	LEU117	N	−6.907	−1.2245	−20.0738
608	LEU117	CA	−6.3848	−0.0669	−20.8005
609	LEU117	C	−7.5499	0.6023	−21.5521
610	LEU117	O	−7.6652	1.8281	−21.6358
611	LEU117	CB	−5.3551	−0.6028	−21.8165
612	LEU117	CG	−4.3222	0.4745	−22.206
613	LEU117	CD1	−4.997	1.6243	−22.9783
614	LEU117	CD2	−3.6102	1.0177	−20.9523
615	SER118	N	−8.4382	−0.2753	−22.1503
616	SER118	CA	−9.527	0.208	−22.9924
617	SER118	C	−10.6426	0.9255	−22.2129
618	SER118	O	−11.551	1.5242	−22.7887
619	SER118	CB	−10.1172	−0.9766	−23.7808
620	SER118	OG	−10.6208	−1.9642	−22.8771
621	ARG119	N	−10.569	0.8227	−20.8375
622	ARG119	CA	−11.4775	1.5553	−19.9688
623	ARG119	C	−11.0002	3.0022	−19.7933
624	ARG119	O	−11.7956	3.914	−19.5696
625	ARG119	CB	−11.4061	0.886	−18.5811
626	ARG119	CG	−11.688	−0.6251	−18.6941
627	ARG119	CD	−11.0396	−1.3648	−17.5085
628	ARG119	NE	−11.8994	−1.2975	−16.3406
629	ARG119	CZ	−11.4917	−0.7479	−15.2328
630	ARG119	NH1	−10.3021	−0.2312	−15.1306
631	ARG119	NH2	−12.2908	−0.7158	−14.2078
632	VAL120	N	−9.631	3.1517	−19.7311
633	VAL120	CA	−8.9808	4.4338	−19.4573
634	VAL120	C	−8.8841	5.2185	−20.7772
635	VAL120	O	−9.0856	6.4347	−20.8279
636	VAL120	CB	−7.5615	4.1665	−18.9181
637	VAL120	CG1	−6.8398	5.5013	−18.6496
638	VAL120	CG2	−7.6483	3.366	−17.6051
639	VAL121	N	−8.408	4.4728	−21.8391
640	VAL121	CA	−8.1138	5.031	−23.1509
641	VAL121	C	−9.1408	4.4303	−24.128
642	VAL121	O	−9.0317	3.276	−24.5517
643	VAL121	CB	−6.6705	4.6559	−23.5399
644	VAL121	CG1	−6.3261	5.265	−24.9123
645	VAL121	CG2	−5.6994	5.2154	−22.4827
646	PRO122	N	−10.1876	5.2355	−24.526
647	PRO122	CA	−11.1232	4.7921	−25.5543
648	PRO122	C	−10.3369	4.6489	−26.8664
649	PRO122	O	−9.7674	5.5885	−27.4207
650	PRO122	CB	−12.0732	6.0025	−25.6621
651	PRO122	CG	−11.4638	7.1599	−24.8396
652	PRO122	CD	−10.3163	6.5757	−23.9929
653	LEU123	N	−10.2932	3.3366	−27.3276
654	LEU123	CA	−9.3386	2.9467	−28.3737
655	LEU123	C	−9.728	3.5064	−29.7541
656	LEU123	O	−8.9297	3.5627	−30.6897
657	LEU123	CB	−9.2619	1.4084	−28.4148
658	LEU123	CG	−8.9489	0.858	−27.008
659	LEU123	CD1	−8.9634	−0.6812	−27.0383
660	LEU123	CD2	−7.5671	1.3497	−26.5353
661	ASN124	N	−11.0556	3.8432	−29.8956
662	ASN124	CA	−11.6259	4.3803	−31.1327
663	ASN124	C	−11.1889	5.8395	−31.3775
664	ASN124	O	−11.9811	6.7626	−31.546
665	ASN124	CB	−13.1634	4.2757	−31.0468
666	ASN124	CG	−13.7008	5.0409	−29.8683
667	ASN124	OD1	−14.4125	6.0144	−30.0524
668	ASN124	ND2	−13.3664	4.6068	−28.6394
669	GLN125	N	−9.822	6.007	−31.4634
670	GLN125	CA	−9.1814	7.3052	−31.6147
671	GLN125	C	−7.9466	7.1256	−32.5074
672	GLN125	O	−7.3304	6.0632	−32.5861
673	GLN125	CB	−8.7584	7.8764	−30.2481
674	GLN125	CG	−10.0007	8.1186	−29.3703
675	GLN125	CD	−9.6273	8.9664	−28.1894
676	GLN125	OE1	−10.1086	10.0807	−28.068
677	GLN125	NE2	−8.7616	8.4413	−27.3035
678	SER126	N	−7.5777	8.2638	−33.2006
679	SER126	CA	−6.5795	8.2017	−34.263
680	SER126	C	−6.11	9.618	−34.6132
681	SER126	O	−6.7578	10.6218	−34.3314
682	SER126	CB	−7.1575	7.5132	−35.5138
683	SER126	OG	−6.1045	7.2436	−36.4424
684	PHE127	N	−4.9112	9.6319	−35.3035
685	PHE127	CA	−4.4128	10.8268	−35.9869
686	PHE127	C	−4.7745	10.8026	−37.4801
687	PHE127	O	−4.6802	11.8116	−38.1782
688	PHE127	CB	−2.8782	10.8614	−35.8505
689	PHE127	CG	−2.4867	11.0463	−34.3877
690	PHE127	CD1	−1.8966	9.9921	−33.6854
691	PHE127	CD2	−2.7169	12.2688	−33.7507
692	PHE127	CE1	−1.54	10.1607	−32.345
693	PHE127	CE2	−2.3612	12.4355	−32.4099
694	PHE127	CZ	−1.7752	11.3805	−31.7056
695	TYR131	N	−9.2595	13.5067	−37.4336
696	TYR131	CA	−8.2461	13.9236	−36.4549
697	TYR131	C	−8.6868	15.1692	−35.6672
698	TYR131	O	−8.571	15.2393	−34.441
699	TYR131	CB	−6.8845	14.1573	−37.1456
700	TYR131	CG	−6.0123	15.0932	−36.3094
701	TYR131	CD1	−5.3854	14.636	−35.1466
702	TYR131	CD2	−5.8567	16.4228	−36.7094
703	TYR131	CE1	−4.671	15.5291	−34.3426
704	TYR131	CE2	−5.138	17.3139	−35.9092
705	TYR131	CZ	−4.5609	16.8715	−34.7166
706	TYR131	OH	−3.879	17.7694	−33.9043
707	ALA132	N	−9.0168	16.2679	−36.4355
708	ALA132	CA	−9.5328	17.5262	−35.8823
709	ALA132	C	−8.5924	18.3499	−34.9607
710	ALA132	O	−8.709	19.5738	−34.8854
711	ALA132	CB	−10.8532	17.2515	−35.1364
712	GLY133	N	−7.752	17.6291	−34.1455
713	GLY133	CA	−6.9354	18.218	−33.0956
714	GLY133	C	−7.6072	18.1022	−31.7283
715	GLY133	O	−7.5639	19.0118	−30.9002
716	ILE134	N	−8.1599	16.8637	−31.4524
717	ILE134	CA	−8.9173	16.6142	−30.2263
718	ILE134	C	−8.8182	15.1192	−29.8913
719	ILE134	O	−8.8813	14.2514	−30.7603
720	ILE134	CB	−10.3593	17.1524	−30.3084
721	ILE134	CG1	−11.0592	17.0133	−28.9422
722	ILE134	CG2	−11.1426	16.4052	−31.4039
723	ILE134	CD1	−12.4559	17.662	−28.9913
724	PHE135	N	−8.7247	14.8401	−28.5422
725	PHE135	CA	−8.6863	13.4695	−28.0316
726	PHE135	C	−9.4508	13.4417	−26.7029
727	PHE135	O	−9.6315	14.454	−26.0215
728	PHE135	CB	−7.2428	12.9517	−27.8844
729	PHE135	CG	−6.5844	12.915	−29.2601
730	PHE135	CD1	−5.5275	13.7812	−29.5524
731	PHE135	CD2	−7.0427	12.0197	−30.2306
732	PHE135	CE1	−4.95	13.7723	−30.8247
733	PHE135	CE2	−6.4702	12.0164	−31.5053
734	PHE135	CZ	−5.4282	12.898	−31.8044
735	ARG136	N	−9.8698	12.1836	−26.3098
736	ARG136	CA	−10.6148	11.9692	−25.0723
737	ARG136	C	−10.1408	10.7089	−24.3312
738	ARG136	O	−9.6374	9.7405	−24.9031
739	ARG136	CB	−12.1337	11.9063	−25.3183
740	ARG136	CG	−12.4699	10.6748	−26.1801
741	ARG136	CD	−13.9424	10.2825	−25.9652
742	ARG136	NE	−14.3499	9.3766	−27.0232
743	ARG136	CZ	−15.4376	9.5841	−27.7076
744	ARG136	NH1	−15.762	8.7525	−28.6524
745	ARG136	NH2	−16.2059	10.6056	−27.4648
746	PHE137	N	−10.3449	10.7858	−22.9627
747	PHE137	CA	−9.8043	9.8128	−22.011
748	PHE137	C	−10.7722	9.7426	−20.8275
749	PHE137	O	−11.4967	10.6948	−20.5245
750	PHE137	CB	−8.4306	10.2588	−21.4726
751	PHE137	CG	−7.3656	10.217	−22.5633
752	PHE137	CD1	−6.7371	11.3995	−22.9629
753	PHE137	CD2	−7.0118	9.0021	−23.1568
754	PHE137	CE1	−5.7247	11.363	−23.9254
755	PHE137	CE2	−6.0012	8.966	−24.1212
756	PHE137	CZ	−5.3502	10.1442	−24.4974
757	TRP138	N	−10.7345	8.5614	−20.1081
758	TRP138	CA	−11.5544	8.3889	−18.9132
759	TRP138	C	−10.6682	8.1648	−17.6812
760	TRP138	O	−9.5891	7.5783	−17.7445
761	TRP138	CB	−12.5733	7.2508	−19.0873
762	TRP138	CG	−13.8737	7.8635	−19.5123
763	TRP138	CD1	−14.1925	8.302	−20.7399
764	TRP138	CD2	−15.0672	8.1022	−18.628
765	TRP138	NE1	−15.4138	8.7761	−20.7391
766	TRP138	CE2	−15.9622	8.6761	−19.5107
767	TRP138	CE3	−15.3643	7.8732	−17.2856
768	TRP138	CZ2	−17.2375	9.072	−19.1083
769	TRP138	CZ3	−16.6435	8.2685	−16.8761
770	TRP138	CH2	−17.5533	8.8666	−17.7597
771	PHE139	N	−11.2525	8.5736	−16.4965
772	PHE139	CA	−10.6047	8.4027	−15.1966
773	PHE139	C	−11.6784	8.0116	−14.1835
774	PHE139	O	−12.8384	8.4183	−14.2702
775	PHE139	CB	−9.764	9.6274	−14.7891
776	PHE139	CG	−8.9383	10.0973	−15.9836
777	PHE139	CD1	−9.3498	11.2112	−16.7203
778	PHE139	CD2	−7.7763	9.4121	−16.3481
779	PHE139	CE1	−8.6151	11.6229	−17.8355
780	PHE139	CE2	−7.043	9.8219	−17.4648
781	PHE139	CZ	−7.4627	10.9269	−18.2099
782	TRP140	N	−11.2181	7.2355	−13.1403
783	TRP140	CA	−12.0124	7.0661	−11.921
784	TRP140	C	−11.6556	8.2532	−11.0208
785	TRP140	O	−10.5299	8.754	−11.0265
786	TRP140	CB	−11.6066	5.7386	−11.2498
787	TRP140	CG	−12.07	5.687	−9.8235
788	TRP140	CD1	−13.2049	5.1441	−9.3567
789	TRP140	CD2	−11.3253	6.2514	−8.6444
790	TRP140	NE1	−13.2639	5.2892	−8.0555
791	TRP140	CE2	−12.1694	5.9322	−7.598
792	TRP140	CE3	−10.1209	6.9325	−8.4733
793	TRP140	CZ2	−11.8562	6.2641	−6.2802
794	TRP140	CZ3	−9.8106	7.2898	−7.1556
795	TRP140	CH2	−10.6545	6.9571	−6.0853
796	HIS141	N	−12.6722	8.7017	−10.2057
797	HIS141	CA	−12.5073	9.8245	−9.2918
798	HIS141	C	−13.4191	9.5704	−8.0813
799	HIS141	O	−14.6246	9.8188	−8.0798
800	HIS141	CB	−12.934	11.1155	−10.0147
801	HIS141	CG	−12.0532	11.287	−11.2152
802	HIS141	ND1	−10.8127	11.7037	−11.1111
803	HIS141	CD2	−12.4213	11.027	−12.4836
804	HIS141	CE1	−10.3183	11.7319	−12.3074
805	HIS141	NE2	−11.1851	11.36	−13.1359
806	TYR142	N	−12.795	8.9359	−7.0187
807	TYR142	CA	−13.3433	9.0058	−5.6555
808	TYR142	C	−14.7314	8.3527	−5.5148
809	TYR142	O	−15.4898	8.5952	−4.5781
810	TYR142	CB	−13.397	10.4616	−5.1484
811	TYR142	CG	−12.0073	11.0876	−5.0786
812	TYR142	CD1	−11.2482	11.2589	−6.2396
813	TYR142	CD2	−11.4964	11.4996	−3.8452
814	TYR142	CE1	−9.9943	11.8714	−6.1724
815	TYR142	CE2	−10.2473	12.1222	−3.7792
816	TYR142	CZ	−9.5014	12.319	−4.9439
817	TYR142	OH	−8.2703	12.9606	−4.8804
818	GLY143	N	−14.9795	7.3789	−6.4539
819	GLY143	CA	−16.2053	6.619	−6.5102
820	GLY143	C	−16.7664	6.5699	−7.9246
821	GLY143	O	−17.2517	5.5321	−8.3758
822	ASN144	N	−16.8015	7.7865	−8.5793
823	ASN144	CA	−17.3743	7.914	−9.9166
824	ASN144	C	−16.2826	7.8175	−10.9978
825	ASN144	O	−15.0873	7.9838	−10.7567
826	ASN144	CB	−18.1095	9.2584	−10.0685
827	ASN144	CG	−19.2871	9.3093	−9.1396
828	ASN144	OD1	−19.3421	10.171	−8.2779
829	ASN144	ND2	−20.2454	8.3803	−9.3069
830	TRP145	N	−16.775	7.5382	−12.2662
831	TRP145	CA	−15.9231	7.6557	−13.4515
832	TRP145	C	−16.1906	9.0373	−14.058
833	TRP145	O	−17.311	9.5466	−14.0413
834	TRP145	CB	−15.9475	6.4578	−14.4157
835	TRP145	CG	−14.7551	5.6159	−14.0635
836	TRP145	CD1	−14.6017	4.8703	−12.9577
837	TRP145	CD2	−13.5001	5.4609	−14.88
838	TRP145	NE1	−13.4297	4.2851	−12.9715
839	TRP145	CE2	−12.7449	4.6195	−14.0844
840	TRP145	CE3	−13.0513	5.9493	−16.1065
841	TRP145	CZ2	−11.4595	4.2199	−14.4501
842	TRP145	CZ3	−11.7677	5.5395	−16.4885
843	TRP145	CH2	−10.9842	4.7093	−15.6732
844	VAL146	N	−15.0943	9.6111	−14.6657
845	VAL146	CA	−15.1323	10.9329	−15.288
846	VAL146	C	−14.4039	10.7866	−16.6377
847	VAL146	O	−13.3481	10.1608	−16.7543
848	VAL146	CB	−14.5062	12.0252	−14.4016
849	VAL146	CG1	−14.5627	13.3836	−15.1259
850	VAL146	CG2	−15.2933	12.1222	−13.0812
851	PRO147	N	−15.0407	11.4243	−17.688
852	PRO147	CA	−14.4421	11.5445	−19.0197
853	PRO147	C	−13.7611	12.9212	−19.0753
854	PRO147	O	−14.2574	13.9182	−18.5488
855	PRO147	CB	−15.7135	11.6846	−19.8787
856	PRO147	CG	−16.8296	12.2116	−18.9477
857	PRO147	CD	−16.3539	12.0027	−17.496
858	VAL148	N	−12.5849	12.9549	−19.7972
859	VAL148	CA	−11.8514	14.1932	−20.0352
860	VAL148	C	−11.4656	14.2299	−21.5245
861	VAL148	O	−11.0249	13.2515	−22.1259
862	VAL148	CB	−10.5713	14.2143	−19.1768
863	VAL148	CG1	−9.8977	15.597	−19.2662
864	VAL148	CG2	−10.922	13.9061	−17.7098
865	VAL149	N	−11.6336	15.4826	−22.0884
866	VAL149	CA	−11.3107	15.8132	−23.4772
867	VAL149	C	−10.0416	16.689	−23.4257
868	VAL149	O	−9.863	17.5286	−22.5332
869	VAL149	CB	−12.4666	16.541	−24.1919
870	VAL149	CG1	−12.0679	16.8561	−25.6467
871	VAL149	CG2	−13.7176	15.6425	−24.1975
872	ILE150	N	−9.1584	16.5193	−24.4747
873	ILE150	CA	−7.8397	17.1654	−24.4977
874	ILE150	C	−7.3708	17.3781	−25.9591
875	ILE150	O	−7.7728	16.6718	−26.8855
876	ILE150	CB	−6.8268	16.3699	−23.645
877	ILE150	CG1	−6.7359	14.8871	−24.0676
878	ILE150	CG2	−5.4384	17.0398	−23.6387
879	ILE150	CD1	−6.0369	14.7232	−25.4318
880	ASP151	N	−6.4562	18.4073	−26.1257
881	ASP151	CA	−5.7667	18.6717	−27.3943
882	ASP151	C	−4.3473	18.0589	−27.3541
883	ASP151	O	−3.8018	17.6815	−26.3105
884	ASP151	CB	−5.6562	20.196	−27.5904
885	ASP151	CG	−4.8288	20.8034	−26.4927
886	ASP151	OD1	−3.5843	20.8953	−26.6713
887	ASP151	OD2	−5.4241	21.2042	−25.4574
888	ASP152	N	−3.6913	18.0528	−28.5621
889	ASP152	CA	−2.4247	17.3595	−28.815
890	ASP152	C	−1.2286	18.3059	−28.9933
891	ASP152	O	−0.1476	17.9427	−29.4503
892	ASP152	CB	−2.5075	16.1388	−29.7539
893	ASP152	CG	−3.1537	16.4648	−31.0697
894	ASP152	OD1	−2.4711	16.2908	−32.1139
895	ASP152	OD2	−4.3413	16.8882	−31.068
896	ARG153	N	−1.4181	19.5454	−28.4233
897	ARG153	CA	−0.3124	20.46	−28.1713
898	ARG153	C	−0.269	20.0286	−26.8111
899	ARG153	O	−0.4419	19.9335	−25.8052
900	ARG153	CB	−0.8267	21.9081	−28.0912
901	ARG153	CG	−1.4103	22.3167	−29.457
902	ARG153	CD	−2.9178	22.6007	−29.3184
903	ARG153	NE	−3.1155	23.7076	−28.3995
904	ARG153	CZ	−4.2574	23.8944	−27.8041
905	ARG153	NH1	−5.2654	23.1038	−28.0233
906	ARG153	NH2	−4.3959	24.8896	−26.9796
907	LEU154	N	1.6045	19.6786	−26.8477
908	LEU154	CA	2.3415	19.1173	−25.7092
909	LEU154	C	3.5418	20.0371	−25.3873
910	LEU154	O	3.993	20.806	−26.2439
911	LEU154	CB	2.8149	17.6876	−26.038
912	LEU154	CG	1.6904	16.8972	−26.7388
913	LEU154	CD1	2.2113	15.5079	−27.1503
914	LEU154	CD2	0.4791	16.7385	−25.7994
915	PRO155	N	4.1317	19.919	−24.1374
916	PRO155	CA	5.2572	20.7795	−23.7535
917	PRO155	C	6.5862	20.1545	−24.2212
918	PRO155	O	7.092	19.1556	−23.7017
919	PRO155	CB	5.1713	20.6814	−22.217
920	PRO155	CG	4.3153	19.4413	−21.8727
921	PRO155	CD	3.5972	18.9944	−23.1615
922	LEU162	N	9.1036	17.1652	−22.7622
923	LEU162	CA	9.5653	17.3634	−21.3825
924	LEU162	C	8.9453	16.3509	−20.3944
925	LEU162	O	9.3738	16.2255	−19.2479
926	LEU162	CB	9.1718	18.7986	−20.9763
927	LEU162	CG	9.5659	19.7972	−22.085
928	LEU162	CD1	8.9469	21.1771	−21.7988
929	LEU162	CD2	11.0986	19.9205	−22.171
930	VAL163	N	7.8059	15.7359	−20.8548
931	VAL163	CA	6.9001	14.9282	−20.0167
932	VAL163	C	6.8592	13.4935	−20.5742
933	VAL163	O	7.6291	13.1371	−21.4753
934	VAL163	CB	5.5354	15.6534	−20.0225
935	VAL163	CG1	4.4405	14.9111	−20.8157
936	VAL163	CG2	5.0723	15.9333	−18.5813
937	PHE164	N	5.9919	12.5981	−19.9705
938	PHE164	CA	6.0594	11.1646	−20.2933
939	PHE164	C	5.6914	10.9875	−21.7803
940	PHE164	O	4.8581	11.6769	−22.3624
941	PHE164	CB	5.033	10.3666	−19.4641
942	PHE164	CG	5.4169	10.3322	−17.9883
943	PHE164	CD1	4.5025	10.7562	−17.0208
944	PHE164	CD2	6.6779	9.8748	−17.5988
945	PHE164	CE1	4.8585	10.7499	−15.6698
946	PHE164	CE2	7.0391	9.8751	−16.2491
947	PHE164	CZ	6.1318	10.3218	−15.2852
948	VAL165	N	6.3903	9.9741	−22.4123
949	VAL165	CA	6.3353	9.7874	−23.8582
950	VAL165	C	6.8056	8.3647	−24.1952
951	VAL165	O	7.4217	7.6684	−23.3901
952	VAL165	CB	7.2052	10.8184	−24.6081
953	VAL165	CG1	6.5458	12.2098	−24.5778
954	VAL165	CG2	8.6119	10.8981	−23.9844
955	SER166	N	6.4785	7.9386	−25.4751
956	SER166	CA	7.1328	6.7639	−26.0454
957	SER166	C	6.838	6.5724	−27.5437
958	SER166	O	5.686	6.5221	−27.96
959	SER166	CB	6.7683	5.4809	−25.2727
960	SER166	OG	7.7074	4.4486	−25.5853
961	PHE173	N	2.2461	8.4031	−28.877
962	PHE173	CA	1.3314	9.4965	−28.6476
963	PHE173	C	0.336	9.3901	−27.514
964	PHE173	O	−0.4975	10.2913	−27.372
965	PHE173	CB	0.6858	10.0229	−29.9456
966	PHE173	CG	1.7267	10.7609	−30.7817
967	PHE173	CD1	1.8293	10.5041	−32.1511
968	PHE173	CD2	2.5797	11.6915	−30.1812
969	PHE173	CE1	2.8202	11.1379	−32.9053
970	PHE173	CE2	3.5614	12.3352	−30.9387
971	PHE173	CZ	3.6865	12.0519	−32.3009
972	TRP174	N	−0.2464	8.1445	−27.29
973	TRP174	CA	−1.3832	8.0441	−26.3625
974	TRP174	C	−0.9695	8.3698	−24.9116
975	TRP174	O	−1.7629	8.8377	−24.0928
976	TRP174	CB	−2.0615	6.6656	−26.4833
977	TRP174	CG	−1.251	5.612	−25.7898
978	TRP174	CD1	−0.0053	5.2237	−26.1002
979	TRP174	CD2	−1.6945	4.7987	−24.6045
980	TRP174	NE1	0.3806	4.2861	−25.2719
981	TRP174	CE2	−0.5919	3.9966	−24.3848
982	TRP174	CE3	−2.847	4.736	−23.8226
983	TRP174	CZ2	−0.5679	3.042	−23.3686
984	TRP174	CZ3	−2.8211	3.7981	−22.7832
985	TRP174	CH2	−1.7146	2.9625	−22.5695
986	GLY175	N	0.319	8.0074	−24.5881
987	GLY175	CA	0.8548	8.0482	−23.229
988	GLY175	C	0.924	9.5013	−22.7453
989	GLY175	O	0.4744	9.8591	−21.6563
990	ALA176	N	1.5527	10.3575	−23.6355
991	ALA176	CA	1.8162	11.7463	−23.2667
992	ALA176	C	0.4778	12.4832	−23.1036
993	ALA176	O	0.3087	13.3923	−22.2917
994	ALA176	CB	2.6081	12.4016	−24.4134
995	LEU177	N	−0.4862	12.0977	−24.0175
996	LEU177	CA	−1.8072	12.7043	−24.039
997	LEU177	C	−2.6026	12.2801	−22.7889
998	LEU177	O	−3.3645	13.0632	−22.2149
999	LEU177	CB	−2.5591	12.1878	−25.2811
1000	LEU177	CG	−2.1824	13.0175	−26.5242
1001	LEU177	CD1	−2.8503	12.4028	−27.7689
1002	LEU177	CD2	−2.6658	14.4711	−26.3557
1003	LEU178	N	−2.4445	10.9664	−22.3884
1004	LEU178	CA	−3.1132	10.4544	−21.1906
1005	LEU178	C	−2.5251	11.1643	−19.9513
1006	LEU178	O	−3.234	11.5138	−19.0034
1007	LEU178	CB	−2.9177	8.9296	−21.0891
1008	LEU178	CG	−3.8563	8.3476	−20.0123
1009	LEU178	CD1	−5.3264	8.6533	−20.3596
1010	LEU178	CD2	−3.6637	6.822	−19.9332
1011	GLU179	N	−1.1524	11.3234	−19.9572
1012	GLU179	CA	−0.4475	11.9849	−18.8542
1013	GLU179	C	−0.9306	13.4538	−18.7813
1014	GLU179	O	−1.217	13.995	−17.7092
1015	GLU179	CB	1.0603	11.9436	−19.1576
1016	GLU179	CG	1.836	12.3442	−17.8903
1017	GLU179	CD	2.7056	13.5265	−18.1976
1018	GLU179	OE1	3.9506	13.3429	−18.2297
1019	GLU179	OE2	2.1484	14.6392	−18.399
1020	LYS180	N	−1.0611	14.1019	−20.0001
1021	LYS180	CA	−1.5294	15.4849	−20.058
1022	LYS180	C	−2.9863	15.5584	−19.5596
1023	LYS180	O	−3.4062	16.527	−18.9195
1024	LYS180	CB	−1.4866	15.9011	−21.5416
1025	LYS180	CG	−1.9217	17.3697	−21.7058
1026	LYS180	CD	−1.7157	17.8037	−23.1693
1027	LYS180	CE	−2.086	19.2905	−23.326
1028	LYS180	NZ	−3.4519	19.4078	−23.8566
1029	ALA181	N	−3.8236	14.534	−19.9589
1030	ALA181	CA	−5.227	14.5397	−19.5633
1031	ALA181	C	−5.3504	14.3395	−18.0377
1032	ALA181	O	−6.2726	14.8376	−17.3882
1033	ALA181	CB	−5.9142	13.3553	−20.2685
1034	TYR182	N	−4.3968	13.5147	−17.4674
1035	TYR182	CA	−4.3378	13.3084	−16.014
1036	TYR182	C	−3.911	14.6397	−15.3604
1037	TYR182	O	−4.4286	15.0613	−14.3233
1038	TYR182	CB	−3.2428	12.2534	−15.7604
1039	TYR182	CG	−3.7672	11.0667	−14.9534
1040	TYR182	CD1	−2.8826	10.3532	−14.1401
1041	TYR182	CD2	−5.1102	10.6836	−15.0169
1042	TYR182	CE1	−3.3303	9.249	−13.4101
1043	TYR182	CE2	−5.5616	9.5844	−14.2811
1044	TYR182	CZ	−4.6681	8.8552	−13.4923
1045	TYR182	OH	−5.1083	7.7388	−12.792
1046	ALA183	N	−2.9084	15.3364	−16.0158
1047	ALA183	CA	−2.4983	16.6457	−15.5238
1048	ALA183	C	−3.6901	17.6177	−15.5866
1049	ALA183	O	−3.8643	18.4841	−14.7264
1050	ALA183	CB	−1.384	17.1672	−16.4469
1051	LYS184	N	−4.5087	17.4934	−16.6977
1052	LYS184	CA	−5.611	18.422	−16.9143
1053	LYS184	C	−6.6679	18.2596	−15.8071
1054	LYS184	O	−7.2776	19.2443	−15.383
1055	LYS184	CB	−6.2294	18.0837	−18.2832
1056	LYS184	CG	−7.201	19.2001	−18.7066
1057	LYS184	CD	−7.6414	18.9677	−20.1633
1058	LYS184	CE	−8.7361	19.9852	−20.5324
1059	LYS184	NZ	−10.0419	19.4944	−20.0694
1060	LEU185	N	−6.9762	16.9651	−15.4203
1061	LEU185	CA	−7.9755	16.7511	−14.3701
1062	LEU185	C	−7.4202	17.1729	−12.997
1063	LEU185	O	−8.1656	17.635	−12.1312
1064	LEU185	CB	−8.3535	15.2591	−14.3817
1065	LEU185	CG	−9.887	15.1102	−14.4244
1066	LEU185	CD1	−10.4851	15.9782	−15.5488
1067	LEU185	CD2	−10.2408	13.6361	−14.6884
1068	SER186	N	−6.0696	16.9426	−12.7748
1069	SER186	CA	−5.4961	17.2909	−11.4717
1070	SER186	C	−5.3992	18.8264	−11.3662
1071	SER186	O	−5.5294	19.4181	−10.296
1072	SER186	CB	−4.0918	16.6661	−11.3442
1073	SER186	OG	−3.2145	17.1663	−12.3577
1074	GLY187	N	−5.0223	19.4634	−12.5324
1075	GLY187	CA	−5.2652	20.8786	−12.7556
1076	GLY187	C	−4.0857	21.5354	−13.4597
1077	GLY187	O	−4.2258	22.4645	−14.2511
1078	SER188	N	−2.8658	21.077	−13.0129
1079	SER188	CA	−1.5882	21.562	−13.5121
1080	SER188	C	−0.6525	20.3481	−13.5519
1081	SER188	O	−0.7697	19.4057	−12.7666
1082	SER188	CB	−0.9967	22.6529	−12.6005
1083	SER188	OG	−1.8848	23.7722	−12.5576
1084	TYR189	N	0.4309	20.4751	−14.4103
1085	TYR189	CA	1.394	19.3635	−14.5345
1086	TYR189	C	2.1028	19.1407	−13.1875
1087	TYR189	O	2.5564	18.0528	−12.8423
1088	TYR189	CB	2.4205	19.6809	−15.6392
1089	TYR189	CG	1.7359	19.639	−17.0026
1090	TYR189	CD1	1.6966	20.7877	−17.7958
1091	TYR189	CD2	1.1439	18.4599	−17.4631
1092	TYR189	CE1	0.9697	20.7938	−18.9882
1093	TYR189	CE2	0.4434	18.4535	−18.6726
1094	TYR189	CZ	0.3372	19.627	−19.4238
1095	TYR189	OH	−0.3958	19.6344	−20.6038
1096	GLU190	N	2.2353	20.2856	−12.4271
1097	GLU190	CA	2.9165	20.2945	−11.1371
1098	GLU190	C	2.0888	19.4928	−10.1168
1099	GLU190	O	2.6117	18.9198	−9.1614
1100	GLU190	CB	2.9576	21.7516	−10.642
1101	GLU190	CG	3.8849	21.8584	−9.417
1102	GLU190	CD	5.0422	22.7653	−9.7201
1103	GLU190	OE1	5.8825	22.393	−10.5826
1104	GLU190	OE2	5.1173	23.8523	−9.0882
1105	ASP191	N	0.7198	19.4753	−10.3209
1106	ASP191	CA	−0.1724	18.7469	−9.4237
1107	ASP191	C	−0.2456	17.2321	−9.7198
1108	ASP191	O	−1.1273	16.5148	−9.243
1109	ASP191	CB	−1.5988	19.335	−9.455
1110	ASP191	CG	−1.6043	20.8365	−9.5147
1111	ASP191	OD1	−2.4983	21.3884	−10.2095
1112	ASP191	OD2	−0.7159	21.4698	−8.8827
1113	LEU192	N	0.819	16.7221	−10.4365
1114	LEU192	CA	1.1802	15.3111	−10.4241
1115	LEU192	C	2.3645	15.0764	−9.4578
1116	LEU192	O	2.6051	13.967	−8.9756
1117	LEU192	CB	1.606	14.9339	−11.8584
1118	LEU192	CG	0.6433	15.546	−12.899
1119	LEU192	CD1	1.1591	15.2637	−14.3222
1120	LEU192	CD2	−0.7731	14.9614	−12.7367
1121	GLN193	N	3.2225	16.1521	−9.2984
1122	GLN193	CA	4.4874	16.0024	−8.5836
1123	GLN193	C	4.1847	15.9215	−7.0888
1124	GLN193	O	3.7016	16.8552	−6.4531
1125	GLN193	CB	5.4245	17.2036	−8.8285
1126	GLN193	CG	5.1253	17.9062	−10.168
1127	GLN193	CD	6.0074	19.112	−10.3215
1128	GLN193	OE1	6.5849	19.3114	−11.3772
1129	GLN193	NE2	6.126	19.9311	−9.2612
1130	SER194	N	4.4584	14.6824	−6.5404
1131	SER194	CA	4.1404	14.3758	−5.1598
1132	SER194	C	2.866	13.5587	−4.9666
1133	SER194	O	2.5222	13.2064	−3.8381
1134	SER194	CB	4.3567	15.493	−4.1172
1135	SER194	OG	3.1377	16.2063	−3.8864
1136	GLY195	N	2.176	13.2208	−6.1141
1137	GLY195	CA	1.1358	12.206	−6.0658
1138	GLY195	C	1.7882	10.822	−6.0921
1139	GLY195	O	2.8873	10.6093	−6.6065
1140	GLU199	N	−2.5121	6.1465	−5.8806
1141	GLU199	CA	−3.5773	7.0891	−6.2252
1142	GLU199	C	−3.8566	6.9376	−7.7303
1143	GLU199	O	−4.9993	6.8618	−8.1812
1144	GLU199	CB	−3.046	8.5117	−5.9794
1145	GLU199	CG	−2.8762	8.7261	−4.4654
1146	GLU199	CD	−1.8341	9.7648	−4.1654
1147	GLU199	OE1	−1.63	10.6852	−5.0024
1148	GLU199	OE2	−1.2058	9.6511	−3.0809
1149	ALA200	N	−2.7213	6.902	−8.5278
1150	ALA200	CA	−2.842	6.7555	−9.9605
1151	ALA200	C	−3.45	5.3988	−10.3097
1152	ALA200	O	−4.3298	5.2754	−11.1633
1153	ALA200	CB	−1.4357	6.8497	−10.5805
1154	LEU201	N	−2.922	4.3129	−9.635
1155	LEU201	CA	−3.3751	2.957	−9.9427
1156	LEU201	C	−4.9075	2.8846	−9.7547
1157	LEU201	O	−5.6129	2.2233	−10.5246
1158	LEU201	CB	−2.7345	2.0008	−8.9179
1159	LEU201	CG	−2.2696	0.7033	−9.6083
1160	LEU201	CD1	−1.7303	−0.2762	−8.5491
1161	LEU201	CD2	−3.4383	0.0413	−10.3625
1162	VAL202	N	−5.4127	3.5437	−8.6455
1163	VAL202	CA	−6.8497	3.6253	−8.4177
1164	VAL202	C	−7.5073	4.5069	−9.4862
1165	VAL202	O	−8.4947	4.0924	−10.1074
1166	VAL202	CB	−7.1239	4.2445	−7.0323
1167	VAL202	CG1	−8.6414	4.274	−6.7649
1168	VAL202	CG2	−6.4384	3.4126	−5.9324
1169	ASP203	N	−6.9576	5.7508	−9.7191
1170	ASP203	CA	−7.662	6.72	−10.568
1171	ASP203	C	−7.7643	6.2123	−12.0267
1172	ASP203	O	−8.6495	6.6136	−12.7897
1173	ASP203	CB	−6.9312	8.0729	−10.5074
1174	ASP203	CG	−7.5182	8.8924	−9.3946
1175	ASP203	OD1	−7.1833	8.6159	−8.2117
1176	ASP203	OD2	−8.3092	9.8239	−9.7007
1177	PHE204	N	−6.7745	5.3288	−12.4181
1178	PHE204	CA	−6.7787	4.6718	−13.7209
1179	PHE204	C	−7.5039	3.3039	−13.7173
1180	PHE204	O	−7.4301	2.5569	−14.6962
1181	PHE204	CB	−5.3233	4.4568	−14.1833
1182	PHE204	CG	−4.7906	5.6489	−14.9755
1183	PHE204	CD1	−5.63	6.3842	−15.8175
1184	PHE204	CD2	−3.4431	6.0012	−14.8599
1185	PHE204	CE1	−5.1116	7.4353	−16.5787
1186	PHE204	CE2	−2.9247	7.0537	−15.619
1187	PHE204	CZ	−3.7573	7.7662	−16.4858
1188	THR205	N	−8.3016	2.9986	−12.63
1189	THR205	CA	−8.952	1.6829	−12.5554
1190	THR205	C	−10.2705	1.7051	−11.7627
1191	THR205	O	−11.295	1.18	−12.1962
1192	THR205	CB	−7.9841	0.6757	−11.8991
1193	THR205	OG1	−6.7587	0.6175	−12.6342
1194	THR205	CG2	−8.6165	−0.7291	−11.8676
1195	GLY206	N	−10.175	2.1806	−10.4697
1196	GLY206	CA	−11.3324	2.192	−9.5885
1197	GLY206	C	−11.5494	0.908	−8.7917
1198	GLY206	O	−12.5236	0.7578	−8.0566
1199	GLY207	N	−10.5234	−0.0067	−8.9016
1200	GLY207	CA	−10.3755	−1.0644	−7.9225
1201	GLY207	C	−9.6759	−0.488	−6.6908
1202	GLY207	O	−9.3188	0.6865	−6.6208
1203	VAL208	N	−9.4719	−1.4096	−5.6779
1204	VAL208	CA	−8.7137	−1.0176	−4.4842
1205	VAL208	C	−7.2252	−1.2657	−4.7591
1206	VAL208	O	−6.8341	−2.3528	−5.1916
1207	VAL208	CB	−9.1628	−1.8	−3.2343
1208	VAL208	CG1	−8.4598	−1.2244	−1.9901
1209	VAL208	CG2	−10.6865	−1.6664	−3.0526
1210	ASN220	N	0.5391	−14.5311	5.7482
1211	ASN220	CA	−0.345	−15.0597	4.7114
1212	ASN220	C	−0.1301	−14.4188	3.3248
1213	ASN220	O	−0.8992	−14.5951	2.3761
1214	ASN220	CB	−1.8072	−14.8271	5.1425
1215	ASN220	CG	−2.0916	−13.3553	5.2608
1216	ASN220	OD1	−2.825	−12.8137	4.4512
1217	ASN220	ND2	−1.5115	−12.6891	6.2753
1218	LEU221	N	1.0815	−13.7713	3.1652
1219	LEU221	CA	1.2715	−12.8164	2.0571
1220	LEU221	C	1.2799	−13.5607	0.7046
1221	LEU221	O	0.9346	−13.036	−0.3535
1222	LEU221	CB	2.6218	−12.1004	2.259
1223	LEU221	CG	2.9041	−11.0458	1.1678
1224	LEU221	CD1	3.4846	−11.7071	−0.0966
1225	LEU221	CD2	1.6457	−10.2237	0.8287
1226	TRP222	N	1.7467	−14.8606	0.7729
1227	TRP222	CA	1.8026	−15.7229	−0.4088
1228	TRP222	C	0.3893	−16.1528	−0.8382
1229	TRP222	O	0.1312	−16.4911	−1.9975
1230	TRP222	CB	2.6551	−16.9633	−0.0895
1231	TRP222	CG	3.514	−17.3207	−1.269
1232	TRP222	CD1	3.9296	−18.551	−1.6056
1233	TRP222	CD2	4.0798	−16.3703	−2.2906
1234	TRP222	NE1	4.6819	−18.4953	−2.6768
1235	TRP222	CE2	4.8057	−17.2219	−3.1023
1236	TRP222	CE3	4.0008	−14.9958	−2.5113
1237	TRP222	CZ2	5.5389	−16.7516	−4.1911
1238	TRP222	CZ3	4.7089	−14.5196	−3.6215
1239	TRP222	CH2	5.4654	−15.3749	−4.4357
1240	ASP223	N	−0.5353	−16.2792	0.1887
1241	ASP223	CA	−1.9239	−16.5685	−0.1432
1242	ASP223	C	−2.5885	−15.3166	−0.7289
1243	ASP223	O	−3.4409	−15.4233	−1.6113
1244	ASP223	CB	−2.6696	−16.9075	1.1623
1245	ASP223	CG	−1.9071	−17.9206	1.9661
1246	ASP223	OD1	−2.0616	−19.1383	1.6831
1247	ASP223	OD2	−1.1616	−17.4999	2.8907
1248	ILE224	N	−2.193	−14.0971	−0.2059
1249	ILE224	CA	−2.7525	−12.8492	−0.744
1250	ILE224	C	−2.3698	−12.7895	−2.2384
1251	ILE224	O	−3.2148	−12.5541	−3.1062
1252	ILE224	CB	−1.9614	−11.7006	−0.08
1253	ILE224	CG1	−2.0125	−11.7554	1.4607
1254	ILE224	CG2	−2.415	−10.3245	−0.6073
1255	ILE224	CD1	−3.3687	−11.2443	1.9805
1256	LEU225	N	−1.0318	−13.0064	−2.5371
1257	LEU225	CA	−0.5436	−12.7541	−3.9012
1258	LEU225	C	−1.2766	−13.7007	−4.8795
1259	LEU225	O	−1.6357	−13.3264	−5.9993
1260	LEU225	CB	0.9715	−13.0185	−3.9213
1261	LEU225	CG	1.7156	−11.6713	−3.8561
1262	LEU225	CD1	1.4854	−10.9979	−2.4897
1263	LEU225	CD2	3.2213	−11.907	−4.0649
1264	ILE226	N	−1.4518	−15.0023	−4.4456
1265	ILE226	CA	−2.0745	−15.9965	−5.3171
1266	ILE226	C	−3.5714	−15.6726	−5.5167
1267	ILE226	O	−4.1698	−15.9481	−6.5587
1268	ILE226	CB	−1.8899	−17.3793	−4.662
1269	ILE226	CG1	−0.3862	−17.718	−4.6364
1270	ILE226	CG2	−2.642	−18.4549	−5.4702
1271	ILE226	CD1	−0.1398	−18.9911	−3.8053
1272	GLU227	N	−4.2228	−15.1961	−4.388
1273	GLU227	CA	−5.6282	−14.8244	−4.443
1274	GLU227	C	−5.8203	−13.5496	−5.2825
1275	GLU227	O	−6.8718	−13.3538	−5.8944
1276	GLU227	CB	−6.0543	−14.5149	−2.9947
1277	GLU227	CG	−6.7645	−15.73	−2.3659
1278	GLU227	CD	−5.8178	−16.8723	−2.1316
1279	GLU227	OE1	−5.5349	−17.1663	−0.9395
1280	GLU227	OE2	−5.3604	−17.486	−3.1329
1281	ALA228	N	−4.7777	−12.6483	−5.2521
1282	ALA228	CA	−4.776	−11.455	−6.0904
1283	ALA228	C	−4.5622	−11.8767	−7.5598
1284	ALA228	O	−5.1727	−11.3474	−8.4919
1285	ALA228	CB	−3.5912	−10.5744	−5.6525
1286	THR229	N	−3.6251	−12.867	−7.7922
1287	THR229	CA	−3.3162	−13.2583	−9.1709
1288	THR229	C	−4.5689	−13.8572	−9.8218
1289	THR229	O	−4.9097	−13.5481	−10.9646
1290	THR229	CB	−2.119	−14.2272	−9.2376
1291	THR229	OG1	−2.3184	−15.3492	−8.3756
1292	THR229	CG2	−0.825	−13.4907	−8.8429
1293	TYR230	N	−5.2918	−14.763	−9.0662
1294	TYR230	CA	−6.4583	−15.4248	−9.6547
1295	TYR230	C	−7.7072	−14.5029	−9.7559
1296	TYR230	O	−8.7572	−14.8875	−10.2748
1297	TYR230	CB	−6.8168	−16.6984	−8.8549
1298	TYR230	CG	−7.4666	−16.426	−7.4941
1299	TYR230	CD1	−6.9898	−17.0901	−6.3609
1300	TYR230	CD2	−8.5379	−15.5367	−7.3628
1301	TYR230	CE1	−7.6126	−16.9041	−5.1237
1302	TYR230	CE2	−9.1241	−15.2992	−6.118
1303	TYR230	CZ	−8.6664	−15.9953	−4.9973
1304	TYR230	OH	−9.2605	−15.7837	−3.7595
1305	ASN231	N	−7.5398	−13.2328	−9.2411
1306	ASN231	CA	−8.4707	−12.1217	−9.4628
1307	ASN231	C	−8.1441	−11.3886	−10.7844
1308	ASN231	O	−8.9255	−10.5879	−11.3015
1309	ASN231	CB	−8.3969	−11.1441	−8.2751
1310	ASN231	CG	−9.4706	−11.4856	−7.2813
1311	ASN231	OD1	−9.169	−11.7934	−6.1402
1312	ASN231	ND2	−10.7455	−11.4325	−7.7086
1313	ARG232	N	−6.8621	−11.5676	−11.2662
1314	ARG232	CA	−6.3853	−10.8097	−12.4064
1315	ARG232	C	−6.0159	−9.4036	−11.9561
1316	ARG232	O	−6.1526	−8.4293	−12.6958
1317	ARG232	CB	−7.3543	−10.8028	−13.6066
1318	ARG232	CG	−7.3407	−12.1783	−14.2961
1319	ARG232	CD	−8.3543	−12.1759	−15.4551
1320	ARG232	NE	−8.1377	−13.349	−16.2813
1321	ARG232	CZ	−7.7623	−13.2361	−17.5225
1322	ARG232	NH1	−7.5672	−14.3083	−18.2308
1323	ARG232	NH2	−7.5763	−12.0707	−18.0691
1324	THR233	N	−5.4344	−9.3387	−10.7042
1325	THR233	CA	−5.0346	−8.0619	−10.1287
1326	THR233	C	−3.7566	−7.5873	−10.8289
1327	THR233	O	−2.8805	−8.3575	−11.2235
1328	THR233	CB	−4.8108	−8.1256	−8.6048
1329	THR233	OG1	−3.7279	−9.0059	−8.2917
1330	THR233	CG2	−6.0987	−8.5817	−7.8934
1331	LEU234	N	−3.6383	−6.2111	−10.9129
1332	LEU234	CA	−2.4212	−5.6082	−11.4445
1333	LEU234	C	−1.4991	−5.4025	−10.2343
1334	LEU234	O	−1.7828	−4.5966	−9.3461
1335	LEU234	CB	−2.7335	−4.2456	−12.0927
1336	LEU234	CG	−3.805	−4.3977	−13.1899
1337	LEU234	CD1	−4.1814	−3.0033	−13.7237
1338	LEU234	CD2	−3.261	−5.2597	−14.3451
1339	ILE235	N	−0.4064	−6.2504	−10.1931
1340	ILE235	CA	0.6017	−6.192	−9.1379
1341	ILE235	C	1.7908	−5.3356	−9.6145
1342	ILE235	O	2.2008	−5.3498	−10.779
1343	ILE235	CB	1.1155	−7.6203	−8.8608
1344	ILE235	CG1	−0.0599	−8.5572	−8.5186
1345	ILE235	CG2	2.1354	−7.6114	−7.7054
1346	ILE235	CD1	0.4306	−10.0168	−8.4676
1347	GLY236	N	2.42	−4.6484	−8.592
1348	GLY236	CA	3.6097	−3.8503	−8.8163
1349	GLY236	C	4.4661	−3.8751	−7.556
1350	GLY236	O	4.0494	−3.4215	−6.4864
1351	CYS237	N	5.5388	−4.1763	−7.1446
1352	CYS237	CA	6.8369	−4.8386	−7.1951
1353	CYS237	C	7.4475	−4.8061	−5.8055
1354	CYS237	O	7.484	−3.758	−5.1653
1355	CYS237	CB	6.5137	−6.2914	−7.605
1356	CYS237	SG	7.8892	−6.9754	−8.5747
1357	VAL250	N	18.7837	−1.06	−3.8757
1358	VAL250	CA	18.7728	−1.6614	−5.1968
1359	VAL250	C	18.1563	−0.7936	−6.2755
1360	VAL250	O	17.5799	0.2563	−6.0141
1361	VAL250	CB	20.2071	−2.0544	−5.6135
1362	VAL250	CG1	21.0983	−0.8042	−5.7459
1363	VAL250	CG2	20.1899	−2.8323	−6.9436
1364	GLU251	N	18.3669	−0.2148	−7.3233
1365	GLU251	CA	17.7665	0.993	−7.8951
1366	GLU251	C	16.217	0.9991	−7.9433
1367	GLU251	O	15.5217	−0.0022	−8.13
1368	GLU251	CB	18.3563	1.2753	−9.292
1369	GLU251	CG	17.7693	0.3208	−10.3534
1370	GLU251	CD	18.039	−1.1153	−10.0041
1371	GLU251	OE1	17.0546	−1.8468	−9.7159
1372	GLU251	OE2	19.2333	−1.5165	−10.0181
1373	GLY252	N	15.6734	2.2759	−7.9118
1374	GLY252	CA	14.2506	2.4954	−7.6952
1375	GLY252	C	13.3542	2.3126	−8.9232
1376	GLY252	O	12.4592	3.1102	−9.1965
1377	HIS253	N	13.5372	1.1273	−9.6114
1378	HIS253	CA	12.566	0.6781	−10.6178
1379	HIS253	C	11.4865	−0.1559	−9.8957
1380	HIS253	O	11.7404	−0.8619	−8.9163
1381	HIS253	CB	13.2764	−0.2088	−11.6594
1382	HIS253	CG	12.3129	−0.6933	−12.7051
1383	HIS253	ND1	11.7376	0.1248	−13.5563
1384	HIS253	CD2	11.9466	−1.9771	−12.8817
1385	HIS253	CE1	10.9773	−0.5897	−14.3244
1386	HIS253	NE2	11.0523	−1.7983	−13.9931
1387	ALA254	N	10.2237	−0.0612	−10.4641
1388	ALA254	CA	9.1262	−0.9262	−10.0465
1389	ALA254	C	8.9691	−2.0231	−11.1058
1390	ALA254	O	8.8789	−1.772	−12.3048
1391	ALA254	CB	7.8177	−0.1351	−9.8715
1392	TYR255	N	8.9631	−3.2945	−10.5799
1393	TYR255	CA	8.7409	−4.4746	−11.4081
1394	TYR255	C	7.2757	−4.8968	−11.1662
1395	TYR255	O	6.6574	−4.578	−10.1462
1396	TYR255	CB	9.7104	−5.6038	−11.0117
1397	TYR255	CG	11.1349	−5.06	−11.0372
1398	TYR255	CD1	11.6245	−4.3411	−9.9433
1399	TYR255	CD2	11.9459	−5.2746	−12.1545
1400	TYR255	CE1	12.9031	−3.7807	−9.9923
1401	TYR255	CE2	13.2324	−4.731	−12.1946
1402	TYR255	CZ	13.7027	−3.9676	−11.1225
1403	TYR255	OH	14.9665	−3.3926	−11.1806
1404	THR256	N	6.7032	−5.6869	−12.1463
1405	THR256	CA	5.4017	−6.3105	−11.8927
1406	THR256	C	5.62	−7.7682	−11.4666
1407	THR256	O	6.704	−8.3411	−11.5791
1408	THR256	CB	4.5349	−6.2455	−13.1674
1409	THR256	OG1	3.2634	−6.8578	−12.933
1410	THR256	CG2	5.2393	−6.9521	−14.3426
1411	LEU257	N	4.4903	−8.3735	−10.956
1412	LEU257	CA	4.4405	−9.7992	−10.6241
1413	LEU257	C	3.3352	−10.3492	−11.5298
1414	LEU257	O	2.3071	−9.7058	−11.7431
1415	LEU257	CB	4.0719	−9.97	−9.1384
1416	LEU257	CG	4.449	−11.3805	−8.6471
1417	LEU257	CD1	4.42	−11.398	−7.1079
1418	LEU257	CD2	3.4361	−12.4093	−9.1829
1419	THR258	N	3.5695	−11.6026	−12.0657
1420	THR258	CA	2.6236	−12.1467	−13.048
1421	THR258	C	2.3296	−13.6435	−12.8472
1422	THR258	O	1.6948	−14.3054	−13.6756
1423	THR258	CB	3.0645	−11.8512	−14.4962
1424	THR258	OG1	4.2124	−12.6355	−14.8271
1425	THR258	CG2	3.3907	−10.3551	−14.6699
1426	GLY259	N	2.8063	−14.1799	−11.6779
1427	GLY259	CA	2.6734	−15.5863	−11.3513
1428	GLY259	C	3.3389	−15.854	−10.0101
1429	GLY259	O	4.304	−15.195	−9.6162
1430	ILE260	N	2.7885	−16.9007	−9.3074
1431	ILE260	CA	3.2533	−17.2739	−7.977
1432	ILE260	C	2.7416	−18.6858	−7.7268
1433	ILE260	O	1.5506	−18.9623	−7.8542
1434	ILE260	CB	2.6632	−16.3002	−6.934
1435	ILE260	CG1	3.4254	−14.9634	−6.9743
1436	ILE260	CG2	2.7358	−16.8838	−5.5089
1437	ILE260	CD1	2.5596	−13.8638	−6.3365
1438	LEU272	N	7.6295	−20.2065	−5.2267
1439	LEU272	CA	8.2148	−19.4305	−6.3034
1440	LEU272	C	7.2661	−18.2935	−6.6979
1441	LEU272	O	6.0434	−18.3602	−6.5699
1442	LEU272	CB	8.5888	−20.3099	−7.5079
1443	LEU272	CG	9.6054	−21.3716	−7.0504
1444	LEU272	CD1	10.0036	−22.2306	−8.2604
1445	LEU272	CD2	10.8588	−20.691	−6.4681
1446	VAL273	N	7.917	−17.2324	−7.2939
1447	VAL273	CA	7.2358	−16.0184	−7.722
1448	VAL273	C	7.8155	−15.6174	−9.0891
1449	VAL273	O	9.0208	−15.6826	−9.3478
1450	VAL273	CB	7.4182	−14.8938	−6.6832
1451	VAL273	CG1	6.7413	−13.5994	−7.1733
1452	VAL273	CG2	8.9137	−14.6382	−6.4113
1453	LYS274	N	6.862	−15.1989	−10.0027
1454	LYS274	CA	7.2322	−14.6979	−11.3118
1455	LYS274	C	7.1133	−13.1683	−11.2835
1456	LYS274	O	6.0599	−12.5689	−11.505
1457	LYS274	CB	6.3006	−15.2891	−12.3851
1458	LYS274	CG	7.1148	−15.4963	−13.6755
1459	LYS274	CD	6.3157	−14.9771	−14.8844
1460	LYS274	CE	7.0223	−13.7456	−15.4826
1461	LYS274	NZ	6.9113	−12.6067	−14.5607
1462	LEU275	N	8.3118	−12.5555	−10.9585
1463	LEU275	CA	8.497	−11.1202	−11.1425
1464	LEU275	C	8.6314	−10.9281	−12.6662
1465	LEU275	O	9.183	−11.7675	−13.3771
1466	LEU275	CB	9.7478	−10.6015	−10.4053
1467	LEU275	CG	9.6452	−10.8357	−8.8828
1468	LEU275	CD1	8.3234	−10.2695	−8.3293
1469	LEU275	CD2	9.7528	−12.336	−8.5475
1470	ARG276	N	8.1358	−9.74	−13.1543
1471	ARG276	CA	8.3421	−9.324	−14.5331
1472	ARG276	C	8.8047	−7.8552	−14.5295
1473	ARG276	O	8.2417	−6.9722	−13.8836
1474	ARG276	CB	7.0444	−9.48	−15.3455
1475	ARG276	CG	7.3659	−9.3005	−16.8396
1476	ARG276	CD	6.062	−9.0872	−17.6287
1477	ARG276	NE	6.3734	−8.4218	−18.8805
1478	ARG276	CZ	6.0922	−7.1638	−19.0603
1479	ARG276	NH1	5.5179	−6.4638	−18.1263
1480	ARG276	NH2	6.3917	−6.5985	−20.192
1481	ASN277	N	9.9012	−7.6359	−15.3508
1482	ASN277	CA	10.3016	−6.298	−15.783
1483	ASN277	C	9.4382	−6.0331	−17.0268
1484	ASN277	O	9.4635	−6.8197	−17.9793
1485	ASN277	CB	11.7935	−6.306	−16.1691
1486	ASN277	CG	12.2067	−4.9569	−16.6844
1487	ASN277	OD1	12.677	−4.8526	−17.8051
1488	ASN277	ND2	12.0325	−3.9052	−15.8638
1489	PRO278	N	8.6591	−4.8959	−17.042
1490	PRO278	CA	7.7978	−4.6224	−18.1928
1491	PRO278	C	8.507	−3.9806	−19.3944
1492	PRO278	O	7.9611	−3.9586	−20.4996
1493	PRO278	CB	6.8446	−3.5703	−17.5903
1494	PRO278	CG	7.4474	−3.0849	−16.2523
1495	PRO278	CD	8.643	−3.9952	−15.9106
1496	TRP279	N	9.717	−3.3532	−19.1338
1497	TRP279	CA	10.5405	−2.8672	−20.2519
1498	TRP279	C	11.0577	−4.086	−21.0245
1499	TRP279	O	11.2024	−4.0388	−22.2464
1500	TRP279	CB	11.702	−2.029	−19.6878
1501	TRP279	CG	11.2019	−0.672	−19.2777
1502	TRP279	CD1	9.9671	−0.1697	−19.4439
1503	TRP279	CD2	12.0185	0.3875	−18.5888
1504	TRP279	NE1	9.9044	1.0428	−18.9516
1505	TRP279	CE2	11.0955	1.4041	−18.4321
1506	TRP279	CE3	13.34	0.4865	−18.1559
1507	TRP279	CZ2	11.43	2.606	−17.8089
1508	TRP279	CZ3	13.6877	1.6954	−17.5407
1509	TRP279	CH2	12.7518	2.7248	−17.3612
1510	GLY280	N	11.4159	−5.1697	−20.251
1511	GLY280	CA	11.5344	−6.5276	−20.7606
1512	GLY280	C	12.812	−7.2554	−20.3432
1513	GLY280	O	12.8765	−8.4845	−20.3353
1514	LYS281	N	13.8767	−6.4156	−20.0798
1515	LYS281	CA	15.2169	−6.9354	−19.7943
1516	LYS281	C	15.1794	−7.7785	−18.5004
1517	LYS281	O	14.3936	−7.5659	−17.5749
1518	LYS281	CB	16.1986	−5.759	−19.6333
1519	LYS281	CG	16.1224	−4.8304	−20.8619
1520	LYS281	CD	14.9288	−3.865	−20.7223
1521	LYS281	CE	14.4474	−3.4386	−22.1215
1522	LYS281	NZ	13.5748	−2.261	−22.0113
1523	VAL282	N	16.105	−8.809	−18.4822
1524	VAL282	CA	16.0955	−9.8258	−17.4338
1525	VAL282	C	17.2642	−9.5236	−16.4779
1526	VAL282	O	17.0861	−9.1222	−15.3283
1527	VAL282	CB	16.1673	−11.2512	−18.0151
1528	VAL282	CG1	16.201	−12.2832	−16.8712
1529	VAL282	CG2	14.9202	−11.505	−18.8829
1530	GLU283	N	18.5204	−9.7489	−17.0297
1531	GLU283	CA	19.7139	−9.1512	−16.4335
1532	GLU283	C	19.9054	−9.6078	−14.9743
1533	GLU283	O	20.3128	−8.8728	−14.0762
1534	GLU283	CB	19.699	−7.6156	−16.554
1535	GLU283	CG	20.3208	−7.2119	−17.9031
1536	GLU283	CD	19.2366	−6.8709	−18.8827
1537	GLU283	OE1	18.5651	−7.8146	−19.3787
1538	GLU283	OE2	19.0641	−5.6559	−19.1671
1539	TRP284	N	19.711	−10.9701	−14.8076
1540	TRP284	CA	19.6307	−11.5979	−13.4902
1541	TRP284	C	20.32	−12.9674	−13.5704
1542	TRP284	O	20.2535	−13.6941	−14.5633
1543	TRP284	CB	18.1888	−11.6225	−12.9414
1544	TRP284	CG	17.91	−12.8763	−12.1649
1545	TRP284	CD1	18.4357	−13.2441	−10.9858
1546	TRP284	CD2	16.9741	−13.97	−12.5976
1547	TRP284	NE1	17.9623	−14.4142	−10.6335
1548	TRP284	CE2	17.0959	−14.874	−11.5596
1549	TRP284	CE3	16.1459	−14.1717	−13.7001
1550	TRP284	CZ2	16.3837	−16.0723	−11.5488
1551	TRP284	CZ3	15.4287	−15.3733	−13.6977
1552	TRP284	CH2	15.5441	−16.2952	−12.6471
1553	LYS285	N	21.0285	−13.3247	−12.4366
1554	LYS285	CA	21.9632	−14.4504	−12.445
1555	LYS285	C	21.1662	−15.7417	−12.2212
1556	LYS285	O	21.0234	−16.2627	−11.1162
1557	LYS285	CB	22.9755	−14.2461	−11.3019
1558	LYS285	CG	23.8492	−13.0142	−11.6019
1559	LYS285	CD	24.8293	−12.7871	−10.4367
1560	LYS285	CE	25.6941	−11.5475	−10.7307
1561	LYS285	NZ	26.6278	−11.3271	−9.6171
1562	GLY286	N	20.6382	−16.2482	−13.3943
1563	GLY286	CA	20.0064	−17.545	−13.4703
1564	GLY286	C	18.5419	−17.4677	−13.887
1565	GLY286	O	18.054	−16.5184	−14.4926
1566	ASP287	N	17.8398	−18.6115	−13.5564
1567	ASP287	CA	16.4395	−18.815	−13.9266
1568	ASP287	C	15.7156	−19.501	−12.7544
1569	ASP287	O	14.7632	−20.2565	−12.9279
1570	ASP287	CB	16.3237	−19.6363	−15.2247
1571	ASP287	CG	17.1292	−18.977	−16.3062
1572	ASP287	OD1	18.2282	−19.5037	−16.6247
1573	ASP287	OD2	16.665	−17.9357	−16.8427
1574	TRP288	N	16.2054	−19.094	−11.5288
1575	TRP288	CA	15.7924	−19.5176	−10.1793
1576	TRP288	C	17.1337	−19.6211	−9.4095
1577	TRP288	O	18.0766	−20.2926	−9.8253
1578	TRP288	CB	15.2243	−20.9457	−10.3028
1579	TRP288	CG	14.4441	−21.4204	−9.1144
1580	TRP288	CD1	14.2846	−20.8147	−7.9271
1581	TRP288	CD2	13.6849	−22.7179	−9.0621
1582	TRP288	NE1	13.5403	−21.5602	−7.1474
1583	TRP288	CE2	13.1744	−22.695	−7.779
1584	TRP288	CE3	13.4595	−23.7651	−9.9543
1585	TRP288	CZ2	12.4059	−23.7469	−7.2824
1586	TRP288	CZ3	12.6885	−24.8255	−9.4629
1587	TRP288	CH2	12.1814	−24.8191	−8.1553
1588	PRO300	N	9.0722	−32.1709	−11.9449
1589	PRO300	CA	8.8932	−32.9359	−13.178
1590	PRO300	C	8.459	−32.0578	−14.3665
1591	PRO300	O	8.001	−30.9196	−14.2469
1592	PRO300	CB	7.7612	−33.9139	−12.8092
1593	PRO300	CG	7.3326	−33.6108	−11.3554
1594	PRO300	CD	8.2537	−32.5064	−10.7989
1595	LYS301	N	8.4755	−32.7272	−15.5917
1596	LYS301	CA	8.2126	−31.983	−16.8382
1597	LYS301	C	6.7948	−31.3758	−16.8074
1598	LYS301	O	6.4991	−30.3471	−17.4149
1599	LYS301	CB	8.3182	−32.9405	−18.0382
1600	LYS301	CG	8.4303	−32.1066	−19.3278
1601	LYS301	CD	8.3916	−33.0374	−20.5528
1602	LYS301	CE	8.5678	−32.1989	−21.8321
1603	LYS301	NZ	8.4029	−33.0622	−23.01
1604	GLU302	N	5.8877	−32.1242	−16.0797
1605	GLU302	CA	4.4635	−31.8098	−16.0248
1606	GLU302	C	4.2584	−30.4583	−15.3122
1607	GLU302	O	3.2774	−29.75	−15.532
1608	GLU302	CB	3.7449	−32.9192	−15.2313
1609	GLU302	CG	4.1635	−34.3169	−15.7328
1610	GLU302	CD	4.0107	−34.4234	−17.2234
1611	GLU302	OE1	5.0311	−34.2282	−17.9371
1612	GLU302	OE2	2.8745	−34.7148	−17.6831
1613	LYS303	N	5.1878	−30.1862	−14.3216
1614	LYS303	CA	5.2839	−28.8544	−13.7347
1615	LYS303	C	6.181	−27.9679	−14.6161
1616	LYS303	O	5.9116	−26.7753	−14.7922
1617	LYS303	CB	5.9465	−29.0006	−12.3508
1618	LYS303	CG	5.9219	−27.6475	−11.6131
1619	LYS303	CD	6.4192	−27.8209	−10.165
1620	LYS303	CE	7.945	−28.0304	−10.1462
1621	LYS303	NZ	8.4058	−28.1127	−8.7527
1622	ILE304	N	7.3301	−28.5457	−15.1282
1623	ILE304	CA	8.3447	−27.6959	−15.7599
1624	ILE304	C	7.7032	−26.9572	−16.9428
1625	ILE304	O	7.8731	−25.7468	−17.1109
1626	ILE304	CB	9.5889	−28.5008	−16.1894
1627	ILE304	CG1	10.7478	−27.5181	−16.4497
1628	ILE304	CG2	9.3053	−29.3183	−17.4652
1629	ILE304	CD1	12.0311	−28.283	−16.8261
1630	LEU305	N	6.8641	−27.6745	−17.7763
1631	LEU305	CA	6.3499	−27.0252	−18.9765
1632	LEU305	C	5.2601	−25.9522	−18.6982
1633	LEU305	O	4.7144	−25.3199	−19.6105
1634	LEU305	CB	5.7725	−28.0968	−19.9225
1635	LEU305	CG	6.7608	−29.2675	−20.0948
1636	LEU305	CD1	6.095	−30.3824	−20.9216
1637	LEU305	CD2	8.0414	−28.7888	−20.8042
1638	LEU306	N	4.9626	−25.7193	−17.3707
1639	LEU306	CA	4.19	−24.5679	−16.915
1640	LEU306	C	5.1086	−23.3267	−16.7981
1641	LEU306	O	4.6468	−22.188	−16.8894
1642	LEU306	CB	3.6932	−24.9148	−15.4982
1643	LEU306	CG	2.3333	−24.2509	−15.2081
1644	LEU306	CD1	1.7749	−24.8107	−13.8871
1645	LEU306	CD2	2.4939	−22.7248	−15.0753
1646	GLU314	N	10.4436	−13.0374	−18.0961
1647	GLU314	CA	9.8608	−14.2598	−17.5136
1648	GLU314	C	10.4432	−14.4897	−16.0917
1649	GLU314	O	9.7656	−14.9325	−15.1632
1650	GLU314	CB	10.1254	−15.473	−18.426
1651	GLU314	CG	9.5111	−16.7608	−17.8338
1652	GLU314	CD	8.0669	−16.6372	−17.4286
1653	GLU314	OE1	7.4432	−15.5794	−17.7046
1654	GLU314	OE2	7.5444	−17.6074	−16.8192
1655	PHE315	N	11.8166	−14.3518	−15.9765
1656	PHE315	CA	12.5183	−14.472	−14.6901
1657	PHE315	C	12.3189	−15.866	−14.031
1658	PHE315	O	13.0269	−16.8309	−14.3115
1659	PHE315	CB	12.1636	−13.3303	−13.7155
1660	PHE315	CG	12.6233	−11.9913	−14.2807
1661	PHE315	CD1	11.6801	−11.0594	−14.7211
1662	PHE315	CD2	13.9865	−11.6939	−14.3555
1663	PHE315	CE1	12.0974	−9.832	−15.2422
1664	PHE315	CE2	14.4035	−10.465	−14.8729
1665	PHE315	CZ	13.4607	−9.5373	−15.3237
1666	TRP316	N	11.2462	−15.886	−13.1565
1667	TRP316	CA	10.9705	−16.8771	−12.1173
1668	TRP316	C	12.0455	−16.8216	−11.0225
1669	TRP316	O	13.2421	−16.9797	−11.2594
1670	TRP316	CB	10.7381	−18.2895	−12.6822
1671	TRP316	CG	9.2689	−18.4001	−12.963
1672	TRP316	CD1	8.6753	−18.4596	−14.1651
1673	TRP316	CD2	8.1813	−18.4517	−11.9242
1674	TRP316	NE1	7.3755	−18.5351	−14.0157
1675	TRP316	CE2	7.0451	−18.521	−12.7075
1676	TRP316	CE3	8.1457	−18.4409	−10.5304
1677	TRP316	CZ2	5.7705	−18.5613	−12.1427
1678	TRP316	CZ3	6.8694	−18.4933	−9.9561
1679	TRP316	CH2	5.7095	−18.5382	−10.7435
1680	MET317	N	11.5608	−16.6275	−9.7416
1681	MET317	CA	12.4538	−16.6791	−8.585
1682	MET317	C	11.7004	−17.2206	−7.3651
1683	MET317	O	10.5025	−17.5005	−7.3973
1684	MET317	CB	13.0084	−15.2749	−8.2814
1685	MET317	CG	14.094	−14.9049	−9.309
1686	MET317	SD	15.4463	−14.1	−8.4033
1687	MET317	CE	15.699	−12.6828	−9.5119
1688	THR318	N	12.492	−17.4183	−6.2542
1689	THR318	CA	11.9454	−17.9342	−5.0041
1690	THR318	C	11.1717	−16.829	−4.282
1691	THR318	O	11.3504	−15.634	−4.5022
1692	THR318	CB	13.0703	−18.454	−4.0877
1693	THR318	OG1	13.9732	−17.3917	−3.7649
1694	THR318	CG2	13.8342	−19.5924	−4.7897
1695	LEU319	N	10.3978	−17.2806	−3.2302
1696	LEU319	CA	9.7894	−16.3701	−2.2475
1697	LEU319	C	10.7846	−16.099	−1.0834
1698	LEU319	O	10.4341	−15.8581	0.0692
1699	LEU319	CB	8.4396	−16.8786	−1.7027
1700	LEU319	CG	7.7041	−15.7551	−0.9471
1701	LEU319	CD1	7.3298	−14.6195	−1.9176
1702	LEU319	CD2	6.4304	−16.3237	−0.2959
1703	GLN320	N	12.099	−15.9985	−1.4967
1704	GLN320	CA	13.173	−15.3505	−0.75
1705	GLN320	C	13.7063	−14.1818	−1.6072
1706	GLN320	O	14.8386	−13.7152	−1.4908
1707	GLN320	CB	14.2955	−16.3545	−0.4343
1708	GLN320	CG	13.8084	−17.3041	0.6748
1709	GLN320	CD	14.5664	−17.039	1.9426
1710	GLN320	OE1	15.1729	−17.9483	2.4844
1711	GLN320	NE2	14.54	−15.7854	2.4298
1712	ASP321	N	12.7319	−13.6055	−2.4064
1713	ASP321	CA	12.9573	−12.3844	−3.173
1714	ASP321	C	13.1776	−11.2013	−2.2097
1715	ASP321	O	13.9278	−10.2725	−2.5109
1716	ASP321	CB	14.1028	−12.5416	−4.1915
1717	ASP321	CG	13.7547	−13.6243	−5.1712
1718	ASP321	OD1	12.8643	−13.3811	−6.0284
1719	ASP321	OD2	14.3754	−14.7177	−5.0903
1720	PHE322	N	12.475	−11.3022	−1.0119
1721	PHE322	CA	12.3354	−10.223	−0.0218
1722	PHE322	C	13.6416	−9.511	0.3661
1723	PHE322	O	13.64	−8.4219	0.9499
1724	PHE322	CB	11.4304	−10.6181	1.1591
1725	PHE322	CG	10.0585	−11.0417	0.6353
1726	PHE322	CD1	9.2605	−11.8954	1.4014
1727	PHE322	CD2	9.5925	−10.5846	−0.6018
1728	PHE322	CE1	7.9945	−12.2709	0.9439
1729	PHE322	CE2	8.3347	−10.9736	−1.0689
1730	PHE322	CZ	7.5274	−11.8041	−0.2875
1731	LYS323	N	14.7997	−10.2081	0.0947
1732	LYS323	CA	16.1194	−9.6218	0.2551
1733	LYS323	C	16.1537	−8.3235	−0.5737
1734	LYS323	O	16.3342	−7.2263	−0.029
1735	LYS323	CB	17.1131	−10.6095	−0.3812
1736	LYS323	CG	18.4795	−9.9502	−0.6458
1737	LYS323	CD	19.1402	−10.6292	−1.8584
1738	LYS323	CE	20.4432	−11.3163	−1.4133
1739	LYS323	NZ	20.5091	−12.6665	−1.99
1740	THR324	N	15.8967	−8.4471	−1.9344
1741	THR324	CA	15.6988	−7.2238	−2.7145
1742	THR324	C	14.1951	−6.8837	−2.728
1743	THR324	O	13.3502	−7.5502	−2.1343
1744	THR324	CB	16.624	−6.0305	−2.3844
1745	THR324	OG1	16.4994	−5.0168	−3.3859
1746	THR324	CG2	18.0917	−6.4955	−2.3306
1747	HIS325	N	13.8893	−5.7011	−3.3613
1748	HIS325	CA	12.5701	−5.0806	−3.2588
1749	HIS325	C	12.3707	−4.4943	−1.8399
1750	HIS325	O	13.062	−4.7926	−0.8612
1751	HIS325	CB	11.4731	−6.1791	−3.1788
1752	HIS325	CG	11.4857	−7.5352	−3.8442
1753	HIS325	ND1	11.8417	−7.758	−5.0868
1754	HIS325	CD2	11.1153	−8.6655	−3.2092
1755	HIS325	CE1	11.7254	−9.0281	−5.3105
1756	HIS325	NE2	11.3146	−9.6062	−4.2763
1757	PHE326	N	11.3365	−3.58	−1.7799
1758	PHE326	CA	11.0657	−2.793	−0.5818
1759	PHE326	C	9.5505	−2.7222	−0.3591
1760	PHE326	O	9.0613	−2.9912	0.741
1761	PHE326	CB	11.643	−1.3757	−0.7791
1762	PHE326	CG	10.9725	−0.3951	0.179
1763	PHE326	CD1	11.4	−0.3051	1.506
1764	PHE326	CD2	9.9205	0.405	−0.2744
1765	PHE326	CE1	10.7388	0.5484	2.393
1766	PHE326	CE2	9.2546	1.2522	0.6146
1767	PHE326	CZ	9.659	1.3172	1.9505
1768	VAL327	N	8.7986	−2.2783	−1.4285
1769	VAL327	CA	7.3404	−2.1816	−1.3464
1770	VAL327	C	6.7208	−3.0375	−2.455
1771	VAL327	O	7.3013	−3.2595	−3.5218
1772	VAL327	CB	6.9055	−0.7062	−1.473
1773	VAL327	CG1	7.4988	−0.0788	−2.749
1774	VAL327	CG2	5.3698	−0.5854	−1.5107
1775	LEU328	N	5.4394	−3.474	−2.1761
1776	LEU328	CA	4.5394	−3.9376	−3.218
1777	LEU328	C	3.2176	−3.1868	−3.0694
1778	LEU328	O	2.724	−2.9298	−1.9697
1779	LEU328	CB	4.3597	−5.4663	−3.2082
1780	LEU328	CG	4.3722	−6.0073	−4.6495
1781	LEU328	CD1	4.7594	−7.4974	−4.632
1782	LEU328	CD2	2.9671	−5.859	−5.2598
1783	LEU329	N	2.6001	−2.9333	−4.2765
1784	LEU329	CA	1.193	−2.6048	−4.3562
1785	LEU329	C	0.5155	−3.4931	−5.4062
1786	LEU329	O	1.1143	−3.9316	−6.3919
1787	LEU329	CB	0.9461	−1.1036	−4.6129
1788	LEU329	CG	1.0817	−0.7385	−6.1049
1789	LEU329	CD1	0.608	0.7114	−6.3194
1790	LEU329	CD2	2.55	−0.8685	−6.5509
1791	VAL330	N	−0.8373	−3.6758	−5.1815
1792	VAL330	CA	−1.6764	−4.2365	−6.2131
1793	VAL330	C	−3.022	−3.5331	−6.2756
1794	VAL330	O	−3.6295	−3.1734	−5.2701
1795	VAL330	CB	−1.869	−5.7582	−6.0539
1796	VAL330	CG1	−0.5085	−6.4754	−6.1294
1797	VAL330	CG2	−2.5427	−6.0744	−4.7044
1798	ILE331	N	−3.4871	−3.4207	−7.5793
1799	ILE331	CA	−4.8617	−3.0387	−7.8651
1800	ILE331	C	−5.6687	−4.3361	−7.8171
1801	ILE331	O	−5.5087	−5.2422	−8.6338
1802	ILE331	CB	−5.0263	−2.2262	−9.166
1803	ILE331	CG1	−6.3283	−1.3981	−9.1359
1804	ILE331	CG2	−4.9675	−3.1137	−10.4239
1805	ILE331	CD1	−7.5711	−2.308	−9.0729
1806	CYS332	N	−6.5239	−4.4163	−6.736
1807	CYS332	CA	−7.5339	−5.4568	−6.6303
1808	CYS332	C	−8.7833	−4.897	−7.3342
1809	CYS332	O	−9.3973	−3.9144	−6.9199
1810	CYS332	CB	−7.8548	−5.6814	−5.1396
1811	CYS332	SG	−6.3101	−6.0673	−4.2608
1812	LYS333	N	−9.0627	−5.5952	−8.5015
1813	LYS333	CA	−9.997	−5.1317	−9.5439
1814	LYS333	C	−11.4423	−5.0121	−9.0241
1815	LYS333	O	−11.8644	−5.6915	−8.0867
1816	LYS333	CB	−9.9678	−6.077	−10.7587
1817	LYS333	CG	−8.7072	−5.8054	−11.5994
1818	LYS333	CD	−8.75	−6.6779	−12.8672
1819	LYS333	CE	−7.6597	−6.2085	−13.8472
1820	LYS333	NZ	−7.6919	−7.0471	−15.0537
1821	LEU334	N	−12.2462	−4.1357	−9.7491
1822	LEU334	CA	−13.6898	−4.1252	−9.5387
1823	LEU334	C	−14.38	−5.1407	−10.4725
1824	LEU334	O	−13.8784	−5.5852	−11.5051
1825	LEU334	CB	−14.2489	−2.7054	−9.764
1826	LEU334	CG	−13.6956	−2.0947	−11.0682
1827	LEU334	CD1	−14.4444	−2.6689	−12.2862
1828	LEU334	CD2	−13.8664	−0.5646	−11.0349
1829	THR335	N	−15.6585	−5.5113	−10.0732
1830	THR335	CA	−16.2374	−6.7494	−10.6099
1831	THR335	C	−16.3599	−6.7425	−12.1466
1832	THR335	O	−16.076	−7.7687	−12.7772
1833	THR335	CB	−17.6125	−6.9852	−9.9543
1834	THR335	OG1	−17.482	−6.932	−8.5303
1835	THR335	CG2	−18.1496	−8.3726	−10.354
1836	PRO336	N	−16.8272	−5.6156	−12.7969
1837	PRO336	CA	−16.8699	−5.5685	−14.2567
1838	PRO336	C	−15.6067	−4.9394	−14.8805
1839	PRO336	O	−15.6777	−4.3094	−15.9435
1840	PRO336	CB	−18.0247	−4.5626	−14.4324
1841	PRO336	CG	−18.0624	−3.6981	−13.1509
1842	PRO336	CD	−17.2995	−4.468	−12.0539
1843	GLY337	N	−14.381	−5.2228	−14.289
1844	GLY337	CA	−13.1765	−4.5548	−14.8168
1845	GLY337	C	−12.9591	−5.1022	−16.2423
1846	GLY337	O	−12.8797	−4.3511	−17.2155
1847	LEU338	N	−12.8761	−6.4767	−16.2809
1848	LEU338	CA	−12.7619	−7.2844	−17.5043
1849	LEU338	C	−14.1606	−7.3168	−18.1368
1850	LEU338	O	−15.1946	−7.3322	−17.4563
1851	LEU338	CB	−12.3491	−8.7238	−17.1402
1852	LEU338	CG	−11.1472	−8.7229	−16.1758
1853	LEU338	CD1	−10.784	−10.1731	−15.8073
1854	LEU338	CD2	−9.9384	−8.0376	−16.8396
1855	LEU339	N	−14.2068	−7.4401	−19.5093
1856	LEU339	CA	−15.4662	−7.5266	−20.2566
1857	LEU339	C	−16.0264	−8.9621	−20.1133
1858	LEU339	O	−16.1698	−9.7391	−21.05
1859	LEU339	CB	−15.192	−7.1854	−21.7343
1860	LEU339	CG	−16.5204	−7.0397	−22.5014
1861	LEU339	CD1	−17.3118	−5.8347	−21.9584
1862	LEU339	CD2	−16.225	−6.8256	−23.9972
1863	LYS346	N	−13.6587	−13.5204	−9.0845
1864	LYS346	CA	−14.0349	−12.0861	−9.0598
1865	LYS346	C	−14.753	−11.721	−7.7248
1866	LYS346	O	−15.5288	−12.4919	−7.1579
1867	LYS346	CB	−14.8475	−11.6676	−10.2994
1868	LYS346	CG	−13.8718	−11.3517	−11.4487
1869	LYS346	CD	−14.6375	−11.2352	−12.7799
1870	LYS346	CE	−13.64	−10.9981	−13.9304
1871	LYS346	NZ	−12.7546	−12.1634	−14.0738
1872	TRP347	N	−14.4396	−10.4473	−7.2103
1873	TRP347	CA	−15.068	−9.9379	−5.9543
1874	TRP347	C	−16.5675	−9.6711	−6.1715
1875	TRP347	O	−17.1483	−9.8828	−7.2403
1876	TRP347	CB	−14.3837	−8.615	−5.5551
1877	TRP347	CG	−12.8879	−8.754	−5.5676
1878	TRP347	CD1	−12.0729	−8.5668	−6.6177
1879	TRP347	CD2	−12.022	−9.1279	−4.3949
1880	TRP347	NE1	−10.8288	−8.7702	−6.2605
1881	TRP347	CE2	−10.7582	−9.1	−4.9544
1882	TRP347	CE3	−12.2542	−9.4434	−3.0568
1883	TRP347	CZ2	−9.6178	−9.3836	−4.2033
1884	TRP347	CZ3	−11.1126	−9.7177	−2.2936
1885	TRP347	CH2	−9.8263	−9.6862	−2.8519
1886	THR348	N	−17.2311	−9.2235	−5.0483
1887	THR348	CA	−18.4309	−8.3907	−5.139
1888	THR348	C	−18.0481	−7.0196	−4.5307
1889	THR348	O	−17.0874	−6.9101	−3.7678
1890	THR348	CB	−19.6085	−9.0273	−4.3772
1891	THR348	OG1	−19.828	−10.3558	−4.8585
1892	THR348	CG2	−20.8846	−8.1903	−4.5859
1893	TYR349	N	−18.8199	−5.9544	−4.9677
1894	TYR349	CA	−18.4601	−4.5467	−4.7233
1895	TYR349	C	−19.692	−3.7778	−4.2256
1896	TYR349	O	−20.7812	−3.8985	−4.7855
1897	TYR349	CB	−17.8732	−3.949	−6.0187
1898	TYR349	CG	−17.9756	−2.4268	−6.045
1899	TYR349	CD1	−17.1307	−1.644	−5.2536
1900	TYR349	CD2	−18.9219	−1.8173	−6.8727
1901	TYR349	CE1	−17.1971	−0.2503	−5.3315
1902	TYR349	CE2	−18.9874	−0.4242	−6.9511
1903	TYR349	CZ	−18.1057	0.3584	−6.2015
1904	TYR349	OH	−18.1316	1.7425	−6.3246
1905	THR350	N	−19.4222	−2.8685	−3.2006
1906	THR350	CA	−20.5275	−2.1332	−2.5886
1907	THR350	C	−20.0198	−0.8124	−1.9808
1908	THR350	O	−19.9987	−0.6341	−0.7551
1909	THR350	CB	−21.212	−3.0091	−1.5168
1910	THR350	CG1	−21.5712	−4.2818	−2.0617
1911	THR350	CG2	−22.4841	−2.3087	−1.0023
1912	GLY354	N	−20.2194	9.6125	1.8171
1913	GLY354	CA	−20.5803	10.3257	3.0207
1914	GLY354	C	−19.8737	11.6442	3.2219
1915	GLY354	O	−18.9396	12.0026	2.4973
1916	ARG355	N	−20.3337	12.3595	4.2427
1917	ARG355	CA	−19.7915	13.6605	4.6068
1918	ARG355	C	−19.4816	13.7208	6.1058
1919	ARG355	O	−20.0338	12.963	6.9176
1920	ARG355	CB	−20.7626	14.7857	4.2022
1921	ARG355	CG	−21.0081	14.7491	2.6824
1922	ARG355	CD	−22.067	15.8029	2.3093
1923	ARG355	NE	−22.3912	15.6855	0.8987
1924	ARG355	CZ	−23.607	15.4387	0.5038
1925	ARG355	NH1	−23.8574	15.3402	−0.7679
1926	ARG355	NH2	−24.5773	15.2885	1.3572
1927	TRP356	N	−18.603	14.6557	6.4408
1928	TRP356	CA	−18.2124	14.9479	7.8044
1929	TRP356	C	−18.4159	16.4559	7.8518
1930	TRP356	O	−17.8369	17.1917	7.0365
1931	TRP356	CB	−16.7627	14.5146	8.0845
1932	TRP356	CG	−16.7726	13.0541	8.4347
1933	TRP356	CD1	−17.5396	12.4499	9.3559
1934	TRP356	CD2	−15.9162	11.9884	7.8061
1935	TRP356	NE1	−17.2804	11.1661	9.3854
1936	TRP356	CE2	−16.3253	10.8559	8.4849
1937	TRP356	CE3	−14.9384	11.9675	6.8125
1938	TRP356	CZ2	−15.7751	9.6027	8.2162
1939	TRP356	CZ3	−14.3821	10.7126	6.5358
1940	TRP356	CH2	−14.7886	9.5589	7.2227
1941	GLU357	N	−19.3203	16.9044	8.7193
1942	GLU357	CA	−19.606	18.3352	8.86
1943	GLU357	C	−19.4842	18.8277	10.2964
1944	GLU357	O	−19.9862	18.1955	11.2342
1945	GLU357	CB	−20.9875	18.6842	8.2767
1946	GLU357	CG	−20.9456	18.5321	6.7443
1947	GLU357	CD	−21.6577	17.277	6.3304
1948	GLU357	OE1	−22.7097	17.3928	5.6473
1949	GLU357	OE2	−21.1669	16.1715	6.6798
1950	LYS358	N	−18.8123	19.9638	10.4456
1951	LYS358	CA	−18.5784	20.596	11.7354
1952	LYS358	C	−19.8825	20.9007	12.4589
1953	LYS358	O	−20.7664	21.5584	11.8981
1954	LYS358	CB	−17.7647	21.8918	11.564
1955	LYS358	CG	−16.3456	21.5481	11.0763
1956	LYS358	CD	−15.5385	22.8472	10.9012
1957	LYS358	CE	−14.1201	22.5052	10.4097
1958	LYS358	NZ	−13.3483	23.7448	10.2417
1959	ARG359	N	−19.988	20.4295	13.7038
1960	ARG359	CA	−21.1878	20.6504	14.4995
1961	ARG359	C	−22.3413	19.688	14.2599
1962	ARG359	O	−23.4525	19.9153	14.7234
1963	ARG359	CB	−21.6403	22.1238	14.5393
1964	ARG359	CG	−20.4707	23.0073	15.0097
1965	ARG359	CD	−20.7762	24.4776	14.6735
1966	ARG359	NE	−19.57	25.2658	14.8468
1967	ARG359	CZ	−18.8966	25.6977	13.8199
1968	ARG359	NH1	−17.8225	26.4039	14.0137
1969	ARG359	NH2	−19.2781	25.435	12.6043
1970	SER360	N	−22.4316	19.0681	13.2168
1971	SER360	CA	−23.2043	17.8403	13.0915
1972	SER360	C	−22.3742	16.6311	12.6699
1973	SER360	O	−22.0803	15.7688	13.4894
1974	SER360	CB	−24.3557	18.0463	12.0893
1975	SER360	OG	−23.823	18.3466	10.7957
1976	THR361	N	−21.9695	15.9826	11.893
1977	THR361	CA	−21.462	14.6538	11.5505
1978	THR361	C	−19.9422	14.4741	11.6396
1979	THR361	O	−19.4294	13.4251	11.272
1980	THR361	CB	−21.9676	14.2128	10.1637
1981	THR361	OG1	−21.4778	15.1108	9.1653
1982	THR361	CG2	−23.5074	14.2041	10.1459
1983	ALA362	N	−19.2276	15.5002	12.0997
1984	ALA362	CA	−17.7763	15.4321	12.2395
1985	ALA362	C	−17.4367	14.8736	13.6251
1986	ALA362	O	−16.9456	15.5913	14.5084
1987	ALA362	CB	−17.2028	16.8495	12.0477
1988	GLY363	N	−17.7217	13.5846	13.8023
1989	GLY363	CA	−17.4981	12.9106	15.0732
1990	GLY363	C	−16.0507	12.7893	15.4886
1991	GLY363	O	−15.6953	13.0821	16.6239
1992	GLY364	N	−15.2455	12.2508	14.5819
1993	GLY364	CA	−13.8274	12.1014	14.8131
1994	GLY364	C	−13.2913	11.0766	15.7932
1995	GLY364	O	−13.3416	11.273	17.0034
1996	GLN365	N	−12.8872	10.5746	16.2587
1997	GLN365	CA	−12.2177	9.3095	16.5323
1998	GLN365	C	−12.7076	8.4801	17.7223
1999	GLN365	O	−13.2636	9.0032	18.6825
2000	GLN365	CB	−10.6929	9.4905	16.6352
2001	GLN365	CG	−10.134	9.9541	15.278
2002	GLN365	CD	−8.6864	10.3147	15.4393
2003	GLN365	OE1	−8.3248	11.4677	15.2723
2004	GLN365	NE2	−7.8401	9.3218	15.7669
2005	ARG366	N	−12.561	7.1283	17.6094
2006	ARG366	CA	−13.1445	6.4723	18.781
2007	ARG366	C	−12.8896	7.1163	20.1587
2008	ARG366	O	−13.7738	7.0958	21.0315
2009	ARG366	CB	−12.7742	4.9759	18.7668
2010	ARG366	CG	−11.2472	4.7882	18.8446
2011	ARG366	CD	−10.8011	4.8256	20.3175
2012	ARG366	NE	−9.3898	5.1547	20.3869
2013	ARG366	CZ	−8.5786	4.479	21.1478
2014	ARG366	NH1	−9.0032	3.4848	21.8713
2015	ARG366	NH2	−7.3211	4.8053	21.1864
2016	PHE373	N	−17.8554	11.3119	17.8872
2017	PHE373	CA	−17.7516	9.8813	17.6262
2018	PHE373	C	−19.0308	9.2625	17.0505
2019	PHE373	O	−19.2568	8.0759	17.2172
2020	PHE373	CB	−16.9451	9.0389	18.6357
2021	PHE373	CG	−16.324	7.8476	17.9125
2022	PHE373	CD1	−15.6237	8.036	16.7175
2023	PHE373	CD2	−16.4628	6.5632	18.4445
2024	PHE373	CE1	−15.099	6.9361	16.0341
2025	PHE373	CE2	−15.9354	5.4632	17.7631
2026	PHE373	CZ	−15.2611	5.6491	16.5535
2027	TRP374	N	−19.9905	8.9731	17.9207
2028	TRP374	CA	−21.2595	8.3699	17.5252
2029	TRP374	C	−22.0245	9.1861	16.4926
2030	TRP374	O	−23.0368	8.7253	15.9713
2031	TRP374	CB	−22.1189	8.2192	18.7948
2032	TRP374	CG	−22.3328	9.5651	19.4249
2033	TRP374	CD1	−21.6289	10.1063	20.4312
2034	TRP374	CD2	−23.3884	10.5621	19.0313
2035	TRP374	NE1	−22.1033	11.2936	20.7179
2036	TRP374	CE2	−23.1461	11.5948	19.9167
2037	TRP374	CE3	−24.4029	10.5999	18.0759
2038	TRP374	CZ2	−23.9217	12.7536	19.9176
2039	TRP374	CZ3	−25.1882	11.759	18.072
2040	TRP374	CH2	−24.9569	12.8067	18.9757
2041	LYS375	N	−21.5275	10.3856	16.1942
2042	LYS375	CA	−22.1675	11.2789	15.2353
2043	LYS375	C	−21.7208	11.1765	13.7615
2044	LYS375	O	−22.3195	11.8173	12.8934
2045	LYS375	CB	−21.7945	12.7149	15.6462
2046	LYS375	CG	−22.7664	13.2608	16.7055
2047	LYS375	CD	−22.5243	14.7753	16.843
2048	LYS375	CE	−23.3317	15.3345	18.0282
2049	LYS375	NZ	−22.4946	15.3293	19.2364
2050	ASN376	N	−20.6742	10.3986	13.4772
2051	ASN376	CA	−20.2178	10.2068	12.0918
2052	ASN376	C	−21.271	9.3361	11.4082
2053	ASN376	O	−22.0253	8.6268	12.0802
2054	ASN376	CB	−18.8283	9.544	12.0305
2055	ASN376	CG	−17.8494	10.3106	12.8735
2056	ASN376	OD1	−17.0336	11.0469	12.3438
2057	ASN376	ND2	−17.9206	10.135	14.2054
2058	PRO377	N	−21.3361	9.3671	10.0661
2059	PRO377	CA	−22.3291	8.5243	9.388
2060	PRO377	C	−22.057	7.0623	9.8079
2061	PRO377	O	−20.9748	6.7568	10.3328
2062	PRO377	CB	−21.9585	8.734	7.9066
2063	PRO377	CG	−20.8463	9.8082	7.8403
2064	PRO377	CD	−20.4446	10.1914	9.2799
2065	GLN378	N	−23.0285	6.1737	9.6047
2066	GLN378	CA	−22.871	4.7658	9.9888
2067	GLN378	C	−23.3197	3.8135	8.888
2068	GLN378	O	−24.2184	4.1278	8.1139
2069	GLN378	CB	−23.5706	4.4419	11.321
2070	GLN378	CG	−22.6472	4.8381	12.4894
2071	GLN378	CD	−23.0559	6.1428	13.1145
2072	GLN378	OE1	−24.035	6.7518	12.7144
2073	GLN378	NE2	−22.2862	6.5883	14.1234
2074	PHE379	N	−22.6924	2.6423	8.8249
2075	PHE379	CA	−23.0117	1.663	7.7884
2076	PHE379	C	−23.1047	0.251	8.3362
2077	PHE379	O	−22.7774	0.007	9.4898
2078	PHE379	CB	−21.964	1.751	6.6627
2079	PHE379	CG	−21.9246	3.1751	6.118
2080	PHE379	CD1	−20.9971	4.0886	6.6254
2081	PHE379	CD2	−22.8168	3.5682	5.1172
2082	PHE379	CE1	−20.9784	5.4026	6.1507
2083	PHE379	CE2	−22.7849	4.8767	4.6284
2084	PHE379	CZ	−21.8707	5.796	5.1499
2085	LEU380	N	−23.5633	−0.6734	7.5016
2086	LEU380	CA	−23.6919	−2.0542	7.9269
2087	LEU380	C	−23.0878	−3.0227	6.9376
2088	LEU380	O	−23.4873	−3.0586	5.7696
2089	LEU380	CB	−25.1596	−2.4145	8.2242
2090	LEU380	CG	−25.2241	−3.7603	8.9722
2091	LEU380	CD1	−24.5552	−3.6241	10.3532
2092	LEU380	CD2	−26.6953	−4.1756	9.1552
2093	LEU381	N	−22.1021	−3.782	7.3987
2094	LEU381	CA	−21.5005	−4.808	6.5733
2095	LEU381	C	−21.4522	−6.0765	7.4047
2096	LEU381	O	−21.1832	−6.0301	8.6037
2097	LEU381	CB	−20.0819	−4.4019	6.1319
2098	LEU381	CG	−20.1459	−3.1452	5.2433
2099	LEU381	CD1	−18.7168	−2.6707	4.9222
2100	LEU381	CD2	−20.8874	−3.4641	3.931
2101	LEU399	N	−11.2797	−5.8873	3.0256
2102	LEU399	CA	−11.0656	−5.2862	1.7318
2103	LEU399	C	−11.7767	−3.9423	1.8086
2104	LEU399	O	−13.0068	−3.8811	1.7749
2105	LEU399	CB	−11.6931	−6.1679	0.6362
2106	LEU399	CG	−10.8241	−6.0892	−0.6329
2107	LEU399	CD1	−9.5321	−6.9031	−0.4288
2108	LEU399	CD2	−11.6064	−6.6564	−1.8309
2109	VAL400	N	−11.7072	−3.2732	2.9534
2110	VAL400	CA	−12.3614	−1.9744	3.0903
2111	VAL400	C	−11.4375	−0.7941	2.7835
2112	VAL400	O	−10.3307	−0.6825	3.325
2113	VAL400	CB	−13.1588	−1.7882	4.3968
2114	VAL400	CG1	−13.8429	−0.4074	4.412
2115	VAL400	CG2	−14.2414	−2.8795	4.4962
2116	SER401	N	−11.9045	0.062	1.8749
2117	SER401	CA	−11.1554	1.2383	1.489
2118	SER401	C	−11.992	2.4792	1.7104
2119	SER401	O	−13.1049	2.5887	1.1756
2120	SER401	CB	−10.6612	1.1333	0.0332
2121	SER401	OG	−11.7598	0.8699	−0.8441
2122	LEU402	N	−11.4823	3.3815	2.5481
2123	LEU402	CA	−12.1437	4.6486	2.8585
2124	LEU402	C	−11.2989	5.7178	2.21
2125	LEU402	O	−10.1325	5.8508	2.551
2126	LEU402	CB	−12.0995	4.7962	4.3943
2127	LEU402	CG	−12.4102	6.2361	4.8492
2128	LEU402	CD1	−13.8656	6.5997	4.5007
2129	LEU402	CD2	−12.2011	6.3444	6.3717
2130	LEU403	N	−11.8967	6.5044	1.3209
2131	LEU403	CA	−11.1725	7.5474	0.6033
2132	LEU403	C	−11.7708	8.9329	0.8565
2133	LEU403	O	−12.9664	9.0696	1.0485
2134	LEU403	CB	−11.117	7.218	−0.9012
2135	LEU403	CG	−9.8589	7.8275	−1.5495
2136	LEU403	CD1	−9.5662	7.0971	−2.873
2137	LEU403	CD2	−10.0937	9.3191	−1.8493
2138	GLN404	N	−10.9207	9.9471	0.9415
2139	GLN404	CA	−11.3855	11.3051	1.1758
2140	GLN404	C	−11.3453	12.1121	−0.1269
2141	GLN404	O	−10.3735	12.0411	−0.8945
2142	GLN404	CB	−10.5112	12.0111	2.228
2143	GLN404	CG	−9.0183	11.8766	1.864
2144	GLN404	CD	−8.1937	12.9899	2.4457
2145	GLN404	OE1	−8.7227	13.9938	2.8945
2146	GLN404	NE2	−6.8613	12.8104	2.4463
2147	LYS405	N	−12.4222	12.8483	−0.3878
2148	LYS405	CA	−12.518	13.6554	−1.5974
2149	LYS405	C	−11.798	14.9875	−1.4256
2150	LYS405	O	−11.5074	15.4054	−0.2997
2151	LYS405	CB	−13.985	13.8319	−2.0291
2152	LYS405	CG	−14.4537	12.5454	−2.7338
2153	LYS405	CD	−15.9403	12.677	−3.112
2154	LYS405	CE	−16.417	11.381	−3.7939
2155	LYS405	NZ	−16.1861	11.4678	−5.243
2156	PRO406	N	−11.5033	15.637	−2.552
2157	PRO406	CA	−10.8137	16.9279	−2.5722
2158	PRO406	C	−11.7236	18.0933	−2.1786
2159	PRO406	O	−12.9466	18.0443	−2.3818
2160	PRO406	CB	−10.4066	17.0777	−4.0502
2161	PRO406	CG	−11.1618	15.9903	−4.8462
2162	PRO406	CD	−11.8425	15.0486	−3.833
2163	ARG407	N	−11.1004	19.1521	−1.6506
2164	ARG407	CA	−11.8132	20.3502	−1.2146
2165	ARG407	C	−10.9625	21.6281	−1.3154
2166	ARG407	O	−10.4741	22.1387	−0.3026
2167	ARG407	CB	−12.2986	20.1195	0.2285
2168	ARG407	CG	−13.75	20.6143	0.3605
2169	ARG407	CD	−14.5797	19.5414	1.0886
2170	ARG407	NE	−14.806	18.4195	0.1955
2171	ARG407	CZ	−14.1296	17.3139	0.3173
2172	ARG407	NH1	−13.2197	17.1765	1.2366
2173	ARG407	NH2	−14.3691	16.33	−0.4971
2174	HIS408	N	−11.0114	22.2129	−2.2375
2175	HIS408	CA	−9.8249	23.0234	−2.5578
2176	HIS408	C	−9.1699	23.7237	−1.3649
2177	HIS408	O	−8.391	24.3342	−1.9958
2178	HIS408	CB	−10.163	23.9089	−3.7771
2179	HIS408	CG	−9.6012	23.2388	−4.9957
2180	HIS408	ND1	−9.9112	22.0018	−5.3088
2181	HIS408	CD2	−8.7462	23.8055	−5.8684
2182	HIS408	CE1	−9.2706	21.7087	−6.3952
2183	HIS408	NE2	−8.5872	22.6943	−6.7667
2184	ARG409	N	−8.5536	23.516	−0.9303
2185	ARG409	CA	−7.3223	23.5699	−0.1401
2186	ARG409	C	−6.0983	22.8344	−0.7238
2187	ARG409	O	−4.9844	23.3773	−0.7235
2188	ARG409	CB	−7.6311	22.9841	1.2506
2189	ARG409	CG	−6.5102	23.363	2.2356
2190	ARG409	CD	−6.7839	22.7001	3.5974
2191	ARG409	NE	−5.682	22.9836	4.4984
2192	ARG409	CZ	−4.9048	22.0328	4.9303
2193	ARG409	NH1	−5.0867	20.7964	4.5691
2194	ARG409	NH2	−3.9285	22.3251	5.7372
2195	CYS410	N	−6.305	21.6096	−1.211
2196	CYS410	CA	−5.2291	20.8006	−1.7878
2197	CYS410	C	−4.7437	21.339	−3.1387
2198	CYS410	O	−5.4734	21.2892	−4.1333
2199	CYS410	CB	−5.7276	19.3545	−1.9733
2200	CYS410	SG	−6.1399	18.6764	−0.3384
2201	ARG411	N	−3.5047	21.8272	−3.1759
2202	ARG411	CA	−2.9217	22.3682	−4.408
2203	ARG411	C	−2.3706	21.2465	−5.2891
2204	ARG411	O	−2.334	21.365	−6.521
2205	ARG411	CB	−1.8105	23.366	−4.0351
2206	ARG411	CG	−2.2844	24.798	−4.3547
2207	ARG411	CD	−3.3099	25.296	−3.315
2208	ARG411	NE	−4.463	24.4149	−3.2693
2209	ARG411	CZ	−5.5445	24.6704	−3.946
2210	ARG411	NH1	−5.6424	25.7334	−4.689
2211	ARG411	NH2	−6.5441	23.8429	−3.8791
2212	LYS412	N	−2.0034	20.155	−4.6818
2213	LYS412	CA	−1.3477	18.9915	−5.2731
2214	LYS412	C	−2.2703	17.8226	−5.6321
2215	LYS412	O	−1.8677	16.889	−6.3285
2216	LYS412	CB	0.1347	18.7248	−4.9605
2217	LYS412	CG	0.9794	19.5622	−5.9387
2218	LYS412	CD	2.4766	19.3549	−5.6505
2219	LYS412	CE	3.3033	20.2142	−6.6246
2220	LYS412	NZ	4.6206	19.5956	−6.8299
2221	ILE419	N	−8.6232	9.5358	6.8454
2222	ILE419	CA	−8.7953	9.3093	8.2832
2223	ILE419	C	−8.8493	7.8239	8.5702
2224	ILE419	O	−9.1131	7.0324	7.6708
2225	ILE419	CB	−10.0831	9.9968	8.7935
2226	ILE419	CG1	−11.3607	9.3761	8.1849
2227	ILE419	CG2	−10.0433	11.5167	8.5429
2228	ILE419	CD1	−11.4138	9.5647	6.6559
2229	GLY420	N	−9.4897	7.282	9.2986
2230	GLY420	CA	−9.5657	5.8953	9.7181
2231	GLY420	C	−11.0225	5.5333	9.9443
2232	GLY420	O	−11.8765	6.3862	9.9751
2233	PHE421	N	−11.2936	4.2932	10.0838
2234	PHE421	CA	−12.6684	3.8445	10.3389
2235	PHE421	C	−12.6373	2.6376	11.2691
2236	PHE421	O	−11.9166	1.7455	11.091
2237	PHE421	CB	−13.5136	3.6209	9.0676
2238	PHE421	CG	−12.8762	2.5907	8.1403
2239	PHE421	CD1	−12.9824	1.2265	8.4257
2240	PHE421	CD2	−12.1885	3.014	6.9999
2241	PHE421	CE1	−12.3816	0.2863	7.5846
2242	PHE421	CE2	−11.6042	2.0735	6.1476
2243	PHE421	CZ	−11.6955	0.7108	6.444
2244	TYR422	N	−13.3419	2.6273	12.2494
2245	TYR422	CA	−13.4014	1.4742	13.1552
2246	TYR422	C	−14.673	0.7253	12.8963
2247	TYR422	O	−15.7005	1.3588	12.6603
2248	TYR422	CB	−13.4225	1.896	14.6386
2249	TYR422	CG	−12.5988	3.1533	14.9
2250	TYR422	CD1	−13.133	4.4116	14.6103
2251	TYR422	CD2	−11.3139	3.0477	15.4382
2252	TYR422	CE1	−12.3958	5.5657	14.8872
2253	TYR422	CE2	−10.5773	4.2018	15.7175
2254	TYR422	CZ	−11.1209	5.4606	15.449
2255	TYR422	OH	−10.3932	6.6081	15.7404
2256	LEU423	N	−14.6042	−0.5562	12.874
2257	LEU423	CA	−15.8179	−1.3236	12.6719
2258	LEU423	C	−16.1001	−2.2128	13.8968
2259	LEU423	O	−15.2057	−2.8951	14.3503
2260	LEU423	CB	−15.6594	−2.222	11.4302
2261	LEU423	CG	−17.0006	−2.3612	10.6831
2262	LEU423	CD1	−16.8092	−3.278	9.4606
2263	LEU423	CD2	−18.0756	−2.9663	11.6055
2264	TYR424	N	−17.3096	−2.1397	14.4203
2265	TYR424	CA	−17.6813	−3.0385	15.5276
2266	TYR424	C	−18.874	−3.8529	15.0155
2267	TYR424	O	−19.8515	−3.2687	14.737
2268	TYR424	CB	−18.0616	−2.1939	16.7602
2269	TYR424	CG	−17.0839	−1.0307	16.913
2270	TYR424	CD1	−17.3766	0.2036	16.3266
2271	TYR424	CD2	−15.8977	−1.1963	17.6328
2272	TYR424	CE1	−16.4886	1.2738	16.4637
2273	TYR424	CE2	−15.0125	−0.1243	17.7756
2274	TYR424	CZ	−15.3148	1.115	17.2048
2275	TYR424	OH	−14.4498	2.1898	17.3759
2276	SER18	N	−29.219	12.2461	−11.1118
2277	SER18	CA	−28.5696	11.9899	−12.3855
2278	SER18	C	−27.2585	11.2838	−12.1555
2279	SER18	O	−27.0026	10.8074	−11.0608
2280	SER18	CB	−28.3112	13.3475	−13.0696
2281	SER18	OG	−27.4242	14.143	−12.2771
2282	ARG19	N	−26.4112	11.2244	−13.2015
2283	ARG19	CA	−25.1037	10.6172	−13.0182
2284	ARG19	C	−24.0502	11.6653	−12.7938
2285	ARG19	O	−24.3376	12.8489	−12.8706
2286	ARG19	CB	−24.7295	9.8228	−14.2824
2287	ARG19	CG	−25.4163	8.4463	−14.2455
2288	ARG19	CD	−24.9216	7.6042	−15.4352
2289	ARG19	NE	−25.384	6.2371	−15.2815
2290	ARG19	CZ	−26.1887	5.697	−16.1504
2291	ARG19	NH1	−26.5806	4.4696	−15.9789
2292	ARG19	NH2	−26.6077	6.3625	−17.1867
2293	ARG20	N	−22.8103	11.2111	−12.5208
2294	ARG20	CA	−21.7107	12.1564	−12.4228
2295	ARG20	C	−21.2983	12.5578	−13.8122
2296	ARG20	O	−20.9314	13.6994	−14.038
2297	ARG20	CB	−20.5429	11.5144	−11.6414
2298	ARG20	CG	−19.6366	10.6438	−12.5364
2299	ARG20	CD	−20.4078	9.4148	−13.0514
2300	ARG20	NE	−20.1989	8.3033	−12.1436
2301	ARG20	CZ	−19.6059	7.215	−12.5401
2302	ARG20	NH1	−19.4299	6.2426	−11.6958
2303	ARG20	NH2	−19.1839	7.0845	−13.764
2304	ALA21	N	−21.3829	11.5916	−14.7481
2305	ALA21	CA	−21.0788	11.8988	−16.1328
2306	ALA21	C	−22.0716	12.8888	−16.6703
2307	ALA21	O	−21.7518	13.5873	−17.6179
2308	ALA21	CB	−21.1798	10.5933	−16.9424
2309	SER22	N	−23.2695	12.9551	−16.0522
2310	SER22	CA	−24.2611	13.9176	−16.5033
2311	SER22	C	−23.6822	15.3066	−16.5316
2312	SER22	O	−23.6389	15.8754	−17.6094
2313	SER22	CB	−25.5205	13.8526	−15.6185
2314	SER22	OG	−26.0658	12.5317	−15.6598
2315	PRO23	N	−23.2183	15.8635	−15.3902
2316	PRO23	CA	−22.571	17.1607	−15.4163
2317	PRO23	C	−21.2635	17.0693	−16.1522
2318	PRO23	O	−20.8147	18.0635	−16.6991
2319	PRO23	CB	−22.2752	17.4063	−13.9234
2320	PRO23	CG	−22.6119	16.1125	−13.1508
2321	PRO23	CD	−23.3322	15.1657	−14.1283
2322	GLN24	N	−20.6603	15.8634	−16.1775
2323	GLN24	CA	−19.433	15.6899	−16.9365
2324	GLN24	C	−19.6586	16.0109	−18.3889
2325	GLN24	O	−18.7296	16.44	−19.0534
2326	GLN24	CB	−18.9822	14.2236	−16.7999
2327	GLN24	CG	−17.8675	14.121	−15.7416
2328	GLN24	CD	−18.1193	12.964	−14.8176
2329	GLN24	OE1	−18.1804	13.1523	−13.6139
2330	GLN24	NE2	−18.2685	11.7477	−15.3715
2331	GLN25	N	−20.9014	15.8186	−18.8769
2332	GLN25	CA	−21.1908	16.151	−20.262
2333	GLN25	C	−20.9543	17.6163	−20.5161
2334	GLN25	O	−20.1181	17.9192	−21.352
2335	GLN25	CB	−22.6443	15.7729	−20.6035
2336	GLN25	CG	−22.7804	14.244	−20.7335
2337	GLN25	CD	−23.9393	13.7462	−19.9172
2338	GLN25	OE1	−23.8042	12.7627	−19.2084
2339	GLN25	NE2	−25.0992	14.4217	−20.0082
2340	PRO26	N	−21.6509	18.5378	−19.8106
2341	PRO26	CA	−21.3793	19.9524	−19.9756
2342	PRO26	C	−19.9355	20.2396	−19.6769
2343	PRO26	O	−19.3935	21.1953	−20.2082
2344	PRO26	CB	−22.2515	20.5874	−18.8742
2345	PRO26	CG	−23.0722	19.4644	−18.2035
2346	PRO26	CD	−22.6617	18.1328	−18.8588
2347	GLN27	N	−19.3128	19.3876	−18.838
2348	GLN27	CA	−17.8868	19.5279	−18.6092
2349	GLN27	C	−17.1497	18.9872	−19.8049
2350	GLN27	O	−16.4509	17.9906	−19.7081
2351	GLN27	CB	−17.5082	18.7621	−17.3247
2352	GLN27	CG	−18.2592	19.3411	−16.11
2353	GLN27	CD	−17.7275	20.7009	−15.7592
2354	GLN27	OE1	−17.1188	20.8629	−14.7145
2355	GLN27	NE2	−17.9586	21.6981	−16.6322
2356	TYR84	N	−1.9756	4.3397	−31.0125
2357	TYR84	CA	−1.4483	3.2203	−30.2523
2358	TYR84	C	−1.5153	2.0149	−31.1458
2359	TYR84	O	−2.2802	1.0942	−30.904
2360	TYR84	CB	−2.2419	3.0353	−28.9389
2361	TYR84	CG	−1.6037	2.0109	−27.9974
2362	TYR84	CD1	−1.8801	2.0732	−26.6288
2363	TYR84	CD2	−0.7562	1.0093	−28.4802
2364	TYR84	CE1	−1.3609	1.1086	−25.7608
2365	TYR84	CE2	−0.2246	0.0498	−27.6157
2366	TYR84	CZ	−0.5326	0.0971	−26.2544
2367	TYR84	OH	−0.0121	−0.8597	−25.3925
2368	PHE85	N	−0.6833	2.0379	−32.2039
2369	PHE85	CA	−0.6605	0.8992	−33.1028
2370	PHE85	C	−0.1316	−0.33	−32.4212
2371	PHE85	O	0.8114	−0.2439	−31.6511
2372	PHE85	CB	0.0502	1.1935	−34.4406
2373	PHE85	CG	1.4461	1.7991	−34.2859
2374	PHE85	CD1	2.2748	1.8618	−35.409
2375	PHE85	CD2	1.908	2.2915	−33.0616
2376	PHE85	CE1	3.5469	2.4316	−35.3173
2377	PHE85	CE2	3.1787	2.8638	−32.9666
2378	PHE85	CZ	4.0037	2.923	−34.0921
2379	ALA86	N	−0.7817	−1.4764	−32.7076
2380	ALA86	CA	−0.3826	−2.7278	−32.0819
2381	ALA86	C	−0.6149	−2.7582	−30.596
2382	ALA86	O	−0.7942	−1.7247	−29.9715
2383	ALA86	CB	1.0752	−3.1029	−32.4117
2384	LYS87	N	−0.6034	−3.9801	−30.027
2385	LYS87	CA	−0.671	−4.0869	−28.5799
2386	LYS87	C	0.6572	−3.6505	−28.0358
2387	LYS87	O	0.704	−2.8939	−27.0799
2388	LYS87	CB	−0.8607	−5.5636	−28.1861
2389	LYS87	CG	−2.293	−6.028	−28.5039
2390	LYS87	CD	−2.4255	−7.5123	−28.1168
2391	LYS87	CE	−3.8179	−8.0329	−28.5167
2392	LYS87	NZ	−3.8456	−9.4955	−28.374
2393	ALA88	N	1.7382	−4.1405	−28.6778
2394	ALA88	CA	3.0742	−3.7712	−28.2453
2395	ALA88	C	3.3688	−4.3514	−26.8917
2396	ALA88	O	3.2386	−3.6715	−25.8867
2397	ALA88	CB	3.2728	−2.2438	−28.2909
2398	LYS89	N	3.7874	−5.6324	−26.884
2399	LYS89	CA	4.1863	−6.2498	−25.6303
2400	LYS89	C	5.3313	−5.4685	−25.0515
2401	LYS89	O	5.3175	−5.1573	−23.8715
2402	LYS89	CB	4.6519	−7.6849	−25.942
2403	LYS89	CG	5.1193	−8.3944	−24.6573
2404	LYS89	CD	5.6894	−9.7741	−25.0329
2405	LYS89	CE	6.2991	−10.443	−23.7881
2406	LYS89	NZ	7.6382	−10.9439	−24.1273
2407	ARG90	N	6.3187	−5.1388	−25.9065
2408	ARG90	CA	7.4058	−4.306	−25.43
2409	ARG90	C	6.9689	−2.8753	−25.2833
2410	ARG90	O	5.8984	−2.4983	−25.7329
2411	ARG90	CB	8.6259	−4.4176	−26.3627
2412	ARG90	CG	9.4794	−5.6297	−25.944
2413	ARG90	CD	10.1419	−5.3493	−24.582
2414	ARG90	NE	10.8092	−6.5457	−24.1021
2415	ARG90	CZ	12.0871	−6.5553	−23.854
2416	ARG90	NH1	12.8194	−5.4944	−24.0281
2417	ARG90	NH2	12.6424	−7.6489	−23.4233
2418	LEU91	N	7.8313	−2.0836	−24.6216
2419	LEU91	CA	7.4596	−0.7216	−24.2832
2420	LEU91	C	8.727	0.0817	−24.2932
2421	LEU91	O	9.7323	−0.4426	−23.8379
2422	LEU91	CB	7.0082	−0.8199	−22.8106
2423	LEU91	CG	6.2327	0.4149	−22.3125
2424	LEU91	CD1	5.823	0.1552	−20.851
2425	LEU91	CD2	7.1128	1.6773	−22.353
2426	HIS113	N	−6.4353	−10.6403	−22.9262
2427	HIS113	CA	−7.4859	−10.5959	−21.9252
2428	HIS113	C	−7.1733	−9.4431	−21.0038
2429	HIS113	O	−6.1033	−8.8622	−21.1001
2430	HIS113	CB	−7.5034	−11.9014	−21.1055
2431	HIS113	CG	−8.8663	−12.5314	−21.1377
2432	HIS113	ND1	−9.3863	−13.1002	−20.0746
2433	HIS113	CD2	−9.6792	−12.5864	−22.2107
2434	HIS113	CE1	−10.5551	−13.5481	−20.4069
2435	HIS113	NE2	−10.784	−13.2858	−21.6127
2436	GLN114	N	−8.1139	−9.1025	−20.0995
2437	GLN114	CA	−7.8701	−8.0172	−19.1592
2438	GLN114	C	−7.7198	−6.6951	−19.8615
2439	GLN114	O	−6.6331	−6.1411	−19.9172
2440	GLN114	CB	−6.6818	−8.3332	−18.2286
2441	GLN114	CG	−7.1246	−9.3496	−17.16
2442	GLN114	CD	−5.9534	−9.736	−16.3046
2443	GLN114	OE1	−5.5315	−10.8802	−16.3337
2444	GLN114	NE2	−5.4155	−8.779	−15.5274
2445	ASP115	N	−8.8445	−6.1879	−20.4018
2446	ASP115	CA	−8.7767	−4.9376	−21.1382
2447	ASP115	C	−9.0522	−3.7711	−20.2284
2448	ASP115	O	−10.0728	−3.1112	−20.3435
2449	ASP115	CB	−9.7649	−5.0067	−22.3186
2450	ASP115	CG	−9.3264	−6.0903	−23.2611
2451	ASP115	OD1	−9.6038	−7.2836	−22.964
2452	ASP115	OD2	−8.7116	−5.7495	−24.3065
2453	THR128	N	−5.2098	9.6232	−37.965
2454	THR128	CA	−5.5784	9.5422	−39.3665
2455	THR128	C	−6.9869	10.0144	−39.5951
2456	THR128	O	−7.1718	11.0374	−40.2344
2457	THR128	CB	−5.3515	8.1277	−39.9358
2458	THR128	OG1	−6.1329	7.164	−39.2253
2459	THR128	CG2	−3.86	7.7591	−39.8286
2460	GLU129	N	−7.9852	9.2654	−39.0864
2461	GLU129	CA	−9.3583	9.6237	−39.4022
2462	GLU129	C	−9.9398	10.5726	−38.3948
2463	GLU129	O	−9.6855	10.4263	−37.2097
2464	GLU129	CB	−10.2665	8.3833	−39.5128
2465	GLU129	CG	−9.5043	7.2117	−40.1622
2466	GLU129	CD	−9.12	6.2042	−39.116
2467	GLU129	OE1	−9.3115	4.9868	−39.3768
2468	GLU129	OE2	−8.6361	6.6199	−38.0287
2469	LYS130	N	−10.7255	11.5496	−38.8967
2470	LYS130	CA	−11.3809	12.5048	−38.0141
2471	LYS130	C	−10.4409	13.0512	−36.976
2472	LYS130	O	−10.7529	13.0495	−35.7953
2473	LYS130	CB	−12.6455	11.888	−37.3856
2474	LYS130	CG	−13.8196	12.0229	−38.3713
2475	LYS130	CD	−15.1017	11.4827	−37.7127
2476	LYS130	CE	−16.286	11.6603	−38.6803
2477	LYS130	NZ	−17.5138	11.1586	−38.0469
2478	VAL156	N	7.1495	20.8156	−25.2479
2479	VAL156	CA	8.4489	20.3852	−25.72
2480	VAL156	C	9.3571	21.5798	−25.7576
2481	VAL156	O	9.3263	22.352	−26.7025
2482	VAL156	CB	8.3542	19.5969	−27.0437
2483	VAL156	CG1	7.3527	18.4379	−26.8884
2484	VAL156	CG2	7.9085	20.4856	−28.2191
2485	ASN157	N	10.171	21.7158	−24.6912
2486	ASN157	CA	11.0863	22.8404	−24.6216
2487	ASN157	C	11.9997	22.83	−25.8116
2488	ASN157	O	12.9363	22.049	−25.8658
2489	ASN157	CB	11.8895	22.7676	−23.3092
2490	ASN157	CG	12.0553	24.1436	−22.7314
2491	ASN157	OD1	11.62	24.3951	−21.62
2492	ASN157	ND2	12.6951	25.0539	−23.4855
2493	GLU158	N	11.693	23.7209	−26.7747
2494	GLU158	CA	12.4778	23.7633	−27.9961
2495	GLU158	C	12.3657	22.4554	−28.7313
2496	GLU158	O	13.3724	21.8341	−29.0331
2497	GLU158	CB	13.9391	24.1693	−27.7137
2498	GLU158	CG	13.9672	25.4183	−26.8115
2499	GLU158	CD	14.8271	25.1819	−25.6025
2500	GLU158	OE1	14.7949	24.0494	−25.0491
2501	GLU158	OE2	15.5511	26.1332	−25.2057
2502	ALA159	N	11.111	22.0446	−29.0137
2503	ALA159	CA	10.8855	20.7997	−29.7342
2504	ALA159	C	11.0809	19.5888	−28.8664
2505	ALA159	O	10.2895	18.6639	−28.9558
2506	ALA159	CB	11.7364	20.701	−31.0156
2507	GLY160	N	12.1317	19.5892	−28.0226
2508	GLY160	CA	12.365	18.4278	−27.1826
2509	GLY160	C	11.2963	18.3167	−26.1341
2510	GLY160	O	11.1203	19.2402	−25.3565
2511	GLN161	N	10.5875	17.1693	−26.1209
2512	GLN161	CA	9.5227	16.9887	−25.1454
2513	GLN161	C	10.032	17.0777	−23.7338
2514	GLN161	O	11.2329	17.0787	−23.5131
2515	GLN161	CB	8.8426	15.6214	−25.3495
2516	GLN161	CG	8.2536	15.5302	−26.7698
2517	GLN161	CD	7.5633	14.2099	−26.952
2518	GLN161	OE1	6.3575	14.1746	−27.1342
2519	GLN161	NE2	8.3294	13.105	−26.907
2520	GLN196	N	1.0418	9.8707	−5.495
2521	GLN196	CA	1.5364	8.5086	−5.399
2522	GLN196	C	1.1702	7.6878	−6.6023
2523	GLN196	O	0.4086	8.1229	−7.4515
2524	GLN196	CB	0.8757	7.8228	−4.1892
2525	GLN196	CG	1.6729	8.1168	−2.9065
2526	GLN196	CD	0.849	7.7169	−1.7172
2527	GLN196	OE1	0.5058	8.5584	−0.9035
2528	GLN196	NE2	0.5223	6.4166	−1.6058
2529	VAL197	N	1.7343	6.4652	−6.6453
2530	VAL197	CA	1.3872	5.552	−7.7192
2531	VAL197	C	0.0529	4.9215	−7.4393
2532	VAL197	O	−0.7245	4.7517	−8.3627
2533	VAL197	CB	2.4636	4.454	−7.8415
2534	VAL197	CG1	3.7945	5.0789	−8.2996
2535	VAL197	CG2	2.6678	3.7271	−6.4975
2536	SER198	N	−0.2095	4.5813	−6.1611
2537	SER198	CA	−1.4755	3.9503	−5.8222
2538	SER198	C	−2.6401	4.8349	−6.17
2539	SER198	O	−3.6232	4.3419	−6.6982
2540	SER198	CB	−1.4875	3.6546	−4.31
2541	SER198	OG	−1.2465	4.856	−3.5706
2542	THR209	N	−6.394	−0.2325	−4.529
2543	THR209	CA	−4.9737	−0.4368	−4.745
2544	THR209	C	−4.3568	−0.8171	−3.4284
2545	THR209	O	−3.5231	−0.1004	−2.8972
2546	THR209	CB	−4.3323	0.7936	−5.4215
2547	THR209	OG1	−4.9557	1.007	−6.6915
2548	THR209	CG2	−2.8285	0.5495	−5.6524
2549	MET210	N	−4.7905	−1.9815	−2.9044
2550	MET210	CA	−4.2352	−2.4464	−1.6446
2551	MET210	C	−2.7662	−2.7233	−1.7981
2552	MET210	O	−2.3122	−2.9945	−2.8983
2553	MET210	CB	−4.9699	−3.7096	−1.1552
2554	MET210	CG	−6.4845	−3.437	−1.0776
2555	MET210	SD	−7.1137	−4.1282	0.4811
2556	MET210	CE	−8.7771	−3.3982	0.4349
2557	THR211	N	−2.0041	−2.6325	−0.692
2558	THR211	CA	−0.5678	−2.7725	−0.8487
2559	THR211	C	0.0522	−3.5941	0.2431
2560	THR211	O	−0.4589	−3.6484	1.3501
2561	THR211	CB	0.1235	−1.3971	−0.9424
2562	THR211	OG1	−0.0089	−0.6997	0.2994
2563	THR211	CG2	−0.5138	−0.5555	−2.064
2564	ILE212	N	1.1822	−4.2356	−0.1071
2565	ILE212	CA	1.9143	−5.0079	0.8786
2566	ILE212	C	3.3518	−4.9831	0.448
2567	ILE212	O	3.6862	−5.6192	−0.538
2568	ILE212	CB	1.419	−6.4711	0.8968
2569	ILE212	CG1	−0.0541	−6.5406	1.3449
2570	ILE212	CG2	2.2939	−7.3081	1.8521
2571	ILE212	CD1	−0.5911	−7.9762	1.1884
2572	ASN213	N	4.2062	−4.2553	1.1941
2573	ASN213	CA	5.6213	−4.2737	0.8628
2574	ASN213	C	6.1141	−5.6779	1.0676
2575	ASN213	O	6.4063	−6.0634	2.1877
2576	ASN213	CB	6.382	−3.3112	1.7973
2577	ASN213	CG	6.0491	−1.8733	1.5174
2578	ASN213	OD1	4.928	−1.5529	1.1578
2579	ASN213	ND2	7.0439	−0.9828	1.6817
2580	LEU214	N	6.1863	−6.4488	−0.0348
2581	LEU214	CA	6.5039	−7.8588	0.1037
2582	LEU214	C	7.8453	−8.0892	0.7358
2583	LEU214	O	8.0269	−9.1297	1.3458
2584	LEU214	CB	6.4267	−8.5505	−1.2667
2585	LEU214	CG	5.1616	−9.4224	−1.339
2586	LEU214	CD1	3.9038	−8.532	−1.3405
2587	LEU214	CD2	5.2104	−10.2479	−2.6365
2588	ALA215	N	8.7748	−7.1182	0.6196
2589	ALA215	CA	10.0464	−7.2635	1.3105
2590	ALA215	C	9.7769	−7.5799	2.7559
2591	ALA215	O	10.3279	−8.5342	3.2802
2592	ALA215	CB	10.8273	−5.9415	1.1907
2593	GLU216	N	8.8848	−6.7868	3.3804
2594	GLU216	CA	8.3903	−7.1675	4.6907
2595	GLU216	C	6.9476	−7.5615	4.5145
2596	GLU216	O	6.0518	−6.9214	5.0423
2597	GLU216	CB	8.5538	−5.9747	5.6525
2598	GLU216	CG	8.2854	−6.4355	7.0971
2599	GLU216	CD	7.3825	−5.4464	7.7742
2600	GLU216	OE1	6.1396	−5.5673	7.6064
2601	GLU216	OE2	7.9134	−4.5541	8.4878
2602	ALA217	N	6.7323	−8.6343	3.7284
2603	ALA217	CA	5.374	−8.9999	3.3618
2604	ALA217	C	4.6093	−9.6378	4.4887
2605	ALA217	O	5.1317	−9.8014	5.5798
2606	ALA217	CB	5.4467	−9.9884	2.1838
2607	HIS218	N	3.3448	−10.0049	4.1972
2608	HIS218	CA	2.5306	−10.6643	5.2054
2609	HIS218	C	3.0285	−12.0533	5.4931
2610	HIS218	O	3.9116	−12.551	4.8126
2611	HIS218	CB	1.1008	−10.8309	4.6532
2612	HIS218	CG	0.3339	−9.543	4.6987
2613	HIS218	ND1	0.8034	−8.4292	4.186
2614	HIS218	CD2	−0.8854	−9.3962	5.2515
2615	HIS218	CE1	−0.0876	−7.51	4.3834
2616	HIS218	NE2	−1.0761	−7.9962	4.9856
2617	GLY219	N	2.427	−12.6904	6.5166
2618	GLY219	CA	2.6988	−14.1014	6.7225
2619	GLY219	C	1.8493	−14.8442	5.733
2620	GLY219	O	2.3558	−15.6498	4.9688
2621	GLN238	N	8.1324	−5.9044	−5.4568
2622	GLN238	CA	9.4731	−5.6833	−4.9464
2623	GLN238	C	10.3999	−5.3856	−6.106
2624	GLN238	O	9.9466	−5.2424	−7.2302
2625	GLN238	CB	9.5334	−4.5688	−3.8746
2626	GLN238	CG	9.065	−5.0445	−2.4777
2627	GLN238	CD	8.29	−6.3276	−2.4983
2628	GLN238	OE1	8.8192	−7.361	−2.1257
2629	GLN238	NE2	7.0217	−6.2661	−2.9362
2630	THR239	N	11.721	−5.3061	−5.8507
2631	THR239	CA	12.6638	−5.2142	−6.9547
2632	THR239	C	13.5003	−6.4648	−6.9995
2633	THR239	O	14.0679	−6.8369	−5.9846
2634	THR239	CB	13.5534	−3.9622	−6.8255
2635	THR239	OG1	14.3086	−4.0071	−5.6115
2636	THR239	CG2	12.6716	−2.7002	−6.8315
2637	HIS240	N	13.555	−7.1173	−8.1813
2638	HIS240	CA	14.163	−8.4408	−8.284
2639	HIS240	C	15.4515	−8.5949	−7.5159
2640	HIS240	O	16.24	−7.6636	−7.4811
2641	HIS240	CB	13.1137	−9.4858	−7.8556
2642	HIS240	CG	12.9509	−10.5509	−8.9012
2643	HIS240	ND1	12.9088	−10.2764	−10.1849
2644	HIS240	CD2	12.8277	−11.8668	−8.6425
2645	HIS240	CE1	12.7565	−11.3982	−10.8156
2646	HIS240	NE2	12.7006	−12.3442	−9.9931
2647	SER241	N	15.6651	−9.7692	−6.8839
2648	SER241	CA	16.8516	−9.9218	−6.0565
2649	SER241	C	17.0666	−11.3541	−5.6464
2650	SER241	O	16.3248	−12.2304	−6.0604
2651	SER241	CB	16.6801	−9.0557	−4.7945
2652	SER241	OG	17.9373	−8.8969	−4.1322
2653	GLY242	N	18.1042	−11.5854	−4.8158
2654	GLY242	CA	18.3714	−12.9407	−4.3647
2655	GLY242	C	17.6696	−13.2358	−3.0682
2656	GLY242	O	16.7701	−12.5049	−2.686
2657	LYS243	N	18.1019	−14.327	−2.4015
2658	LYS243	CA	17.4273	−14.7712	−1.1904
2659	LYS243	C	17.4213	−13.7439	−0.0914
2660	LYS243	O	18.3491	−12.9592	0.0143
2661	LYS243	CB	18.0192	−16.1181	−0.7284
2662	LYS243	CG	17.3879	−16.5818	0.5981
2663	LYS243	CD	18.3077	−16.1674	1.7608
2664	LYS243	CE	17.5847	−16.3813	3.1027
2665	LYS243	NZ	18.4874	−16.0221	4.2054
2666	ILE244	N	16.3402	−13.7602	0.7163
2667	ILE244	CA	16.1453	−12.7149	1.7103
2668	ILE244	C	17.1361	−12.8139	2.8396
2669	ILE244	O	18.1547	−13.4736	2.7099
2670	ILE244	CB	14.7014	−12.7747	2.2575
2671	ILE244	CG1	13.6943	−13.0402	1.1243
2672	ILE244	CG2	14.3331	−11.4383	2.9311
2673	ILE244	CD1	12.3433	−13.4901	1.7111
2674	LEU245	N	16.8305	−12.1362	3.9658
2675	LEU245	CA	17.7767	−12.0954	5.0684
2676	LEU245	C	19.0752	−11.5053	4.5937
2677	LEU245	O	20.1429	−11.8815	5.0504
2678	LEU245	CB	17.9564	−13.4847	5.7113
2679	LEU245	CG	17.7838	−13.383	7.2384
2680	LEU245	CD1	17.8183	−14.7962	7.8482
2681	LEU245	CD2	18.9207	−12.5377	7.8437
2682	GLU246	N	18.9479	−10.5707	3.6328
2683	GLU246	CA	20.1286	−10.0014	3.0152
2684	GLU246	C	19.7591	−8.6028	2.6115
2685	GLU246	O	19.5634	−8.3168	1.4408
2686	GLU246	CB	20.5101	−10.8743	1.8014
2687	GLU246	CG	21.0342	−12.237	2.2939
2688	GLU246	CD	21.314	−13.1542	1.1397
2689	GLU246	OE1	20.5724	−14.1619	0.9912
2690	GLU246	OE2	22.2844	−12.8773	0.3853
2691	ASN247	N	19.6554	−7.7232	3.6269
2692	ASN247	CA	19.1986	−6.3703	3.359
2693	ASN247	C	20.1145	−5.6244	2.4312
2694	ASN247	O	21.2558	−6.0137	2.2406
2695	ASN247	CB	19.0449	−5.5882	4.6782
2696	ASN247	CG	17.9811	−6.1721	5.5668
2697	ASN247	OD1	17.3251	−7.1387	5.2131
2698	ASN247	ND2	17.8028	−5.571	6.7565
2699	GLY248	N	19.5829	−4.5368	1.8424
2700	GLY248	CA	20.3934	−3.7772	0.9097
2701	GLY248	C	19.509	−3.0448	−0.0569
2702	GLY248	O	18.429	−3.5146	−0.3793
2703	LEU249	N	19.9903	−1.8762	−0.523
2704	LEU249	CA	19.2109	−1.1168	−1.4862
2705	LEU249	C	19.2242	−1.7899	−2.8314
2706	LEU249	O	19.6245	−2.9387	−2.9378
2707	LEU249	CB	19.8299	0.2894	−1.6043
2708	LEU249	CG	19.594	1.0806	−0.3035
2709	LEU249	CD1	20.3173	2.4368	−0.3936
2710	LEU249	CD2	18.0856	1.3192	−0.1002
2711	ARG261	N	3.7075	−19.562	−7.3982
2712	ARG261	CA	3.3851	−20.9648	−7.232
2713	ARG261	C	4.5578	−21.6483	−6.5934
2714	ARG261	O	5.6844	−21.4482	−7.02
2715	ARG261	CB	3.0626	−21.6256	−8.5898
2716	ARG261	CG	4.1152	−21.2544	−9.6556
2717	ARG261	CD	3.7549	−21.9296	−10.9934
2718	ARG261	NE	4.2205	−21.1202	−12.1063
2719	ARG261	CZ	4.8263	−21.6535	−13.1285
2720	ARG261	NH1	5.223	−20.8915	−14.1043
2721	ARG261	NH2	5.0417	−22.9348	−13.1953
2722	LYS262	N	4.2857	−22.4607	−5.5544
2723	LYS262	CA	5.3856	−23.1774	−4.9376
2724	LYS262	C	5.8255	−24.3186	−5.8107
2725	LYS262	O	5.1828	−24.6312	−6.8005
2726	LYS262	CB	5.0152	−23.6907	−3.5336
2727	LYS262	CG	6.3179	−23.9676	−2.7607
2728	LYS262	CD	6.0206	−24.5326	−1.3622
2729	LYS262	CE	7.3098	−24.4656	−0.5237
2730	LYS262	NZ	7.0732	−25.0873	0.7867
2731	VAL263	N	6.9519	−24.9407	−5.421
2732	VAL263	CA	7.4726	−26.0424	−6.208
2733	VAL263	C	8.1395	−26.9928	−5.254
2734	VAL263	O	7.6714	−27.148	−4.138
2735	VAL263	CB	8.4965	−25.4823	−7.2192
2736	VAL263	CG1	7.793	−24.6213	−8.2849
2737	VAL263	CG2	9.5603	−24.642	−6.489
2738	THR264	N	9.2483	−27.6135	−5.6989
2739	THR264	CA	10.0418	−28.4341	−4.8006
2740	THR264	C	11.2795	−28.8729	−5.5293
2741	THR264	O	11.3053	−28.8214	−6.7478
2742	THR264	CB	9.2501	−29.5985	−4.168
2743	THR264	OG1	10.1114	−30.4496	−3.4071
2744	THR264	CG2	8.5052	−30.4127	−5.2411
2745	CYS265	N	12.3218	−29.2873	−4.785
2746	CYS265	CA	13.5704	−29.6047	−5.4528
2747	CYS265	C	14.2751	−30.6831	−4.6861
2748	CYS265	O	15.0484	−30.3903	−3.7888
2749	CYS265	CB	14.4239	−28.3244	−5.5279
2750	CYS265	SG	14.0005	−27.4808	−7.0785
2751	LYS266	N	13.9958	−31.9498	−5.0519
2752	LYS266	CA	14.5391	−33.0536	−4.2768
2753	LYS266	C	13.9713	−32.9782	−2.887
2754	LYS266	O	14.7014	−32.9772	−1.9084
2755	LYS266	CB	16.0807	−33.0492	−4.2688
2756	LYS266	CG	16.6181	−33.1531	−5.7063
2757	LYS266	CD	18.0988	−32.7333	−5.7235
2758	LYS266	CE	18.6018	−32.6909	−7.1778
2759	LYS266	NZ	19.5072	−31.5454	−7.3469
2760	HIS267	N	12.6274	−32.897	−2.8274
2761	HIS267	CA	11.9625	−32.7559	−1.5442
2762	HIS267	C	12.337	−31.4426	−0.9165
2763	HIS267	O	12.6154	−31.3721	0.2699
2764	HIS267	CB	12.2143	−33.9712	−0.6284
2765	HIS267	CG	11.8724	−35.2288	−1.3735
2766	HIS267	ND1	12.6398	−35.6975	−2.3307
2767	HIS267	CD2	10.7805	−35.9862	−1.1556
2768	HIS267	CE1	12.0809	−36.7767	−2.7783
2769	HIS267	NE2	11.0167	−36.9974	−2.1501
2770	ARG268	N	12.3454	−30.3879	−1.7539
2771	ARG268	CA	12.6959	−29.0768	−1.2425
2772	ARG268	C	11.7987	−28.073	−1.9135
2773	ARG268	O	12.2499	−27.365	−2.7992
2774	ARG268	CB	14.1807	−28.7867	−1.539
2775	ARG268	CG	15.075	−29.8563	−0.8856
2776	ARG268	CD	16.5401	−29.5911	−1.2753
2777	ARG268	NE	17.2372	−30.8544	−1.429
2778	ARG268	CZ	17.8507	−31.1554	−2.5371
2779	ARG268	NH1	18.4688	−32.2949	−2.6312
2780	ARG268	NH2	17.8589	−30.3395	−3.5505
2781	PRO269	N	10.5143	−27.9991	−1.5013
2782	PRO269	CA	9.5866	−27.0874	−2.1375
2783	PRO269	C	10.0311	−25.6531	−2.0958
2784	PRO269	O	10.8327	−25.2856	−1.2517
2785	PRO269	CB	8.2973	−27.2861	−1.3184
2786	PRO269	CG	8.542	−28.4617	−0.3464
2787	PRO269	CD	10.0307	−28.8527	−0.437
2788	GLU270	N	9.5086	−24.8419	−3.0367
2789	GLU270	CA	9.9543	−23.4606	−3.0969
2790	GLU270	C	8.8187	−22.5727	−3.5148
2791	GLU270	O	8.3378	−22.6901	−4.6302
2792	GLU270	CB	11.1188	−23.3223	−4.0978
2793	GLU270	CG	12.3248	−24.1506	−3.6156
2794	GLU270	CD	12.7022	−25.1816	−4.6395
2795	GLU270	OE1	13.9116	−25.2456	−4.9833
2796	GLU270	OE2	11.7987	−25.9258	−5.1071
2797	TYR271	N	8.3949	−21.6792	−2.5981
2798	TYR271	CA	7.3008	−20.7762	−2.9181
2799	TYR271	C	7.7473	−19.7883	−3.9578
2800	TYR271	O	8.189	−18.6948	−3.6512
2801	TYR271	CB	6.8694	−20.0676	−1.6213
2802	TYR271	CG	5.9845	−21.0113	−0.8158
2803	TYR271	CD1	6.4001	−21.4533	0.4424
2804	TYR271	CD2	4.7584	−21.4337	−1.3361
2805	TYR271	CE1	5.5858	−22.3144	1.1823
2806	TYR271	CE2	3.9429	−22.2927	−0.5958
2807	TYR271	CZ	4.3602	−22.7375	0.6613
2808	TYR271	OH	3.5561	−23.6022	1.3935
2809	SER289	N	17.1658	−18.93	−8.2538
2810	SER289	CA	18.3422	−19.0254	−7.408
2811	SER289	C	18.4464	−20.4103	−6.8327
2812	SER289	O	17.4627	−21.1337	−6.8167
2813	SER289	CB	18.2595	−17.9919	−6.2689
2814	SER289	OG	18.1295	−16.678	−6.8191
2815	ASP290	N	19.658	−20.783	−6.3711
2816	ASP290	CA	19.8478	−22.1303	−5.8561
2817	ASP290	C	19.5131	−23.1241	−6.936
2818	ASP290	O	18.616	−23.9338	−6.7705
2819	ASP290	CB	19.0064	−22.3277	−4.577
2820	ASP290	CG	19.3558	−23.6273	−3.9113
2821	ASP290	OD1	18.5143	−24.5634	−3.9658
2822	ASP290	OD2	20.4676	−23.7127	−3.3249
2823	SER291	N	20.2476	−23.0372	−8.0632
2824	SER291	CA	19.905	−23.8381	−9.229
2825	SER291	C	18.8119	−23.1601	−10.0025
2826	SER291	O	17.6878	−23.0693	−9.5349
2827	SER291	CB	19.5625	−25.3123	−8.9314
2828	SER291	OG	20.6095	−25.9035	−8.1578
2829	SER292	N	19.1722	−22.6698	−11.2039
2830	SER292	CA	18.195	−21.9554	−12.0051
2831	SER292	C	17.2106	−22.9225	−12.5874
2832	SER292	O	16.0318	−22.843	−12.2828
2833	SER292	CB	18.9224	−21.2063	−13.1356
2834	SER292	OG	19.8986	−20.3309	−12.5658
2835	SER293	N	17.7122	−23.8448	−13.4301
2836	SER293	CA	16.8111	−24.8207	−14.0092
2837	SER293	C	16.5572	−25.9456	−13.0439
2838	SER293	O	16.5136	−27.0961	−13.4508
2839	SER293	CB	17.3567	−25.3185	−15.3625
2840	SER293	OG	18.6298	−25.9437	−15.1757
2841	LYS294	N	16.3779	−25.616	−11.7479
2842	LYS294	CA	16.0064	−26.6686	−10.8191
2843	LYS294	C	14.5559	−27.0301	−10.9887
2844	LYS294	O	13.9326	−27.5604	−10.0834
2845	LYS294	CB	16.4338	−26.4047	−9.3613
2846	LYS294	CG	15.808	−25.1066	−8.8236
2847	LYS294	CD	16.0614	−25.0134	−7.3086
2848	LYS294	CE	15.7922	−23.576	−6.8301
2849	LYS294	NZ	16.1052	−23.4562	−5.3993
2850	TRP295	N	14.0282	−26.7341	−12.1931
2851	TRP295	CA	12.6821	−27.1618	−12.5219
2852	TRP295	C	12.7817	−28.5945	−12.97
2853	TRP295	O	12.2805	−28.9619	−14.0205
2854	TRP295	CB	12.1207	−26.2722	−13.6524
2855	TRP295	CG	12.5239	−24.8384	−13.4586
2856	TRP295	CD1	13.5624	−24.2088	−14.0301
2857	TRP295	CD2	11.8326	−23.8389	−12.5722
2858	TRP295	NE1	13.6136	−22.9662	−13.6174
2859	TRP295	CE2	12.6152	−22.7131	−12.7465
2860	TRP295	CE3	10.7148	−23.8688	−11.7405
2861	TRP295	CZ2	12.3445	−21.5215	−12.0738
2862	TRP295	CZ3	10.4389	−22.6751	−11.0637
2863	TRP295	CH2	11.2391	−21.5329	−11.2145
2864	GLU296	N	13.4637	−29.41	−12.1425
2865	GLU296	CA	13.6738	−30.7962	−12.5123
2866	GLU296	C	13.6457	−31.6147	−11.2556
2867	GLU296	O	12.8159	−32.5018	−11.1372
2868	GLU296	CB	15.0491	−30.9523	−13.1904
2869	GLU296	CG	15.1797	−29.9717	−14.3715
2870	GLU296	CD	16.5672	−30.0476	−14.9383
2871	GLU296	OE1	16.7082	−30.5508	−16.0845
2872	GLU296	OE2	17.5189	−29.5994	−14.2445
2873	LEU297	N	14.5557	−31.3013	−10.3102
2874	LEU297	CA	14.5168	−31.9927	−9.0331
2875	LEU297	C	13.2502	−31.6135	−8.3246
2876	LEU297	O	13.0172	−30.4371	−8.0988
2877	LEU297	CB	15.7497	−31.6707	−8.1644
2878	LEU297	CG	15.873	−30.1595	−7.8868
2879	LEU297	CD1	16.8503	−29.9272	−6.7201
2880	LEU297	CD2	16.3974	−29.4396	−9.1426
2881	LEU298	N	12.4169	−32.625	−8.0092
2882	LEU298	CA	11.1004	−32.3273	−7.4683
2883	LEU298	C	10.3621	−31.3525	−8.3461
2884	LEU298	O	9.5067	−30.6151	−7.8844
2885	LEU298	CB	11.1792	−31.8834	−5.9944
2886	LEU298	CG	10.8252	−33.0547	−5.057
2887	LEU298	CD1	9.3623	−33.4862	−5.2695
2888	LEU298	CD2	11.7632	−34.252	−5.3043
2889	SER299	N	10.7258	−31.3566	−9.6423
2890	SER299	CA	10.0974	−30.4369	−10.5692
2891	SER299	C	9.9685	−31.1633	−11.8808
2892	SER299	O	10.6607	−30.836	−12.8313
2893	SER299	CB	10.9557	−29.1635	−10.7041
2894	SER299	OG	11.1545	−28.5444	−9.4319
2895	LEU307	N	6.3956	−23.5665	−16.4828
2896	LEU307	CA	7.1931	−22.4652	−15.9735
2897	LEU307	C	8.1083	−21.8538	−16.9929
2898	LEU307	O	8.3851	−22.46	−18.0157
2899	LEU307	CB	7.9177	−22.8665	−14.6752
2900	LEU307	CG	8.3099	−21.5991	−13.8939
2901	LEU307	CD1	7.8988	−21.7401	−12.417
2902	LEU307	CD2	9.832	−21.3982	−13.9943
2903	ARG308	N	8.5562	−20.6166	−16.6943
2904	ARG308	CA	9.3351	−19.8688	−17.6684
2905	ARG308	C	8.5562	−19.7259	−18.9476
2906	ARG308	O	9.1174	−19.6909	−20.0309
2907	ARG308	CB	10.7482	−20.4495	−17.8796
2908	ARG308	CG	11.4783	−20.6338	−16.5325
2909	ARG308	CD	11.4961	−19.3378	−15.6937
2910	ARG308	NE	11.6184	−18.1599	−16.534
2911	ARG308	CZ	12.7781	−17.7454	−16.9542
2912	ARG308	NH1	13.8705	−18.3784	−16.6421
2913	ARG308	NH2	12.8453	−16.6798	−17.6953
2914	LYS309	N	7.2213	−19.6664	−18.7836
2915	LYS309	CA	6.3417	−19.6382	−19.9366
2916	LYS309	C	6.2972	−18.2635	−20.5374
2917	LYS309	O	6.5442	−18.1125	−21.723
2918	LYS309	CB	4.9278	−20.0332	−19.4624
2919	LYS309	CG	4.57	−19.2689	−18.1717
2920	LYS309	CD	3.2022	−19.7298	−17.6396
2921	LYS309	CE	3.0136	−19.1806	−16.2137
2922	LYS309	NZ	1.7878	−19.7429	−15.6295
2923	ASP310	N	5.9687	−17.2608	−19.7008
2924	ASP310	CA	5.7973	−15.9166	−20.2246
2925	ASP310	C	7.1124	−15.3194	−20.6424
2926	ASP310	O	8.1451	−15.9616	−20.5346
2927	ASP310	CB	5.1166	−15.0146	−19.1735
2928	ASP310	CG	4.1993	−15.8056	−18.2838
2929	ASP310	OD1	4.64	−16.1806	−17.1645
2930	ASP310	OD2	3.0342	−16.0465	−18.6978
2931	ASN311	N	7.063	−14.0653	−21.1296
2932	ASN311	CA	8.2916	−13.436	−21.579
2933	ASN311	C	8.2891	−11.9763	−21.2219
2934	ASN311	O	7.4456	−11.544	−20.4518
2935	ASN311	CB	8.4266	−13.6396	−23.0999
2936	ASN311	CG	8.7356	−15.0815	−23.3811
2937	ASN311	OD1	7.923	−15.7738	−23.9713
2938	ASN311	ND2	9.9227	−15.549	−22.9547
2939	ASP312	N	9.2508	−11.2127	−21.7825
2940	ASP312	CA	9.3773	−9.8163	−21.3949
2941	ASP312	C	9.6773	−9.7719	−19.9227
2942	ASP312	O	9.0169	−9.0665	−19.1788
2943	ASP312	CB	8.0958	−9.0254	−21.7348
2944	ASP312	CG	8.4216	−7.784	−22.5124
2945	ASP312	OD1	8.8258	−6.7738	−21.8768
2946	ASP312	OD2	8.2557	−7.8116	−23.7612
2947	GLY313	N	10.6782	−10.5659	−19.4978
2948	GLY313	CA	10.9377	−10.6612	−18.074
2949	GLY313	C	10.2069	−11.8465	−17.5087
2950	GLY313	O	9.4537	−11.6938	−16.5604
2951	SER340	N	−16.3887	−9.2646	−18.8495
2952	SER340	CA	−16.9789	−10.5545	−18.5298
2953	SER340	C	−16.0382	−11.6865	−18.8295
2954	SER340	O	−16.4224	−12.6865	−19.4149
2955	SER340	CB	−18.4188	−10.7336	−19.0575
2956	SER340	OG	−18.4344	−10.989	−20.4645
2957	GLN341	N	−14.7733	−11.4999	−18.409
2958	GLN341	CA	−13.7872	−12.5328	−18.6593
2959	GLN341	C	−13.9973	−13.6595	−17.6917
2960	GLN341	O	−13.8658	−13.4705	−16.4927
2961	GLN341	CB	−12.3744	−11.9426	−18.5055
2962	GLN341	CG	−12.2671	−10.6679	−19.3635
2963	GLN341	CD	−10.834	−10.3784	−19.6945
2964	GLN341	OE1	−10.5296	−10.0819	−20.8379
2965	GLN341	NE2	−9.9401	−10.467	−18.693
2966	GLU342	N	−14.334	−14.842	−18.2416
2967	GLU342	CA	−14.5771	−15.9907	−17.3859
2968	GLU342	C	−13.3583	−16.3002	−16.5657
2969	GLU342	O	−13.4924	−16.6687	−15.4098
2970	GLU342	CB	−14.8923	−17.2033	−18.2805
2971	GLU342	CG	−16.2407	−16.984	−18.991
2972	GLU342	CD	−16.5529	−18.1797	−19.8423
2973	GLU342	OE1	−17.2257	−19.1124	−19.3278
2974	GLU342	OE2	−16.1387	−18.1828	−21.0323
2975	ALA343	N	−12.1655	−16.1326	−17.171
2976	ALA343	CA	−10.9489	−16.4186	−16.4325
2977	ALA343	C	−10.8286	−15.5741	−15.1958
2978	ALA343	O	−11.2757	−14.4382	−15.1781
2979	ALA343	CB	−9.7128	−16.2208	−17.3284
2980	ALA344	N	−10.219	−16.1722	−14.1534
2981	ALA344	CA	−10.0489	−15.4662	−12.8946
2982	ALA344	C	−11.3061	−15.3614	−12.0785
2983	ALA344	O	−12.3777	−15.1106	−12.6066
2984	ALA344	CB	−9.3563	−14.097	−13.0367
2985	GLN345	N	−11.1378	−15.5569	−10.756
2986	GLN345	CA	−12.2396	−15.3412	−9.8354
2987	GLN345	C	−12.542	−13.8713	−9.7507
2988	GLN345	O	−11.7928	−13.0625	−10.2761
2989	GLN345	CB	−11.831	−15.8247	−8.4297
2990	GLN345	CG	−11.2853	−17.2639	−8.4949
2991	GLN345	CD	−11.6086	−17.9922	−7.2226
2992	GLN345	OE1	−12.3591	−18.9535	−7.2488
2993	GLN345	NE2	−11.0403	−17.5418	−6.0899
2994	MET351	N	−19.5867	0.1074	−2.8647
2995	MET351	CA	−18.9917	1.333	−2.3614
2996	MET351	C	−19.9682	2.156	−1.5709
2997	MET351	O	−21.1705	2.0284	−1.7395
2998	MET351	CB	−18.407	2.171	−3.5126
2999	MET351	CG	−17.4789	3.2657	−2.9539
3000	MET351	SD	−17.0298	4.3543	−4.336
3001	MET351	CE	−16.091	5.592	−3.3933
3002	ARG352	N	−19.4169	3.0104	−0.69
3003	ARG352	CA	−20.2817	3.8805	0.083
3004	ARG352	C	−19.7783	5.2922	−0.0036
3005	ARG352	O	−18.6784	5.5259	−0.4795
3006	ARG352	CB	−20.3127	3.4321	1.5564
3007	ARG352	CG	−21.0796	2.1031	1.6878
3008	ARG352	CD	−21.145	1.7058	3.174
3009	ARG352	NE	−22.4189	1.0678	3.4523
3010	ARG352	CZ	−22.4939	0.0081	4.2047
3011	ARG352	NH1	−23.6545	−0.5309	4.4316
3012	ARG352	NH2	−21.4306	−0.5187	4.7369
3013	GLU353	N	−20.6069	6.243	0.4656
3014	GLU353	CA	−20.1771	7.6289	0.4297
3015	GLU353	C	−20.6351	8.3401	1.6712
3016	GLU353	O	−21.3374	7.7625	2.4861
3017	GLU353	CB	−20.7213	8.3284	−0.8312
3018	GLU353	CG	−20.068	7.729	−2.0919
3019	GLU353	CD	−18.7307	8.3669	−2.3365
3020	GLU353	OE1	−17.7195	7.8583	−1.7831
3021	GLU353	OE2	−18.6851	9.3703	−3.097
3022	SER58	N	4.5161	25.4695	−12.0448
3023	SER58	CA	5.8856	25.2609	−11.6081
3024	SER58	C	6.2426	26.247	−10.5313
3025	SER58	O	5.5046	27.1917	−10.3003
3026	SER58	CB	6.8662	25.352	−12.7932
3027	SER58	OG	6.8328	26.6638	−13.3617
3028	GLY59	N	7.3844	26.0176	−9.8559
3029	GLY59	CA	7.7292	26.9004	−8.758
3030	GLY59	C	9.2172	27.0021	−8.6004
3031	GLY59	O	9.8707	26.0045	−8.3406
3032	SER60	N	9.7354	28.2375	−8.7523
3033	SER60	CA	11.1556	28.4693	−8.5488
3034	SER60	C	12.0012	27.647	−9.479
3035	SER60	O	12.1356	28.0169	−10.6331
3036	SER60	CB	11.5573	28.3065	−7.0694
3037	SER60	OG	12.8944	28.7744	−6.8761
3038	LEU61	N	12.5734	26.5358	−8.974
3039	LEU61	CA	13.3703	25.6829	−9.8388
3040	LEU61	C	12.4744	25.0345	−10.8529
3041	LEU61	O	12.8547	24.9202	−12.0066
3042	LEU61	CB	14.0287	24.5946	−8.9719
3043	LEU61	CG	15.1543	25.2173	−8.1245
3044	LEU61	CD1	15.6818	24.1691	−7.1278
3045	LEU61	CD2	16.3056	25.6736	−9.0407
3046	LEU62	N	11.2662	24.634	−10.4083
3047	LEU62	CA	10.2797	24.1384	−11.3521
3048	LEU62	C	9.9899	25.2189	−12.3535
3049	LEU62	O	9.8158	24.9231	−13.5236
3050	LEU62	CB	8.9859	23.8371	−10.5738
3051	LEU62	CG	9.182	22.6054	−9.6716
3052	LEU62	CD1	8.0821	22.5794	−8.5947
3053	LEU62	CD2	9.0985	21.3253	−10.5239
3054	GLN63	N	9.9609	26.4803	−11.8805
3055	GLN63	CA	9.7744	27.5884	−12.8005
3056	GLN63	C	11.0014	27.8053	−13.6448
3057	GLN63	O	10.9047	28.4324	−14.6872
3058	GLN63	CB	9.4807	28.8481	−11.9674
3059	GLN63	CG	8.0421	28.7587	−11.4255
3060	GLN63	CD	7.7327	29.9547	−10.5738
3061	GLN63	OE1	7.4182	29.8047	−9.4048
3062	GLN63	NE2	7.817	31.1633	−11.1583
3063	LYS64	N	12.1588	27.2732	−13.205
3064	LYS64	CA	13.3485	27.3597	−14.0352
3065	LYS64	C	13.3802	26.1327	−14.9053
3066	LYS64	O	14.3184	25.3531	−14.8528
3067	LYS64	CB	14.5923	27.3962	−13.124
3068	LYS64	CG	14.4931	28.5556	−12.1168
3069	LYS64	CD	15.2394	28.156	−10.8313
3070	LYS64	CE	14.8604	29.1181	−9.6912
3071	LYS64	NZ	15.2794	28.5352	−8.4089
3072	SER167	N	7.9208	6.4674	−28.337
3073	SER167	CA	7.7436	6.2762	−29.7669
3074	SER167	C	7.2045	4.9046	−30.0648
3075	SER167	O	6.6653	4.6971	−31.1401
3076	SER167	CB	9.1172	6.4355	−30.4468
3077	SER167	OG	10.0458	5.4869	−29.9125
3078	THR168	N	7.3541	3.9725	−29.1012
3079	THR168	CA	6.9327	2.6017	−29.3399
3080	THR168	C	5.4961	2.4848	−29.7688
3081	THR168	O	4.7103	3.393	−29.5518
3082	THR168	CB	7.1788	1.756	−28.0715
3083	THR168	OG1	6.9197	0.3739	−28.3344
3084	THR168	CG2	6.2787	2.2329	−26.9143
3085	TYR169	N	5.1649	1.3297	−30.3795
3086	TYR169	CA	3.7747	1.0608	−30.7045
3087	TYR169	C	2.9715	1.1166	−29.4396
3088	TYR169	O	1.8501	1.5972	−29.4459
3089	TYR169	CB	3.6924	−0.3845	−31.2285
3090	TYR169	CG	3.9609	−0.4244	−32.7279
3091	TYR169	CD1	5.1791	0.031	−33.2386
3092	TYR169	CD2	2.9836	−0.9252	−33.5914
3093	TYR169	CE1	5.4261	−0.0331	−34.6122
3094	TYR169	CE2	3.2344	−0.9998	−34.9634
3095	TYR169	CZ	4.4537	−0.5472	−35.4739
3096	TYR169	OH	4.6986	−0.6064	−36.8403
3097	LYS170	N	3.5939	0.6215	−28.3524
3098	LYS170	CA	2.9178	0.5885	−27.0715
3099	LYS170	C	2.4888	1.9565	−26.6329
3100	LYS170	O	1.4293	2.063	−26.0395
3101	LYS170	CB	3.9232	0.0584	−26.0343
3102	LYS170	CG	3.2029	−0.8809	−25.0516
3103	LYS170	CD	4.1528	−1.1913	−23.8821
3104	LYS170	CE	3.8263	−2.5692	−23.2794
3105	LYS170	NZ	4.6894	−2.8051	−22.1133
3106	ASN171	N	3.2958	3.0014	−26.9052
3107	ASN171	CA	2.9531	4.2881	−26.3252
3108	ASN171	C	3.0516	5.4378	−27.2882
3109	ASN171	O	3.275	6.5589	−26.8592
3110	ASN171	CB	3.7789	4.5344	−25.0468
3111	ASN171	CG	3.7314	3.3219	−24.1631
3112	ASN171	OD1	4.7508	2.6909	−23.9381
3113	ASN171	ND2	2.5327	2.9848	−23.6566
3114	LEU172	N	2.8874	5.1636	−28.5976
3115	LEU172	CA	3.054	6.2303	−29.5703
3116	LEU172	C	1.9448	7.2373	−29.4526
3117	LEU172	O	0.8427	6.9818	−29.8769
3118	LEU172	CB	3.1005	5.6355	−30.9919
3119	LEU172	CG	3.9739	6.524	−31.8995
3120	LEU172	CD1	4.1502	5.8546	−33.2738
3121	LEU172	CD2	3.3112	7.8991	−32.0998
3122	GLN367	N	−11.6584	7.6284	20.3537
3123	GLN367	CA	−11.2992	8.0993	21.678
3124	GLN367	C	−11.1646	9.5941	21.7165
3125	GLN367	O	−10.6996	10.1277	22.7113
3126	GLN367	CB	−9.9796	7.4354	22.1105
3127	GLN367	CG	−10.0225	7.1421	23.6209
3128	GLN367	CD	−9.0236	6.071	23.9479
3129	GLN367	OE1	−9.4052	4.9967	24.3819
3130	GLN367	NE2	−7.7255	6.3572	23.7419
3131	LEU368	N	−11.5754	10.2737	20.6282
3132	LEU368	CA	−11.5262	11.7231	20.6468
3133	LEU368	C	−12.5936	12.2155	21.5826
3134	LEU368	O	−13.7453	12.3421	21.199
3135	LEU368	CB	−11.7396	12.2486	19.2157
3136	LEU368	CG	−10.4387	12.1409	18.3976
3137	LEU368	CD1	−10.6866	12.677	16.9756
3138	LEU368	CD2	−9.3312	12.9809	19.061
3139	LEU369	N	−12.1779	12.482	22.8363
3140	LEU369	CA	−13.138	12.8724	23.8552
3141	LEU369	C	−13.8092	14.1623	23.484
3142	LEU369	O	−15.0241	14.2483	23.5615
3143	LEU369	CB	−12.4024	13.0369	25.1993
3144	LEU369	CG	−11.5478	11.7894	25.5007
3145	LEU369	CD1	−10.7853	11.9967	26.8221
3146	LEU369	CD2	−12.4446	10.5419	25.6142
3147	GLN370	N	−13.0054	15.1578	23.0591
3148	GLN370	CA	−13.5889	16.3942	22.5661
3149	GLN370	C	−14.5276	16.0684	21.4381
3150	GLN370	O	−15.6099	16.6268	21.3618
3151	GLN370	CB	−12.4205	17.2626	22.0543
3152	GLN370	CG	−12.9177	18.3894	21.1269
3153	GLN370	CD	−12.7668	17.9552	19.697
3154	GLN370	OE1	−13.7564	17.7562	19.0116
3155	GLN370	NE2	−11.5136	17.8049	19.2321
3156	ASP371	N	−14.091	15.14	20.567
3157	ASP371	CA	−14.927	14.755	19.446
3158	ASP371	C	−16.1654	14.0217	19.8864
3159	ASP371	O	−16.2572	13.6014	21.0288
3160	ASP371	CB	−14.0746	13.845	18.5474
3161	ASP371	CG	−13.1204	14.6693	17.7345
3162	ASP371	OD1	−13.3767	14.8343	16.5124
3163	ASP371	OD2	−12.1057	15.1415	18.3128
3164	THR372	N	−17.1371	13.8795	18.9632
3165	THR372	CA	−18.3719	13.2085	19.3346
3166	THR372	C	−18.2847	11.727	19.0963
3167	THR372	O	−18.5994	10.9685	19.9991
3168	THR372	CB	−19.5853	13.834	18.6163
3169	THR372	OG1	−20.7591	13.0569	18.8679
3170	THR372	CG2	−19.3433	13.9323	17.0994
3171	SER382	N	−21.7125	−7.2387	6.7774
3172	SER382	CA	−21.778	−8.4416	7.5895
3173	SER382	C	−21.0627	−9.6106	6.9768
3174	SER382	O	−20.601	−9.5345	5.8495
3175	SER382	CB	−23.2573	−8.7962	7.832
3176	SER382	OG	−23.8439	−7.8212	8.6971
3177	VAL383	N	−20.9902	−10.709	7.7553
3178	VAL383	CA	−20.4217	−11.9392	7.2332
3179	VAL383	C	−20.6916	−13.0827	8.177
3180	VAL383	O	−20.5488	−12.9223	9.3776
3181	VAL383	CB	−18.9717	−11.7586	6.7422
3182	VAL383	CG1	−18.0495	−11.3373	7.8991
3183	VAL383	CG2	−18.4529	−13.0195	6.0279
3184	TRP384	N	−21.1268	−14.2369	7.6338
3185	TRP384	CA	−21.5699	−15.3172	8.4986
3186	TRP384	C	−20.5212	−16.3844	8.6219
3187	TRP384	O	−19.4864	−16.2913	7.9853
3188	TRP384	CB	−22.9502	−15.8666	8.0679
3189	TRP384	CG	−22.8699	−16.9451	7.0232
3190	TRP384	CD1	−22.4297	−18.2021	7.1924
3191	TRP384	CD2	−23.2752	−16.8131	5.5799
3192	TRP384	NE1	−22.4732	−18.8513	6.0555
3193	TRP384	CE2	−22.9321	−18.0508	5.0717
3194	TRP384	CE3	−23.8559	−15.8137	4.8008
3195	TRP384	CZ2	−23.0663	−18.3452	3.7152
3196	TRP384	CZ3	−23.9929	−16.1	3.4379
3197	TRP384	CH2	−23.5757	−17.3263	2.9019
3198	ARG385	N	−20.7905	−17.4056	9.4551
3199	ARG385	CA	−19.783	−18.4237	9.6788
3200	ARG385	C	−20.499	−19.7103	9.9672
3201	ARG385	O	−20.8957	−19.9297	11.0998
3202	ARG385	CB	−19.0048	−17.9882	10.929
3203	ARG385	CG	−17.802	−18.8999	11.2165
3204	ARG385	CD	−17.0898	−18.3609	12.4697
3205	ARG385	NE	−16.5758	−17.0295	12.204
3206	ARG385	CZ	−16.7127	−16.069	13.0714
3207	ARG385	NH1	−16.2541	−14.8854	12.7933
3208	ARG385	NH2	−17.3001	−16.2711	14.2137
3209	PRO386	N	−20.6675	−20.5782	8.95
3210	PRO386	CA	−21.3959	−21.8131	9.1558
3211	PRO386	C	−20.581	−22.7837	9.9612
3212	PRO386	O	−19.5745	−22.4162	10.546
3213	PRO386	CB	−21.4659	−22.3502	7.7145
3214	PRO386	CG	−20.3189	−21.6558	6.9529
3215	PRO386	CD	−20.1001	−20.302	7.6479
3216	GLU387	N	−21.0369	−24.0503	9.9733
3217	GLU387	CA	−20.2927	−25.0616	10.6974
3218	GLU387	C	−19.1297	−25.5457	9.8748
3219	GLU387	O	−19.0221	−26.7281	9.5903
3220	GLU387	CB	−21.2503	−26.2048	11.0833
3221	GLU387	CG	−22.2475	−25.6974	12.1415
3222	GLU387	CD	−22.8692	−26.8624	12.8554
3223	GLU387	OE1	−24.1183	−27.0029	12.7751
3224	GLU387	OE2	−22.1147	−27.6381	13.5014
3225	GLU388	N	−18.2392	−24.6075	9.495
3226	GLU388	CA	−17.0442	−25.0076	8.7712
3227	GLU388	C	−16.0882	−25.7088	9.7019
3228	GLU388	O	−16.4578	−26.0365	10.8183
3229	GLU388	CB	−16.4101	−23.7532	8.1366
3230	GLU388	CG	−15.4454	−24.1537	7.0037
3231	GLU388	CD	−14.086	−23.5654	7.2516
3232	GLU388	OE1	−13.8775	−22.379	6.8856
3233	GLU388	OE2	−13.2139	−24.2894	7.8016
3234	GLY389	N	−14.8465	−25.9491	9.2367
3235	GLY389	CA	−13.9185	−26.7395	10.0294
3236	GLY389	C	−13.4262	−26.0583	11.2769
3237	GLY389	O	−14.1027	−25.2136	11.8411
3238	ARG390	N	−12.2154	−26.4654	11.7068
3239	ARG390	CA	−11.6679	−25.9396	12.9458
3240	ARG390	C	−11.1701	−24.5349	12.7423
3241	ARG390	O	−9.9756	−24.313	12.6198
3242	ARG390	CB	−10.5192	−26.8764	13.3736
3243	ARG390	CG	−9.9781	−26.4675	14.7567
3244	ARG390	CD	−10.9625	−26.9213	15.8493
3245	ARG390	NE	−10.6131	−26.2863	17.1065
3246	ARG390	CZ	−11.4716	−25.5413	17.74
3247	ARG390	NH1	−11.1289	−24.9888	18.8657
3248	ARG390	NH2	−12.6657	−25.3433	17.2635
3249	ARG391	N	−12.1066	−23.5686	12.7144
3250	ARG391	CA	−11.675	−22.2004	12.5066
3251	ARG391	C	−11.4618	−21.4903	13.8149
3252	ARG391	O	−11.8324	−20.3359	13.9572
3253	ARG391	CB	−12.6535	−21.4511	11.5852
3254	ARG391	CG	−12.843	−22.2182	10.2661
3255	ARG391	CD	−14.3048	−22.0698	9.8165
3256	ARG391	NE	−15.1209	−22.9598	10.6179
3257	ARG391	CZ	−16.3317	−22.6372	10.9653
3258	ARG391	NH1	−17.0163	−23.463	11.6969
3259	ARG391	NH2	−16.8663	−21.511	10.597
3260	SER392	N	−10.8434	−22.1905	14.7859
3261	SER392	CA	−10.515	−21.5197	16.0313
3262	SER392	C	−9.222	−20.7737	15.8405
3263	SER392	O	−8.3233	−20.8575	16.6623
3264	SER392	CB	−10.4051	−22.5746	17.1487
3265	SER392	OG	−10.2618	−21.9278	18.4149
3266	LEU393	N	−9.1362	−20.0478	14.7083
3267	LEU393	CA	−7.9112	−19.3408	14.3803
3268	LEU393	C	−8.1089	−18.7172	13.0275
3269	LEU393	O	−7.5096	−19.1761	12.0691
3270	LEU393	CB	−6.6802	−20.2771	14.4205
3271	LEU393	CG	−6.7754	−21.4227	13.3897
3272	LEU393	CD1	−5.3984	−22.0945	13.2432
3273	LEU393	CD2	−7.8166	−22.4742	13.8166
3274	ARG394	N	−8.9651	−17.68	12.9082
3275	ARG394	CA	−9.2458	−17.2221	11.5567
3276	ARG394	C	−9.8339	−15.8452	11.3865
3277	ARG394	O	−10.9908	−15.7473	11.0116
3278	ARG394	CB	−10.0227	−18.2562	10.7129
3279	ARG394	CG	−10.3857	−19.5313	11.4973
3280	ARG394	CD	−9.5337	−20.7126	10.9953
3281	ARG394	NE	−9.8601	−20.9951	9.6108
3282	ARG394	CZ	−10.0667	−22.2112	9.1969
3283	ARG394	NH1	−9.9769	−23.2251	10.0066
3284	ARG394	NH2	−10.3721	−22.4142	7.9499
3285	PRO395	N	−9.0548	−14.7611	11.5918
3286	PRO395	CA	−9.4903	−13.4464	11.1559
3287	PRO395	C	−9.5917	−13.3885	9.6519
3288	PRO395	O	−9.9065	−14.3791	9.0115
3289	PRO395	CB	−8.2908	−12.5759	11.5797
3290	PRO395	CG	−7.1218	−13.5207	11.9251
3291	PRO395	CD	−7.7492	−14.8988	12.1934
3292	CYS396	N	−9.3171	−12.2055	9.0684
3293	CYS396	CA	−9.4524	−12.0876	7.6277
3294	CYS396	C	−8.5442	−11.0416	7.0407
3295	CYS396	O	−7.3374	−11.1843	7.1501
3296	CYS396	CB	−10.9109	−11.954	7.1431
3297	CYS396	SG	−12.0989	−11.6904	8.4891
3298	SER397	N	−9.1188	−10.002	6.3994
3299	SER397	CA	−8.292	−9.0079	5.7316
3300	SER397	C	−9.2078	−8.0129	5.0712
3301	SER397	O	−9.3854	−8.057	3.8638
3302	SER397	CB	−7.3971	−9.7028	4.683
3303	SER397	OG	−6.675	−8.7363	3.9151
3304	VAL398	N	−9.8078	−7.1174	5.8837
3305	VAL398	CA	−10.8301	−6.221	5.3628
3306	VAL398	C	−10.4519	−5.596	4.0479
3307	VAL398	O	−9.4642	−4.8841	3.9578
3308	VAL398	CB	−11.2403	−5.1829	6.4317
3309	VAL398	CG1	−12.403	−5.6985	7.2958
3310	VAL398	CG2	−11.6695	−3.8377	5.8196
3311	ARG413	N	−3.5361	17.9222	−5.1779
3312	ARG413	CA	−4.5546	17.0011	−5.6605
3313	ARG413	C	−4.175	15.563	−5.4325
3314	ARG413	O	−4.4415	14.7133	−6.2676
3315	ARG413	CB	−4.8684	17.2711	−7.1488
3316	ARG413	CG	−4.846	18.782	−7.4541
3317	ARG413	CD	−6.0673	19.4646	−6.8115
3318	ARG413	NE	−6.5687	20.4804	−7.7188
3319	ARG413	CZ	−7.587	20.2434	−8.4945
3320	ARG413	NH1	−8.0068	21.1749	−9.298
3321	ARG413	NH2	−8.1897	19.0908	−8.4829
3322	LYS414	N	−3.5268	15.2994	−4.2827
3323	LYS414	CA	−3.097	13.9421	−4.006
3324	LYS414	C	−4.2566	13.1746	−3.4316
3325	LYS414	O	−4.6461	13.4592	−2.3106
3326	LYS414	CB	−1.9473	14.0374	−2.985
3327	LYS414	CG	−1.2538	12.6735	−2.821
3328	LYS414	CD	−0.0276	12.8477	−1.9063
3329	LYS414	CE	0.6361	11.4828	−1.6484
3330	LYS414	NZ	1.2619	10.9927	−2.8844
3331	PRO415	N	−4.8245	12.1984	−4.1729
3332	PRO415	CA	−5.9278	11.4283	−3.6376
3333	PRO415	C	−5.4305	10.5023	−2.5651
3334	PRO415	O	−4.2473	10.2053	−2.5311
3335	PRO415	CB	−6.3697	10.5929	−4.8548
3336	PRO415	CG	−5.305	10.7827	−5.9589
3337	PRO415	CD	−4.336	11.8912	−5.4999
3338	LEU416	N	−6.342	10.0587	−1.676
3339	LEU416	CA	−5.9331	9.1489	−0.6153
3340	LEU416	C	−7.0039	9.0322	0.4323
3341	LEU416	O	−8.0493	9.6537	0.3288
3342	LEU416	CB	−4.619	9.587	0.0726
3343	LEU416	CG	−4.8195	10.827	0.9679
3344	LEU416	CD1	−3.5219	11.1125	1.7452
3345	LEU416	CD2	−5.1887	12.0586	0.1214
3346	LEU417	N	−6.7043	8.2312	1.4723
3347	LEU417	CA	−7.5794	8.2124	2.6286
3348	LEU417	C	−7.3649	9.4743	3.4198
3349	LEU417	O	−6.304	10.0736	3.3467
3350	LEU417	CB	−7.2075	6.9839	3.4839
3351	LEU417	CG	−7.7439	7.1108	4.9227
3352	LEU417	CD1	−9.2749	6.9509	4.9301
3353	LEU417	CD2	−7.0885	6.0397	5.8121
3354	ALA418	N	−8.3933	9.879	4.1866
3355	ALA418	CA	−8.2268	11.0604	5.0123
3356	ALA418	C	−7.9996	10.6567	6.4381
3357	ALA418	O	−7.305	11.3518	7.1618
3358	ALA418	CB	−9.5567	11.8355	5.0052
3359	ARG425	N	−18.6675	−5.1801	14.9036
3360	ARG425	CA	−19.6778	−6.0328	14.305
3361	ARG425	C	−20.1478	−7.0639	15.2912
3362	ARG425	O	−19.5831	−7.1842	16.3665
3363	ARG425	CB	−19.0115	−6.8061	13.1523
3364	ARG425	CG	−18.7297	−5.8777	11.9577
3365	ARG425	CD	−17.818	−6.6277	10.9718
3366	ARG425	NE	−18.051	−6.2043	9.6033
3367	ARG425	CZ	−18.372	−7.0629	8.6781
3368	ARG425	NH1	−18.5431	−6.6498	7.4579
3369	ARG425	NH2	−18.5244	−8.3268	8.944
3370	MET426	N	−21.1986	−7.8127	14.8983
3371	MET426	CA	−21.719	−8.856	15.767
3372	MET426	C	−22.8919	−9.499	15.0809
3373	MET426	O	−23.8577	−8.8107	14.7916
3374	MET426	CB	−22.1868	−8.2571	17.1093
3375	MET426	CG	−21.173	−8.61	18.2132
3376	MET426	SD	−21.2964	−7.3602	19.5248
3377	MET426	CE	−20.4539	−8.2886	20.841
3378	ASN427	N	−22.806	−10.8212	14.8188
3379	ASN427	CA	−23.905	−11.506	14.1492
3380	ASN427	C	−24.0585	−11.0112	12.7365
3381	ASN427	O	−24.7822	−10.0552	12.5058
3382	ASN427	CB	−25.2258	−11.3252	14.9271
3383	ASN427	CG	−25.2255	−12.1399	16.1864
3384	ASN427	OD1	−25.405	−11.5895	17.2602
3385	ASN427	ND2	−25.0267	−13.4644	16.0609
3386	LYS428	N	−23.3755	−11.6623	11.7736
3387	LYS428	CA	−23.4068	−11.1175	10.4257
3388	LYS428	C	−23.5166	−12.1736	9.3602
3389	LYS428	O	−23.4093	−13.3498	9.6638
3390	LYS428	CB	−22.1673	−10.2258	10.2332
3391	LYS428	CG	−22.2938	−9.0068	11.1642
3392	LYS428	CD	−21.2459	−7.9416	10.8006
3393	LYS428	CE	−21.6234	−6.6238	11.5019
3394	LYS428	NZ	−22.8771	−6.1019	10.9393
3395	GLU429	N	−23.745	−11.7541	8.0983
3396	GLU429	CA	−23.9137	−12.747	7.0487
3397	GLU429	C	−22.9051	−12.5924	5.9465
3398	GLU429	O	−22.5972	−11.4855	5.5365
3399	GLU429	CB	−25.3542	−12.7924	6.5111
3400	GLU429	CG	−26.2967	−13.2913	7.6237
3401	GLU429	CD	−26.9033	−14.6167	7.2596
3402	GLU429	OE1	−27.0373	−15.4729	8.1741
3403	GLU429	OE2	−27.2415	−14.815	6.0614
3404	MET430	N	−22.3773	−13.7516	5.5091
3405	MET430	CA	−21.1515	−13.7997	4.7279
3406	MET430	C	−21.1395	−12.9922	3.4604
3407	MET430	O	−22.144	−12.4203	3.0689
3408	MET430	CB	−20.8649	−15.2791	4.4098
3409	MET430	CG	−19.7321	−15.8442	5.2888
3410	MET430	SD	−19.858	−17.6607	5.2326
3411	MET430	CE	−18.4284	−18.1405	6.2421
3412	THR431	N	−19.9535	−12.9534	2.8209
3413	THR431	CA	−19.8141	−12.1439	1.6235
3414	THR431	C	−18.9606	−12.8425	0.6023
3415	THR431	O	−19.3568	−12.9109	−0.5498
3416	THR431	CB	−19.1786	−10.7891	1.993
3417	THR431	OG1	−17.9959	−10.9957	2.7711
3418	THR431	CG2	−20.1824	−9.9446	2.7992
3419	TRP432	N	−17.7884	−13.3659	1.0154
3420	TRP432	CA	−16.9162	−13.9906	0.0315
3421	TRP432	C	−16.2157	−15.1928	0.5972
3422	TRP432	O	−16.0256	−15.2741	1.7998
3423	TRP432	CB	−15.8778	−13.0153	−0.563
3424	TRP432	CG	−16.01	−11.6374	0.0191
3425	TRP432	CD1	−15.3432	−11.1416	1.072
3426	TRP432	CD2	−16.9314	−10.5544	−0.4726
3427	TRP432	NE1	−15.7193	−9.9077	1.3003
3428	TRP432	CE2	−16.6595	−9.5272	0.4111
3429	TRP432	CE3	−17.862	−10.4442	−1.5046
3430	TRP432	CZ2	−17.3132	−8.298	0.3284
3431	TRP432	CZ3	−18.5277	−9.2155	−1.589
3432	TRP432	CH2	−18.2584	−8.1672	−0.6968
3433	SER433	N	−15.8397	−16.141	−0.2841
3434	SER433	CA	−15.2145	−17.355	0.2126
3435	SER433	C	−13.7172	−17.2481	0.2387
3436	SER433	O	−13.1583	−16.9925	1.2919
3437	SER433	CB	−15.6441	−18.5942	−0.5969
3438	SER433	OG	−15.599	−18.3117	−1.9987
3439	SER434	N	−13.0628	−17.4649	−0.9199
3440	SER434	CA	−11.6091	−17.4594	−0.9335
3441	SER434	C	−11.0874	−16.1277	−0.4781
3442	SER434	O	−10.2951	−16.0703	0.4479
3443	SER434	CB	−11.1082	−17.7736	−2.3567
3444	SER434	OG	−11.7692	−16.9353	−3.3093
3445	LEU435	N	−11.5575	−15.0507	−1.1328
3446	LEU435	CA	−11.1601	−13.7259	−0.6947
3447	LEU435	C	−11.8117	−13.4319	0.6284
3448	LEU435	O	−11.2044	−12.7876	1.4682
3449	LEU435	CB	−11.6284	−12.7274	−1.7707
3450	LEU435	CG	−10.9874	−13.0944	−3.1253
3451	LEU435	CD1	−11.6377	−12.2835	−4.2611
3452	LEU435	CD2	−9.4715	−12.8205	−3.0924
3453	GLY436	N	−13.0511	−13.9301	0.8141
3454	GLY436	CA	−13.7463	−13.6783	2.0645
3455	GLY436	C	−13.1259	−14.3795	3.2413
3456	GLY436	O	−13.5195	−14.0929	4.3606
3457	SER437	N	−12.1546	−15.2873	3.0096
3458	SER437	CA	−11.4627	−15.8787	4.1435
3459	SER437	C	−10.6472	−14.7866	4.7693
3460	SER437	O	−10.7271	−14.5724	5.9678
3461	SER437	CB	−10.5753	−17.0588	3.6991
3462	SER437	OG	−9.4322	−16.605	2.9694
3463	ARG438	N	−9.8934	−14.0746	3.9088
3464	ARG438	CA	−9.3036	−12.8255	4.3441
3465	ARG438	C	−10.3339	−11.7506	4.1274
3466	ARG438	O	−10.0357	−10.6992	3.5855
3467	ARG438	CB	−8.0394	−12.5637	3.5059
3468	ARG438	CG	−6.799	−12.675	4.4085
3469	ARG438	CD	−5.537	−12.5279	3.5426
3470	ARG438	NE	−5.2563	−13.78	2.8628
3471	ARG438	CZ	−5.3165	−13.8747	1.5657
3472	ARG438	NH1	−5.0562	−15.0148	0.9988
3473	ARG438	NH2	−5.633	−12.8539	0.824
3474	GLN439	N	−11.5724	−12.058	4.5619
3475	GLN439	CA	−12.6718	−11.1177	4.4405
3476	GLN439	C	−12.5563	−9.9071	5.3258
3477	GLN439	O	−11.6003	−9.1648	5.1923
3478	GLN439	CB	−13.1032	−10.8149	2.9912
3479	GLN439	CG	−12.3806	−9.5842	2.4087
3480	GLN439	CD	−11.8279	−9.9146	1.0526
3481	GLN439	OE1	−10.6294	−9.8213	0.8458
3482	GLN439	NE2	−12.7032	−10.308	0.1101
3483	PRO440	N	−13.5257	−9.6736	6.2322
3484	PRO440	CA	−13.5102	−8.454	7.0132
3485	PRO440	C	−12.5963	−8.5337	8.2091
3486	PRO440	O	−13.0331	−8.9057	9.2865
3487	PRO440	CB	−14.9903	−8.3729	7.4378
3488	PRO440	CG	−15.6183	−9.7669	7.2251
3489	PRO440	CD	−14.6348	−10.5924	6.3754
3490	PHE441	N	−11.313	−8.154	8.0356
3491	PHE441	CA	−10.4329	−8.1481	9.1921
3492	PHE441	C	−9.3135	−7.1499	9.079
3493	PHE441	O	−9.307	−6.1968	9.8411
3494	PHE441	CB	−9.9065	−9.5694	9.4543
3495	PHE441	CG	−8.8765	−9.5947	10.5777
3496	PHE441	CD1	−9.2572	−9.3263	11.8948
3497	PHE441	CD2	−7.5447	−9.8939	10.2826
3498	PHE441	CE1	−8.3156	−9.4054	12.9238
3499	PHE441	CE2	−6.598	−9.9539	11.3085
3500	PHE441	CZ	−6.9871	−9.7246	12.6308
3501	PHE442	N	−8.3614	−7.3754	8.1492
3502	PHE442	CA	−7.2088	−6.4892	8.0549
3503	PHE442	C	−7.6206	−5.0488	7.9371
3504	PHE442	O	−7.2474	−4.2463	8.7776
3505	PHE442	CB	−6.3572	−6.8537	6.8211
3506	PHE442	CG	−5.5543	−8.1401	7.0126
3507	PHE442	CD1	−5.6384	−8.883	8.1934
3508	PHE442	CD2	−4.7192	−8.579	5.9815
3509	PHE442	CE1	−4.9145	−10.0705	8.3302
3510	PHE442	CE2	−4.0186	−9.7817	6.1024
3511	PHE442	CZ	−4.1198	−10.531	7.2771
3512	SER443	N	−8.4024	−4.7357	6.8855
3513	SER443	CA	−8.8934	−3.3779	6.7157
3514	SER443	C	−7.8833	−2.4435	6.119
3515	SER443	O	−6.7567	−2.3707	6.5828
3516	SER443	CB	−9.561	−2.7648	7.9634
3517	SER443	OG	−10.3106	−3.7529	8.6741
3518	LEU444	N	−8.3187	−1.7202	5.0706
3519	LEU444	CA	−7.4518	−0.7066	4.4982
3520	LEU444	C	−7.9637	0.6451	4.9117
3521	LEU444	O	−9.1482	0.7865	5.1681
3522	LEU444	CB	−7.4563	−0.8342	2.9623
3523	LEU444	CG	−6.0403	−0.638	2.3877
3524	LEU444	CD1	−6.0698	−0.9038	0.8713
3525	LEU444	CD2	−5.5569	0.8034	2.6349
3526	GLU445	N	−7.0692	1.6498	4.9992
3527	GLU445	CA	−7.5102	2.9652	5.4428
3528	GLU445	C	−8.0475	2.9437	6.8497
3529	GLU445	O	−8.5261	3.9614	7.3251
3530	GLU445	CB	−8.5437	3.576	4.4742
3531	GLU445	CG	−7.8604	3.8923	3.1312
3532	GLU445	CD	−8.464	3.0583	2.0392
3533	GLU445	OE1	−9.0241	3.6551	1.0816
3534	GLU445	OE2	−8.378	1.8052	2.1324
3535	ALA446	N	−7.9729	1.7703	7.5121
3536	ALA446	CA	−8.5063	1.6526	8.8598
3537	ALA446	C	−8.4942	0.2083	9.2776
3538	ALA446	O	−7.7962	−0.5946	8.679
3539	ALA446	CB	−9.9677	2.1342	8.881
3540	CYS447	N	−9.2914	−0.1215	10.3135
3541	CYS447	CA	−9.3604	−1.5071	10.7405
3542	CYS447	C	−10.7781	−1.9128	11.0309
3543	CYS447	O	−11.6868	−1.1019	10.9439
3544	CYS447	CB	−8.4782	−1.7215	11.9838
3545	CYS447	SG	−6.7617	−1.8849	11.4129
3546	GLN448	N	−10.9572	−3.2019	11.3776
3547	GLN448	CA	−12.2997	−3.6777	11.6568
3548	GLN448	C	−12.3559	−4.3087	13.021
3549	GLN448	O	−11.3584	−4.3311	13.7248
3550	GLN448	CB	−12.6929	−4.7019	10.576
3551	GLN448	CG	−14.2214	−4.8842	10.5841
3552	GLN448	CD	−14.5629	−6.3446	10.5964
3553	GLN448	OE1	−15.2139	−6.8123	9.6784
3554	GLN448	NE2	−14.1306	−7.0806	11.6362
3555	GLY449	N	−13.5408	−4.8271	13.3986
3556	GLY449	CA	−13.6521	−5.4298	14.7135
3557	GLY449	C	−14.9421	−6.1864	14.8275
3558	GLY449	O	−15.9341	−5.6338	15.2739
3559	ILE450	N	−14.9096	−7.4713	14.4265
3560	ILE450	CA	−16.0878	−8.305	14.5969
3561	ILE450	C	−16.2627	−8.6601	16.0492
3562	ILE450	O	−15.3354	−8.5252	16.8306
3563	ILE450	CB	−15.9326	−9.5842	13.7436
3564	ILE450	CG1	−16.9995	−10.6372	14.115
3565	ILE450	CG2	−14.5118	−10.1605	13.8955
3566	ILE450	CD1	−16.6492	−12.0235	13.5415
3567	LEU451	N	−17.4745	−9.1269	16.4041
3568	LEU451	CA	−17.6792	−9.6277	17.7517
3569	LEU451	C	−18.6535	−10.7714	17.7146
3570	LEU451	O	−19.1634	−11.1042	16.6561
3571	LEU451	CB	−18.2011	−8.5351	18.7037
3572	LEU451	CG	−17.1039	−7.5035	19.0253
3573	LEU451	CD1	−17.0986	−6.4004	17.9498
3574	LEU451	CD2	−17.4062	−6.8647	20.3934
3575	ALA452	N	−18.914	−11.3855	18.8851
3576	ALA452	CA	−19.811	−12.5274	18.8843
3577	ALA452	C	−20.4078	−12.7757	20.2417
3578	ALA452	O	−20.1361	−12.0456	21.1807
3579	ALA452	CB	−19.0467	−13.7711	18.3944
3580	LEU453	N	−21.2402	−13.8306	20.3318
3581	LEU453	CA	−21.8105	−14.1856	21.6185
3582	LEU453	C	−22.2269	−15.6259	21.5694
3583	LEU453	O	−22.9677	−16.0119	20.6803
3584	LEU453	CB	−23.0163	−13.2979	21.9873
3585	LEU453	CG	−23.0743	−13.0848	23.5134
3586	LEU453	CD1	−24.2561	−12.1686	23.8721
3587	LEU453	CD2	−23.2629	−14.4276	24.2404
3588	LEU454	N	−21.7179	−16.4119	22.538
3589	LEU454	CA	−22.0031	−17.8374	22.5715
3590	LEU454	C	−21.7649	−18.549	21.2663
3591	LEU454	O	−20.6473	−18.9583	20.9962
3592	LEU454	CB	−23.3365	−18.2012	23.258
3593	LEU454	CG	−24.5355	−17.7682	22.3939
3594	LEU454	CD1	−25.5516	−18.9204	22.3016
3595	LEU454	CD2	−25.1978	−16.5096	22.9825
3596	ASP455	N	−22.8361	−18.7049	20.4641
3597	ASP455	CA	−22.7111	−19.4269	19.2122
3598	ASP455	C	−23.9693	−19.2033	18.4191
3599	ASP455	O	−24.6883	−20.1354	18.0977
3600	ASP455	CB	−22.4065	−20.918	19.468
3601	ASP455	CG	−23.4556	−21.5473	20.3398
3602	ASP455	OD1	−23.4766	−21.2391	21.5615
3603	ASP455	OD2	−24.2562	−22.3595	19.8046
3604	LEU456	N	−24.2256	−17.9188	18.1067
3605	LEU456	CA	−25.4488	−17.5741	17.4012
3606	LEU456	C	−25.4177	−18.0274	15.967
3607	LEU456	O	−24.4079	−18.5308	15.5021
3608	LEU456	CB	−25.7189	−16.0568	17.4969
3609	LEU456	CG	−25.2245	−15.4804	18.8418
3610	LEU456	CD1	−25.6259	−14.0019	18.9853
3611	LEU456	CD2	−25.8085	−16.2725	20.0232
3612	ASN457	N	−26.5491	−17.8556	15.2576
3613	ASN457	CA	−26.6035	−18.3511	13.894
3614	ASN457	C	−26.5348	−17.2364	12.8876
3615	ASN457	O	−26.5467	−17.5147	11.6988
3616	ASN457	CB	−27.8819	−19.1935	13.7129
3617	ASN457	CG	−27.8633	−19.9255	12.4013
3618	ASN457	OD1	−28.7526	−19.734	11.5886
3619	ASN457	ND2	−26.8431	−20.773	12.1774
3620	ALA458	N	−26.4576	−15.9721	13.3534
3621	ALA458	CA	−26.3819	−14.8723	12.4045
3622	ALA458	C	−25.196	−15.0739	11.5085
3623	ALA458	O	−25.3624	−15.2125	10.3077
3624	ALA458	CB	−26.2713	−13.5328	13.1531
3625	SER459	N	−23.9936	−15.1232	12.1096
3626	SER459	CA	−22.8526	−15.5511	11.3264
3627	SER459	C	−22.8258	−17.0523	11.3748
3628	SER459	O	−22.1743	−17.633	12.2275
3629	SER459	CB	−21.5611	−14.9004	11.8587
3630	SER459	OG	−21.5207	−14.9551	13.2861
3631	GLY460	N	−23.5545	−17.6907	10.4395
3632	GLY460	CA	−23.5731	−19.1412	10.4551
3633	GLY460	C	−24.3729	−19.7103	9.3209
3634	GLY460	O	−24.3669	−20.9183	9.1474
3635	THR461	N	−25.0651	−18.8512	8.5476
3636	THR461	CA	−25.8776	−19.3898	7.4721
3637	THR461	C	−25.8192	−18.4964	6.2644
3638	THR461	O	−25.8752	−17.2885	6.42
3639	THR461	CB	−27.332	−19.5874	7.9424
3640	THR461	OG1	−27.3496	−20.3788	9.1335
3641	THR461	CG2	−28.1507	−20.3002	6.8495
3642	MET462	N	−25.7015	−19.054	5.0422
3643	MET462	CA	−25.6916	−20.5014	4.9115
3644	MET462	C	−24.3543	−21.0862	5.2649
3645	MET462	O	−24.2089	−21.6013	6.3617
3646	MET462	CB	−26.1399	−20.9827	3.5177
3647	MET462	CG	−27.1019	−19.9726	2.8654
3648	MET462	SD	−26.0964	−18.9071	1.7923
3649	MET462	CE	−26.8403	−17.3145	2.2498
3650	SER463	N	−23.3742	−21.0232	4.3405
3651	SER463	CA	−22.0933	−21.625	4.6622
3652	SER463	C	−20.9364	−21.0807	3.872
3653	SER463	O	−21.1103	−20.3086	2.9427
3654	SER463	CB	−22.1673	−23.1539	4.4857
3655	SER463	OG	−22.2279	−23.478	3.0943
3656	ILE464	N	−19.7303	−21.5325	4.2705
3657	ILE464	CA	−18.5404	−21.2112	3.5042
3658	ILE464	C	−18.6579	−21.8491	2.1499
3659	ILE464	O	−18.1781	−21.2911	1.1766
3660	ILE464	CB	−17.2947	−21.7419	4.2472
3661	ILE464	CG1	−16.0361	−21.5244	3.3814
3662	ILE464	CG2	−17.4593	−23.237	4.5853
3663	ILE464	CD1	−14.7779	−22.0522	4.0959
3664	GLN465	N	−19.3259	−23.0197	2.1078
3665	GLN465	CA	−19.5831	−23.6643	0.8319
3666	GLN465	C	−20.3295	−22.7182	−0.0651
3667	GLN465	O	−20.1229	−22.7443	−1.2678
3668	GLN465	CB	−20.444	−24.9165	1.0837
3669	GLN465	CG	−19.7163	−25.8538	2.0665
3670	GLN465	CD	−20.3202	−25.7168	3.434
3671	GLN465	OE1	−19.6984	−25.1588	4.3229
3672	GLN465	NE2	−21.5492	−26.2327	3.6147
3673	GLU466	N	−21.1788	−21.8589	0.5345
3674	GLU466	CA	−21.8194	−20.8295	−0.2637
3675	GLU466	C	−20.8655	−19.7025	−0.5599
3676	GLU466	O	−21.2575	−18.5461	−0.5642
3677	GLU466	CB	−23.1093	−20.3379	0.4195
3678	GLU466	CG	−24.3071	−21.0682	−0.2163
3679	GLU466	CD	−25.0033	−21.9491	0.7812
3680	GLU466	OE1	−26.2538	−22.0654	0.6838
3681	GLU466	OE2	−24.3149	−22.5175	1.6704
3682	PHE467	N	−19.5915	−20.0647	−0.8145
3683	PHE467	CA	−18.6057	−19.0773	−1.2217
3684	PHE467	C	−18.4992	−17.9348	−0.2519
3685	PHE467	O	−18.7028	−16.7913	−0.6279
3686	PHE467	CB	−18.8775	−18.6158	−2.6665
3687	PHE467	CG	−18.8007	−19.8306	−3.5833
3688	PHE467	CD1	−19.9284	−20.6341	−3.7711
3689	PHE467	CD2	−17.6019	−20.1412	−4.2302
3690	PHE467	CE1	−19.8486	−21.7681	−4.5831
3691	PHE467	CE2	−17.5246	−21.272	−5.0471
3692	PHE467	CZ	−18.6458	−22.0883	−5.2185
3693	ARG468	N	−18.1687	−18.2613	1.013
3694	ARG468	CA	−18.1013	−17.2092	2.0099
3695	ARG468	C	−17.3189	−17.6414	3.219
3696	ARG468	O	−17.2096	−18.8309	3.4638
3697	ARG468	CB	−19.5391	−16.8	2.3619
3698	ARG468	CG	−19.9077	−15.5407	1.5629
3699	ARG468	CD	−21.3668	−15.6322	1.0869
3700	ARG468	NE	−21.6434	−14.468	0.2675
3701	ARG468	CZ	−22.7141	−13.7518	0.4496
3702	ARG468	NH1	−22.9115	−12.7039	−0.2936
3703	ARG468	NH2	−23.5889	−14.0641	1.3599
3704	ASP469	N	−16.745	−16.6834	3.9761
3705	ASP469	CA	−15.8373	−17.0925	5.037
3706	ASP469	C	−16.05	−16.3696	6.3396
3707	ASP469	O	−16.7493	−15.3712	6.3862
3708	ASP469	CB	−14.3729	−16.9777	4.571
3709	ASP469	CG	−13.9302	−18.2086	3.8302
3710	ASP469	OD1	−12.8642	−18.7671	4.199
3711	ASP469	OD2	−14.6368	−18.6322	2.8782
3712	LEU470	N	−15.4321	−16.918	7.4061
3713	LEU470	CA	−15.6036	−16.3798	8.7478
3714	LEU470	C	−14.9378	−15.0526	8.9918
3715	LEU470	O	−14.3951	−14.435	8.0894
3716	LEU470	CB	−17.0618	−16.4526	9.2636
3717	LEU470	CG	−17.5558	−15.1004	9.8279
3718	LEU470	CD1	−17.8156	−14.1091	8.6845
3719	LEU470	CD2	−18.8011	−15.2192	10.7218
3720	TRP471	N	−15.0438	−14.6356	10.2697
3721	TRP471	CA	−14.7445	−13.2703	10.6552
3722	TRP471	C	−13.4505	−13.1482	11.3955
3723	TRP471	O	−12.402	−13.4452	10.8476
3724	TRP471	CB	−15.0216	−12.1778	9.6047
3725	TRP471	CG	−15.8341	−11.0716	10.2105
3726	TRP471	CD1	−15.4677	−9.7868	10.3272
3727	TRP471	CD2	−17.2153	−11.191	10.7969
3728	TRP471	NE1	−16.4268	−9.0953	10.8891
3729	TRP471	CE2	−17.4766	−9.8896	11.18
3730	TRP471	CE3	−18.1224	−12.2308	10.9936
3731	TRP471	CZ2	−18.6801	−9.5302	11.7862
3732	TRP471	CZ3	−19.3127	−11.8885	11.647
3733	TRP471	CH2	−19.5898	−10.5671	12.0256
3734	LYS472	N	−13.5592	−12.7134	12.6662
3735	LYS472	CA	−12.3709	−12.5281	13.4815
3736	LYS472	C	−11.6523	−11.2652	13.091
3737	LYS472	O	−11.6137	−10.9442	11.9143
3738	LYS472	CB	−11.4541	−13.7677	13.4882
3739	LYS472	CG	−10.8259	−13.9547	14.8805
3740	LYS472	CD	−9.2936	−13.8414	14.7855
3741	LYS472	CE	−8.8943	−12.4087	14.3892
3742	LYS472	NZ	−7.4446	−12.2088	14.5154
3743	GLN473	N	−11.084	−10.5054	14.0493
3744	GLN473	CA	−11.1305	−10.9221	15.4406
3745	GLN473	C	−12.4414	−10.5471	16.0684
3746	GLN473	O	−12.6715	−9.3793	16.3395
3747	GLN473	CB	−9.9771	−10.2204	16.1819
3748	GLN473	CG	−9.5473	−11.0699	17.3916
3749	GLN473	CD	−8.0631	−11.2939	17.3549
3750	GLN473	OE1	−7.6174	−12.4275	17.289
3751	GLN473	NE2	−7.2768	−10.2035	17.398
3752	LEU474	N	−13.3061	−11.5517	16.3078
3753	LEU474	CA	−14.5666	−11.2333	16.9532
3754	LEU474	C	−14.5097	−11.4497	18.434
3755	LEU474	O	−13.7945	−12.319	18.9057
3756	LEU474	CB	−15.7761	−11.9997	16.3893
3757	LEU474	CG	−15.5066	−13.5144	16.358
3758	LEU474	CD1	−16.8462	−14.2616	16.2372
3759	LEU474	CD2	−14.6256	−13.8496	15.1415
3760	LYS475	N	−15.2938	−10.6362	19.1639
3761	LYS475	CA	−15.3764	−10.8467	20.594
3762	LYS475	C	−16.4623	−11.8534	20.8451
3763	LYS475	O	−17.5127	−11.5008	21.3576
3764	LYS475	CB	−15.6919	−9.5072	21.2876
3765	LYS475	CG	−14.3884	−8.7313	21.5514
3766	LYS475	CD	−14.1716	−7.6716	20.455
3767	LYS475	CE	−13.5279	−8.3139	19.2124
3768	LYS475	NZ	−13.2432	−7.2662	18.2213
3769	LEU476	N	−16.2048	−13.1274	20.4849
3770	LEU476	CA	−17.2074	−14.1368	20.7723
3771	LEU476	C	−17.3598	−14.2772	22.2538
3772	LEU476	O	−16.3956	−14.5589	22.9461
3773	LEU476	CB	−16.9055	−15.5044	20.1284
3774	LEU476	CG	−17.8622	−16.6139	20.6251
3775	LEU476	CD1	−17.4057	−17.2144	21.9679
3776	LEU476	CD2	−19.3105	−16.1055	20.7499
3777	SER477	N	−18.6041	−14.0714	22.7157
3778	SER477	CA	−18.8438	−14.1521	24.1395
3779	SER477	C	−18.8661	−15.576	24.6074
3780	SER477	O	−19.5043	−16.4165	23.9951
3781	SER477	CB	−20.1921	−13.484	24.4527
3782	SER477	OG	−20.1822	−12.1688	23.8963
3783	GLN478	N	−18.1605	−15.8375	25.7225
3784	GLN478	CA	−18.2964	−17.1389	26.3477
3785	GLN478	C	−19.6603	−17.184	26.9769
3786	GLN478	O	−20.2313	−18.2527	27.1221
3787	GLN478	CB	−17.1909	−17.314	27.4078
3788	GLN478	CG	−15.7783	−17.1447	26.8039
3789	GLN478	CD	−15.6896	−17.5401	25.3558
3790	GLN478	OE1	−15.394	−16.7066	24.5156
3791	GLN478	NE2	−15.947	−18.8243	25.0503
3792	LYS479	N	−20.1891	−15.989	27.3126
3793	LYS479	CA	−21.5695	−15.9001	27.7579
3794	LYS479	C	−22.518	−16.3006	26.6601
3795	LYS479	O	−22.093	−16.6819	25.5809
3796	LYS479	CB	−21.9024	−14.4684	28.2308
3797	LYS479	CG	−21.1271	−13.4064	27.4257
3798	LYS479	CD	−19.8093	−13.0884	28.158
3799	LYS479	CE	−18.6074	−13.3679	27.239
3800	LYS479	NZ	−18.3823	−12.2219	26.3474
3801	VAL480	N	−23.8296	−16.2161	26.9525
3802	VAL480	CA	−24.8025	−16.6796	25.9789
3803	VAL480	C	−25.9383	−15.6936	25.8977
3804	VAL480	O	−25.7435	−14.5235	26.1871
3805	VAL480	CB	−25.2777	−18.0865	26.4048
3806	VAL480	CG1	−26.1324	−18.7344	25.2995
3807	VAL480	CG2	−24.0673	−18.9988	26.6815
3808	PHE481	N	−27.1361	−16.1703	25.5011
3809	PHE481	CA	−28.2686	−15.2688	25.3871
3810	PHE481	C	−28.5777	−14.6108	26.6995
3811	PHE481	O	−28.5512	−15.2663	27.7289
3812	PHE481	CB	−29.5093	−16.0464	24.9095
3813	PHE481	CG	−30.6455	−15.1135	24.4987
3814	PHE481	CD1	−30.405	−13.7648	24.2231
3815	PHE481	CD2	−31.9427	−15.622	24.3969
3816	PHE481	CE1	−31.4575	−12.929	23.8457
3817	PHE481	CE2	−32.9913	−14.7928	23.9908
3818	PHE481	CZ	−32.7463	−13.4484	23.7008
3819	HIS482	N	−28.8647	−13.2949	26.6425
3820	HIS482	CA	−29.1666	−12.5712	27.8657
3821	HIS482	C	−28.0142	−12.6683	28.827
3822	HIS482	O	−28.2101	−12.9102	30.0076
3823	HIS482	CB	−30.4761	−13.0913	28.4932
3824	HIS482	CG	−31.6535	−12.8039	27.6078
3825	HIS482	ND1	−32.6249	−13.674	27.4567
3826	HIS482	CD2	−31.8465	−11.6717	26.9044
3827	HIS482	CE1	−33.4945	−13.1446	26.6562
3828	HIS482	NE2	−33.1102	−12.0025	26.3046
3829	LYS483	N	−26.7884	−12.4904	28.299
3830	LYS483	CA	−25.6324	−12.6329	29.1636
3831	LYS483	C	−24.6189	−11.5883	28.8083
3832	LYS483	O	−24.1628	−11.5435	27.6766
3833	LYS483	CB	−25.0266	−14.0442	29.0363
3834	LYS483	CG	−26.1005	−15.0942	29.3754
3835	LYS483	CD	−25.5128	−16.5103	29.2494
3836	LYS483	CE	−26.6608	−17.5359	29.2614
3837	LYS483	NZ	−26.1029	−18.8953	29.2265
3838	GLN484	N	−24.2798	−10.7423	29.8014
3839	GLN484	CA	−23.3352	−9.6715	29.5338
3840	GLN484	C	−22.0236	−10.2252	29.0607
3841	GLN484	O	−21.606	−11.2796	29.5116
3842	GLN484	CB	−23.0998	−8.8037	30.7839
3843	GLN484	CG	−24.4469	−8.3374	31.367
3844	GLN484	CD	−24.5187	−8.7226	32.8162
3845	GLN484	OE1	−25.3451	−9.5393	33.1882
3846	GLN484	NE2	−23.647	−8.1311	33.6529
3847	ASP485	N	−21.3878	−9.501	28.1225
3848	ASP485	CA	−20.1652	−10.0221	27.5422
3849	ASP485	C	−18.9649	−9.6997	28.3851
3850	ASP485	O	−19.0926	−9.0979	29.4395
3851	ASP485	CB	−20.0082	−9.4693	26.1131
3852	ASP485	CG	−21.1396	−9.9172	25.2301
3853	ASP485	OD1	−21.3088	−9.3015	24.1442
3854	ASP485	OD2	−21.8672	−10.8755	25.6106
3855	ARG486	N	−17.7821	−10.1222	27.8996
3856	ARG486	CA	−16.5738	−9.915	28.6768
3857	ARG486	C	−15.3703	−10.2696	27.8524
3858	ARG486	O	−15.4489	−11.148	27.0082
3859	ARG486	CB	−16.5904	−10.7773	29.9554
3860	ARG486	CG	−16.9669	−12.2259	29.5918
3861	ARG486	CD	−16.9507	−13.1163	30.8459
3862	ARG486	NE	−16.4438	−14.423	30.4703
3863	ARG486	CZ	−15.4104	−14.941	31.0681
3864	ARG486	NH1	−14.9709	−16.1035	30.6875
3865	ARG486	NH2	−14.8105	−14.3159	32.0383
3866	GLY487	N	−14.2495	−9.5658	28.1093
3867	GLY487	CA	−13.0384	−9.848	27.3574
3868	GLY487	C	−12.5891	−11.2554	27.6213
3869	GLY487	O	−12.254	−11.9666	26.6877
3870	SER488	N	−12.6035	−11.6512	28.91
3871	SER488	CA	−12.2732	−13.0265	29.2428
3872	SER488	C	−13.2361	−13.9456	28.5477
3873	SER488	O	−12.8507	−15.0267	28.1324
3874	SER488	CB	−12.4306	−13.2055	30.7638
3875	SER488	OG	−11.5098	−12.3537	31.45
3876	GLY489	N	−14.4953	−13.4891	28.4056
3877	GLY489	CA	−15.4568	−14.2927	27.6778
3878	GLY489	C	−15.3005	−14.0949	26.1974
3879	GLY489	O	−16.3031	−13.9565	25.519
3880	TYR490	N	−14.0514	−14.0902	25.6914
3881	TYR490	CA	−13.8643	−13.9993	24.2515
3882	TYR490	C	−12.8647	−15.0311	23.8046
3883	TYR490	O	−12.3326	−15.7491	24.6367
3884	TYR490	CB	−13.3901	−12.5919	23.8351
3885	TYR490	CG	−14.4754	−11.5416	24.0603
3886	TYR490	CD1	−14.1148	−10.2607	24.4856
3887	TYR490	CD2	−15.8226	−11.8443	23.8463
3888	TYR490	CE1	−15.0993	−9.2996	24.7292
3889	TYR490	CE2	−16.8117	−10.8964	24.1178
3890	TYR490	CZ	−16.4483	−9.6229	24.562
3891	TYR490	OH	−17.4285	−8.6779	24.8375
3892	LEU491	N	−12.6034	−15.1172	22.4831
3893	LEU491	CA	−11.6638	−16.129	22.0245
3894	LEU491	C	−11.252	−15.9456	20.5902
3895	LEU491	O	−12.0934	−15.9191	19.7064
3896	LEU491	CB	−12.1723	−17.5602	22.3026
3897	LEU491	CG	−13.3597	−17.9456	21.3964
3898	LEU491	CD1	−13.8683	−19.3391	21.8069
3899	LEU491	CD2	−14.5076	−16.9305	21.5416
3900	ASN492	N	−9.9258	−15.8234	20.3828
3901	ASN492	CA	−9.4009	−15.6758	19.0336
3902	ASN492	C	−7.9853	−16.1958	19.0018
3903	ASN492	O	−7.4131	−16.4041	20.0593
3904	ASN492	CB	−9.4086	−14.1987	18.5951
3905	ASN492	CG	−10.7986	−13.6329	18.5686
3906	ASN492	OD1	−11.1194	−12.7871	19.3868
3907	ASN492	ND2	−11.639	−14.0921	17.6246
3908	TRP493	N	−7.3666	−16.4405	17.8269
3909	TRP493	CA	−8.033	−16.1681	16.5662
3910	TRP493	C	−6.9975	−15.7789	15.5421
3911	TRP493	O	−6.647	−14.6114	15.4916
3912	TRP493	CB	−8.8914	−17.3508	16.0732
3913	TRP493	CG	−9.8756	−17.8632	17.0855
3914	TRP493	CD1	−11.2065	−17.7072	17.0457
3915	TRP493	CD2	−9.5666	−18.6484	18.3321
3916	TRP493	NE1	−11.7642	−18.2709	18.0878
3917	TRP493	CE2	−10.8239	−18.8265	18.8777
3918	TRP493	CE3	−8.4064	−19.1398	18.9289
3919	TRP493	CZ2	−11.0102	−19.4955	20.0863
3920	TRP493	CZ3	−8.5843	−19.8107	20.1447
3921	TRP493	CH2	−9.8554	−19.9779	20.7139
3922	GLU494	N	−6.4974	−16.7269	14.7194
3923	GLU494	CA	−5.4994	−16.3438	13.7304
3924	GLU494	C	−5.5057	−17.2435	12.5255
3925	GLU494	O	−5.0583	−18.3768	12.5985
3926	GLU494	CB	−4.0923	−16.2476	14.3499
3927	GLU494	CG	−3.7763	−14.7825	14.7135
3928	GLU494	CD	−4.102	−13.8467	13.5824
3929	GLU494	OE1	−4.7641	−12.8096	13.8511
3930	GLU494	OE2	−3.7091	−14.1469	12.4229
3931	GLN495	N	−6.0364	−16.7035	11.4097
3932	GLN495	CA	−6.304	−17.5166	10.2357
3933	GLN495	C	−7.2692	−16.8182	9.3166
3934	GLN495	O	−7.338	−15.5996	9.3254
3935	GLN495	CB	−5.0369	−17.9776	9.493
3936	GLN495	CG	−4.8209	−19.4876	9.7211
3937	GLN495	CD	−6.0634	−20.2775	9.4157
3938	GLN495	OE1	−6.5357	−21.0175	10.2627
3939	GLN495	NE2	−6.6081	−20.1267	8.195
3940	LEU496	N	−8.0384	−17.6007	8.5327
3941	LEU496	CA	−9.0582	−16.9857	7.6964
3942	LEU496	C	−10.3289	−17.7858	7.7509
3943	LEU496	O	−10.2675	−18.9972	7.8842
3944	LEU496	CB	−8.595	−16.8349	6.2352
3945	LEU496	CG	−7.3993	−15.8684	6.14
3946	LEU496	CD1	−6.9522	−15.7636	4.6704
3947	LEU496	CD2	−7.801	−14.4732	6.6533
3948	HIS497	N	−11.4883	−17.0996	7.6701
3949	HIS497	CA	−12.7528	−17.7812	7.9109
3950	HIS497	C	−12.8025	−18.1945	9.3541
3951	HIS497	O	−12.5097	−19.3358	9.6708
3952	HIS497	CB	−12.9863	−18.9953	6.9865
3953	HIS497	CG	−14.3988	−19.4985	7.1136
3954	HIS497	ND1	−14.9898	−19.6536	8.2769
3955	HIS497	CD2	−15.1943	−19.8347	6.0807
3956	HIS497	CE1	−16.1873	−20.0931	8.0516
3957	HIS497	NE2	−16.3672	−20.2202	6.8162
3958	ALA498	N	−13.1814	−17.2429	10.2272
3959	ALA498	CA	−13.1595	−17.515	11.6542
3960	ALA498	C	−14.0791	−18.6291	12.0738
3961	ALA498	O	−14.7686	−19.2061	11.248
3962	ALA498	CB	−13.566	−16.215	12.3713
3963	ALA499	N	−14.0686	−18.913	13.3931
3964	ALA499	CA	−14.9678	−19.9106	13.9554
3965	ALA499	C	−14.5838	−20.2116	15.3775
3966	ALA499	O	−13.7636	−19.5168	15.957
3967	ALA499	CB	−14.9691	−21.2336	13.1688
3968	MET500	N	−15.1933	−21.276	15.9393
3969	MET500	CA	−14.9391	−21.6046	17.3319
3970	MET500	C	−15.426	−20.4754	18.1944
3971	MET500	O	−14.7312	−20.0207	19.0888
3972	MET500	CB	−13.4583	−21.9532	17.5791
3973	MET500	CG	−13.3701	−22.9759	18.7261
3974	MET500	SD	−12.7965	−22.1232	20.2218
3975	MET500	CE	−12.6583	−23.5784	21.2996
3976	ARG501	N	−16.6523	−20.0177	17.8795
3977	ARG501	CA	−17.1808	−18.8494	18.5571
3978	ARG501	C	−18.6731	−18.8256	18.3681
3979	ARG501	O	−19.2805	−19.8657	18.1664
3980	ARG501	CB	−16.5623	−17.6128	17.8727
3981	ARG501	CG	−15.1516	−17.3417	18.4288
3982	ARG501	CD	−14.67	−15.9651	17.9376
3983	ARG501	NE	−13.573	−16.1153	16.9993
3984	ARG501	CZ	−13.7607	−16.5824	15.7985
3985	ARG501	NH1	−12.7489	−16.6829	14.9893
3986	ARG501	NH2	−14.9398	−16.9495	15.3919
3987	GLU502	N	−19.2645	−17.6149	18.4157
3988	GLU502	CA	−20.6847	−17.4904	18.1419
3989	GLU502	C	−20.9358	−18.0166	16.7604
3990	GLU502	O	−21.946	−18.6548	16.5213
3991	GLU502	CB	−21.034	−15.992	18.1823
3992	GLU502	CG	−22.4733	−15.7624	17.6882
3993	GLU502	CD	−22.4706	−15.3281	16.2512
3994	GLU502	OE1	−23.0568	−16.062	15.4118
3995	GLU502	OE2	−21.886	−14.2506	15.958
3996	ALA503	N	−19.9852	−17.7542	15.8483
3997	ALA503	CA	−20.1081	−18.3485	14.5355
3998	ALA503	C	−19.1648	−19.5142	14.4331
3999	ALA503	O	−18.1384	−19.5231	15.095
4000	ALA503	CB	−19.7811	−17.2624	13.5006
4001	GLY504	N	−19.5234	−20.5169	13.6078
4002	GLY504	CA	−18.6468	−21.6697	13.4978
4003	GLY504	C	−19.42	−22.9573	13.4308
4004	GLY504	O	−20.6152	−22.9529	13.1838
4005	ARG505	N	−18.7084	−24.0801	13.6488
4006	ARG505	CA	−19.3631	−25.3686	13.5231
4007	ARG505	C	−20.074	−25.7173	14.7992
4008	ARG505	O	−19.5131	−26.3771	15.6597
4009	ARG505	CB	−18.3277	−26.4418	13.1353
4010	ARG505	CG	−19.0675	−27.7271	12.72
4011	ARG505	CD	−18.0544	−28.8431	12.4085
4012	ARG505	NE	−17.7481	−28.8403	10.9898
4013	ARG505	CZ	−16.5174	−28.8541	10.5667
4014	ARG505	NH1	−16.2831	−28.843	9.2882
4015	ARG505	NH2	−15.5188	−28.8751	11.4002
4016	HIS506	N	−21.3392	−25.2707	14.9051
4017	HIS506	CA	−22.0972	−25.5838	16.103
4018	HIS506	C	−23.5704	−25.4112	15.8649
4019	HIS506	O	−23.9709	−24.8666	14.8481
4020	HIS506	CB	−21.6667	−24.6447	17.2475
4021	HIS506	CG	−21.8326	−23.2214	16.8002
4022	HIS506	ND1	−20.8661	−22.5673	16.199
4023	HIS506	CD2	−22.9533	−22.4907	16.9519
4024	HIS506	CE1	−21.3155	−21.3829	15.9318
4025	HIS506	NE2	−22.4991	−21.2742	16.3357
4026	ARG507	N	−24.3781	−25.8767	16.8381
4027	ARG507	CA	−25.8061	−25.6307	16.7529
4028	ARG507	C	−26.0315	−24.1818	17.0795
4029	ARG507	O	−26.2062	−23.8255	18.2343
4030	ARG507	CB	−26.5313	−26.5302	17.7719
4031	ARG507	CG	−26.5793	−27.9768	17.2479
4032	ARG507	CD	−27.4735	−28.8176	18.177
4033	ARG507	NE	−27.5376	−30.1818	17.6859
4034	ARG507	CZ	−27.1634	−31.1823	18.4298
4035	ARG507	NH1	−27.2354	−32.3908	17.9563
4036	ARG507	NH2	−26.7191	−30.9953	19.6381
4037	LYS508	N	−26.0164	−23.3389	16.03
4038	LYS508	CA	−26.1133	−21.911	16.2711
4039	LYS508	C	−27.4339	−21.5158	16.8629
4040	LYS508	O	−28.4323	−22.1882	16.6601
4041	LYS508	CB	−25.8432	−21.1072	14.9894
4042	LYS508	CG	−24.5525	−21.6192	14.3297
4043	LYS508	CD	−24.0293	−20.5702	13.3357
4044	LYS508	CE	−22.737	−21.1133	12.7056
4045	LYS508	NZ	−23.0643	−22.1664	11.7337
4046	SER509	N	−27.4122	−20.4063	17.6226
4047	SER509	CA	−28.6287	−19.9826	18.289
4048	SER509	C	−29.2901	−18.8631	17.5368
4049	SER509	O	−28.8376	−18.4787	16.4708
4050	SER509	CB	−28.2766	−19.5319	19.7192
4051	SER509	OG	−27.1009	−18.72	19.6917
4052	TRP510	N	−30.3851	−18.3351	18.1135
4053	TRP510	CA	−31.0793	−17.2525	17.4436
4054	TRP510	C	−31.6712	−16.3401	18.4731
4055	TRP510	O	−31.5666	−15.1336	18.328
4056	TRP510	CB	−32.1576	−17.8213	16.506
4057	TRP510	CG	−31.5471	−17.9643	15.1431
4058	TRP510	CD1	−31.6121	−19.0333	14.3346
4059	TRP510	CD2	−30.7387	−16.9143	14.4302
4060	TRP510	NE1	−30.9688	−18.7879	13.2198
4061	TRP510	CE2	−30.4479	−17.5436	13.235
4062	TRP510	CE3	−30.3098	−15.6237	14.7371
4063	TRP510	CZ2	−29.7138	−16.9085	12.2337
4064	TRP510	CZ3	−29.5691	−14.98	13.7383
4065	TRP510	CH2	−29.298	−15.6002	12.5096
4066	SER511	N	−32.2648	−16.9216	19.5345
4067	SER511	CA	−32.6247	−16.0935	20.6714
4068	SER511	C	−31.3371	−15.569	21.239
4069	SER511	O	−31.1914	−14.3694	21.4039
4070	SER511	CB	−33.3761	−16.9395	21.7181
4071	SER511	OG	−32.5848	−18.0604	22.1239
4072	CYS512	N	−30.3893	−16.4931	21.4971
4073	CYS512	CA	−29.054	−16.0688	21.8787
4074	CYS512	C	−28.4923	−15.2338	20.7654
4075	CYS512	O	−27.8063	−14.2613	21.0336
4076	CYS512	CB	−28.1483	−17.3054	22.0288
4077	CYS512	SG	−28.9325	−18.5393	23.1075
4078	GLY513	N	−28.8125	−15.6145	19.5098
4079	GLY513	CA	−28.404	−14.7965	18.3796
4080	GLY513	C	−28.8091	−13.3673	18.6036
4081	GLY513	O	−28.0983	−12.4604	18.202
4082	HIS514	N	−29.9612	−13.1856	19.2758
4083	HIS514	CA	−30.3937	−11.8433	19.6082
4084	HIS514	C	−29.7979	−11.4447	20.9308
4085	HIS514	O	−30.5143	−11.0878	21.8524
4086	HIS514	CB	−31.9352	−11.8143	19.6557
4087	HIS514	CG	−32.5477	−12.2504	18.3549
4088	HIS514	ND1	−33.8314	−12.5101	18.2644
4089	HIS514	CD2	−31.8956	−12.4211	17.188
4090	HIS514	CE1	−34.0697	−12.8611	17.0406
4091	HIS514	NE2	−33.0067	−12.8313	16.3739
4092	THR515	N	−28.4557	−11.5005	21.0224
4093	THR515	CA	−27.8194	−11.0067	22.2293
4094	THR515	C	−26.3531	−10.7594	22.0257
4095	THR515	O	−25.697	−11.4658	21.2771
4096	THR515	CB	−28.1139	−11.8416	23.4938
4097	THR515	OG1	−27.8485	−11.0635	24.664
4098	THR515	CG2	−27.2537	−13.119	23.5284
4099	ARG516	N	−25.8659	−9.7201	22.7263
4100	ARG516	CA	−24.4561	−9.3847	22.6753
4101	ARG516	C	−24.1989	−8.6831	23.9778
4102	ARG516	O	−24.063	−7.4704	24.0149
4103	ARG516	CB	−24.1919	−8.4614	21.4679
4104	ARG516	CG	−24.1308	−9.2883	20.1698
4105	ARG516	CD	−22.8747	−10.1783	20.1863
4106	ARG516	NE	−23.2084	−11.5089	19.7188
4107	ARG516	CZ	−22.8227	−11.9319	18.5506
4108	ARG516	NH1	−23.1216	−13.1413	18.1896
4109	ARG516	NH2	−22.1438	−11.1823	17.7344
4110	ALA517	N	−24.1631	−9.4895	25.0588
4111	ALA517	CA	−24.1682	−8.9339	26.4027
4112	ALA517	C	−25.5826	−8.5546	26.7308
4113	ALA517	O	−26.1868	−9.1632	27.5994
4114	ALA517	CB	−23.1864	−7.7643	26.6139
4115	GLY518	N	−26.1113	−7.5522	26.0026
4116	GLY518	CA	−27.5232	−7.2444	26.1371
4117	GLY518	C	−28.2821	−7.9608	25.0554
4118	GLY518	O	−27.716	−8.263	24.0173
4119	CYS519	N	−29.5766	−8.2345	25.3095
4120	CYS519	CA	−30.3711	−8.9519	24.3259
4121	CYS519	C	−30.5352	−8.1275	23.0777
4122	CYS519	O	−31.4697	−7.3499	22.9756
4123	CYS519	CB	−31.7408	−9.2363	24.9747
4124	CYS519	SG	−32.8006	−10.1165	23.7893
4125	THR520	N	−29.6209	−8.3119	22.1069
4126	THR520	CA	−29.7507	−7.5622	20.8694
4127	THR520	C	−30.8517	−8.1256	20.0101
4128	THR520	O	−31.5034	−9.0805	20.3994
4129	THR520	CB	−28.394	−7.5683	20.1334
4130	THR520	OG1	−28.4296	−6.678	19.0149
4131	THR520	CG2	−28.0242	−8.9858	19.6561
4132	LEU521	N	−31.0527	−7.5245	18.8205
4133	LEU521	CA	−31.9831	−8.0999	17.8603
4134	LEU521	C	−33.3511	−8.3288	18.4454
4135	LEU521	O	−33.9048	−9.4088	18.3106
4136	LEU521	CB	−31.4089	−9.3888	17.2327
4137	LEU521	CG	−29.882	−9.2979	17.0395
4138	LEU521	CD1	−29.3723	−10.6018	16.3999
4139	LEU521	CD2	−29.53	−8.1117	16.1223
4140	ILE522	N	−33.8974	−7.2926	19.1103
4141	ILE522	CA	−35.2004	−7.4612	19.7307
4142	ILE522	C	−35.9061	−6.1378	19.7926
4143	ILE522	O	−37.0732	−6.0582	19.4452
4144	ILE522	CB	−35.056	−8.0071	21.1669
4145	ILE522	CG1	−34.1288	−7.105	22.0053
4146	ILE522	CG2	−34.5223	−9.4518	21.1494
4147	ILE522	CD1	−34.4094	−7.314	23.5048
4148	ARG523	N	−35.1764	−5.0996	20.246
4149	ARG523	CA	−35.7994	−3.8012	20.4375
4150	ARG523	C	−36.2561	−3.1922	19.1412
4151	ARG523	O	−35.9804	−3.7237	18.0772
4152	ARG523	CB	−34.7842	−2.8678	21.1238
4153	ARG523	CG	−34.2737	−3.5234	22.4214
4154	ARG523	CD	−32.8623	−2.9969	22.7386
4155	ARG523	NE	−32.2864	−3.7696	23.8244
4156	ARG523	CZ	−31.2373	−4.5175	23.6371
4157	ARG523	NH1	−30.746	−5.1887	24.6364
4158	ARG523	NH2	−30.6715	−4.607	22.4691
4159	GLN524	N	−36.9758	−2.0581	19.2423
4160	GLN524	CA	−37.4924	−1.4475	18.0298
4161	GLN524	C	−37.3061	0.0429	18.0658
4162	GLN524	O	−36.8246	0.5916	19.0436
4163	GLN524	CB	−38.9918	−1.7647	17.8648
4164	GLN524	CG	−39.2516	−3.2706	18.0637
4165	GLN524	CD	−39.684	−3.5165	19.4805
4166	GLN524	OE1	−38.9756	−4.1667	20.231
4167	GLN524	NE2	−40.8636	−2.9935	19.8606

TABLE V


Atom No	Residue	Atom name	x coord	y coord	z coord

1	LYS12	CA	−26.781	19.61	−5.574
2	LYS12	C	−27.853	18.529	−5.301
3	LYS12	N	−27.307	20.9	−5.159
4	LYS12	O	−27.815	17.778	−4.324
5	LYS12	CB	−25.462	19.387	−4.819
6	LYS12	CG	−24.469	18.447	−5.526
7	LYS12	CD	−24.861	16.966	−5.511
8	LYS12	CE	−23.82	16.057	−6.165
9	LYS12	NZ	−22.573	16.005	−5.369
10	LEU13	N	−28.901	18.567	−6.21
11	LEU13	CA	−30.014	17.612	−6.169
12	LEU13	C	−29.844	16.617	−7.329
13	LEU13	O	−29.62	15.424	−7.12
14	LEU13	CB	−31.372	18.325	−6.222
15	LEU13	CG	−31.708	19.142	−4.957
16	LEU13	CD1	−33.003	19.928	−5.179
17	LEU13	CD2	−31.852	18.264	−3.713
18	ALA14	N	−29.896	17.156	−8.611
19	ALA14	CA	−30.062	16.233	−9.738
20	ALA14	C	−28.974	15.148	−9.82
21	ALA14	O	−29.304	13.976	−10.052
22	ALA14	CB	−30.199	16.966	−11.065
23	PRO15	N	−27.648	15.468	−9.615
24	PRO15	CA	−26.614	14.433	−9.739
25	PRO15	C	−26.577	13.384	−8.613
26	PRO15	O	−25.683	12.539	−8.564
27	PRO15	CB	−25.291	15.205	−9.757
28	PRO15	CG	−25.689	16.611	−10.179
29	PRO15	CD	−27.049	16.79	−9.529
30	ARG16	N	−27.611	13.432	−7.698
31	ARG16	CA	−27.889	12.313	−6.808
32	ARG16	C	−28.719	11.222	−7.523
33	ARG16	O	−28.811	10.092	−7.04
34	ARG16	CB	−28.669	12.759	−5.565
35	ARG16	CG	−27.998	13.889	−4.779
36	ARG16	CD	−28.841	14.271	−3.563
37	ARG16	NE	−28.553	15.641	−3.125
38	ARG16	CZ	−28.879	16.152	−1.919
39	ARG16	NH1	−28.577	17.443	−1.675
40	ARG16	NH2	−29.498	15.43	−0.966
41	TYR17	N	−29.447	11.628	−8.629
42	TYR17	CA	−30.387	10.734	−9.307
43	TYR17	C	−30.365	10.753	−10.847
44	TYR17	O	−30.892	9.844	−11.493
45	TYR17	CB	−31.82	10.861	−8.763
46	TYR17	CG	−32.42	12.247	−8.83
47	TYR17	CD1	−33.026	12.713	−10.005
48	TYR17	CD2	−32.399	13.079	−7.7
49	TYR17	CE1	−33.605	13.985	−10.05
50	TYR17	CE2	−32.976	14.348	−7.743
51	TYR17	CZ	−33.575	14.791	−8.915
52	TYR17	OH	−34.113	16.045	−8.919
53	SER18	N	−29.786	11.842	−11.463
54	SER18	CA	−29.362	11.756	−12.86
55	SER18	C	−28.037	10.969	−12.906
56	SER18	O	−27.418	10.622	−11.898
57	SER18	CB	−29.251	13.135	−13.513
58	SER18	OG	−28.18	13.894	−12.961
59	ARG19	N	−27.608	10.612	−14.178
60	ARG19	CA	−26.407	9.799	−14.302
61	ARG19	C	−25.186	10.715	−14.173
62	ARG19	O	−25.093	11.798	−14.749
63	ARG19	CB	−26.36	9.052	−15.639
64	ARG19	CG	−27.385	7.915	−15.684
65	ARG19	CD	−27.352	7.176	−17.017
66	ARG19	NE	−28.229	5.993	−17.009
67	ARG19	CZ	−29.584	6.014	−17.06
68	ARG19	NH1	−30.264	4.849	−17.011
69	ARG19	NH2	−30.28	7.164	−17.165
70	ARG20	N	−24.137	10.172	−13.44
71	ARG20	CA	−23.074	11.059	−12.946
72	ARG20	C	−22.105	11.601	−14.025
73	ARG20	O	−21.196	12.384	−13.749
74	ARG20	CB	−22.298	10.391	−11.796
75	ARG20	CG	−21.451	9.183	−12.222
76	ARG20	CD	−21.741	7.92	−11.41
77	ARG20	NE	−21.225	7.976	−10.034
78	ARG20	CZ	−21.316	6.918	−9.177
79	ARG20	NH1	−20.707	6.953	−7.974
80	ARG20	NH2	−22.006	5.801	−9.504
81	ALA21	N	−22.342	11.167	−15.311
82	ALA21	CA	−21.702	11.786	−16.466
83	ALA21	C	−22.401	13.094	−16.884
84	ALA21	O	−21.802	13.973	−17.513
85	ALA21	CB	−21.7	10.815	−17.638
86	SER22	N	−23.748	13.189	−16.596
87	SER22	CA	−24.595	14.209	−17.204
88	SER22	C	−24.187	15.655	−16.886
89	SER22	O	−24.338	16.517	−17.76
90	SER22	CB	−26.076	14.009	−16.861
91	SER22	OG	−26.564	12.755	−17.346
92	PRO23	N	−23.727	15.993	−15.627
93	PRO23	CA	−23.271	17.363	−15.354
94	PRO23	C	−21.806	17.591	−15.778
95	PRO23	O	−21.311	18.716	−15.797
96	PRO23	CB	−23.413	17.512	−13.839
97	PRO23	CG	−23.201	16.095	−13.327
98	PRO23	CD	−23.881	15.239	−14.385
99	GLN24	N	−21.082	16.438	−16.016
100	GLN24	CA	−19.678	16.471	−16.414
101	GLN24	C	−19.544	16.633	−17.939
102	GLN24	O	−18.62	17.275	−18.441
103	GLN24	CB	−18.95	15.209	−15.942
104	GLN24	CG	−18.96	15.085	−14.417
105	GLN24	CD	−18.096	13.933	−13.964
106	GLN24	OE1	−16.973	14.097	−13.504
107	GLN24	NE2	−18.651	12.698	−14.155
108	GLN25	N	−20.485	15.967	−18.706
109	GLN25	CA	−20.388	15.96	−20.167
110	GLN25	C	−20.253	17.391	−20.748
111	GLN25	O	−19.364	17.623	−21.581
112	GLN25	CB	−21.577	15.23	−20.803
113	GLN25	CG	−21.36	13.719	−20.86
114	GLN25	CD	−22.653	13.001	−21.172
115	GLN25	OE1	−23.272	12.342	−20.342
116	GLN25	NE2	−23.097	13.155	−22.455
117	PRO26	N	−21.115	18.387	−20.32
118	PRO26	CA	−20.991	19.766	−20.799
119	PRO26	C	−19.946	20.596	−20.02
120	PRO26	O	−20.004	21.825	−19.936
121	PRO26	CB	−22.395	20.35	−20.632
122	PRO26	CG	−22.91	19.622	−19.398
123	PRO26	CD	−22.368	18.214	−19.591
124	GLN27	N	−18.849	19.895	−19.569
125	GLN27	CA	−17.613	20.528	−19.115
126	GLN27	C	−16.463	19.898	−19.929
127	GLN27	O	−15.331	19.73	−19.484
128	GLN27	CB	−17.416	20.396	−17.603
129	GLN27	CG	−18.523	21.051	−16.774
130	GLN27	CD	−18.442	22.561	−16.807
131	GLN27	OE1	−17.735	23.205	−16.038
132	GLN27	NE2	−19.197	23.17	−17.767
133	GLN28	N	−16.808	19.71	−21.262
134	GLN28	CA	−15.945	19.054	−22.232
135	GLN28	C	−16.691	18.924	−23.579
136	GLN28	O	−16.447	19.642	−24.544
137	GLN28	CB	−14.567	19.723	−22.419
138	GLN28	CG	−14.595	21.247	−22.555
139	GLN28	CD	−13.26	21.765	−23.043
140	GLN28	OE1	−13.065	22.155	−24.189
141	GLN28	NE2	−12.242	21.746	−22.12
142	ASP29	N	−17.633	17.907	−23.602
143	ASP29	CA	−18.243	17.418	−24.852
144	ASP29	C	−17.114	16.809	−25.738
145	ASP29	O	−15.94	16.761	−25.373
146	ASP29	CB	−19.095	18.476	−25.541
147	ASP29	CG	−20.157	17.743	−26.352
148	ASP29	OD1	−21.32	17.755	−25.873
149	ASP29	OD2	−19.723	17.189	−27.416
150	PHE30	N	−17.515	16.262	−26.934
151	PHE30	CA	−16.585	15.682	−27.911
152	PHE30	C	−17.336	15.656	−29.244
153	PHE30	O	−16.883	16.173	−30.268
154	PHE30	CB	−16.091	14.297	−27.475
155	PHE30	CG	−15.177	13.595	−28.452
156	PHE30	CD1	−13.793	13.795	−28.41
157	PHE30	CD2	−15.69	12.688	−29.387
158	PHE30	CE1	−12.94	13.077	−29.25
159	PHE30	CE2	−14.838	11.962	−30.221
160	PHE30	CZ	−13.461	12.156	−30.153
161	GLU31	N	−18.571	15.047	−29.206
162	GLU31	CA	−19.371	14.861	−30.403
163	GLU31	C	−19.84	16.233	−30.919
164	GLU31	O	−19.914	16.467	−32.129
165	GLU31	CB	−20.581	13.945	−30.146
166	GLU31	CG	−20.204	12.473	−29.906
167	GLU31	CD	−19.422	12.179	−28.631
168	GLU31	OE1	−18.905	11.018	−28.535
169	GLU31	OE2	−19.275	13.143	−27.826
170	ALA32	N	−20.23	17.156	−29.959
171	ALA32	CA	−20.695	18.481	−30.361
172	ALA32	C	−19.529	19.381	−30.805
173	ALA32	O	−19.719	20.395	−31.476
174	ALA32	CB	−21.475	19.175	−29.256
175	LEU33	N	−18.285	19.002	−30.336
176	LEU33	CA	−17.064	19.644	−30.808
177	LEU33	C	−16.734	19.113	−32.211
178	LEU33	O	−16.436	19.883	−33.13
179	LEU33	CB	−15.891	19.427	−29.85
180	LEU33	CG	−16.068	20.121	−28.484
181	LEU33	CD1	−14.97	19.656	−27.528
182	LEU33	CD2	−16.034	21.645	−28.603
183	LEU34	N	−16.806	17.743	−32.408
184	LEU34	CA	−16.484	17.2	−33.728
185	LEU34	C	−17.468	17.783	−34.767
186	LEU34	O	−17.124	18.027	−35.925
187	LEU34	CB	−16.582	15.672	−33.798
188	LEU34	CG	−15.405	14.894	−33.178
189	LEU34	CD1	−15.661	13.393	−33.352
190	LEU34	CD2	−14.056	15.246	−33.805
191	ALA35	N	−18.765	17.953	−34.31
192	ALA35	CA	−19.818	18.481	−35.163
193	ALA35	C	−19.724	20.006	−35.38
194	ALA35	O	−20.487	20.59	−36.158
195	ALA35	CB	−21.195	18.145	−34.609
196	GLU36	N	−18.78	20.682	−34.645
197	GLU36	CA	−18.303	22.015	−35.021
198	GLU36	C	−17.186	21.785	−36.053
199	GLU36	O	−17.234	22.262	−37.192
200	GLU36	CB	−17.861	22.801	−33.778
201	GLU36	CG	−17.443	24.24	−34.073
202	GLU36	CD	−16.03	24.44	−34.627
203	GLU36	OE1	−15.172	23.573	−34.311
204	GLU36	OE2	−15.902	25.463	−35.368
205	CYS37	N	−16.146	20.978	−35.621
206	CYS37	CA	−14.854	21.039	−36.301
207	CYS37	C	−14.982	20.521	−37.736
208	CYS37	O	−14.408	21.084	−38.67
209	CYS37	CB	−13.796	20.222	−35.56
210	CYS37	SG	−13.318	20.986	−33.971
211	LEU38	N	−15.724	19.365	−37.893
212	LEU38	CA	−15.839	18.71	−39.193
213	LEU38	C	−16.86	19.396	−40.122
214	LEU38	O	−16.939	19.086	−41.311
215	LEU38	CB	−16.189	17.219	−39.063
216	LEU38	CG	−14.967	16.304	−38.849
217	LEU38	CD1	−14.173	16.629	−37.586
218	LEU38	CD2	−15.421	14.844	−38.807
219	ARG39	N	−17.699	20.323	−39.529
220	ARG39	CA	−18.489	21.215	−40.371
221	ARG39	C	−17.655	22.435	−40.786
222	ARG39	O	−17.723	22.893	−41.927
223	ARG39	CB	−19.765	21.696	−39.67
224	ARG39	CG	−20.879	20.645	−39.722
225	ARG39	CD	−22.192	21.193	−39.164
226	ARG39	NE	−22.073	21.461	−37.731
227	ARG39	CZ	−22.814	22.311	−37
228	ARG39	NH1	−22.502	22.479	−35.699
229	ARG39	NH2	−23.855	22.991	−37.519
230	ASN40	N	−16.956	23.056	−39.765
231	ASN40	CA	−16.365	24.376	−39.99
232	ASN40	C	−15.001	24.28	−40.69
233	ASN40	O	−14.541	25.23	−41.324
234	ASN40	CB	−16.187	25.141	−38.682
235	ASN40	CG	−17.481	25.764	−38.204
236	ASN40	OD1	−18.559	25.676	−38.782
237	ASN40	ND2	−17.346	26.478	−37.045
238	GLY41	N	−14.267	23.143	−40.414
239	GLY41	CA	−12.89	23.005	−40.854
240	GLY41	C	−11.898	23.611	−39.86
241	GLY41	O	−10.739	23.874	−40.176
242	CYS42	N	−12.38	23.728	−38.571
243	CYS42	CA	−11.519	24.131	−37.462
244	CYS42	C	−10.8	22.878	−36.938
245	CYS42	O	−11.253	21.744	−37.079
246	CYS42	CB	−12.322	24.733	−36.301
247	CYS42	SG	−13.088	26.325	−36.732
248	LEU43	N	−9.636	23.151	−36.236
249	LEU43	CA	−9.096	22.166	−35.302
250	LEU43	C	−9.601	22.594	−33.916
251	LEU43	O	−9.9	23.761	−33.659
252	LEU43	CB	−7.562	22.161	−35.29
253	LEU43	CG	−6.909	21.736	−36.619
254	LEU43	CD1	−5.39	21.902	−36.521
255	LEU43	CD2	−7.243	20.293	−36.997
256	PHE44	N	−9.616	21.585	−32.979
257	PHE44	CA	−10.012	21.847	−31.602
258	PHE44	C	−8.796	22.437	−30.875
259	PHE44	O	−7.718	21.842	−30.797
260	PHE44	CB	−10.433	20.557	−30.896
261	PHE44	CG	−10.682	20.72	−29.416
262	PHE44	CD1	−11.728	21.521	−28.942
263	PHE44	CD2	−9.854	20.066	−28.491
264	PHE44	CE1	−11.942	21.656	−27.57
265	PHE44	CE2	−10.09	20.179	−27.123
266	PHE44	CZ	−11.134	20.973	−26.663
267	GLU45	N	−9.013	23.684	−30.325
268	GLU45	CA	−8.117	24.256	−29.33
269	GLU45	C	−8.867	24.183	−27.994
270	GLU45	O	−10.022	24.587	−27.87
271	GLU45	CB	−7.785	25.72	−29.653
272	GLU45	CG	−6.759	25.864	−30.78
273	GLU45	CD	−5.434	25.234	−30.385
274	GLU45	OE1	−4.99	25.529	−29.227
275	GLU45	OE2	−4.95	24.381	−31.195
276	ASP46	N	−8.134	23.636	−26.957
277	ASP46	CA	−8.68	23.586	−25.607
278	ASP46	C	−8.389	24.957	−24.968
279	ASP46	O	−7.252	25.424	−24.869
280	ASP46	CB	−8.053	22.452	−24.808
281	ASP46	CG	−8.813	22.241	−23.517
282	ASP46	OD1	−8.966	23.266	−22.785
283	ASP46	OD2	−9.206	21.058	−23.267
284	THR47	N	−9.545	25.589	−24.549
285	THR47	CA	−9.557	26.883	−23.87
286	THR47	C	−9.369	26.703	−22.354
287	THR47	O	−9.032	27.646	−21.638
288	THR47	CB	−10.923	27.57	−24.096
289	THR47	OG1	−11.445	27.165	−25.369
290	THR47	CG2	−10.813	29.09	−24.071
291	SER48	N	−9.771	25.476	−21.842
292	SER48	CA	−9.616	25.195	−20.406
293	SER48	C	−8.158	24.878	−20.053
294	SER48	O	−7.708	25.08	−18.927
295	SER48	CB	−10.481	23.997	−20.005
296	SER48	OG	−11.808	24.115	−20.522
297	PHE49	N	−7.444	24.246	−21.05
298	PHE49	CA	−6.033	23.891	−20.932
299	PHE49	C	−5.262	24.642	−22.045
300	PHE49	O	−4.968	24.108	−23.124
301	PHE49	CB	−5.884	22.368	−21.029
302	PHE49	CG	−4.649	21.869	−20.32
303	PHE49	CD1	−4.611	21.823	−18.919
304	PHE49	CD2	−3.528	21.454	−21.044
305	PHE49	CE1	−3.482	21.342	−18.255
306	PHE49	CE2	−2.404	20.966	−20.375
307	PHE49	CZ	−2.382	20.901	−18.983
308	PRO50	N	−4.967	25.977	−21.797
309	PRO50	CA	−4.379	26.826	−22.826
310	PRO50	C	−2.846	26.69	−22.873
311	PRO50	O	−2.174	26.135	−22.003
312	PRO50	CB	−4.778	28.243	−22.411
313	PRO50	CG	−4.787	28.153	−20.889
314	PRO50	CD	−5.383	26.774	−20.646
315	ALA51	N	−2.291	27.32	−23.972
316	ALA51	CA	−0.862	27.308	−24.27
317	ALA51	C	−0.137	28.512	−23.632
318	ALA51	O	0.623	29.243	−24.266
319	ALA51	CB	−0.667	27.281	−25.782
320	THR52	N	−0.343	28.652	−22.272
321	THR52	CA	0.396	29.632	−21.46
322	THR52	C	1.448	28.845	−20.633
323	THR52	O	1.924	27.777	−21.027
324	THR52	CB	−0.56	30.482	−20.595
325	THR52	OG1	−1.056	29.691	−19.513
326	THR52	CG2	−1.734	31.065	−21.37
327	LEU53	N	1.866	29.437	−19.452
328	LEU53	CA	2.574	28.681	−18.418
329	LEU53	C	1.6	28.189	−17.317
330	LEU53	O	1.975	27.466	−16.389
331	LEU53	CB	3.658	29.545	−17.754
332	LEU53	CG	4.727	30.104	−18.714
333	LEU53	CD1	5.702	30.996	−17.939
334	LEU53	CD2	5.501	29	−19.432
335	SER54	N	0.291	28.613	−17.427
336	SER54	CA	−0.681	28.479	−16.33
337	SER54	C	−1.097	27.015	−16.162
338	SER54	O	−1.541	26.571	−15.109
339	SER54	CB	−1.966	29.285	−16.585
340	SER54	OG	−1.692	30.559	−17.187
341	SER55	N	−1.042	26.303	−17.334
342	SER55	CA	−1.355	24.887	−17.495
343	SER55	C	−0.139	24.005	−17.146
344	SER55	O	−0.226	22.783	−17.014
345	SER55	CB	−1.784	24.668	−18.951
346	SER55	OG	−1.052	25.563	−19.803
347	ILE56	N	1.066	24.675	−17.053
348	ILE56	CA	2.278	24.001	−16.601
349	ILE56	C	2.33	24.124	−15.072
350	ILE56	O	2.39	23.12	−14.359
351	ILE56	CB	3.557	24.544	−17.287
352	ILE56	CG1	3.357	24.643	−18.815
353	ILE56	CG2	4.77	23.668	−16.944
354	ILE56	CD1	4.592	25.09	−19.577
355	GLY57	N	2.367	25.424	−14.596
356	GLY57	CA	2.7	25.698	−13.211
357	GLY57	C	4.2	25.489	−12.997
358	GLY57	O	4.879	24.744	−13.702
359	SER58	N	4.733	26.171	−11.92
360	SER58	CA	6.169	26.079	−11.675
361	SER58	C	6.538	26.776	−10.355
362	SER58	O	6.339	27.974	−10.161
363	SER58	CB	6.996	26.674	−12.826
364	SER58	OG	6.453	27.908	−13.297
365	GLY59	N	7.11	25.938	−9.41
366	GLY59	CA	7.488	26.438	−8.1
367	GLY59	C	8.997	26.684	−7.979
368	GLY59	O	9.805	25.782	−7.739
369	SER60	N	9.412	27.977	−8.223
370	SER60	CA	10.761	28.487	−7.965
371	SER60	C	11.834	27.742	−8.76
372	SER60	O	12.293	28.182	−9.813
373	SER60	CB	11.133	28.6	−6.484
374	SER60	OG	12.351	29.365	−6.324
375	LEU61	N	12.211	26.514	−8.255
376	LEU61	CA	13.268	25.74	−8.903
377	LEU61	C	12.877	25.429	−10.352
378	LEU61	O	13.704	25.396	−11.26
379	LEU61	CB	13.547	24.434	−8.149
380	LEU61	CG	14.084	24.624	−6.716
381	LEU61	CD1	14.196	23.264	−6.022
382	LEU61	CD2	15.443	25.326	−6.691
383	LEU62	N	11.543	25.141	−10.533
384	LEU62	CA	11.016	24.696	−11.813
385	LEU62	C	10.583	25.875	−12.708
386	LEU62	O	10.123	25.694	−13.835
387	LEU62	CB	9.873	23.701	−11.583
388	LEU62	CG	10.292	22.481	−10.728
389	LEU62	CD1	9.07	21.645	−10.371
390	LEU62	CD2	11.333	21.609	−11.429
391	GLN63	N	10.796	27.141	−12.19
392	GLN63	CA	10.621	28.352	−12.999
393	GLN63	C	11.905	28.667	−13.786
394	GLN63	O	11.905	29.443	−14.74
395	GLN63	CB	10.28	29.576	−12.132
396	GLN63	CG	8.901	29.468	−11.493
397	GLN63	CD	8.643	30.538	−10.459
398	GLN63	OE1	8.52	30.29	−9.262
399	GLN63	NE2	8.574	31.811	−10.947
400	LYS64	N	13.058	28.134	−13.247
401	LYS64	CA	14.394	28.557	−13.668
402	LYS64	C	14.973	27.615	−14.741
403	LYS64	O	15.964	27.927	−15.399
404	LYS64	CB	15.316	28.621	−12.437
405	LYS64	CG	14.863	29.702	−11.435
406	LYS64	CD	15.239	29.355	−9.991
407	LYS64	CE	14.471	30.217	−8.988
408	LYS64	NZ	14.487	29.568	−7.652
409	LEU65	N	14.366	26.377	−14.827
410	LEU65	CA	14.828	25.348	−15.748
411	LEU65	C	14.299	25.454	−17.198
412	LEU65	O	15.052	25.083	−18.109
413	LEU65	CB	14.562	23.925	−15.22
414	LEU65	CG	15.32	23.551	−13.931
415	LEU65	CD1	14.885	22.157	−13.472
416	LEU65	CD2	16.838	23.581	−14.107
417	PRO66	N	12.985	25.815	−17.464
418	PRO66	CA	12.437	25.65	−18.822
419	PRO66	C	12.087	26.965	−19.575
420	PRO66	O	10.97	27.132	−20.083
421	PRO66	CB	11.167	24.841	−18.536
422	PRO66	CG	10.629	25.517	−17.279
423	PRO66	CD	11.893	25.854	−16.493
424	PRO67	N	13.064	27.918	−19.775
425	PRO67	CA	12.782	29.095	−20.588
426	PRO67	C	12.822	28.72	−22.081
427	PRO67	O	13.391	27.717	−22.509
428	PRO67	CB	13.909	30.063	−20.243
429	PRO67	CG	15.085	29.13	−19.974
430	PRO67	CD	14.441	27.918	−19.309
431	ARG68	N	12.179	29.621	−22.908
432	ARG68	CA	12.145	29.51	−24.371
433	ARG68	C	11.217	28.398	−24.898
434	ARG68	O	11.203	28.074	−26.089
435	ARG68	CB	13.525	29.367	−25.038
436	ARG68	CG	14.512	30.474	−24.677
437	ARG68	CD	15.852	30.291	−25.393
438	ARG68	NE	16.958	30.148	−24.436
439	ARG68	CZ	17.252	29.02	−23.743
440	ARG68	NH1	18.155	29.105	−22.735
441	ARG68	NH2	16.701	27.826	−23.987
442	LEU69	N	10.338	27.861	−23.97
443	LEU69	CA	9.584	26.677	−24.351
444	LEU69	C	8.561	27.022	−25.444
445	LEU69	O	8.092	28.147	−25.609
446	LEU69	CB	8.961	25.93	−23.163
447	LEU69	CG	8.04	26.719	−22.211
448	LEU69	CD1	6.717	27.144	−22.848
449	LEU69	CD2	7.734	25.845	−20.99
450	GLN70	N	8.218	25.944	−26.234
451	GLN70	CA	7.169	26.042	−27.225
452	GLN70	C	6.106	25.004	−26.851
453	GLN70	O	6.376	23.939	−26.294
454	GLN70	CB	7.673	25.793	−28.652
455	GLN70	CG	8.902	26.625	−29.028
456	GLN70	CD	10.18	25.816	−28.919
457	GLN70	OE1	10.366	24.784	−29.564
458	GLN70	NE2	11.135	26.334	−28.095
459	TRP71	N	4.834	25.353	−27.245
460	TRP71	CA	3.735	24.391	−27.223
461	TRP71	C	3.541	24.022	−28.697
462	TRP71	O	3.381	24.894	−29.553
463	TRP71	CB	2.456	25.02	−26.68
464	TRP71	CG	2.454	25.224	−25.191
465	TRP71	CD1	2.869	26.355	−24.508
466	TRP71	CD2	1.907	24.336	−24.212
467	TRP71	NE1	2.537	26.21	−23.186
468	TRP71	CE2	1.903	25.006	−22.992
469	TRP71	CE3	1.334	23.052	−24.262
470	TRP71	CZ2	1.291	24.491	−21.846
471	TRP71	CZ3	0.686	22.535	−23.136
472	TRP71	CH2	0.656	23.252	−21.947
473	LYS72	N	3.624	22.679	−28.983
474	LYS72	CA	3.494	22.16	−30.348
475	LYS72	C	2.42	21.068	−30.347
476	LYS72	O	2.092	20.469	−29.321
477	LYS72	CB	4.83	21.592	−30.849
478	LYS72	CG	5.858	22.692	−31.13
479	LYS72	CD	7.126	22.114	−31.759
480	LYS72	CE	8.191	23.186	−31.926
481	LYS72	NZ	9.428	22.568	−32.457
482	ARG73	N	1.871	20.801	−31.584
483	ARG73	CA	1.094	19.583	−31.812
484	ARG73	C	2.115	18.449	−32.15
485	ARG73	O	3.335	18.648	−32.191
486	ARG73	CB	0.057	19.819	−32.924
487	ARG73	CG	−1	20.889	−32.607
488	ARG73	CD	−2.153	20.364	−31.751
489	ARG73	NE	−3.204	21.384	−31.608
490	ARG73	CZ	−4.491	21.125	−31.262
491	ARG73	NH1	−4.929	19.909	−30.901
492	ARG73	NH2	−5.372	22.129	−31.273
493	PRO74	N	1.613	17.176	−32.386
494	PRO74	CA	2.532	16.075	−32.691
495	PRO74	C	3.204	16.037	−34.08
496	PRO74	O	4.339	15.537	−34.178
497	PRO74	CB	1.692	14.815	−32.46
498	PRO74	CG	0.722	15.264	−31.377
499	PRO74	CD	0.364	16.664	−31.838
500	PRO75	N	2.518	16.432	−35.214
501	PRO75	CA	3.119	16.245	−36.536
502	PRO75	C	4.309	17.176	−36.837
503	PRO75	O	5.012	17.021	−37.836
504	PRO75	CB	1.974	16.435	−37.528
505	PRO75	CG	1.003	17.338	−36.786
506	PRO75	CD	1.147	16.902	−35.335
507	GLU76	N	4.55	18.16	−35.895
508	GLU76	CA	5.745	18.993	−35.974
509	GLU76	C	6.967	18.157	−35.531
510	GLU76	O	8.116	18.48	−35.833
511	GLU76	CB	5.704	20.206	−35.021
512	GLU76	CG	4.624	21.241	−35.323
513	GLU76	CD	3.326	20.806	−34.659
514	GLU76	OE1	2.924	21.524	−33.694
515	GLU76	OE2	2.825	19.744	−35.135
516	LEU77	N	6.674	17.174	−34.601
517	LEU77	CA	7.698	16.407	−33.913
518	LEU77	C	7.993	15.098	−34.656
519	LEU77	O	9.151	14.751	−34.894
520	LEU77	CB	7.281	16.104	−32.465
521	LEU77	CG	7.016	17.359	−31.608
522	LEU77	CD1	6.472	16.945	−30.239
523	LEU77	CD2	8.274	18.211	−31.436
524	HIS78	N	6.902	14.283	−34.897
525	HIS78	CA	7.056	12.996	−35.577
526	HIS78	C	6.321	13.054	−36.919
527	HIS78	O	5.266	13.665	−37.072
528	HIS78	CB	6.494	11.831	−34.748
529	HIS78	CG	7.297	11.485	−33.533
530	HIS78	ND1	6.849	10.582	−32.599
531	HIS78	CD2	8.531	11.874	−33.051
532	HIS78	CE1	7.78	10.503	−31.605
533	HIS78	NE2	8.817	11.265	−31.855
534	SER79	N	6.896	12.292	−37.918
535	SER79	CA	6.39	12.314	−39.296
536	SER79	C	5.22	11.341	−39.492
537	SER79	O	4.445	11.438	−40.442
538	SER79	CB	7.51	11.959	−40.277
539	SER79	OG	8.239	10.828	−39.793
540	ASN80	N	5.183	10.301	−38.59
541	ASN80	CA	4.117	9.304	−38.557
542	ASN80	C	3.642	9.227	−37.097
543	ASN80	O	4.05	8.344	−36.336
544	ASN80	CB	4.652	7.957	−39.044
545	ASN80	CG	3.555	6.92	−39.15
546	ASN80	OD1	2.364	7.137	−38.952
547	ASN80	ND2	4.004	5.678	−39.506
548	PRO81	N	2.793	10.222	−36.654
549	PRO81	CA	2.231	10.173	−35.311
550	PRO81	C	1.077	9.157	−35.31
551	PRO81	O	0.264	9.064	−36.229
552	PRO81	CB	1.737	11.592	−35.063
553	PRO81	CG	1.329	12.074	−36.452
554	PRO81	CD	2.346	11.41	−37.374
555	GLN82	N	1.042	8.343	−34.196
556	GLN82	CA	0.079	7.257	−34.071
557	GLN82	C	−0.393	7.201	−32.612
558	GLN82	O	0.262	7.684	−31.692
559	GLN82	CB	0.687	5.91	−34.478
560	GLN82	CG	1.026	5.81	−35.967
561	GLN82	CD	−0.208	5.864	−36.841
562	GLN82	OE1	−1.346	5.673	−36.422
563	GLN82	NE2	0.056	6.091	−38.163
564	PHE83	N	−1.617	6.574	−32.457
565	PHE83	CA	−2.289	6.467	−31.154
566	PHE83	C	−2.105	5.038	−30.619
567	PHE83	O	−1.725	4.817	−29.472
568	PHE83	CB	−3.772	6.838	−31.314
569	PHE83	CG	−4.503	7.095	−30.019
570	PHE83	CD1	−4.162	8.191	−29.213
571	PHE83	CD2	−5.58	6.284	−29.64
572	PHE83	CE1	−4.89	8.472	−28.055
573	PHE83	CE2	−6.306	6.572	−28.483
574	PHE83	CZ	−5.965	7.666	−27.693
575	TYR84	N	−2.472	4.037	−31.493
576	TYR84	CA	−2.292	2.612	−31.215
577	TYR84	C	−2.348	1.943	−32.592
578	TYR84	O	−3.064	2.387	−33.49
579	TYR84	CB	−3.419	2.024	−30.349
580	TYR84	CG	−3.01	1.729	−28.926
581	TYR84	CD1	−3.451	2.547	−27.877
582	TYR84	CD2	−2.2	0.621	−28.633
583	TYR84	CE1	−3.109	2.253	−26.556
584	TYR84	CE2	−1.846	0.336	−27.314
585	TYR84	CZ	−2.305	1.151	−26.286
586	TYR84	OH	−1.942	0.839	−25.008
587	PHE85	N	−1.583	0.803	−32.722
588	PHE85	CA	−1.557	0.054	−33.976
589	PHE85	C	−1.018	−1.353	−33.675
590	PHE85	O	0.157	−1.675	−33.809
591	PHE85	CB	−0.869	0.752	−35.162
592	PHE85	CG	0.567	1.2	−35.04
593	PHE85	CD1	1.526	0.717	−35.943
594	PHE85	CD2	0.952	2.184	−34.122
595	PHE85	CE1	2.828	1.22	−35.939
596	PHE85	CE2	2.257	2.675	−34.109
597	PHE85	CZ	3.192	2.205	−35.026
598	ALA86	N	−2.007	−2.183	−33.163
599	ALA86	CA	−1.777	−3.512	−32.605
600	ALA86	C	−1.378	−3.41	−31.12
601	ALA86	O	−1.093	−2.352	−30.564
602	ALA86	CB	−0.813	−4.383	−33.405
603	LYS87	N	−1.392	−4.628	−30.458
604	LYS87	CA	−1.132	−4.726	−29.017
605	LYS87	C	0.379	−4.824	−28.732
606	LYS87	O	0.853	−4.568	−27.626
607	LYS87	CB	−1.863	−5.937	−28.42
608	LYS87	CG	−3.355	−5.643	−28.213
609	LYS87	CD	−4.1	−6.829	−27.594
610	LYS87	CE	−5.384	−6.375	−26.912
611	LYS87	NZ	−6.065	−7.531	−26.331
612	ALA88	N	1.123	−5.333	−29.781
613	ALA88	CA	2.584	−5.318	−29.775
614	ALA88	C	3.128	−6.294	−28.7
615	ALA88	O	2.602	−7.389	−28.494
616	ALA88	CB	3.115	−3.89	−29.745
617	LYS89	N	4.291	−5.911	−28.057
618	LYS89	CA	4.945	−6.782	−27.079
619	LYS89	C	5.754	−5.89	−26.126
620	LYS89	O	5.385	−5.663	−24.971
621	LYS89	CB	5.825	−7.83	−27.788
622	LYS89	CG	6.606	−8.738	−26.825
623	LYS89	CD	7.485	−9.72	−27.607
624	LYS89	CE	8.029	−10.844	−26.742
625	LYS89	NZ	9.338	−10.527	−26.142
626	ARG90	N	6.881	−5.351	−26.721
627	ARG90	CA	7.917	−4.637	−25.989
628	ARG90	C	7.456	−3.225	−25.613
629	ARG90	O	6.494	−2.665	−26.128
630	ARG90	CB	9.216	−4.573	−26.818
631	ARG90	CG	10.046	−5.857	−26.712
632	ARG90	CD	10.86	−5.887	−25.426
633	ARG90	NE	11.405	−7.218	−25.152
634	ARG90	CZ	12.701	−7.569	−25.276
635	ARG90	NH1	13.526	−6.914	−26.122
636	ARG90	NH2	13.203	−8.576	−24.542
637	LEU91	N	8.235	−2.676	−24.617
638	LEU91	CA	7.904	−1.434	−23.919
639	LEU91	C	9.226	−0.664	−23.931
640	LEU91	O	10.235	−1.123	−23.392
641	LEU91	CB	7.475	−1.815	−22.498
642	LEU91	CG	6.487	−0.843	−21.846
643	LEU91	CD1	5.945	−1.465	−20.56
644	LEU91	CD2	7.114	0.509	−21.527
645	ASP92	N	9.214	0.48	−24.704
646	ASP92	CA	10.404	1.319	−24.881
647	ASP92	C	11.496	0.478	−25.612
648	ASP92	O	11.245	−0.57	−26.213
649	ASP92	CB	10.877	1.95	−23.572
650	ASP92	CG	9.809	2.796	−22.89
651	ASP92	OD1	9.046	3.441	−23.668
652	ASP92	OD2	9.837	2.781	−21.626
653	LEU93	N	12.768	1.028	−25.618
654	LEU93	CA	13.922	0.28	−26.105
655	LEU93	C	14.741	−0.175	−24.876
656	LEU93	O	14.723	−1.344	−24.474
657	LEU93	CB	14.753	1.108	−27.099
658	LEU93	CG	14.023	1.446	−28.416
659	LEU93	CD1	14.893	2.381	−29.261
660	LEU93	CD2	13.669	0.2	−29.23
661	CYS94	N	15.447	0.831	−24.233
662	CYS94	CA	16.324	0.577	−23.09
663	CYS94	C	16.131	1.7	−22.062
664	CYS94	O	15.357	1.583	−21.113
665	CYS94	CB	17.803	0.423	−23.481
666	CYS94	SG	18.154	−1.146	−24.337
667	GLN95	N	16.809	2.875	−22.309
668	GLN95	CA	16.828	3.977	−21.343
669	GLN95	C	15.595	4.88	−21.519
670	GLN95	O	15.666	6.101	−21.667
671	GLN95	CB	18.128	4.782	−21.468
672	GLN95	CG	19.35	3.971	−21.037
673	GLN95	CD	20.631	4.716	−21.332
674	GLN95	OE1	21.398	4.387	−22.23
675	GLN95	NE2	20.868	5.796	−20.532
676	GLY96	N	14.387	4.241	−21.336
677	GLY96	CA	13.113	4.941	−21.412
678	GLY96	C	12.794	5.775	−20.17
679	GLY96	O	11.648	5.904	−19.733
680	ILE97	N	13.836	6.527	−19.667
681	ILE97	CA	13.733	7.329	−18.442
682	ILE97	C	13.041	8.673	−18.791
683	ILE97	O	13.56	9.771	−18.609
684	ILE97	CB	15.122	7.531	−17.772
685	ILE97	CG1	15.926	6.211	−17.692
686	ILE97	CG2	14.949	8.124	−16.362
687	ILE97	CD1	17.34	6.378	−17.148
688	VAL98	N	11.751	8.517	−19.265
689	VAL98	CA	10.927	9.621	−19.757
690	VAL98	C	9.429	9.286	−19.558
691	VAL98	O	8.531	9.843	−20.19
692	VAL98	CB	11.221	9.994	−21.234
693	VAL98	CG1	12.475	10.859	−21.382
694	VAL98	CG2	11.335	8.767	−22.145
695	GLY99	N	9.185	8.415	−18.514
696	GLY99	CA	7.875	7.865	−18.235
697	GLY99	C	7.657	7.649	−16.74
698	GLY99	O	8.578	7.612	−15.928
699	ASP100	N	6.324	7.452	−16.42
700	ASP100	CA	5.893	7.06	−15.077
701	ASP100	C	5.918	5.515	−15.027
702	ASP100	O	5.893	4.805	−16.031
703	ASP100	CB	4.487	7.593	−14.83
704	ASP100	CG	4.12	7.565	−13.351
705	ASP100	OD1	3.671	8.643	−12.877
706	ASP100	OD2	4.264	6.429	−12.793
707	CYS101	N	5.865	4.992	−13.749
708	CYS101	CA	5.769	3.556	−13.534
709	CYS101	C	4.373	3.044	−13.937
710	CYS101	O	4.226	1.887	−14.348
711	CYS101	CB	6.133	3.221	−12.089
712	CYS101	SG	6.148	1.438	−11.724
713	TRP102	N	3.29	3.904	−13.831
714	TRP102	CA	1.957	3.399	−14.203
715	TRP102	C	1.878	3.08	−15.714
716	TRP102	O	1.076	2.263	−16.17
717	TRP102	CB	0.751	4.239	−13.763
718	TRP102	CG	0.781	5.685	−14.133
719	TRP102	CD1	1.058	6.715	−13.259
720	TRP102	CD2	0.498	6.294	−15.401
721	TRP102	NE1	1.069	7.893	−13.955
722	TRP102	CE2	0.713	7.668	−15.262
723	TRP102	CE3	0.088	5.808	−16.661
724	TRP102	CZ2	0.584	8.568	−16.325
725	TRP102	CZ3	−0.042	6.694	−17.735
726	TRP102	CH2	0.205	8.052	−17.567
727	PHE103	N	2.748	3.808	−16.5
728	PHE103	CA	2.834	3.674	−17.967
729	PHE103	C	3.477	2.303	−18.263
730	PHE103	O	3.184	1.626	−19.246
731	PHE103	CB	3.697	4.823	−18.521
732	PHE103	CG	3.647	5.182	−19.989
733	PHE103	CD1	2.98	4.439	−20.966
734	PHE103	CD2	4.345	6.338	−20.391
735	PHE103	CE1	3.011	4.843	−22.306
736	PHE103	CE2	4.371	6.743	−21.725
737	PHE103	CZ	3.701	5.994	−22.684
738	LEU104	N	4.471	1.954	−17.371
739	LEU104	CA	5.153	0.669	−17.416
740	LEU104	C	4.232	−0.452	−16.89
741	LEU104	O	4.292	−1.604	−17.331
742	LEU104	CB	6.453	0.729	−16.608
743	LEU104	CG	7.691	1.153	−17.425
744	LEU104	CD1	7.519	2.468	−18.183
745	LEU104	CD2	8.893	1.266	−16.486
746	ALA105	N	3.406	−0.101	−15.834
747	ALA105	CA	2.56	−1.101	−15.193
748	ALA105	C	1.429	−1.517	−16.151
749	ALA105	O	1.062	−2.692	−16.237
750	ALA105	CB	1.951	−0.585	−13.898
751	ALA106	N	0.819	−0.484	−16.85
752	ALA106	CA	−0.432	−0.724	−17.57
753	ALA106	C	−0.204	−1.675	−18.754
754	ALA106	O	−1.054	−2.479	−19.141
755	ALA106	CB	−1.013	0.582	−18.097
756	LEU107	N	1.009	−1.53	−19.398
757	LEU107	CA	1.321	−2.176	−20.673
758	LEU107	C	1.718	−3.678	−20.575
759	LEU107	O	2.258	−4.293	−21.501
760	LEU107	CB	2.384	−1.374	−21.433
761	LEU107	CG	1.878	−0.061	−22.059
762	LEU107	CD1	3.035	0.663	−22.753
763	LEU107	CD2	0.74	−0.303	−23.051
764	GLN108	N	1.244	−4.303	−19.443
765	GLN108	CA	1.063	−5.742	−19.321
766	GLN108	C	−0.337	−6.131	−19.827
767	GLN108	O	−0.557	−7.222	−20.355
768	GLN108	CB	1.183	−6.173	−17.853
769	GLN108	CG	2.621	−6.422	−17.41
770	GLN108	CD	3.582	−5.274	−17.628
771	GLN108	OE1	4.64	−5.424	−18.24
772	GLN108	NE2	3.248	−4.089	−17.05
773	ALA109	N	−1.359	−5.255	−19.525
774	ALA109	CA	−2.772	−5.614	−19.668
775	ALA109	C	−3.291	−5.495	−21.117
776	ALA109	O	−4.39	−5.022	−21.399
777	ALA109	CB	−3.596	−4.765	−18.713
778	LEU110	N	−2.462	−6.089	−22.051
779	LEU110	CA	−2.756	−6.142	−23.476
780	LEU110	C	−2.279	−7.502	−24.017
781	LEU110	O	−1.669	−7.63	−25.075
782	LEU110	CB	−2.121	−5.001	−24.287
783	LEU110	CG	−2.629	−3.591	−23.935
784	LEU110	CD1	−1.731	−2.934	−22.892
785	LEU110	CD2	−2.658	−2.71	−25.186
786	ALA111	N	−2.72	−8.579	−23.265
787	ALA111	CA	−2.753	−9.915	−23.857
788	ALA111	C	−4.101	−10.024	−24.608
789	ALA111	O	−4.773	−9.031	−24.91
790	ALA111	CB	−2.558	−10.955	−22.762
791	LEU112	N	−4.465	−11.29	−25.015
792	LEU112	CA	−5.86	−11.58	−25.354
793	LEU112	C	−6.414	−12.161	−24.046
794	LEU112	O	−5.918	−13.188	−23.572
795	LEU112	CB	−5.952	−12.64	−26.461
796	LEU112	CG	−5.261	−12.256	−27.785
797	LEU112	CD1	−5.34	−13.427	−28.769
798	LEU112	CD2	−5.866	−11.005	−28.417
799	HIS113	N	−7.43	−11.444	−23.451
800	HIS113	CA	−7.821	−11.611	−22.048
801	HIS113	C	−6.923	−10.665	−21.229
802	HIS113	O	−5.785	−10.341	−21.566
803	HIS113	CB	−7.778	−13.012	−21.426
804	HIS113	CG	−8.611	−14.02	−22.134
805	HIS113	ND1	−8.095	−14.823	−23.122
806	HIS113	CD2	−9.929	−14.406	−22.032
807	HIS113	CE1	−9.092	−15.645	−23.555
808	HIS113	NE2	−10.212	−15.424	−22.906
809	GLN114	N	−7.504	−10.22	−20.054
810	GLN114	CA	−6.884	−9.215	−19.194
811	GLN114	C	−6.983	−7.797	−19.8
812	GLN114	O	−6.316	−6.849	−19.385
813	GLN114	CB	−5.444	−9.521	−18.747
814	GLN114	CG	−5.258	−10.965	−18.283
815	GLN114	CD	−4.009	−11.203	−17.462
816	GLN114	OE1	−4.009	−11.949	−16.481
817	GLN114	NE2	−2.866	−10.614	−17.913
818	ASP115	N	−7.986	−7.644	−20.739
819	ASP115	CA	−8.063	−6.457	−21.592
820	ASP115	C	−8.661	−5.284	−20.776
821	ASP115	O	−9.86	−5.006	−20.77
822	ASP115	CB	−8.938	−6.751	−22.812
823	ASP115	CG	−8.176	−7.678	−23.745
824	ASP115	OD1	−8.126	−8.901	−23.421
825	ASP115	OD2	−7.654	−7.126	−24.764
826	ILE116	N	−7.728	−4.588	−20.009
827	ILE116	CA	−8.165	−3.488	−19.133
828	ILE116	C	−8.463	−2.254	−20.018
829	ILE116	O	−9.325	−1.423	−19.726
830	ILE116	CB	−7.086	−3.125	−18.066
831	ILE116	CG1	−6.977	−4.238	−16.998
832	ILE116	CG2	−7.362	−1.775	−17.38
833	ILE116	CD1	−5.89	−3.988	−15.958
834	LEU117	N	−7.566	−2.046	−21.058
835	LEU117	CA	−7.268	−0.678	−21.504
836	LEU117	C	−8.491	0.028	−22.113
837	LEU117	O	−8.609	1.255	−22.1
838	LEU117	CB	−6.122	−0.667	−22.53
839	LEU117	CG	−4.72	−0.396	−21.943
840	LEU117	CD1	−4.559	1.055	−21.489
841	LEU117	CD2	−4.346	−1.348	−20.81
842	SER118	N	−9.408	−0.805	−22.71
843	SER118	CA	−10.624	−0.306	−23.348
844	SER118	C	−11.65	0.289	−22.366
845	SER118	O	−12.654	0.877	−22.767
846	SER118	CB	−11.286	−1.413	−24.169
847	SER118	OG	−11.43	−2.584	−23.363
848	ARG119	N	−11.355	0.143	−21.025
849	ARG119	CA	−12.143	0.802	−19.988
850	ARG119	C	−11.707	2.275	−19.855
851	ARG119	O	−12.455	3.127	−19.381
852	ARG119	CB	−11.894	0.161	−18.62
853	ARG119	CG	−12.335	−1.297	−18.522
854	ARG119	CD	−11.739	−1.974	−17.293
855	ARG119	NE	−12.298	−1.457	−16.042
856	ARG119	CZ	−11.803	−0.482	−15.244
857	ARG119	NH1	−10.711	0.247	−15.555
858	ARG119	NH2	−12.452	−0.258	−14.089
859	VAL120	N	−10.362	2.488	−20.094
860	VAL120	CA	−9.706	3.779	−19.884
861	VAL120	C	−9.694	4.555	−21.214
862	VAL120	O	−9.932	5.766	−21.253
863	VAL120	CB	−8.269	3.569	−19.347
864	VAL120	CG1	−7.529	4.896	−19.144
865	VAL120	CG2	−8.281	2.797	−18.023
866	VAL121	N	−9.242	3.824	−22.299
867	VAL121	CA	−8.985	4.393	−23.615
868	VAL121	C	−10.137	3.956	−24.546
869	VAL121	O	−10.169	2.831	−25.059
870	VAL121	CB	−7.629	3.889	−24.177
871	VAL121	CG1	−7.284	4.624	−25.476
872	VAL121	CG2	−6.481	4.066	−23.177
873	PRO122	N	−11.151	4.858	−24.793
874	PRO122	CA	−12.257	4.516	−25.681
875	PRO122	C	−11.727	4.64	−27.122
876	PRO122	O	−11.655	5.707	−27.736
877	PRO122	CB	−13.34	5.54	−25.351
878	PRO122	CG	−12.56	6.749	−24.854
879	PRO122	CD	−11.376	6.132	−24.132
880	LEU123	N	−11.265	3.444	−27.648
881	LEU123	CA	−10.478	3.372	−28.894
882	LEU123	C	−11.285	3.658	−30.198
883	LEU123	O	−11.001	3.142	−31.278
884	LEU123	CB	−9.802	1.984	−29.007
885	LEU123	CG	−8.58	1.794	−28.085
886	LEU123	CD1	−8.246	0.307	−27.948
887	LEU123	CD2	−7.356	2.544	−28.613
888	ASN124	N	−12.211	4.68	−30.117
889	ASN124	CA	−12.829	5.29	−31.3
890	ASN124	C	−12.104	6.607	−31.619
891	ASN124	O	−12.689	7.68	−31.747
892	ASN124	CB	−14.341	5.477	−31.162
893	ASN124	CG	−14.79	6.026	−29.825
894	ASN124	OD1	−14.992	7.218	−29.597
895	ASN124	ND2	−14.98	5.078	−28.858
896	GLN125	N	−10.738	6.468	−31.766
897	GLN125	CA	−9.826	7.606	−31.832
898	GLN125	C	−8.631	7.22	−32.717
899	GLN125	O	−8.277	6.051	−32.867
900	GLN125	CB	−9.337	7.976	−30.426
901	GLN125	CG	−10.449	8.556	−29.557
902	GLN125	CD	−9.954	8.892	−28.177
903	GLN125	OE1	−9.565	10.014	−27.87
904	GLN125	NE2	−9.96	7.87	−27.275
905	SER126	N	−7.977	8.292	−33.292
906	SER126	CA	−6.903	8.121	−34.272
907	SER126	C	−6.247	9.491	−34.529
908	SER126	O	−6.72	10.537	−34.089
909	SER126	CB	−7.434	7.571	−35.601
910	SER126	OG	−6.393	7.517	−36.579
911	PHE127	N	−5.107	9.413	−35.315
912	PHE127	CA	−4.435	10.605	−35.843
913	PHE127	C	−4.861	10.96	−37.282
914	PHE127	O	−4.327	11.876	−37.907
915	PHE127	CB	−2.908	10.456	−35.78
916	PHE127	CG	−2.362	10.971	−34.47
917	PHE127	CD1	−2.161	10.109	−33.388
918	PHE127	CD2	−2.091	12.334	−34.312
919	PHE127	CE1	−1.653	10.589	−32.181
920	PHE127	CE2	−1.612	12.819	−33.096
921	PHE127	CZ	−1.378	11.946	−32.035
922	THR128	N	−5.945	10.264	−37.77
923	THR128	CA	−6.502	10.531	−39.093
924	THR128	C	−8.034	10.544	−38.983
925	THR128	O	−8.707	11.526	−39.309
926	THR128	CB	−5.992	9.555	−40.178
927	THR128	OG1	−5.571	8.303	−39.635
928	THR128	CG2	−4.785	10.13	−40.915
929	GLU129	N	−8.608	9.393	−38.494
930	GLU129	CA	−10.035	9.112	−38.601
931	GLU129	C	−10.811	10.009	−37.614
932	GLU129	O	−11.205	9.625	−36.517
933	GLU129	CB	−10.335	7.629	−38.31
934	GLU129	CG	−9.759	6.652	−39.339
935	GLU129	CD	−8.271	6.311	−39.226
936	GLU129	OE1	−7.929	5.198	−39.697
937	GLU129	OE2	−7.53	7.208	−38.687
938	LYS130	N	−11.005	11.296	−38.093
939	LYS130	CA	−11.731	12.34	−37.373
940	LYS130	C	−10.877	12.95	−36.242
941	LYS130	O	−11.355	13.334	−35.177
942	LYS130	CB	−13.126	11.929	−36.873
943	LYS130	CG	−13.995	11.284	−37.956
944	LYS130	CD	−15.417	11.04	−37.448
945	LYS130	CE	−16.274	10.388	−38.522
946	LYS130	NZ	−17.636	10.16	−37.984
947	TYR131	N	−9.562	13.204	−36.609
948	TYR131	CA	−8.563	13.568	−35.589
949	TYR131	C	−8.9	14.925	−34.947
950	TYR131	O	−8.836	15.109	−33.729
951	TYR131	CB	−7.175	13.608	−36.26
952	TYR131	CG	−6.046	14.306	−35.541
953	TYR131	CD1	−5.831	14.159	−34.166
954	TYR131	CD2	−5.155	15.104	−36.281
955	TYR131	CE1	−4.787	14.844	−33.54
956	TYR131	CE2	−4.101	15.77	−35.655
957	TYR131	CZ	−3.927	15.641	−34.285
958	TYR131	OH	−2.881	16.305	−33.712
959	ALA132	N	−9.049	15.977	−35.833
960	ALA132	CA	−9.562	17.291	−35.428
961	ALA132	C	−8.797	18.069	−34.324
962	ALA132	O	−9.201	19.152	−33.891
963	ALA132	CB	−11.05	17.221	−35.103
964	GLY133	N	−7.619	17.503	−33.892
965	GLY133	CA	−6.856	18.065	−32.802
966	GLY133	C	−7.431	17.783	−31.41
967	GLY133	O	−7.144	18.534	−30.47
968	ILE134	N	−8.174	16.624	−31.274
969	ILE134	CA	−8.908	16.289	−30.044
970	ILE134	C	−8.793	14.785	−29.724
971	ILE134	O	−8.765	13.926	−30.603
972	ILE134	CB	−10.397	16.728	−30.16
973	ILE134	CG1	−11.124	16.686	−28.801
974	ILE134	CG2	−11.169	15.94	−31.224
975	ILE134	CD1	−12.511	17.313	−28.847
976	PHE135	N	−8.83	14.489	−28.371
977	PHE135	CA	−8.833	13.117	−27.856
978	PHE135	C	−9.685	13.073	−26.568
979	PHE135	O	−10.012	14.085	−25.944
980	PHE135	CB	−7.413	12.624	−27.534
981	PHE135	CG	−6.553	12.377	−28.746
982	PHE135	CD1	−5.529	13.267	−29.088
983	PHE135	CD2	−6.777	11.255	−29.554
984	PHE135	CE1	−4.741	13.032	−30.215
985	PHE135	CE2	−6.003	11.033	−30.691
986	PHE135	CZ	−4.989	11.924	−31.023
987	ARG136	N	−10.009	11.798	−26.139
988	ARG136	CA	−10.796	11.546	−24.941
989	ARG136	C	−10.416	10.231	−24.233
990	ARG136	O	−9.984	9.248	−24.834
991	ARG136	CB	−12.308	11.593	−25.207
992	ARG136	CG	−12.821	10.558	−26.217
993	ARG136	CD	−14.35	10.528	−26.238
994	ARG136	NE	−14.865	9.659	−27.303
995	ARG136	CZ	−16.136	9.742	−27.779
996	ARG136	NH1	−16.503	8.988	−28.829
997	ARG136	NH2	−17.054	10.552	−27.241
998	PHE137	N	−10.687	10.254	−22.871
999	PHE137	CA	−10.321	9.168	−21.959
1000	PHE137	C	−11.354	9.108	−20.824
1001	PHE137	O	−11.947	10.111	−20.421
1002	PHE137	CB	−8.944	9.385	−21.316
1003	PHE137	CG	−7.793	9.34	−22.287
1004	PHEI37	CD1	−7.236	10.527	−22.776
1005	PHE137	CD2	−7.258	8.117	−22.706
1006	PHE137	CE1	−6.147	10.489	−23.643
1007	PHE137	CE2	−6.162	8.083	−23.57
1008	PHE137	CZ	−5.604	9.269	−24.035
1009	TRP138	N	−11.51	7.859	−20.254
1010	TRP138	CA	−12.345	7.665	−19.064
1011	TRP138	C	−11.447	7.497	−17.827
1012	TRP138	O	−10.396	6.86	−17.86
1013	TRP138	CB	−13.233	6.421	−19.182
1014	TRP138	CG	−14.47	6.651	−19.997
1015	TRP138	CD1	−14.621	6.385	−21.343
1016	TRP138	CD2	−15.731	7.146	−19.529
1017	TRP138	NE1	−15.902	6.7	−21.705
1018	TRP138	CE2	−16.607	7.155	−20.616
1019	TRP138	CE3	−16.213	7.579	−18.276
1020	TRP138	CZ2	−17.942	7.566	−20.511
1021	TRP138	CZ3	−17.545	7.994	−18.155
1022	TRP138	CH2	−18.395	7.984	−19.257
1023	PHE139	N	−11.973	8.052	−16.675
1024	PHE139	CA	−11.306	7.973	−15.369
1025	PHE139	C	−12.384	7.696	−14.306
1026	PHE139	O	−13.538	8.118	−14.423
1027	PHE139	CB	−10.641	9.303	−14.98
1028	PHE139	CG	−9.331	9.613	−15.658
1029	PHE139	CD1	−9.286	10.091	−16.972
1030	PHE139	CD2	−8.136	9.507	−14.936
1031	PHE139	CE1	−8.072	10.481	−17.539
1032	PHE139	CE2	−6.928	9.916	−15.495
1033	PHE139	CZ	−6.896	10.405	−16.798
1034	TRP140	N	−11.939	7.013	−13.188
1035	TRP140	CA	−12.701	7.014	−11.929
1036	TRP140	C	−12.057	8.105	−11.072
1037	TRP140	O	−10.834	8.217	−11.006
1038	TRP140	CB	−12.571	5.667	−11.208
1039	TRP140	CG	−13.383	5.54	−9.948
1040	TRP140	CD1	−14.64	4.977	−9.842
1041	TRP140	CD2	−12.984	5.899	−8.615
1042	TRP140	NE1	−14.998	4.949	−8.521
1043	TRP140	CE2	−14.019	5.533	−7.753
1044	TRP140	CE3	−11.825	6.474	−8.053
1045	TRP140	CZ2	−13.959	5.73	−6.367
1046	TRP140	CZ3	−11.752	6.684	−6.671
1047	TRP140	CH2	−12.806	6.317	−5.841
1048	HIS141	N	−12.917	8.884	−10.331
1049	HIS141	CA	−12.408	9.837	−9.352
1050	HIS141	C	−13.458	10.025	−8.248
1051	HIS141	O	−14.635	10.302	−8.48
1052	HIS141	CB	−12.141	11.23	−9.934
1053	HIS141	CG	−11.032	11.334	−10.919
1054	HIS141	ND1	−9.75	10.877	−10.725
1055	HIS141	CD2	−11.005	11.901	−12.174
1056	HIS141	CE1	−9.024	11.237	−11.83
1057	HIS141	NE2	−9.754	11.864	−12.721
1058	TYR142	N	−12.985	9.871	−6.958
1059	TYR142	CA	−13.632	10.526	−5.814
1060	TYR142	C	−15.113	10.164	−5.57
1061	TYR142	O	−15.861	10.868	−4.883
1062	TYR142	CB	−13.428	12.057	−5.844
1063	TYR142	CG	−11.989	12.464	−5.617
1064	TYR142	CD1	−11.292	13.263	−6.54
1065	TYR142	CD2	−11.335	12.063	−4.448
1066	TYR142	CE1	−9.96	13.636	−6.306
1067	TYR142	CE2	−10.014	12.428	−4.224
1068	TYR142	CZ	−9.338	13.215	−5.142
1069	TYR142	OH	−8.073	13.56	−4.798
1070	GLY143	N	−15.476	8.919	−6.043
1071	GLY143	CA	−16.832	8.412	−5.971
1072	GLY143	C	−17.655	8.645	−7.241
1073	GLY143	O	−18.883	8.482	−7.266
1074	ASN144	N	−16.955	8.951	−8.384
1075	ASN144	CA	−17.596	9.151	−9.681
1076	ASN144	C	−16.688	8.618	−10.801
1077	ASN144	O	−15.464	8.573	−10.704
1078	ASN144	CB	−17.922	10.63	−9.913
1079	ASN144	CG	−19.33	11.081	−9.576
1080	ASN144	OD1	−19.774	12.166	−9.944
1081	ASN144	ND2	−20.118	10.253	−8.821
1082	TRP145	N	−17.387	8.237	−11.939
1083	TRP145	CA	−16.681	8.01	−13.204
1084	TRP145	C	−16.708	9.355	−13.94
1085	TRP145	O	−17.634	10.157	−13.793
1086	TRP145	CB	−17.356	6.938	−14.068
1087	TRP145	CG	−16.948	5.548	−13.681
1088	TRP145	CD1	−17.601	4.704	−12.805
1089	TRP145	CD2	−15.793	4.843	−14.152
1090	TRP145	NE1	−16.89	3.535	−12.723
1091	TRP145	CE2	−15.768	3.603	−13.515
1092	TRP145	CE3	−14.756	5.154	−15.056
1093	TRP145	CZ2	−14.741	2.671	−13.713
1094	TRP145	CZ3	−13.723	4.237	−15.269
1095	TRP145	CH2	−13.715	3.018	−14.599
1096	VAL146	N	−15.638	9.562	−14.788
1097	VAL146	CA	−15.39	10.871	−15.385
1098	VAL146	C	−14.852	10.644	−16.812
1099	VAL146	O	−13.837	9.963	−17.003
1100	VAL146	CB	−14.358	11.689	−14.569
1101	VAL146	CG1	−14.207	13.097	−15.155
1102	VAL146	CG2	−14.719	11.766	−13.081
1103	PRO147	N	−15.549	11.254	−17.843
1104	PRO147	CA	−15.043	11.286	−19.215
1105	PRO147	C	−14.214	12.571	−19.39
1106	PRO147	O	−14.723	13.693	−19.373
1107	PRO147	CB	−16.31	11.349	−20.067
1108	PRO147	CG	−17.313	12.106	−19.2
1109	PRO147	CD	−16.917	11.758	−17.771
1110	VAL148	N	−12.848	12.386	−19.479
1111	VAL148	CA	−11.973	13.532	−19.714
1112	VAL148	C	−11.758	13.62	−21.235
1113	VAL148	O	−11.456	12.641	−21.92
1114	VAL148	CB	−10.63	13.404	−18.97
1115	VAL148	CG1	−9.735	14.62	−19.234
1116	VAL148	CG2	−10.856	13.273	−17.459
1117	VAL149	N	−11.918	14.896	−21.747
1118	VAL149	CA	−11.758	15.208	−23.164
1119	VAL149	C	−10.691	16.306	−23.249
1120	VAL149	O	−10.646	17.256	−22.459
1121	VAL149	CB	−13.078	15.659	−23.809
1122	VAL149	CG1	−12.86	15.9	−25.303
1123	VAL149	CG2	−14.181	14.618	−23.583
1124	ILE150	N	−9.774	16.139	−24.272
1125	ILE150	CA	−8.459	16.763	−24.18
1126	ILE150	C	−7.864	16.991	−25.584
1127	ILE150	O	−8.123	16.277	−26.552
1128	ILE150	CB	−7.579	15.931	−23.204
1129	ILE150	CG1	−7.164	14.539	−23.729
1130	ILE150	CG2	−6.394	16.721	−22.679
1131	ILE150	CD1	−5.892	14.481	−24.561
1132	ASP151	N	−7.006	18.07	−25.645
1133	ASP151	CA	−6.201	18.426	−26.811
1134	ASP151	C	−4.805	17.775	−26.706
1135	ASP151	O	−4.388	17.224	−25.683
1136	ASP151	CB	−6.032	19.945	−26.887
1137	ASP151	CG	−5.19	20.42	−25.713
1138	ASP151	OD1	−4.284	21.264	−25.964
1139	ASP151	OD2	−5.384	19.863	−24.585
1140	ASP152	N	−4.025	17.991	−27.816
1141	ASP152	CA	−2.677	17.478	−27.987
1142	ASP152	C	−1.662	18.603	−28.256
1143	ASP152	O	−0.641	18.39	−28.91
1144	ASP152	CB	−2.65	16.414	−29.084
1145	ASP152	CG	−3.094	16.94	−30.439
1146	ASP152	OD1	−2.806	16.218	−31.441
1147	ASP152	OD2	−3.733	18.037	−30.439
1148	ARG153	N	−1.855	19.793	−27.564
1149	ARG153	CA	−0.747	20.739	−27.456
1150	ARG153	C	0.246	20.139	−26.438
1151	ARG153	O	−0.025	20.021	−25.241
1152	ARG153	CB	−1.151	22.137	−26.954
1153	ARG153	CG	−1.674	23.084	−28.031
1154	ARG153	CD	−3.156	22.953	−28.326
1155	ARG153	NE	−3.946	24.076	−27.803
1156	ARG153	CZ	−4.465	24.31	−26.588
1157	ARG153	NH1	−4.293	23.508	−25.537
1158	ARG153	NH2	−5.257	25.397	−26.457
1159	LEU154	N	1.429	19.688	−26.982
1160	LEU154	CA	2.505	19.144	−26.165
1161	LEU154	C	3.541	20.264	−25.943
1162	LEU154	O	3.869	21.026	−26.862
1163	LEU154	CB	3.194	17.955	−26.85
1164	LEU154	CG	2.267	16.758	−27.143
1165	LEU154	CD1	3.043	15.674	−27.891
1166	LEU154	CD2	1.648	16.167	−25.875
1167	PRO155	N	4.111	20.347	−24.685
1168	PRO155	CA	5.113	21.362	−24.364
1169	PRO155	C	6.512	20.815	−24.687
1170	PRO155	O	6.887	19.688	−24.347
1171	PRO155	CB	4.968	21.566	−22.858
1172	PRO155	CG	4.548	20.186	−22.366
1173	PRO155	CD	3.654	19.668	−23.481
1174	VAL156	N	7.327	21.709	−25.354
1175	VAL156	CA	8.68	21.349	−25.743
1176	VAL156	C	9.633	22.52	−25.452
1177	VAL156	O	9.287	23.695	−25.577
1178	VAL156	CB	8.788	20.883	−27.216
1179	VAL156	CG1	7.781	19.785	−27.562
1180	VAL156	CG2	8.635	22.013	−28.231
1181	ASN157	N	10.914	22.138	−25.11
1182	ASN157	CA	12.032	23.084	−25.07
1183	ASN157	C	12.942	22.663	−26.224
1184	ASN157	O	13.399	21.524	−26.322
1185	ASN157	CB	12.82	23.005	−23.768
1186	ASN157	CG	12.778	24.303	−22.999
1187	ASN157	OD1	12.36	24.37	−21.846
1188	ASN157	ND2	13.288	25.39	−23.643
1189	GLU158	N	13.114	23.635	−27.193
1190	GLU158	CA	14.111	23.471	−28.257
1191	GLU158	C	13.939	22.143	−29.043
1192	GLU158	O	14.864	21.58	−29.619
1193	GLU158	CB	15.539	23.711	−27.735
1194	GLU158	CG	15.875	25.194	−27.503
1195	GLU158	CD	15.163	25.954	−26.384
1196	GLU158	OE1	14.01	25.532	−26.053
1197	GLU158	OE2	15.803	26.929	−25.881
1198	ALA159	N	12.617	21.738	−29.135
1199	ALA159	CA	12.147	20.492	−29.732
1200	ALA159	C	12.26	19.213	−28.87
1201	ALA159	O	11.813	18.139	−29.278
1202	ALA159	CB	12.681	20.149	−31.14
1203	GLY160	N	12.833	19.347	−27.627
1204	GLY160	CA	12.798	18.281	−26.635
1205	GLY160	C	11.549	18.465	−25.774
1206	GLY160	O	11.176	19.573	−25.389
1207	GLN161	N	10.863	17.298	−25.497
1208	GLN161	CA	9.536	17.315	−24.877
1209	GLN161	C	9.627	17.448	−23.346
1210	GLN161	O	10.562	16.979	−22.701
1211	GLN161	CB	8.748	16.054	−25.271
1212	GLN161	CG	8.339	16.079	−26.748
1213	GLN161	CD	7.893	14.73	−27.264
1214	GLN161	OE1	6.724	14.462	−27.521
1215	GLN161	NE2	8.897	13.818	−27.434
1216	LEU162	N	8.539	18.094	−22.773
1217	LEU162	CA	8.479	18.432	−21.343
1218	LEU162	C	7.22	17.826	−20.681
1219	LEU162	O	6.667	18.336	−19.709
1220	LEU162	CB	8.471	19.955	−21.132
1221	LEU162	CG	9.695	20.714	−21.671
1222	LEU162	CD1	9.416	22.218	−21.608
1223	LEU162	CD2	10.961	20.379	−20.884
1224	VAL163	N	6.852	16.608	−21.195
1225	VAL163	CA	5.733	15.782	−20.703
1226	VAL163	C	6.309	14.359	−20.709
1227	VAL163	O	7.323	14.092	−21.364
1228	VAL163	CB	4.505	16.045	−21.609
1229	VAL163	CG1	3.751	14.822	−22.116
1230	VAL163	CG2	3.528	16.978	−20.898
1231	PHE164	N	5.641	13.412	−19.96
1232	PHE164	CA	6.097	12.023	−19.996
1233	PHE164	C	5.696	11.42	−21.353
1234	PHE164	O	4.599	11.614	−21.871
1235	PHE164	CB	5.51	11.16	−18.874
1236	PHE164	CG	6.08	11.364	−17.485
1237	PHE164	CD1	5.246	11.166	−16.374
1238	PHE164	CD2	7.433	11.648	−17.25
1239	PHE164	CE1	5.739	11.272	−15.071
1240	PHE164	CE2	7.928	11.755	−15.947
1241	PHE164	CZ	7.081	11.568	−14.858
1242	VAL165	N	6.676	10.652	−21.956
1243	VAL165	CA	6.617	10.292	−23.37
1244	VAL165	C	7.345	8.961	−23.613
1245	VAL165	O	8.126	8.471	−22.805
1246	VAL165	CB	7.223	11.381	−24.302
1247	VAL165	CG1	6.328	12.612	−24.414
1248	VAL165	CG2	8.64	11.794	−23.892
1249	SER166	N	7.079	8.398	−24.848
1250	SER166	CA	7.919	7.347	−25.421
1251	SER166	C	7.498	7.205	−26.902
1252	SER166	O	6.799	8.053	−27.462
1253	SER166	CB	7.877	6.045	−24.628
1254	SER166	OG	8.632	5.033	−25.298
1255	SER167	N	8.079	6.144	−27.575
1256	SER167	CA	8.464	6.287	−28.98
1257	SER167	C	8.573	4.942	−29.715
1258	SER167	O	9.355	4.741	−30.642
1259	SER167	CB	9.76	7.099	−29.116
1260	SER167	OG	10.789	6.523	−28.311
1261	THR168	N	7.578	4.041	−29.385
1262	THR168	CA	7.352	2.822	−30.159
1263	THR168	C	5.861	2.433	−30.103
1264	THR168	O	4.999	3.146	−29.584
1265	THR168	CB	8.308	1.676	−29.773
1266	THR168	OG1	8.098	0.575	−30.674
1267	THR168	CG2	8.139	1.18	−28.343
1268	TYR169	N	5.555	1.224	−30.708
1269	TYR169	CA	4.235	0.981	−31.326
1270	TYR169	C	3.133	0.935	−30.247
1271	TYR169	O	1.952	1.171	−30.479
1272	TYR169	CB	4.186	−0.38	−32.048
1273	TYR169	CG	4.901	−0.542	−33.373
1274	TYR169	CD1	5.939	0.286	−33.819
1275	TYR169	CD2	4.514	−1.623	−34.188
1276	TYR169	CE1	6.569	0.048	−35.046
1277	TYR169	CE2	5.137	−1.86	−35.414
1278	TYR169	CZ	6.16	−1.023	−35.835
1279	TYR169	OH	6.741	−1.283	−37.041
1280	LYS170	N	3.604	0.444	−29.05
1281	LYS170	CA	2.798	0.211	−27.851
1282	LYS170	C	2.744	1.483	−26.973
1283	LYS170	O	1.97	1.612	−26.026
1284	LYS170	CB	3.513	−0.903	−27.078
1285	LYS170	CG	2.592	−1.758	−26.213
1286	LYS170	CD	3.445	−2.788	−25.473
1287	LYS170	CE	2.645	−3.746	−24.614
1288	LYS170	NZ	3.578	−4.381	−23.651
1289	ASN171	N	3.768	2.368	−27.234
1290	ASN171	CA	4.157	3.465	−26.36
1291	ASN171	C	3.766	4.86	−26.88
1292	ASN171	O	3.772	5.831	−26.123
1293	ASN171	CB	5.673	3.426	−26.177
1294	ASN171	CG	6.106	2.591	−24.996
1295	ASN171	OD1	6.751	1.552	−25.119
1296	ASN171	ND2	5.736	3.079	−23.775
1297	LEU172	N	3.56	4.976	−28.242
1298	LEU172	CA	3.524	6.295	−28.877
1299	LEU172	C	2.334	7.141	−28.378
1300	LEU172	O	1.156	6.825	−28.533
1301	LEU172	CB	3.413	6.16	−30.41
1302	LEU172	CG	4.79	6.179	−31.103
1303	LEU172	CD1	4.705	5.551	−32.493
1304	LEU172	CD2	5.329	7.605	−31.219
1305	PHE173	N	2.709	8.282	−27.695
1306	PHE173	CA	1.821	9.402	−27.38
1307	PHE173	C	0.666	9.132	−26.405
1308	PHE173	O	0.273	10.019	−25.637
1309	PHE173	CB	1.276	10.121	−28.626
1310	PHE173	CG	2.341	10.844	−29.423
1311	PHE173	CD1	2.506	10.572	−30.787
1312	PHE173	CD2	3.148	11.827	−28.829
1313	PHE173	CE1	3.466	11.258	−31.533
1314	PHE173	CE2	4.118	12.501	−29.574
1315	PHE173	CZ	4.274	12.219	−30.927
1316	TRP174	N	0.023	7.916	−26.48
1317	TRP174	CA	−1.29	7.75	−25.846
1318	TRP174	C	−1.183	8.012	−24.328
1319	TRP174	O	−2.097	8.524	−23.68
1320	TRP174	CB	−1.927	6.381	−26.136
1321	TRP174	CG	−1.211	5.23	−25.501
1322	TRP174	CD1	−0.168	4.507	−26.045
1323	TRP174	CD2	−1.461	4.683	−24.201
1324	TRP174	NE1	0.247	3.582	−25.124
1325	TRP174	CE2	−0.482	3.72	−23.964
1326	TRP174	CE3	−2.406	4.949	−23.187
1327	TRP174	CZ2	−0.367	3.063	−22.735
1328	TRP174	CZ3	−2.329	4.256	−21.975
1329	TRP174	CH2	−1.31	3.34	−21.745
1330	GLY175	N	0.002	7.587	−23.752
1331	GLY175	CA	0.236	7.682	−22.325
1332	GLY175	C	0.629	9.101	−21.926
1333	GLY175	O	0.44	9.536	−20.791
1334	ALA176	N	1.254	9.821	−22.928
1335	ALA176	CA	1.572	11.239	−22.756
1336	ALA176	C	0.261	12.029	−22.715
1337	ALA176	O	0.075	12.979	−21.955
1338	ALA176	CB	2.429	11.734	−23.913
1339	LEU177	N	−0.676	11.617	−23.645
1340	LEU177	CA	−1.98	12.249	−23.72
1341	LEU177	C	−2.785	11.896	−22.451
1342	LEU177	O	−3.58	12.7	−21.959
1343	LEU177	CB	−2.706	11.834	−25
1344	LEU177	CG	−2.049	12.401	−26.277
1345	LEU177	CD1	−2.554	11.649	−27.507
1346	LEU177	CD2	−2.312	13.898	−26.435
1347	LEU178	N	−2.592	10.625	−21.934
1348	LEU178	CA	−3.28	10.202	−20.707
1349	LEU178	C	−2.707	10.975	−19.493
1350	LEU178	O	−3.431	11.346	−18.565
1351	LEU178	CB	−3.153	8.688	−20.509
1352	LEU178	CG	−3.953	8.098	−19.331
1353	LEU178	CD1	−5.449	8.386	−19.425
1354	LEU178	CD2	−3.751	6.582	−19.292
1355	GLU179	N	−1.337	11.179	−19.493
1356	GLU179	CA	−0.666	11.977	−18.452
1357	GLU179	C	−1.269	13.394	−18.502
1358	GLU179	O	−1.645	13.991	−17.49
1359	GLU179	CB	0.855	11.976	−18.681
1360	GLU179	CG	1.656	12.821	−17.692
1361	GLU179	CD	2.28	14.011	−18.424
1362	GLU179	OE1	3.48	13.853	−18.808
1363	GLU179	OE2	1.514	15.003	−18.568
1364	LYS180	N	−1.378	13.922	−19.776
1365	LYS180	CA	−1.856	15.282	−19.97
1366	LYS180	C	−3.353	15.369	−19.581
1367	LYS180	O	−3.847	16.402	−19.118
1368	LYS180	CB	−1.6	15.696	−21.418
1369	LYS180	CG	−2.039	17.119	−21.728
1370	LYS180	CD	−1.673	17.5	−23.169
1371	LYS180	CE	−2.371	18.765	−23.644
1372	LYS180	NZ	−3.814	18.529	−23.623
1373	ALA181	N	−4.117	14.246	−19.843
1374	ALA181	CA	−5.532	14.194	−19.486
1375	ALA181	C	−5.69	14.205	−17.958
1376	ALA181	O	−6.638	14.765	−17.406
1377	ALA181	CB	−6.199	12.946	−20.04
1378	TYR182	N	−4.741	13.478	−17.262
1379	TYR182	CA	−4.747	13.443	−15.796
1380	TYR182	C	−4.457	14.878	−15.312
1381	TYR182	O	−5.095	15.396	−14.394
1382	TYR182	CB	−3.706	12.448	−15.261
1383	TYR182	CG	−3.807	11.985	−13.822
1384	TYR182	CD1	−2.727	11.247	−13.3
1385	TYR182	CD2	−4.931	12.18	−13.008
1386	TYR182	CE1	−2.776	10.707	−12.013
1387	TYR182	CE2	−4.982	11.63	−11.721
1388	TYR182	CZ	−3.915	10.88	−11.242
1389	TYR182	OH	−3.965	10.288	−10.012
1390	ALA183	N	−3.434	15.531	−15.988
1391	ALA183	CA	−3.038	16.884	−15.608
1392	ALA183	C	−4.202	17.867	−15.839
1393	ALA183	O	−4.4	18.834	−15.1
1394	ALA183	CB	−1.832	17.348	−16.408
1395	LYS184	N	4.981	17.64	−16.97
1396	LYS184	CA	−5.99	18.628	−17.348
1397	LYS184	C	−7.037	18.763	−16.233
1398	LYS184	O	−7.581	19.841	−15.989
1399	LYS184	CB	−6.679	18.274	−18.681
1400	LYS184	CG	−7.882	19.184	−18.979
1401	LYS184	CD	−8.472	18.994	−20.377
1402	LYS184	CE	−9.794	19.745	−20.487
1403	LYS184	NZ	−10.324	19.656	−21.85
1404	LEU185	N	−7.438	17.574	−15.652
1405	LEU185	CA	−8.502	17.579	−14.661
1406	LEU185	C	−7.993	18.015	−13.276
1407	LEU185	O	−8.758	18.521	−12.443
1408	LEU185	CB	−9.19	16.216	−14.574
1409	LEU185	CG	−10.637	16.334	−14.048
1410	LEU185	CD1	−11.596	16.865	−15.116
1411	LEU185	CD2	−11.116	14.98	−13.546
1412	SER186	N	−6.679	17.712	−12.974
1413	SER186	CA	−6.082	18.2	−11.73
1414	SER186	C	−5.982	19.731	−11.809
1415	SER186	O	−6.408	20.446	−10.901
1416	SER186	CB	−4.744	17.544	−11.396
1417	SER186	OG	−3.726	17.894	−12.319
1418	GLY187	N	−5.389	20.215	−12.954
1419	GLY187	CA	−5.295	21.628	−13.244
1420	GLY187	C	−4.073	21.958	−14.088
1421	GLY187	O	−4.101	22.845	−14.942
1422	SER188	N	−2.929	21.274	−13.737
1423	SER188	CA	−1.635	21.633	−14.299
1424	SER188	C	−0.634	20.464	−14.245
1425	SER188	O	−0.86	19.395	−13.679
1426	SER188	CB	−1.08	22.931	−13.683
1427	SER188	OG	−1.317	23.08	−12.285
1428	TYR189	N	0.559	20.686	−14.923
1429	TYR189	CA	1.584	19.634	−14.924
1430	TYR189	C	2.307	19.583	−13.564
1431	TYR189	O	2.837	18.545	−13.161
1432	TYR189	CB	2.64	19.809	−16.024
1433	TYR189	CG	2.104	19.641	−17.427
1434	TYR189	CD1	2.367	20.614	−18.397
1435	TYR189	CD2	1.388	18.501	−17.808
1436	TYR189	CE1	1.864	20.5	−19.69
1437	TYR189	CE2	0.894	18.376	−19.107
1438	TYR189	CZ	1.125	19.379	−20.036
1439	TYR189	OH	0.641	19.22	−21.301
1440	GLU190	N	2.384	20.786	−12.876
1441	GLU190	CA	3.168	20.899	−11.632
1442	GLU190	C	2.609	19.903	−10.583
1443	GLU190	O	3.317	19.387	−9.715
1444	GLU190	CB	3.086	22.345	−11.112
1445	GLU190	CG	3.854	22.683	−9.83
1446	GLU190	CD	5.371	22.781	−9.988
1447	GLU190	OE1	5.914	21.822	−10.607
1448	GLU190	OE2	5.92	23.794	−9.457
1449	ASP191	N	1.247	19.682	−10.688
1450	ASP191	CA	0.426	19.049	−9.662
1451	ASP191	C	0.887	17.614	−9.366
1452	ASP191	O	0.648	17.058	−8.296
1453	ASP191	CB	−1.033	18.939	−10.123
1454	ASP191	CG	−1.597	20.089	−10.935
1455	ASP191	OD1	−2.749	19.891	−11.438
1456	ASP191	OD2	−0.842	21.096	−11.106
1457	LEU192	N	1.439	16.958	−10.45
1458	LEU192	CA	1.598	15.508	−10.482
1459	LEU192	C	2.801	15.062	−9.63
1460	LEU192	O	2.951	13.901	−9.246
1461	LEU192	CB	1.795	15.005	−11.92
1462	LEU192	CG	0.622	15.292	−12.882
1463	LEU192	CD1	0.972	14.781	−14.281
1464	LEU192	CD2	−0.69	14.659	−12.415
1465	GLN193	N	3.748	16.036	−9.4
1466	GLN193	CA	4.968	15.752	−8.659
1467	GLN193	C	4.574	15.629	−7.176
1468	GLN193	O	4.262	16.607	−6.498
1469	GLN193	CB	5.987	16.876	−8.833
1470	GLN193	CG	6.408	17.051	−10.293
1471	GLN193	CD	6.947	18.435	−10.567
1472	GLN193	OE1	8.067	18.62	−11.029
1473	GLN193	NE2	6.061	19.431	−10.274
1474	SER194	N	4.539	14.329	−6.718
1475	SER194	CA	3.995	13.858	−5.439
1476	SER194	C	2.514	13.433	−5.505
1477	SER194	O	1.863	13.182	−4.484
1478	SER194	CB	4.308	14.701	−4.189
1479	SER194	OG	3.332	15.722	−3.907
1480	GLY195	N	2.041	13.202	−6.782
1481	GLY195	CA	1.101	12.127	−7.04
1482	GLY195	C	1.928	10.845	−7.219
1483	GLY195	O	3.15	10.857	−7.377
1484	GLN196	N	1.186	9.688	−7.163
1485	GLN196	CA	1.797	8.377	−7.026
1486	GLN196	C	0.965	7.331	−7.793
1487	GLN196	O	−0.253	7.41	−7.969
1488	GLN196	CB	1.808	7.923	−5.553
1489	GLN196	CG	2.661	8.755	−4.601
1490	GLN196	CD	2.095	8.793	−3.196
1491	GLN196	OE1	1.964	9.849	−2.572
1492	GLN196	NE2	1.777	7.59	−2.63
1493	VAL197	N	1.681	6.168	−8.084
1494	VAL197	CA	1.01	5.06	−8.774
1495	VAL197	C	−0.116	4.492	−7.891
1496	VAL197	O	−1.095	3.91	−8.354
1497	VAL197	CB	1.965	3.914	−9.166
1498	VAL197	CG1	2.893	4.344	−10.297
1499	VAL197	CG2	2.792	3.356	−8.003
1500	SER198	N	0.102	4.647	−6.537
1501	SER198	CA	−0.789	4.109	−5.523
1502	SER198	C	−2.164	4.782	−5.597
1503	SER198	O	−3.17	4.207	−5.178
1504	SER198	CB	−0.199	4.308	−4.12
1505	SER198	OG	0.213	5.664	−3.897
1506	GLU199	N	−2.127	6.1	−6.024
1507	GLU199	CA	−3.344	6.848	−6.325
1508	GLU199	C	−3.753	6.559	−7.786
1509	GLU199	O	−4.916	6.304	−8.108
1510	GLU199	CB	−3.127	8.357	−6.167
1511	GLU199	CG	−2.568	8.791	−4.805
1512	GLU199	CD	−1.449	9.818	−4.893
1513	GLU199	OE1	−0.876	9.96	−6.007
1514	GLU199	OE2	−1.141	10.415	−3.814
1515	ALA200	N	−2.74	6.657	−8.734
1516	ALA200	CA	−3.103	6.771	−10.149
1517	ALA200	C	−3.797	5.49	−10.633
1518	ALA200	O	−4.67	5.494	−11.502
1519	ALA200	CB	−1.874	7.023	−11.007
1520	LEU201	N	−3.322	4.319	−10.067
1521	LEU201	CA	−3.888	3.038	−10.447
1522	LEU201	C	−5.325	2.882	−9.906
1523	LEU201	O	−6.103	2.079	−10.432
1524	LEU201	CB	−3.011	1.864	−9.998
1525	LEU201	CG	−1.642	1.78	−10.709
1526	LEU201	CD1	−0.767	0.732	−10.018
1527	LEU201	CD2	−1.779	1.433	−12.193
1528	VAL202	N	−5.667	3.646	−8.801
1529	VAL202	CA	−7.065	3.737	−8.368
1530	VAL202	C	−7.818	4.513	−9.456
1531	VAL202	O	−8.867	4.067	−9.927
1532	VAL202	CB	−7.246	4.416	−6.992
1533	VAL202	CG1	−8.72	4.645	−6.65
1534	VAL202	CG2	−6.578	3.6	−5.892
1535	ASP203	N	−7.254	5.713	−9.85
1536	ASP203	CA	−8.006	6.652	−10.692
1537	ASP203	C	−8.397	5.992	−12.047
1538	ASP203	O	−9.411	6.314	−12.673
1539	ASP203	CB	−7.187	7.906	−10.97
1540	ASP203	CG	−7.174	8.932	−9.842
1541	ASP203	OD1	−8.293	9.238	−9.338
1542	ASP203	OD2	−6.035	9.446	−9.607
1543	PHE204	N	−7.478	5.086	−12.557
1544	PHE204	CA	−7.72	4.42	−13.845
1545	PHE204	C	−8.653	3.196	−13.671
1546	PHE204	O	−9.343	2.734	−14.585
1547	PHE204	CB	−6.418	3.901	−14.479
1548	PHE204	CG	−5.392	4.96	−14.801
1549	PHE204	CD1	−5.721	6.123	−15.505
1550	PHE204	CD2	−4.062	4.771	−14.4
1551	PHE204	CE1	−4.747	7.093	−15.756
1552	PHE204	CE2	−3.099	5.749	−14.641
1553	PHE204	CZ	−3.439	6.913	−15.318
1554	THR205	N	−8.498	2.525	−12.471
1555	THR205	CA	−9.181	1.26	−12.237
1556	THR205	C	−10.57	1.499	−11.629
1557	THR205	O	−11.559	0.925	−12.094
1558	THR205	CB	−8.375	0.292	−11.355
1559	THR205	OG1	−7.023	0.205	−11.813
1560	THR205	CG2	−8.953	−1.117	−11.399
1561	GLY206	N	−10.57	2.248	−10.471
1562	GLY206	CA	−11.736	2.417	−9.612
1563	GLY206	C	−11.773	1.408	−8.46
1564	GLY206	O	−12.704	1.354	−7.66
1565	GLY207	N	−10.674	0.575	−8.411
1566	GLY207	CA	−10.481	−0.4	−7.359
1567	GLY207	C	−9.701	0.191	−6.19
1568	GLY207	O	−9.342	1.364	−6.144
1569	VAL208	N	−9.446	−0.722	−5.185
1570	VAL208	CA	−8.721	−0.33	−3.977
1571	VAL208	C	−7.241	−0.71	−4.177
1572	VAL208	O	−6.892	−1.808	−4.614
1573	VAL208	CB	−9.326	−1.027	−2.736
1574	VAL208	CG1	−8.569	−0.683	−1.451
1575	VAL208	CG2	−10.802	−0.645	−2.557
1576	THR209	N	−6.341	0.271	−3.793
1577	THR209	CA	−4.903	−0.012	−3.717
1578	THR209	C	−4.681	−0.655	−2.348
1579	THR209	O	−5.049	−0.111	−1.308
1580	THR209	CB	−4.045	1.266	−3.824
1581	THR209	OG1	−4.047	1.727	−5.178
1582	THR209	CG2	−2.587	1.029	−3.45
1583	MET210	N	−4.037	−1.872	−2.386
1584	MET210	CA	−3.614	−2.558	−1.167
1585	MET210	C	−2.079	−2.547	−1.22
1586	MET210	O	−1.446	−3.248	−2.012
1587	MET210	CB	−4.185	−3.983	−1.139
1588	MET210	CG	−4.994	−4.283	0.122
1589	MET210	SD	−3.932	−4.483	1.581
1590	MET210	CE	−5.23	−4.677	2.828
1591	THR211	N	−1.497	−1.595	−0.405
1592	THR211	CA	−0.058	−1.513	−0.171
1593	THR211	C	0.329	−2.517	0.925
1594	THR211	O	−0.404	−2.788	1.874
1595	THR211	CB	0.362	−0.109	0.315
1596	THR211	OG1	−0.653	0.417	1.181
1597	THR211	CG2	0.568	0.868	−0.834
1598	ILE212	N	1.606	−3.032	0.784
1599	ILE212	CA	2.161	−3.992	1.731
1600	ILE212	C	3.653	−3.623	1.842
1601	ILE212	O	4.439	−3.679	0.89
1602	ILE212	CB	1.996	−5.467	1.269
1603	ILE212	CG1	0.544	−5.787	0.842
1604	ILE212	CG2	2.459	−6.409	2.391
1605	ILE212	CD1	0.323	−7.224	0.389
1606	ASN213	N	4.051	−3.196	3.097
1607	ASN213	CA	5.445	−2.852	3.377
1608	ASN213	C	6.198	−4.176	3.565
1609	ASN213	O	6.374	−4.712	4.655
1610	ASN213	CB	5.591	−1.95	4.597
1611	ASN213	CG	5.543	−0.495	4.194
1612	ASN213	OD1	4.56	0.217	4.374
1613	ASN213	ND2	6.679	−0.011	3.606
1614	LEU214	N	6.631	−4.743	2.372
1615	LEU214	CA	6.849	−6.196	2.257
1616	LEU214	C	7.948	−6.786	3.164
1617	LEU214	O	8.071	−8	3.325
1618	LEU214	CB	7.21	−6.524	0.797
1619	LEU214	CG	6.781	−7.924	0.322
1620	LEU214	CD1	5.264	−8.091	0.35
1621	LEU214	CD2	7.299	−8.169	−1.097
1622	ALA215	N	8.799	−5.866	3.743
1623	ALA215	CA	9.746	−6.28	4.771
1624	ALA215	C	9.004	−6.749	6.035
1625	ALA215	O	9.443	−7.658	6.743
1626	ALA215	CB	10.69	−5.138	5.114
1627	GLU216	N	7.856	−6.051	6.362
1628	GLU216	CA	6.965	−6.512	7.432
1629	GLU216	C	6.126	−7.656	6.824
1630	GLU216	O	4.964	−7.535	6.437
1631	GLU216	CB	6.095	−5.354	7.928
1632	GLU216	CG	5.351	−5.703	9.216
1633	GLU216	CD	4.479	−4.568	9.76
1634	GLU216	OE1	4.149	−4.705	10.973
1635	GLU216	OE2	4.185	−3.649	8.948
1636	ALA217	N	6.837	−8.845	6.702
1637	ALA217	CA	6.304	−9.929	5.891
1638	ALA217	C	5.088	−10.522	6.619
1639	ALA217	O	5.13	−10.958	7.768
1640	ALA217	CB	7.341	−11.017	5.666
1641	HIS218	N	3.921	−10.47	5.876
1642	HIS218	CA	2.657	−10.92	6.446
1643	HIS218	C	2.558	−12.444	6.27
1644	HIS218	O	3.016	−13.043	5.297
1645	HIS218	CB	1.464	−10.242	5.762
1646	HIS218	CG	1.262	−8.812	6.152
1647	HIS218	ND1	2.268	−7.905	6.403
1648	HIS218	CD2	0.101	−8.086	6.327
1649	HIS218	CE1	1.678	−6.716	6.736
1650	HIS218	NE2	0.368	−6.792	6.683
1651	GLY219	N	1.808	−13.081	7.253
1652	GLY219	CA	1.951	−14.522	7.447
1653	GLY219	C	1.238	−15.416	6.431
1654	GLY219	O	1.25	−16.64	6.533
1655	ASN220	N	0.606	−14.742	5.413
1656	ASN220	CA	−0.097	−15.41	4.321
1657	ASN220	C	0.08	−14.582	3.033
1658	ASN220	O	−0.798	−14.425	2.188
1659	ASN220	CB	−1.55	−15.706	4.677
1660	ASN220	CG	−2.411	−14.472	4.791
1661	ASN220	OD1	−3.291	−14.195	3.978
1662	ASN220	ND2	−2.161	−13.678	5.875
1663	LEU221	N	1.381	−14.127	2.838
1664	LEU221	CA	1.657	−13.17	1.758
1665	LEU221	C	1.537	−13.871	0.395
1666	LEU221	O	1.097	−13.298	−0.603
1667	LEU221	CB	3.045	−12.546	1.961
1668	LEU221	CG	3.463	−11.398	1.019
1669	LEU221	CD1	4.053	−11.903	−0.296
1670	LEU221	CD2	2.349	−10.38	0.782
1671	TRP222	N	2.049	−15.157	0.345
1672	TRP222	CA	1.983	−15.898	−0.921
1673	TRP222	C	0.519	−16.115	−1.317
1674	TRP222	O	0.144	−16.108	−2.489
1675	TRP222	CB	2.717	−17.243	−0.832
1676	TRP222	CG	3.702	−17.433	−1.948
1677	TRP222	CD1	3.681	−18.413	−2.923
1678	TRP222	CD2	4.845	−16.612	−2.211
1679	TRP222	NE1	4.778	−18.245	−3.728
1680	TRP222	CE2	5.502	−17.153	−3.312
1681	TRP222	CE3	5.376	−15.448	−1.617
1682	TRP222	CZ2	6.677	−16.608	−3.831
1683	TRP222	CZ3	6.521	−14.861	−2.163
1684	TRP222	CH2	7.167	−15.439	−3.249
1685	ASP223	N	−0.329	−16.336	−0.26
1686	ASP223	CA	−1.732	−16.674	−0.436
1687	ASP223	C	−2.439	−15.457	−1.071
1688	ASP223	O	−3.327	−15.585	−1.915
1689	ASP223	CB	−2.365	−17.05	0.899
1690	ASP223	CG	−1.492	−17.936	1.793
1691	ASP223	OD1	−2.122	−18.723	2.549
1692	ASP223	OD2	−0.241	−17.716	1.707
1693	ILE224	N	−2.011	−14.218	−0.61
1694	ILE224	CA	−2.557	−12.964	−1.141
1695	ILE224	C	−2.205	−12.885	−2.64
1696	ILE224	O	−3.014	−12.473	−3.475
1697	ILE224	CB	−2.02	−11.714	−0.388
1698	ILE224	CG1	−2.573	−11.676	1.05
1699	ILE224	CG2	−2.385	−10.409	−1.116
1700	ILE224	CD1	−1.906	−10.642	1.947
1701	LEU225	N	−0.9	−13.202	−2.961
1702	LEU225	CA	−0.423	−13.139	−4.339
1703	LEU225	C	−1.047	−14.261	−5.206
1704	LEU225	O	−1.184	−14.141	−6.428
1705	LEU225	CB	1.105	−13.173	−4.41
1706	LEU225	CG	1.79	−11.79	−4.36
1707	LEU225	CD1	1.338	−10.9	−3.204
1708	LEU225	CD2	3.308	−11.981	−4.304
1709	ILE226	N	−1.398	−15.419	−4.543
1710	ILE226	CA	−2.158	−16.482	−5.211
1711	ILE226	C	−3.569	−15.908	−5.494
1712	ILE226	O	−4.099	−16.024	−6.603
1713	ILE226	CB	−2.153	−17.799	−4.401
1714	ILE226	CG1	−0.769	−18.483	−4.52
1715	ILE226	CG2	−3.251	−18.76	−4.869
1716	ILE226	CD1	−0.545	−19.602	−3.512
1717	GLU227	N	−4.195	−15.25	−4.447
1718	GLU227	CA	−5.531	−14.679	−4.634
1719	GLU227	C	−5.479	−13.627	−5.764
1720	GLU227	O	−6.373	−13.499	−6.603
1721	GLU227	CB	−6.066	−13.998	−3.364
1722	GLU227	CG	−7.271	−14.723	−2.779
1723	GLU227	CD	−6.902	−15.919	−1.909
1724	GLU227	OE1	−6.286	−15.606	−0.837
1725	GLU227	OE2	−7.305	−17.042	−2.336
1726	ALA228	N	−4.374	−12.795	−5.728
1727	ALA228	CA	−4.271	−11.646	−6.618
1728	ALA228	C	−4.181	−12.118	−8.08
1729	ALA228	O	−4.639	−11.458	−9.016
1730	ALA228	CB	−3.029	−10.841	−6.274
1731	THR229	N	−3.461	−13.284	−8.258
1732	THR229	CA	−3.262	−13.877	−9.57
1733	THR229	C	−4.478	−14.672	−10.083
1734	THR229	O	−4.599	−14.866	−11.298
1735	THR229	CB	−1.972	−14.717	−9.672
1736	THR229	OG1	−1.776	−15.533	−8.52
1737	THR229	CG2	−0.729	−13.851	−9.858
1738	TYR230	N	−5.375	−15.178	−9.154
1739	TYR230	CA	−6.665	−15.726	−9.594
1740	TYR230	C	−7.839	−14.716	−9.578
1741	TYR230	O	−8.954	−15.009	−10.022
1742	TYR230	CB	−7.009	−17.12	−9.043
1743	TYR230	CG	−7.495	−17.25	−7.62
1744	TYR230	CD1	−6.708	−17.904	−6.661
1745	TYR230	CD2	−8.779	−16.824	−7.252
1746	TYR230	CE1	−7.187	−18.111	−5.364
1747	TYR230	CE2	−9.252	−17.022	−5.955
1748	TYR230	CZ	−8.453	−17.658	−5.02
1749	TYR230	OH	−8.933	−17.814	−3.747
1750	ASN231	N	−7.53	−13.45	−9.12
1751	ASN231	CA	−8.366	−12.287	−9.435
1752	ASN231	C	−7.876	−11.655	−10.756
1753	ASN231	O	−8.659	−11.094	−11.523
1754	ASN231	CB	−8.348	−11.242	−8.326
1755	ASN231	CG	−9.222	−10.039	−8.619
1756	ASN231	OD1	−8.789	−8.888	−8.614
1757	ASN231	ND2	−10.546	−10.28	−8.837
1758	ARG232	N	−6.507	−11.647	−10.936
1759	ARG232	CA	−5.833	−11	−12.059
1760	ARG232	C	−5.824	−9.478	−11.843
1761	ARG232	O	−6.255	−8.671	−12.672
1762	ARG232	CB	−6.345	−11.407	−13.448
1763	ARG232	CG	−6.11	−12.893	−13.724
1764	ARG232	CD	−6.664	−13.312	−15.077
1765	ARG232	NE	−6.351	−14.717	−15.369
1766	ARG232	CZ	−5.132	−15.175	−15.748
1767	ARG232	NH1	−4.95	−16.509	−15.884
1768	ARG232	NH2	−4.091	−14.362	−16.01
1769	THR233	N	−5.206	−9.094	−10.668
1770	THR233	CA	−4.818	−7.708	−10.41
1771	THR233	C	−3.502	−7.41	−11.169
1772	THR233	O	−2.874	−8.268	−11.79
1773	THR233	CB	−4.613	−7.455	−8.905
1774	THR233	OG1	−3.54	−8.265	−8.436
1775	THR233	CG2	−5.843	−7.771	−8.071
1776	LEU234	N	−3.086	−6.084	−11.103
1777	LEU234	CA	−1.673	−5.76	−11.307
1778	LEU234	C	−1.027	−5.808	−9.904
1779	LEU234	O	−1.659	−5.515	−8.885
1780	LEU234	CB	−1.478	−4.37	−11.924
1781	LEU234	CG	−2.119	−4.193	−13.316
1782	LEU234	CD1	−1.98	−2.738	−13.772
1783	LEU234	CD2	−1.502	−5.121	−14.364
1784	ILE235	N	0.303	−6.192	−9.912
1785	ILE235	CA	1.03	−6.564	−8.693
1786	ILE235	C	2.451	−6.001	−8.879
1787	ILE235	O	3.222	−6.474	−9.721
1788	ILE235	CB	1.117	−8.11	−8.538
1789	ILE235	CG1	−0.254	−8.808	−8.634
1790	ILE235	CG2	1.846	−8.473	−7.234
1791	ILE235	CD1	−0.173	−10.328	−8.602
1792	GLY236	N	2.759	−4.917	−8.08
1793	GLY236	CA	4.011	−4.19	−8.235
1794	GLY236	C	4.421	−3.56	−6.91
1795	GLY236	O	3.603	−3.04	−6.148
1796	CYS237	N	5.725	−3.547	−6.529
1797	CYS237	CA	6.857	−4.283	−7.087
1798	CYS237	C	8.063	−3.78	−6.284
1799	CYS237	O	7.91	−3.319	−5.153
1800	CYS237	CB	6.703	−5.791	−6.864
1801	CYS237	SG	7.471	−6.738	−8.199
1802	GLN238	N	9.292	−3.94	−6.884
1803	GLN238	CA	10.552	−3.904	−6.105
1804	GLN238	C	11.444	−4.893	−6.899
1805	GLN238	O	10.903	−5.735	−7.626
1806	GLN238	CB	11.161	−2.511	−5.884
1807	GLN238	CG	10.422	−1.538	−4.952
1808	GLN238	CD	10.355	−1.958	−3.489
1809	GLN238	OE1	11.034	−1.48	−2.583
1810	GLN238	NE2	9.443	−2.924	−3.213
1811	THR239	N	12.807	−4.866	−6.7
1812	THR239	CA	13.655	−5.762	−7.512
1813	THR239	C	13.612	−7.169	−6.901
1814	THR239	O	13.197	−8.155	−7.508
1815	THR239	CB	15.092	−5.205	−7.588
1816	THR239	OG1	14.982	−3.829	−7.972
1817	THR239	CG2	15.953	−5.988	−8.57
1818	HIS240	N	14.09	−7.248	−5.61
1819	HIS240	CA	14.281	−8.527	−4.914
1820	HIS240	C	12.978	−8.912	−4.203
1821	HIS240	O	12.88	−9.04	−2.985
1822	HIS240	CB	15.458	−8.418	−3.936
1823	HIS240	CG	16.743	−8.341	−4.684
1824	HIS240	ND1	17.417	−9.453	−5.129
1825	HIS240	CD2	17.479	−7.272	−5.144
1826	HIS240	CE1	18.495	−9.005	−5.838
1827	HIS240	NE2	18.56	−7.693	−5.865
1828	SER241	N	11.916	−9.112	−5.071
1829	SER241	CA	10.542	−9.313	−4.607
1830	SER241	C	10.161	−10.799	−4.555
1831	SER241	O	9.027	−11.208	−4.801
1832	SER241	CB	9.546	−8.498	−5.441
1833	SER241	OG	8.59	−7.843	−4.604
1834	GLY242	N	11.138	−11.604	−4.005
1835	GLY242	CA	10.886	−12.991	−3.685
1836	GLY242	C	11.518	−13.984	−4.646
1837	GLY242	O	11.058	−15.12	−4.768
1838	GLU243	N	12.701	−13.574	−5.222
1839	GLU243	CA	13.646	−14.531	−5.795
1840	GLU243	C	14.662	−14.811	−4.652
1841	GLU243	O	14.268	−14.833	−3.474
1842	GLU243	CB	14.083	−14.032	−7.179
1843	GLU243	CG	12.878	−14.115	−8.149
1844	GLU243	CD	13.078	−14.26	−9.661
1845	GLU243	OE1	13.914	−13.477	−10.185
1846	GLU243	OE2	12.38	−15.174	−10.223
1847	LYS244	N	15.967	−15.146	−4.963
1848	LYS244	CA	16.915	−15.495	−3.891
1849	LYS244	C	18.043	−14.481	−3.601
1850	LYS244	O	18.714	−14.546	−2.563
1851	LYS244	CB	17.58	−16.87	−4.126
1852	LYS244	CG	16.585	−18.033	−4.084
1853	LYS244	CD	17.202	−19.34	−3.557
1854	LYS244	CE	16.229	−20.489	−3.773
1855	LYS244	NZ	16.694	−21.744	−3.179
1856	ILE245	N	18.33	−13.6	−4.621
1857	ILE245	CA	19.704	−13.1	−4.817
1858	ILE245	C	20.105	−12.091	−3.705
1859	ILE245	O	19.301	−11.591	−2.912
1860	ILE245	CB	19.866	−12.645	−6.297
1861	ILE245	CG1	19.798	−13.898	−7.213
1862	ILE245	CG2	21.135	−11.843	−6.591
1863	ILE245	CD1	19.972	−13.612	−8.696
1864	LEU246	N	21.47	−11.909	−3.553
1865	LEU246	CA	22.04	−11.035	−2.526
1866	LEU246	C	21.875	−9.555	−2.918
1867	LEU246	O	21.604	−9.188	−4.057
1868	LEU246	CB	23.526	−11.357	−2.297
1869	LEU246	CG	23.787	−12.752	−1.695
1870	LEU246	CD1	25.282	−13.077	−1.762
1871	LEU246	CD2	23.311	−12.848	−0.244
1872	GLU247	N	22.06	−8.689	−1.856
1873	GLU247	CA	21.764	−7.257	−1.941
1874	GLU247	C	22.653	−6.574	−0.877
1875	GLU247	O	23.403	−7.203	−0.125
1876	GLU247	CB	20.257	−7.031	−1.687
1877	GLU247	CG	19.585	−6.072	−2.672
1878	GLU247	CD	19.777	−4.615	−2.3
1879	GLU247	OE1	18.747	−3.984	−1.931
1880	GLU247	OE2	20.979	−4.202	−2.313
1881	ASN248	N	22.551	−5.209	−0.847
1882	ASN248	CA	23.218	−4.331	0.109
1883	ASN248	C	22.391	−3.063	0.424
1884	ASN248	O	22.479	−2.526	1.53
1885	ASN248	CB	24.616	−3.956	−0.38
1886	ASN248	CG	25.697	−4.59	0.469
1887	ASN248	OD1	26.558	−3.934	1.047
1888	ASN248	ND2	25.696	−5.956	0.515
1889	GLY249	N	21.675	−2.496	−0.615
1890	GLY249	CA	20.704	−1.458	−0.326
1891	GLY249	C	19.996	−0.918	−1.571
1892	GLY249	O	20.588	−0.612	−2.604
1893	LEU250	N	18.631	−0.761	−1.405
1894	LEU250	CA	17.733	−0.144	−2.382
1895	LEU250	C	17.442	−1.024	−3.615
1896	LEU250	O	16.294	−1.159	−4.043
1897	LEU250	CB	18.165	1.266	−2.829
1898	LEU250	CG	18.419	2.269	−1.685
1899	LEU250	CD1	18.916	3.597	−2.264
1900	LEU250	CD2	17.174	2.512	−0.832
1901	VAL251	N	18.555	−1.5	−4.273
1902	VAL251	CA	18.487	−2.051	−5.63
1903	VAL251	C	18.664	−0.876	−6.614
1904	VAL251	O	19.193	0.189	−6.286
1905	VAL251	CB	19.538	−3.173	−5.802
1906	VAL251	CG1	20.982	−2.666	−5.845
1907	VAL251	CG2	19.264	−4.06	−7.016
1908	GLU252	N	18.244	−1.121	−7.904
1909	GLU252	CA	18.313	−0.099	−8.931
1910	GLU252	C	17.064	0.805	−8.898
1911	GLU252	O	16.015	0.521	−8.32
1912	GLU252	CB	18.559	−0.724	−10.313
1913	GLU252	CG	17.381	−1.462	−10.947
1914	GLU252	CD	16.937	−2.788	−10.337
1915	GLU252	OE1	16.583	−3.673	−11.176
1916	GLU252	OE2	16.902	−2.853	−9.067
1917	GLY253	N	17.189	1.972	−9.635
1918	GLY253	CA	16.143	2.98	−9.629
1919	GLY253	C	15.005	2.579	−10.561
1920	GLY253	O	14.857	3.072	−11.678
1921	HIS254	N	14.186	1.593	−10.045
1922	HIS254	CA	13.164	0.948	−10.863
1923	HIS254	C	12.181	0.227	−9.928
1924	HIS254	O	12.461	−0.07	−8.768
1925	HIS254	CB	13.814	−0.05	−11.838
1926	HIS254	CG	13.022	−0.295	−13.075
1927	HIS254	ND1	12.803	0.665	−14.031
1928	HIS254	CD2	12.416	−1.427	−13.576
1929	HIS254	CE1	12.073	0.088	−15.029
1930	HIS254	NE2	11.843	−1.182	−14.795
1931	ALA255	N	10.976	−0.101	−10.519
1932	ALA255	CA	10.04	−1.017	−9.884
1933	ALA255	C	9.562	−1.974	−10.98
1934	ALA255	O	9.432	−1.627	−12.151
1935	ALA255	CB	8.88	−0.293	−9.228
1936	TYR256	N	9.353	−3.256	−10.524
1937	TYR256	CA	9.061	−4.376	−11.419
1938	TYR256	C	7.558	−4.671	−11.306
1939	TYR256	O	6.814	−4.032	−10.559
1940	TYR256	CB	9.918	−5.601	−11.046
1941	TYR256	CG	11.385	−5.477	−11.403
1942	TYR256	CD1	12.185	−4.472	−10.843
1943	TYR256	CD2	11.987	−6.408	−12.262
1944	TYR256	CE1	13.533	−4.357	−11.178
1945	TYR256	CE2	13.343	−6.305	−12.583
1946	TYR256	CZ	14.104	−5.269	−12.053
1947	TYR256	OH	15.418	−5.186	−12.413
1948	THR257	N	7.096	−5.697	−12.106
1949	THR257	CA	5.751	−6.245	−11.912
1950	THR257	C	5.882	−7.767	−11.761
1951	THR257	O	6.862	−8.39	−12.172
1952	THR257	CB	4.785	−5.826	−13.033
1953	THR257	OG1	3.438	−6.226	−12.762
1954	THR257	CG2	5.178	−6.346	−14.404
1955	LEU258	N	4.796	−8.365	−11.156
1956	LEU258	CA	4.758	−9.801	−10.82
1957	LEU258	C	3.657	−10.421	−11.703
1958	LEU258	O	2.578	−9.849	−11.876
1959	LEU258	CB	4.477	−9.917	−9.316
1960	LEU258	CG	5.035	−11.17	−8.621
1961	LEU258	CD1	5.351	−10.838	−7.159
1962	LEU258	CD2	4.036	−12.324	−8.672
1963	THR259	N	3.969	−11.634	−12.313
1964	THR259	CA	3.099	−12.191	−13.367
1965	THR259	C	2.945	−13.737	−13.36
1966	THR259	O	2.503	−14.372	−14.326
1967	THR259	CB	3.424	−11.686	−14.802
1968	THR259	OG1	4.665	−12.213	−15.307
1969	THR259	CG2	3.491	−10.171	−14.94
1970	GLY260	N	3.177	−14.344	−12.141
1971	GLY260	CA	3.017	−15.778	−11.933
1972	GLY260	C	3.617	−16.24	−10.601
1973	GLY260	O	4.34	−15.515	−9.914
1974	ILE261	N	3.277	−17.542	−10.254
1975	ILE261	CA	3.485	−18.055	−8.884
1976	ILE261	C	3.328	−19.605	−8.878
1977	ILE261	O	2.85	−20.201	−9.849
1978	ILE261	CB	2.487	−17.336	−7.933
1979	ILE261	CG1	2.935	−17.375	−6.465
1980	ILE261	CG2	1.049	−17.849	−8.094
1981	ILE261	CD1	2.357	−16.213	−5.673
1982	ARG262	N	3.751	−20.227	−7.708
1983	ARG262	CA	3.281	−21.535	−7.183
1984	ARG262	C	4.264	−22.043	−6.083
1985	ARG262	O	5.187	−21.344	−5.659
1986	ARG262	CB	3.005	−22.6	−8.255
1987	ARG262	CG	4.269	−23.015	−9.005
1988	ARG262	CD	3.951	−23.82	−10.255
1989	ARG262	NE	3.381	−22.955	−11.296
1990	ARG262	CZ	3.328	−23.325	−12.598
1991	ARG262	NH1	2.901	−22.461	−13.537
1992	ARG262	NH2	3.705	−24.548	−13.015
1993	LYS263	N	4.005	−23.303	−5.575
1994	LYS263	CA	4.937	−24.024	−4.693
1995	LYS263	C	5.465	−25.246	−5.468
1996	LYS263	O	4.778	−25.813	−6.32
1997	LYS263	CB	4.195	−24.458	−3.421
1998	LYS263	CG	5.102	−25.081	−2.357
1999	LYS263	CD	4.339	−25.363	−1.061
2000	LYS263	CE	5.27	−25.934	0
2001	LYS263	NZ	4.644	−25.802	1.335
2002	VAL264	N	6.718	−25.681	−5.083
2003	VAL264	CA	7.326	−26.916	−5.581
2004	VAL264	C	8.106	−27.592	−4.428
2005	VAL264	O	8.335	−27.036	−3.352
2006	VAL264	CB	8.248	−26.692	−6.807
2007	VAL264	CG1	7.47	−26.229	−8.04
2008	VAL264	CG2	9.396	−25.727	−6.504
2009	THR265	N	8.554	−28.869	−4.72
2010	THR265	CA	9.554	−29.535	−3.884
2011	THR265	C	10.787	−29.809	−4.756
2012	THR265	O	10.694	−30.025	−5.965
2013	THR265	CB	8.991	−30.793	−3.206
2014	THR265	OG1	9.877	−31.233	−2.175
2015	THR265	CG2	8.73	−31.956	−4.153
2016	CYS266	N	11.978	−29.812	−4.061
2017	CYS266	CA	13.284	−29.87	−4.714
2018	CYS266	C	14.097	−30.88	−3.895
2019	CYS266	O	14.691	−30.581	−2.86
2020	CYS266	CB	13.989	−28.509	−4.7
2021	CYS266	SG	13.206	−27.305	−5.819
2022	LYS267	N	13.961	−32.195	−4.31
2023	LYS267	CA	14.689	−33.299	−3.673
2024	LYS267	C	14.286	−33.478	−2.192
2025	LYS267	O	15.01	−34.029	−1.367
2026	LYS267	CB	16.213	−33.232	−3.892
2027	LYS267	CG	16.663	−34.184	−5.011
2028	LYS267	CD	18.157	−34.036	−5.317
2029	LYS267	CE	18.571	−34.928	−6.481
2030	LYS267	NZ	20.005	−34.709	−6.786
2031	HIS268	N	12.97	−33.132	−1.95
2032	HIS268	CA	12.274	−33.231	−0.667
2033	HIS268	C	12.482	−32.006	0.241
2034	HIS268	O	11.993	−31.959	1.37
2035	HIS268	CB	12.498	−34.526	0.134
2036	HIS268	CG	12.281	−35.759	−0.675
2037	HIS268	ND1	13.322	−36.468	−1.223
2038	HIS268	CD2	11.152	−36.446	−1.061
2039	HIS268	CE1	12.789	−37.516	−1.915
2040	HIS268	NE2	11.478	−37.53	−1.837
2041	ARG269	N	13.176	−30.951	−0.316
2042	ARG269	CA	13.144	−29.618	0.276
2043	ARG269	C	12.021	−28.86	−0.454
2044	ARG269	O	12.043	−28.758	−1.688
2045	ARG269	CB	14.46	−28.861	0.046
2046	ARG269	CG	15.662	−29.559	0.684
2047	ARG269	CD	16.953	−28.794	0.405
2048	ARG269	NE	18.104	−29.474	1.018
2049	ARG269	CZ	19.385	−29.046	0.903
2050	ARG269	NH1	20.373	−29.744	1.502
2051	ARG269	NH2	19.706	−27.938	0.204
2052	PRO270	N	11.014	−28.293	0.298
2053	PRO270	CA	9.987	−27.465	−0.337
2054	PRO270	C	10.563	−26.091	−0.7
2055	PRO270	O	11.354	−25.501	0.037
2056	PRO270	CB	8.9	−27.291	0.729
2057	PRO270	CG	9.144	−28.441	1.697
2058	PRO270	CD	10.654	−28.613	1.67
2059	GLU271	N	10.059	−25.54	−1.864
2060	GLU271	CA	10.405	−24.182	−2.273
2061	GLU271	C	9.161	−23.549	−2.941
2062	GLU271	O	8.302	−24.202	−3.534
2063	GLU271	CB	11.647	−24.106	−3.183
2064	GLU271	CG	12.953	−24.496	−2.481
2065	GLU271	CD	14.184	−24.134	−3.291
2066	GLU271	OE1	14.842	−23.104	−2.938
2067	GLU271	OE2	14.491	−24.885	−4.272
2068	TYR272	N	9.087	−22.177	−2.79
2069	TYR272	CA	7.922	−21.389	−3.21
2070	TYR272	C	8.368	−20.54	−4.407
2071	TYR272	O	9.178	−19.715	−4.286
2072	TYR272	CB	7.46	−20.467	−2.073
2073	TYR272	CG	6.69	−21.185	−0.988
2074	TYR272	CD1	7.353	−21.902	0.018
2075	TYR272	CD2	5.289	−21.136	−0.972
2076	TYR272	CE1	6.629	−22.565	1.011
2077	TYR272	CE2	4.562	−21.773	0.033
2078	TYR272	CZ	5.241	−22.492	1.008
2079	TYR272	OH	4.541	−23.204	1.947
2080	LEU273	N	7.728	−20.835	−5.606
2081	LEU273	CA	8.076	−20.123	−6.831
2082	LEU273	C	7.317	−18.786	−6.864
2083	LEU273	O	6.155	−18.677	−6.464
2084	LEU273	CB	7.669	−20.839	−8.137
2085	LEU273	CG	8.109	−22.294	−8.354
2086	LEU273	CD1	7.859	−22.713	−9.808
2087	LEU273	CD2	9.577	−22.515	−8.047
2088	VAL274	N	8.026	−17.773	−7.475
2089	VAL274	CA	7.432	−16.492	−7.872
2090	VAL274	C	7.657	−16.369	−9.387
2091	VAL274	O	8.406	−17.132	−9.998
2092	VAL274	CB	8.092	−15.34	−7.081
2093	VAL274	CG1	7.194	−14.107	−6.99
2094	VAL274	CG2	9.471	−14.952	−7.61
2095	LYS275	N	7.044	−15.285	−9.983
2096	LYS275	CA	7.464	−14.86	−11.304
2097	LYS275	C	7.322	−13.332	−11.429
2098	LYS275	O	6.235	−12.753	−11.4
2099	LYS275	CB	6.633	−15.505	−12.406
2100	LYS275	CG	7.255	−15.212	−13.764
2101	LYS275	CD	6.224	−15.112	−14.878
2102	LYS275	CE	6.828	−14.538	−16.146
2103	LYS275	NZ	7.123	−13.098	−16.004
2104	LEU276	N	8.531	−12.693	−11.629
2105	LEU276	CA	8.634	−11.276	−11.95
2106	LEU276	C	8.444	−11.093	−13.474
2107	LEU276	O	8.563	−12.011	−14.289
2108	LEU276	CB	10.025	−10.736	−11.562
2109	LEU276	CG	10.275	−10.475	−10.064
2110	LEU276	CD1	9.462	−9.29	−9.559
2111	LEU276	CD2	10.031	−11.687	−9.173
2112	ARG277	N	8.207	−9.79	−13.855
2113	ARG277	CA	8.528	−9.281	−15.18
2114	ARG277	C	9.232	−7.926	−14.936
2115	ARG277	O	8.825	−7.1	−14.115
2116	ARG277	CB	7.289	−9.089	−16.071
2117	ARG277	CG	7.647	−8.941	−17.558
2118	ARG277	CD	6.742	−7.976	−18.331
2119	ARG277	NE	7.367	−7.619	−19.624
2120	ARG277	CZ	7.335	−6.381	−20.171
2121	ARG277	NH1	6.551	−5.395	−19.697
2122	ARG277	NH2	8.113	−6.055	−21.217
2123	ASN278	N	10.335	−7.719	−15.749
2124	ASN278	CA	10.937	−6.394	−15.894
2125	ASN278	C	10.093	−5.697	−16.971
2126	ASN278	O	9.954	−6.213	−18.087
2127	ASN278	CB	12.38	−6.516	−16.379
2128	ASN278	CG	12.947	−5.17	−16.753
2129	ASN278	OD1	13.081	−4.819	−17.922
2130	ASN278	ND2	13.226	−4.345	−15.705
2131	PRO279	N	9.541	−4.466	−16.682
2132	PRO279	CA	8.574	−3.868	−17.602
2133	PRO279	C	9.153	−3.581	−18.99
2134	PRO279	O	8.444	−3.571	−19.998
2135	PRO279	CB	8.162	−2.571	−16.921
2136	PRO279	CG	8.254	−2.911	−15.445
2137	PRO279	CD	9.464	−3.83	−15.371
2138	TRP280	N	10.496	−3.269	−18.983
2139	TRP280	CA	11.252	−2.927	−20.181
2140	TRP280	C	11.773	−4.151	−20.963
2141	TRP280	O	12.441	−4.014	−21.997
2142	TRP280	CB	12.425	−1.995	−19.834
2143	TRP280	CG	12.051	−0.559	−19.634
2144	TRP280	CD1	11.011	0.111	−20.238
2145	TRP280	CD2	12.756	0.413	−18.852
2146	TRP280	NE1	11.062	1.433	−19.885
2147	TRP280	CE2	12.088	1.633	−18.993
2148	TRP280	CE3	13.913	0.373	−18.044
2149	TRP280	CZ2	12.486	2.793	−18.323
2150	TRP280	CZ3	14.341	1.535	−17.391
2151	TRP280	CH2	13.626	2.723	−17.518
2152	GLY281	N	11.411	−5.382	−20.457
2153	GLY281	CA	11.425	−6.582	−21.268
2154	GLY281	C	12.714	−7.392	−21.278
2155	GLY281	O	12.875	−8.304	−22.095
2156	LYS282	N	13.616	−7.04	−20.301
2157	LYS282	CA	14.908	−7.695	−20.115
2158	LYS282	C	14.766	−8.868	−19.119
2159	LYS282	O	13.791	−9.007	−18.381
2160	LYS282	CB	15.966	−6.676	−19.648
2161	LYS282	CG	16.466	−5.772	−20.791
2162	LYS282	CD	15.559	−4.566	−21.044
2163	LYS282	CE	15.714	−4.002	−22.451
2164	LYS282	NZ	14.659	−2.99	−22.664
2165	VAL283	N	15.825	−9.754	−19.149
2166	VAL283	CA	15.911	−10.959	−18.324
2167	VAL283	C	17.177	−10.793	−17.449
2168	VAL283	O	17.109	−10.559	−16.244
2169	VAL283	CB	15.921	−12.225	−19.213
2170	VAL283	CG1	15.998	−13.494	−18.366
2171	VAL283	CG2	14.674	−12.299	−20.104
2172	GLU284	N	18.379	−10.886	−18.141
2173	GLU284	CA	19.583	−10.233	−17.632
2174	GLU284	C	20.03	−10.638	−16.205
2175	GLU284	O	20.417	−9.814	−15.373
2176	GLU284	CB	19.515	−8.702	−17.837
2177	GLU284	CG	19.846	−8.253	−19.27
2178	GLU284	CD	18.886	−8.593	−20.412
2179	GLU284	OE1	18.035	−9.506	−20.171
2180	GLU284	OE2	19.025	−7.906	−21.459
2181	TRP285	N	20.155	−12.004	−15.985
2182	TRP285	CA	20.932	−12.497	−14.842
2183	TRP285	C	21.761	−13.735	−15.237
2184	TRP285	O	21.657	−14.301	−16.323
2185	TRP285	CB	20.117	−12.623	−13.545
2186	TRP285	CG	19.575	−13.984	−13.239
2187	TRP285	CD1	20.1	−14.89	−12.334
2188	TRP285	CD2	18.396	−14.581	−13.783
2189	TRP285	NE1	19.303	−16.003	−12.321
2190	TRP285	CE2	18.241	−15.827	−13.175
2191	TRP285	CE3	17.431	−14.167	−14.72
2192	TRP285	CZ2	17.159	−16.669	−13.441
2193	TRP285	CZ3	16.37	−15.024	−15.03
2194	TRP285	CH2	16.238	−16.257	−14.401
2195	LYS286	N	22.694	−14.106	−14.287
2196	LYS286	CA	23.764	−15.067	−14.54
2197	LYS286	C	23.324	−16.517	−14.251
2198	LYS286	O	23.999	−17.284	−13.562
2199	LYS286	CB	24.995	−14.701	−13.688
2200	LYS286	CG	25.529	−13.29	−13.965
2201	LYS286	CD	26.7	−12.953	−13.039
2202	LYS286	CE	27.191	−11.531	−13.271
2203	LYS286	NZ	28.293	−11.237	−12.323
2204	GLY287	N	22.185	−16.93	−14.91
2205	GLY287	CA	21.742	−18.311	−14.832
2206	GLY287	C	20.289	−18.461	−15.26
2207	GLY287	O	19.729	−17.631	−15.971
2208	ASP288	N	19.681	−19.612	−14.781
2209	ASP288	CA	18.234	−19.828	−14.932
2210	ASP288	C	17.649	−20.449	−13.637
2211	ASP288	O	16.602	−21.088	−13.602
2212	ASP288	CB	17.889	−20.638	−16.168
2213	ASP288	CG	16.404	−20.537	−16.444
2214	ASP288	OD1	15.815	−21.581	−16.865
2215	ASP288	OD2	15.809	−19.437	−16.223
2216	TRP289	N	18.357	−20.074	−12.511
2217	TRP289	CA	17.877	−20.166	−11.129
2218	TRP289	C	19.03	−19.498	−10.338
2219	TRP289	O	19.797	−18.685	−10.878
2220	TRP289	CB	17.536	−21.597	−10.681
2221	TRP289	CG	16.279	−21.693	−9.854
2222	TRP289	CD1	15.892	−20.886	−8.8
2223	TRP289	CD2	15.271	−22.707	−9.975
2224	TRP289	NE1	14.706	−21.358	−8.3
2225	TRP289	CE2	14.29	−22.451	−9.016
2226	TRP289	CE3	15.125	−23.852	−10.78
2227	TRP289	CZ2	13.16	−23.26	−8.852
2228	TRP289	CZ3	14.018	−24.69	−10.599
2229	TRP289	CH2	13.041	−24.39	−9.659
2230	SER290	N	19.145	−19.799	−9.004
2231	SER290	CA	20.299	−19.398	−8.195
2232	SER290	C	20.188	−20.151	−6.87
2233	SER290	O	19.141	−20.146	−6.229
2234	SER290	CB	20.369	−17.892	−7.938
2235	SER290	OG	20.955	−17.227	−9.058
2236	ASP291	N	21.338	−20.833	−6.53
2237	ASP291	CA	21.456	−21.969	−5.601
2238	ASP291	C	22.001	−23.178	−6.401
2239	ASP291	O	22.889	−23.913	−5.967
2240	ASP291	CB	20.238	−22.329	−4.751
2241	ASP291	CG	19.008	−22.859	−5.469
2242	ASP291	OD1	17.956	−22.999	−4.741
2243	ASP291	OD2	19.125	−23.137	−6.697
2244	SER292	N	21.344	−23.438	−7.59
2245	SER292	CA	21.699	−24.591	−8.422
2246	SER292	C	20.931	−24.457	−9.74
2247	SER292	O	19.801	−24.911	−9.91
2248	SER292	CB	21.375	−25.923	−7.735
2249	SER292	OG	22.418	−26.301	−6.832
2250	SER293	N	21.606	−23.736	−10.724
2251	SER293	CA	20.846	−23.111	−11.818
2252	SER293	C	20.023	−24.1	−12.653
2253	SER293	O	19.033	−23.732	−13.285
2254	SER293	CB	21.757	−22.335	−12.787
2255	SER293	OG	21.239	−21.017	−13.017
2256	SER294	N	20.552	−25.369	−12.751
2257	SER294	CA	19.9	−26.41	−13.529
2258	SER294	C	18.95	−27.304	−12.721
2259	SER294	O	18.437	−28.308	−13.229
2260	SER294	CB	20.925	−27.265	−14.275
2261	SER294	OG	21.73	−27.996	−13.353
2262	LYS295	N	18.479	−26.832	−11.502
2263	LYS295	CA	17.627	−27.699	−10.671
2264	LYS295	C	16.164	−27.847	−11.19
2265	LYS295	O	15.295	−28.434	−10.547
2266	LYS295	CB	17.676	−27.362	−9.169
2267	LYS295	CG	16.925	−26.088	−8.807
2268	LYS295	CD	16.949	−25.757	−7.309
2269	LYS295	CE	16.153	−24.479	−7.118
2270	LYS295	NZ	16.053	−24.049	−5.734
2271	TRP296	N	15.972	−27.431	−12.491
2272	TRP296	CA	14.872	−27.88	−13.336
2273	TRP296	C	15.035	−29.371	−13.716
2274	TRP296	O	14.133	−29.981	−14.29
2275	TRP296	CB	14.8	−27.078	−14.65
2276	TRP296	CG	14.669	−25.595	−14.468
2277	TRP296	CD1	15.671	−24.659	−14.644
2278	TRP296	CD2	13.501	−24.875	−14.057
2279	TRP296	NE1	15.173	−23.419	−14.334
2280	TRP296	CE2	13.856	−23.529	−13.95
2281	TRP296	CE3	12.188	−25.254	−13.707
2282	TRP296	CZ2	12.979	−22.546	−13.477
2283	TRP296	CZ3	11.306	−24.286	−13.215
2284	TRP296	CH2	11.7	−22.956	−13.095
2285	GLU297	N	16.247	−29.961	−13.388
2286	GLU297	CA	16.539	−31.354	−13.743
2287	GLU297	C	15.677	−32.306	−12.884
2288	GLU297	O	15.277	−33.393	−13.297
2289	GLU297	CB	18.004	−31.703	−13.427
2290	GLU297	CG	19.007	−31.221	−14.472
2291	GLU297	CD	20.397	−31.304	−13.831
2292	GLU297	OE1	20.987	−30.192	−13.67
2293	GLU297	OE2	20.785	−32.465	−13.524
2294	LEU298	N	15.589	−31.926	−11.556
2295	LEU298	CA	14.981	−32.77	−10.54
2296	LEU298	C	13.519	−32.377	−10.28
2297	LEU298	O	13.091	−31.231	−10.421
2298	LEU298	CB	15.78	−32.784	−9.222
2299	LEU298	CG	16.265	−31.396	−8.743
2300	LEU298	CD1	15.964	−31.174	−7.264
2301	LEU298	CD2	17.769	−31.245	−8.989
2302	LEU299	N	12.717	−33.443	−9.879
2303	LEU299	CA	11.365	−33.264	−9.327
2304	LEU299	C	10.5	−32.443	−10.305
2305	LEU299	O	9.686	−31.581	−9.965
2306	LEU299	CB	11.374	−32.701	−7.897
2307	LEU299	CG	11.447	−33.753	−6.767
2308	LEU299	CD1	10.173	−34.597	−6.679
2309	LEU299	CD2	12.675	−34.655	−6.851
2310	SER300	N	10.666	−32.827	−11.626
2311	SER300	CA	10.223	−31.96	−12.711
2312	SER300	C	9.831	−32.797	−13.923
2313	SER300	O	10.551	−32.865	−14.918
2314	SER300	CB	11.302	−30.919	−13.047
2315	SER300	OG	11.474	−30.005	−11.938
2316	PRO301	N	8.614	−33.454	−13.838
2317	PRO301	CA	8.076	−34.181	−14.985
2318	PRO301	C	7.562	−33.163	−16.023
2319	PRO301	O	7.523	−31.942	−15.833
2320	PRO301	CB	6.982	−35.058	−14.38
2321	PRO301	CG	6.479	−34.227	−13.206
2322	PRO301	CD	7.752	−33.593	−12.669
2323	LYS302	N	7.147	−33.74	−17.215
2324	LYS302	CA	6.877	−32.868	−18.363
2325	LYS302	C	5.645	−31.983	−18.078
2326	LYS302	O	5.484	−30.869	−18.57
2327	LYS302	CB	6.672	−33.716	−19.625
2328	LYS302	CG	7.287	−33.047	−20.856
2329	LYS302	CD	7.05	−33.874	−22.119
2330	LYS302	CE	7.704	−33.225	−23.329
2331	LYS302	NZ	7.445	−34.06	−24.526
2332	GLU303	N	4.738	−32.591	−17.247
2333	GLU303	CA	3.395	−32.129	−16.953
2334	GLU303	C	3.494	−30.92	−16.005
2335	GLU303	O	2.6	−30.081	−15.915
2336	GLU303	CB	2.6	−33.255	−16.265
2337	GLU303	CG	2.426	−34.527	−17.11
2338	GLU303	CD	3.625	−35.48	−17.279
2339	GLU303	OE1	4.772	−34.963	−17.076
2340	GLU303	OE2	3.337	−36.652	−17.636
2341	LYS304	N	4.62	−30.928	−15.189
2342	LYS304	CA	4.963	−29.757	−14.397
2343	LYS304	C	5.639	−28.73	−15.32
2344	LYS304	O	5.311	−27.541	−15.299
2345	LYS304	CB	5.895	−30.123	−13.229
2346	LYS304	CG	5.971	−29.01	−12.169
2347	LYS304	CD	7.075	−29.248	−11.128
2348	LYS304	CE	8.442	−28.814	−11.643
2349	LYS304	NZ	9.501	−29.237	−10.709
2350	ILE305	N	6.707	−29.199	−16.076
2351	ILE305	CA	7.642	−28.242	−16.673
2352	ILE305	C	7.027	−27.482	−17.864
2353	ILE305	O	7.481	−26.394	−18.219
2354	ILE305	CB	9.006	−28.894	−17.018
2355	ILE305	CG1	10.144	−27.856	−16.892
2356	ILE305	CG2	9.013	−29.558	−18.397
2357	ILE305	CD1	11.534	−28.452	−17.059
2358	LEU306	N	5.999	−28.115	−18.541
2359	LEU306	CA	5.39	−27.484	−19.715
2360	LEU306	C	4.473	−26.329	−19.282
2361	LEU306	O	4.199	−25.396	−20.035
2362	LEU306	CB	4.555	−28.471	−20.543
2363	LEU306	CG	5.396	−29.398	−21.441
2364	LEU306	CD1	4.513	−30.523	−21.988
2365	LEU306	CD2	6.043	−28.646	−22.606
2366	LEU307	N	3.896	−26.466	−18.035
2367	LEU307	CA	3.011	−25.447	−17.465
2368	LEU307	C	3.873	−24.476	−16.624
2369	LEU307	O	3.586	−24.139	−15.471
2370	LEU307	CB	1.901	−26.13	−16.649
2371	LEU307	CG	0.531	−25.423	−16.742
2372	LEU307	CD1	−0.532	−26.257	−16.022
2373	LEU307	CD2	0.541	−24.005	−16.174
2374	LEU308	N	4.949	−23.941	−17.302
2375	LEU308	CA	6.035	−23.215	−16.647
2376	LEU308	C	6.876	−22.584	−17.768
2377	LEU308	O	7.035	−23.144	−18.85
2378	LEU308	CB	6.897	−24.178	−15.807
2379	LEU308	CG	6.948	−23.815	−14.312
2380	LEU308	CD1	7.362	−25.033	−13.486
2381	LEU308	CD2	7.91	−22.662	−14.059
2382	ARG309	N	7.444	−21.368	−17.44
2383	ARG309	CA	8.209	−20.556	−18.396
2384	ARG309	C	7.215	−20.097	−19.499
2385	ARG309	O	7.211	−20.57	−20.633
2386	ARG309	CB	9.475	−21.225	−18.956
2387	ARG309	CG	10.305	−22.098	−18.003
2388	ARG309	CD	10.578	−21.562	−16.604
2389	ARG309	NE	11.464	−20.401	−16.533
2390	ARG309	CZ	12.822	−20.438	−16.568
2391	ARG309	NH1	13.482	−21.55	−16.944
2392	ARG309	NH2	13.489	−19.33	−16.193
2393	LYS310	N	6.259	−19.194	−19.052
2394	LYS310	CA	4.947	−19.081	−19.688
2395	LYS310	C	4.447	−17.723	−20.214
2396	LYS310	O	3.452	−17.693	−20.947
2397	LYS310	CB	3.872	−19.736	−18.785
2398	LYS310	CG	3.685	−19.061	−17.42
2399	LYS310	CD	2.648	−17.934	−17.42
2400	LYS310	CE	2.777	−17.078	−16.165
2401	LYS310	NZ	2.02	−15.832	−16.318
2402	ASP311	N	5.044	−16.569	−19.735
2403	ASP311	CA	4.74	−15.299	−20.408
2404	ASP311	C	5.793	−15.231	−21.565
2405	ASP311	O	6.162	−16.217	−22.208
2406	ASP311	CB	4.787	−14.092	−19.465
2407	ASP311	CG	3.902	−13.966	−18.244
2408	ASP311	OD1	4.261	−13.082	−17.402
2409	ASP311	OD2	2.909	−14.74	−18.122
2410	ASN312	N	6.275	−13.977	−21.862
2411	ASN312	CA	7.564	−13.796	−22.526
2412	ASN312	C	8.052	−12.481	−21.892
2413	ASN312	O	7.388	−11.907	−21.023
2414	ASN312	CB	7.365	−13.694	−24.032
2415	ASN312	CG	8.686	−13.81	−24.752
2416	ASN312	OD1	9.415	−12.829	−24.929
2417	ASN312	ND2	9.044	−15.07	−25.117
2418	ASP313	N	9.252	−11.973	−22.353
2419	ASP313	CA	9.486	−10.538	−22.209
2420	ASP313	C	9.702	−10.172	−20.729
2421	ASP313	O	9.242	−9.152	−20.219
2422	ASP313	CB	8.356	−9.744	−22.863
2423	ASP313	CG	9.03	−8.547	−23.469
2424	ASP313	OD1	8.905	−7.459	−22.843
2425	ASP313	OD2	9.677	−8.778	−24.555
2426	GLY314	N	10.58	−11.035	−20.099
2427	GLY314	CA	10.855	−10.98	−18.679
2428	GLY314	C	10.158	−12.165	−18.019
2429	GLY314	O	9.038	−12.091	−17.509
2430	GLU315	N	10.88	−13.349	−18.127
2431	GLU315	CA	10.289	−14.613	−17.709
2432	GLU315	C	10.687	−14.949	−16.262
2433	GLU315	O	9.83	−15.216	−15.422
2434	GLU315	CB	10.576	−15.729	−18.718
2435	GLU315	CG	9.7	−16.972	−18.527
2436	GLU315	CD	8.191	−16.747	−18.473
2437	GLU315	OE1	7.717	−15.846	−19.212
2438	GLU315	OE2	7.517	−17.508	−17.705
2439	PHE316	N	12.03	−15.012	−15.961
2440	PHE316	CA	12.47	−15.188	−14.559
2441	PHE316	C	12.08	−16.622	−14.073
2442	PHE316	O	12.298	−17.599	−14.803
2443	PHE316	CB	12.074	−14.002	−13.659
2444	PHE316	CG	12.736	−12.706	−14.072
2445	PHE316	CD1	12.085	−11.8	−14.916
2446	PHE316	CD2	14.029	−12.408	−13.619
2447	PHE316	CE1	12.726	−10.628	−15.319
2448	PHE316	CE2	14.666	−11.235	−14.021
2449	PHE316	CZ	14.015	−10.347	−14.874
2450	TRP317	N	11.496	−16.719	−12.823
2451	TRP317	CA	11.006	−17.943	−12.169
2452	TRP317	C	12.038	−18.518	−11.196
2453	TRP317	O	12.339	−19.713	−11.19
2454	TRP317	CB	10.477	−19.098	−13.034
2455	TRP317	CG	9.177	−18.858	−13.717
2456	TRP317	CD1	9.014	−18.346	−14.977
2457	TRP317	CD2	7.867	−19.138	−13.223
2458	TRP317	NE1	7.687	−18.372	−15.307
2459	TRP317	CE2	6.957	−18.85	−14.244
2460	TRP317	CE3	7.371	−19.616	−11.994
2461	TRP317	CZ2	5.578	−19.027	−14.082
2462	TRP317	CZ3	5.996	−19.79	−11.817
2463	TRP317	CH2	5.113	−19.497	−12.85
2464	MET318	N	12.499	−17.644	−10.234
2465	MET318	CA	13.197	−18.156	−9.071
2466	MET318	C	12.185	−18.312	−7.905
2467	MET318	O	10.996	−18.594	−8.078
2468	MET318	CB	14.469	−17.368	−8.77
2469	MET318	CG	15.331	−17.18	−10.014
2470	MET318	SD	16.964	−16.506	−9.576
2471	MET318	CE	16.727	−14.789	−10.107
2472	THR319	N	12.757	−18.296	−6.644
2473	THR319	CA	12.098	−18.913	−5.49
2474	THR319	C	12.337	−18.067	−4.244
2475	THR319	O	13.343	−17.371	−4.135
2476	THR319	CB	12.703	−20.312	−5.228
2477	THR319	OG1	14.001	−20.424	−5.84
2478	THR319	CG2	11.866	−21.415	−5.826
2479	LEU320	N	11.403	−18.247	−3.239
2480	LEU320	CA	11.304	−17.264	−2.158
2481	LEU320	C	12.534	−17.265	−1.231
2482	LEU320	O	12.769	−18.16	−0.422
2483	LEU320	CB	10.046	−17.551	−1.315
2484	LEU320	CG	9.838	−16.663	−0.069
2485	LEU320	CD1	9.894	−15.167	−0.371
2486	LEU320	CD2	8.505	−17.02	0.596
2487	GLN321	N	13.316	−16.124	−1.361
2488	GLN321	CA	14.259	−15.744	−0.311
2489	GLN321	C	14.594	−14.23	−0.308
2490	GLN321	O	15.433	−13.779	0.472
2491	GLN321	CB	15.548	−16.59	−0.36
2492	GLN321	CG	15.855	−17.306	0.958
2493	GLN321	CD	16.904	−16.635	1.819
2494	GLN321	OE1	17.909	−17.223	2.211
2495	GLN321	NE2	16.654	−15.35	2.201
2496	ASP322	N	13.848	−13.446	−1.164
2497	ASP322	CA	14.248	−12.103	−1.611
2498	ASP322	C	14.249	−10.983	−0.562
2499	ASP322	O	14.875	−9.931	−0.719
2500	ASP322	CB	15.535	−12.116	−2.438
2501	ASP322	CG	15.34	−11.89	−3.933
2502	ASP322	OD1	14.15	−11.758	−4.35
2503	ASP322	OD2	16.426	−11.883	−4.604
2504	PHE323	N	13.365	−11.161	0.484
2505	PHE323	CA	12.822	−10.018	1.237
2506	PHE323	C	13.889	−9.399	2.172
2507	PHE323	O	13.821	−9.433	3.397
2508	PHE323	CB	11.584	−10.422	2.054
2509	PHE323	CG	10.362	−10.884	1.285
2510	PHE323	CD1	9.301	−11.438	2.022
2511	PHE323	CD2	10.212	−10.75	−0.103
2512	PHE323	CE1	8.125	−11.847	1.393
2513	PHE323	CE2	9.041	−11.175	−0.733
2514	PHE323	CZ	7.997	−11.712	0.016
2515	LYS324	N	14.906	−8.775	1.479
2516	LYS324	CA	16.059	−8.143	2.097
2517	LYS324	C	15.945	−6.647	1.722
2518	LYS324	O	15.583	−5.78	2.514
2519	LYS324	CB	17.384	−8.77	1.596
2520	LYS324	CG	17.441	−10.305	1.664
2521	LYS324	CD	18.492	−10.874	0.693
2522	LYS324	CE	18.146	−12.301	0.28
2523	LYS324	NZ	19.058	−12.75	−0.782
2524	THR325	N	16.254	−6.375	0.404
2525	THR325	CA	16.294	−5.05	−0.205
2526	THR325	C	14.889	−4.694	−0.708
2527	THR325	O	14.517	−5.055	−1.83
2528	THR325	CB	17.024	−3.925	0.565
2529	THR325	OG1	17.428	−2.899	−0.365
2530	THR325	CG2	18.294	−4.377	1.287
2531	HIS326	N	14.032	−4.082	0.182
2532	HIS326	CA	12.651	−3.745	−0.185
2533	HIS326	C	12.125	−2.627	0.715
2534	HIS326	O	12.506	−2.495	1.878
2535	HIS326	CB	11.71	−4.964	−0.039
2536	HIS326	CG	11.237	−5.489	−1.35
2537	HIS326	ND1	12.091	−5.768	−2.384
2538	HIS326	CD2	9.99	−5.805	−1.841
2539	HIS326	CE1	11.334	−6.159	−3.442
2540	HIS326	NE2	10.057	−6.237	−3.142
2541	PHE327	N	11.119	−1.865	0.135
2542	PHE327	CA	10.379	−0.87	0.91
2543	PHE327	C	8.879	−1.214	0.924
2544	PHE327	O	8.273	−1.356	1.992
2545	PHE327	CB	10.648	0.554	0.401
2546	PHE327	CG	9.97	1.626	1.224
2547	PHE327	CD1	10.239	1.762	2.594
2548	PHE327	CD2	9.058	2.506	0.626
2549	PHE327	CE1	9.585	2.734	3.353
2550	PHE327	CE2	8.404	3.477	1.386
2551	PHE327	CZ	8.663	3.588	2.75
2552	VAL328	N	8.241	−1.278	−0.304
2553	VAL328	CA	6.78	−1.405	−0.381
2554	VAL328	C	6.367	−2.087	−1.695
2555	VAL328	O	6.835	−1.75	−2.78
2556	VAL328	CB	6.081	−0.035	−0.188
2557	VAL328	CG1	6.279	0.921	−1.369
2558	VAL328	CG2	4.586	−0.186	0.103
2559	LEU329	N	5.431	−3.088	−1.541
2560	LEU329	CA	4.722	−3.699	−2.671
2561	LEU329	C	3.282	−3.186	−2.586
2562	LEU329	O	2.749	−2.915	−1.507
2563	LEU329	CB	4.817	−5.23	−2.55
2564	LEU329	CG	4.055	−6.055	−3.609
2565	LEU329	CD1	4.816	−7.344	−3.94
2566	LEU329	CD2	2.655	−6.451	−3.126
2567	LEU330	N	2.607	−3.134	−3.785
2568	LEU330	CA	1.18	−2.918	−3.839
2569	LEU330	C	0.517	−3.852	−4.863
2570	LEU330	O	1.06	−4.232	−5.901
2571	LEU330	CB	0.768	−1.455	−4.071
2572	LEU330	CG	1.329	−0.785	−5.344
2573	LEU330	CD1	0.345	0.266	−5.863
2574	LEU330	CD2	2.677	−0.104	−5.08
2575	VAL331	N	−0.782	−4.176	−4.512
2576	VAL331	CA	−1.727	−4.683	−5.49
2577	VAL331	C	−2.826	−3.621	−5.632
2578	VAL331	O	−3.195	−2.922	−4.684
2579	VAL331	CB	−2.31	−6.064	−5.13
2580	VAL331	CG1	−1.224	−7.136	−5.153
2581	VAL331	CG2	−3.01	−6.103	−3.774
2582	ILE332	N	−3.381	−3.556	−6.897
2583	ILE332	CA	−4.589	−2.77	−7.148
2584	ILE332	C	−5.721	−3.807	−7.228
2585	ILE332	O	−5.873	−4.569	−8.182
2586	ILE332	CB	−4.423	−1.846	−8.371
2587	ILE332	CG1	−5.382	−0.638	−8.329
2588	ILE332	CG2	−4.482	−2.546	−9.731
2589	ILE332	CD1	−6.855	−0.957	−8.158
2590	CYS333	N	−6.476	−3.899	−6.073
2591	CYS333	CA	−7.589	−4.832	−5.976
2592	CYS333	C	−8.783	−4.185	−6.689
2593	CYS333	O	−9.295	−3.132	−6.309
2594	CYS333	CB	−7.977	−5.116	−4.53
2595	CYS333	SG	−6.842	−6.3	−3.743
2596	LYS334	N	−9.182	−4.88	−7.816
2597	LYS334	CA	−10.111	−4.323	−8.792
2598	LYS334	C	−11.546	−4.279	−8.235
2599	LYS334	O	−11.95	−5.048	−7.365
2600	LYS334	CB	−10.11	−5.186	−10.065
2601	LYS334	CG	−8.863	−4.966	−10.925
2602	LYS334	CD	−8.707	−6.065	−11.976
2603	LYS334	CE	−7.611	−5.715	−12.971
2604	LYS334	NZ	−7.367	−6.877	−13.845
2605	LEU335	N	−12.355	−3.345	−8.864
2606	LEU335	CA	−13.81	−3.365	−8.711
2607	LEU335	C	−14.436	−4.308	−9.769
2608	LEU335	O	−13.753	−4.987	−10.536
2609	LEU335	CB	−14.404	−1.947	−8.725
2610	LEU335	CG	−14.005	−0.994	−9.87
2611	LEU335	CD1	−14.051	−1.609	−11.257
2612	LEU335	CD2	−14.918	0.238	−9.852
2613	THR336	N	−15.823	−4.328	−9.782
2614	THR336	CA	−16.561	−5.327	−10.569
2615	THR336	C	−16.279	−5.158	−12.078
2616	THR336	O	−15.707	−6.075	−12.687
2617	THR336	CB	−18.053	−5.318	−10.19
2618	THR336	OG1	−18.175	−5.305	−8.755
2619	THR336	CG2	−18.802	−6.527	−10.736
2620	PRO337	N	−16.567	−3.978	−12.736
2621	PRO337	CA	−16.139	−3.762	−14.13
2622	PRO337	C	−14.623	−3.439	−14.198
2623	PRO337	O	−14.151	−2.408	−14.688
2624	PRO337	CB	−17.009	−2.603	−14.62
2625	PRO337	CG	−17.292	−1.815	−13.346
2626	PRO337	CD	−17.465	−2.912	−12.306
2627	GLY338	N	−13.819	−4.463	−13.734
2628	GLY338	CA	−12.369	−4.376	−13.653
2629	GLY338	C	−11.676	−4.999	−14.864
2630	GLY338	O	−10.467	−4.881	−15.049
2631	LEU339	N	−12.517	−5.75	−15.649
2632	LEU339	CA	−12.165	−6.392	−16.909
2633	LEU339	C	−13.452	−6.384	−17.749
2634	LEU339	O	−14.556	−6.131	−17.257
2635	LEU339	CB	−11.709	−7.847	−16.695
2636	LEU339	CG	−10.31	−8	−16.073
2637	LEU339	CD1	−10.003	−9.479	−15.823
2638	LEU339	CD2	−9.234	−7.392	−16.971
2639	LEU340	N	−13.287	−6.756	−19.068
2640	LEU340	CA	−14.439	−6.983	−19.944
2641	LEU340	C	−14.996	−8.379	−19.597
2642	LEU340	O	−14.778	−9.384	−20.267
2643	LEU340	CB	−14.015	−6.863	−21.414
2644	LEU340	CG	−15.167	−6.977	−22.434
2645	LEU340	CD1	−16.231	−5.895	−22.245
2646	LEU340	CD2	−14.603	−6.904	−23.856
2647	SER341	N	−15.653	−8.423	−18.375
2648	SER341	CA	−16.715	−9.391	−18.078
2649	SER341	C	−16.338	−10.882	−18.154
2650	SER341	O	−17.186	−11.77	−18.227
2651	SER341	CB	−18.025	−9.094	−18.827
2652	SER341	OG	−17.834	−9.05	−20.236
2653	GLN342	N	−14.988	−11.143	−17.948
2654	GLN342	CA	−14.489	−12.484	−18.241
2655	GLN342	C	−15.049	−13.524	−17.241
2656	GLN342	O	−15.217	−13.291	−16.038
2657	GLN342	CB	−12.955	−12.548	−18.112
2658	GLN342	CG	−12.195	−11.889	−19.267
2659	GLN342	CD	−10.69	−11.852	−19.048
2660	GLN342	OE1	−9.978	−10.911	−19.398
2661	GLN342	NE2	−10.135	−12.963	−18.478
2662	GLU343	N	−15.17	−14.791	−17.779
2663	GLU343	CA	−15.785	−15.89	−17.033
2664	GLU343	C	−14.716	−16.454	−16.084
2665	GLU343	O	−14.982	−16.976	−15.005
2666	GLU343	CB	−16.253	−17.031	−17.956
2667	GLU343	CG	−17.318	−16.631	−18.981
2668	GLU343	CD	−16.779	−15.923	−20.232
2669	GLU343	OE1	−17.457	−16.091	−21.28
2670	GLU343	OE2	−15.72	−15.245	−20.048
2671	ALA344	N	−13.443	−16.438	−16.632
2672	ALA344	CA	−12.281	−16.858	−15.876
2673	ALA344	C	−11.844	−15.727	−14.926
2674	ALA344	O	−12.048	−14.533	−15.162
2675	ALA344	CB	−11.13	−17.223	−16.802
2676	ALA345	N	−11.171	−16.198	−13.811
2677	ALA345	CA	−10.751	−15.378	−12.674
2678	ALA345	C	−11.96	−14.979	−11.811
2679	ALA345	O	−13.024	−14.585	−12.3
2680	ALA345	CB	−9.926	−14.16	−13.056
2681	GLN346	N	−11.726	−15.069	−10.444
2682	GLN346	CA	−12.774	−14.685	−9.503
2683	GLN346	C	−12.841	−13.153	−9.405
2684	GLN346	O	−11.88	−12.418	−9.633
2685	GLN346	CB	−12.573	−15.264	−8.103
2686	GLN346	CG	−12.839	−16.767	−8.015
2687	GLN346	CD	−13.021	−17.178	−6.565
2688	GLN346	OE1	−13.722	−16.548	−5.776
2689	GLN346	NE2	−12.378	−18.327	−6.205
2690	LYS347	N	−14.088	−12.681	−9.037
2691	LYS347	CA	−14.468	−11.268	−9.084
2692	LYS347	C	−15.517	−11.068	−7.972
2693	LYS347	O	−15.975	−12.017	−7.333
2694	LYS347	CB	−15.011	−10.884	−10.479
2695	LYS347	CG	−14.023	−11.248	−11.595
2696	LYS347	CD	−14.491	−10.939	−13.015
2697	LYS347	CE	−13.508	−11.511	−14.04
2698	LYS347	NZ	−13.675	−12.969	−14.176
2699	TRP348	N	−15.889	−9.76	−7.745
2700	TRP348	CA	−16.666	−9.383	−6.56
2701	TRP348	C	−17.466	−8.095	−6.815
2702	TRP348	O	−17.235	−7.335	−7.758
2703	TRP348	CB	−15.803	−9.263	−5.289
2704	TRP348	CG	−14.525	−8.481	−5.421
2705	TRP348	CD1	−14.24	−7.437	−6.28
2706	TRP348	CD2	−13.347	−8.677	−4.623
2707	TRP348	NE1	−12.934	−7.071	−6.103
2708	TRP348	CE2	−12.359	−7.828	−5.115
2709	TRP348	CE3	−13.023	−9.513	−3.536
2710	TRP348	CZ2	−11.051	−7.818	−4.623
2711	TRP348	CZ3	−11.735	−9.471	−2.99
2712	TRP348	CH2	−10.755	−8.653	−3.544
2713	THR349	N	−18.461	−7.859	−5.885
2714	THR349	CA	−19.45	−6.792	−6.055
2715	THR349	C	−18.885	−5.466	−5.521
2716	THR349	O	−18.407	−5.345	−4.393
2717	THR349	CB	−20.729	−7.127	−5.256
2718	THR349	OG1	−20.996	−8.532	−5.384
2719	THR349	CG2	−21.937	−6.338	−5.745
2720	TYR350	N	−19.048	−4.388	−6.376
2721	TYR350	CA	−18.566	−3.041	−6.011
2722	TYR350	C	−19.482	−2.397	−4.937
2723	TYR350	O	−20.094	−1.341	−5.106
2724	TYR350	CB	−18.5	−2.155	−7.273
2725	TYR350	CG	−17.919	−0.767	−7.101
2726	TYR350	CD1	−16.742	−0.538	−6.376
2727	TYR350	CD2	−18.552	0.325	−7.718
2728	TYR350	CE1	−16.227	0.755	−6.246
2729	TYR350	CE2	−18.041	1.618	−7.586
2730	TYR350	CZ	−16.891	1.825	−6.839
2731	TYR350	OH	−16.434	3.104	−6.694
2732	THR351	N	−19.482	−3.031	−3.709
2733	THR351	CA	−20.34	−2.593	−2.598
2734	THR351	C	−19.646	−1.44	−1.834
2735	THR351	O	−19.535	−1.419	−0.609
2736	THR351	CB	−20.687	−3.766	−1.655
2737	THR351	OG1	−21.089	−4.902	−2.437
2738	THR351	CG2	−21.844	−3.438	−0.709
2739	MET352	N	−19.26	−0.379	−2.635
2740	MET352	CA	−18.829	0.901	−2.074
2741	MET352	C	−20.09	1.772	−1.929
2742	MET352	O	−21.078	1.635	−2.649
2743	MET352	CB	−17.791	1.551	−3
2744	MET352	CG	−17.031	2.699	−2.336
2745	MET352	SD	−15.698	3.301	−3.427
2746	MET352	CE	−14.9	4.439	−2.265
2747	ARG353	N	−20.012	2.736	−0.941
2748	ARG353	CA	−21.065	3.727	−0.741
2749	ARG353	C	−20.409	5.118	−0.686
2750	ARG353	O	−19.193	5.265	−0.578
2751	ARG353	CB	−21.866	3.44	0.534
2752	ARG353	CG	−22.613	2.105	0.469
2753	ARG353	CD	−23.63	1.995	1.594
2754	ARG353	NE	−24.282	0.679	1.613
2755	ARG353	CZ	−23.829	−0.389	2.309
2756	ARG353	NH1	−24.598	−1.488	2.418
2757	ARG353	NH2	−22.633	−0.385	2.934
2758	GLU354	N	−21.313	6.159	−0.787
2759	GLU354	CA	−20.907	7.561	−0.682
2760	GLU354	C	−21.009	8.025	0.787
2761	GLU354	O	−21.52	7.333	1.669
2762	GLU354	CB	−21.795	8.418	−1.607
2763	GLU354	CG	−21.021	8.966	−2.806
2764	GLU354	CD	−19.941	9.964	−2.419
2765	GLU354	OE1	−19.803	10.212	−1.187
2766	GLU354	OE2	−19.268	10.453	−3.384
2767	GLY355	N	−20.49	9.288	1.024
2768	GLY355	CA	−20.444	9.829	2.368
2769	GLY355	C	−20.044	11.301	2.472
2770	GLY355	O	−19.549	11.947	1.552
2771	ARG356	N	−20.278	11.835	3.732
2772	ARG356	CA	−19.973	13.22	4.076
2773	ARG356	C	−19.435	13.261	5.517
2774	ARG356	O	−19.822	12.473	6.377
2775	ARG356	CB	−21.22	14.11	4.019
2776	ARG356	CG	−21.71	14.391	2.6
2777	ARG356	CD	−23.015	15.181	2.591
2778	ARG356	NE	−24.15	14.34	3.001
2779	ARG356	CZ	−24.814	14.395	4.183
2780	ARG356	NH1	−25.871	13.564	4.361
2781	ARG356	NH2	−24.496	15.216	5.191
2782	TRP357	N	−18.54	14.291	5.734
2783	TRP357	CA	−17.903	14.556	7.028
2784	TRP357	C	−17.788	16.092	7.12
2785	TRP357	O	−16.762	16.695	6.804
2786	TRP357	CB	−16.508	13.905	7.125
2787	TRP357	CG	−16.539	12.489	7.622
2788	TRP357	CD1	−16.376	12.093	8.934
2789	TRP357	CD2	−16.724	11.29	6.859
2790	TRP357	NE1	−16.508	10.732	9.009
2791	TRP357	CE2	−16.721	10.216	7.753
2792	TRP357	CE3	−16.909	11.018	5.486
2793	TRP357	CZ2	−16.913	8.891	7.346
2794	TRP357	CZ3	−17.116	9.701	5.064
2795	TRP357	CH2	−17.119	8.656	5.983
2796	GLU358	N	−18.962	16.734	7.473
2797	GLU358	CA	−19.026	18.171	7.728
2798	GLU358	C	−18.992	18.415	9.253
2799	GLU358	O	−19.324	17.567	10.083
2800	GLU358	CB	−20.231	18.902	7.101
2801	GLU358	CG	−21.012	18.159	6.019
2802	GLU358	CD	−22.066	17.165	6.495
2803	GLU358	OE1	−22.758	16.626	5.577
2804	GLU358	OE2	−22.149	16.952	7.747
2805	LYS359	N	−18.551	19.678	9.621
2806	LYS359	CA	−18.467	20.053	11.028
2807	LYS359	C	−19.789	20.701	11.473
2808	LYS359	O	−20.366	21.552	10.792
2809	LYS359	CB	−17.316	21.036	11.288
2810	LYS359	CG	−15.946	20.357	11.18
2811	LYS359	CD	−14.806	21.321	11.516
2812	LYS359	CE	−13.46	20.611	11.454
2813	LYS359	NZ	−12.388	21.56	11.839
2814	ARG360	N	−20.185	20.303	12.74
2815	ARG360	CA	−21.453	20.645	13.397
2816	ARG360	C	−22.571	19.638	13.049
2817	ARG360	O	−23.753	19.862	13.306
2818	ARG360	CB	−21.969	22.07	13.156
2819	ARG360	CG	−20.949	23.176	13.424
2820	ARG360	CD	−21.441	24.49	12.83
2821	ARG360	NE	−20.344	25.336	12.347
2822	ARG360	CZ	−19.678	25.135	11.181
2823	ARG360	NH1	−18.825	26.093	10.748
2824	ARG360	NH2	−19.827	24.028	10.422
2825	SER361	N	−22.132	18.433	12.534
2826	SER361	CA	−23.092	17.492	11.962
2827	SER361	C	−22.527	16.07	12.006
2828	SER361	O	−23.052	15.2	12.699
2829	SER361	CB	−23.45	17.878	10.521
2830	SER361	OG	−22.244	18.109	9.793
2831	THR362	N	−21.427	15.849	11.197
2832	THR362	CA	−20.967	14.494	10.88
2833	THR362	C	−19.496	14.261	11.248
2834	THR362	O	−19.07	13.144	11.557
2835	THR362	CB	−21.194	14.156	9.395
2836	THR362	OG1	−20.74	15.237	8.575
2837	THR362	CG2	−22.654	13.848	9.079
2838	ALA363	N	−18.642	15.337	11.141
2839	ALA363	CA	−17.212	15.227	11.413
2840	ALA363	C	−17	15.136	12.933
2841	ALA363	O	−16.552	16.058	13.607
2842	ALA363	CB	−16.435	16.394	10.824
2843	GLY364	N	−17.37	13.903	13.457
2844	GLY364	CA	−17.585	13.743	14.883
2845	GLY364	C	−16.35	13.324	15.673
2846	GLY364	O	−16.398	13.133	16.887
2847	GLY365	N	−15.212	13.151	14.922
2848	GLY365	CA	−13.952	12.741	15.498
2849	GLY365	C	−13.734	11.229	15.406
2850	GLY365	O	−14.527	10.457	14.872
2851	GLN366	N	−12.522	10.847	15.957
2852	GLN366	CA	−12.075	9.46	16.036
2853	GLN366	C	−12.217	9.004	17.5
2854	GLN366	O	−12.316	9.799	18.438
2855	GLN366	CB	−10.612	9.339	15.598
2856	GLN366	CG	−10.4	9.744	14.14
2857	GLN366	CD	−8.968	9.503	13.72
2858	GLN366	OE1	−8.143	10.402	13.608
2859	GLN366	NE2	−8.653	8.196	13.472
2860	ARG367	N	−12.109	7.634	17.701
2861	ARG367	CA	−12.65	7.006	18.916
2862	ARG367	C	−11.849	7.197	20.228
2863	ARG367	O	−11.982	6.425	21.184
2864	ARG367	CB	−12.905	5.498	18.688
2865	ARG367	CG	−11.635	4.631	18.755
2866	ARG367	CD	−11.779	3.48	19.753
2867	ARG367	NE	−10.476	3.073	20.296
2868	ARG367	CZ	−9.887	3.665	21.366
2869	ARG367	NH1	−10.429	4.7	22.025
2870	ARG367	NH2	−8.69	3.226	21.806
2871	GLN368	N	−11.05	8.313	20.291
2872	GLN368	CA	−10.37	8.732	21.509
2873	GLN368	C	−10.05	10.237	21.513
2874	GLN368	O	−9.328	10.743	22.375
2875	GLN368	CB	−9.092	7.906	21.735
2876	GLN368	CG	−8.796	7.688	23.221
2877	GLN368	CD	−7.888	6.49	23.4
2878	GLN368	OE1	−8.313	5.334	23.354
2879	GLN368	NE2	−6.565	6.78	23.545
2880	LEU369	N	−10.702	11.007	20.568
2881	LEU369	CA	−10.55	12.461	20.559
2882	LEU369	C	−11.611	13.036	21.509
2883	LEU369	O	−12.578	13.703	21.135
2884	LEU369	CB	−10.684	13.044	19.146
2885	LEU369	CG	−9.617	12.561	18.142
2886	LEU369	CD1	−9.783	13.301	16.811
2887	LEU369	CD2	−8.184	12.745	18.645
2888	LEU370	N	−11.352	12.814	22.853
2889	LEU370	CA	−12.372	12.994	23.897
2890	LEU370	C	−12.689	14.478	24.253
2891	LEU370	O	−13.159	14.812	25.339
2892	LEU370	CB	−12.003	12.227	25.183
2893	LEU370	CG	−11.831	10.701	25.034
2894	LEU370	CD1	−11.47	10.087	26.39
2895	LEU370	CD2	−13.076	10.017	24.47
2896	GLN371	N	−12.58	15.354	23.193
2897	GLN371	CA	−13.322	16.61	23.121
2898	GLN371	C	−14.711	16.324	22.524
2899	GLN371	O	−15.701	16.945	22.907
2900	GLN371	CB	−12.536	17.591	22.228
2901	GLN371	CG	−13.169	18.976	22.049
2902	GLN371	CD	−14.262	19.089	20.995
2903	GLN371	OE1	−15.348	19.606	21.238
2904	GLN371	NE2	−13.921	18.669	19.739
2905	ASP372	N	−14.704	15.511	21.402
2906	ASP372	CA	−15.799	15.527	20.44
2907	ASP372	C	−16.76	14.328	20.615
2908	ASP372	O	−16.66	13.492	21.511
2909	ASP372	CB	−15.203	15.594	19.035
2910	ASP372	CG	−16.111	16.58	18.31
2911	ASP372	OD1	−17.287	16.145	18.107
2912	ASP372	OD2	−15.588	17.709	18.076
2913	THR373	N	−17.792	14.315	19.688
2914	THR373	CA	−18.977	13.493	19.86
2915	THR373	C	−18.777	12.024	19.482
2916	THR373	O	−19.582	11.178	19.884
2917	THR373	CB	−20.195	14.049	19.085
2918	THR373	OG1	−21.431	13.373	19.431
2919	THR373	CG2	−20.056	13.997	17.571
2920	PHE374	N	−17.84	11.738	18.522
2921	PHE374	CA	−17.508	10.392	18.051
2922	PHE374	C	−18.642	9.76	17.217
2923	PHE374	O	−18.488	9.453	16.032
2924	PHE374	CB	−17.011	9.462	19.177
2925	PHE374	CG	−16.836	8.017	18.764
2926	PHE374	CD1	−16.167	7.669	17.583
2927	PHE374	CD2	−17.375	6.996	19.561
2928	PHE374	CE1	−16.096	6.336	17.182
2929	PHE374	CE2	−17.284	5.66	19.166
2930	PHE374	CZ	−16.653	5.331	17.97
2931	TRP375	N	−19.817	9.514	17.898
2932	TRP375	CA	−20.82	8.557	17.424
2933	TRP375	C	−21.755	9.084	16.323
2934	TRP375	O	−22.61	8.367	15.804
2935	TRP375	CB	−21.629	7.912	18.564
2936	TRP375	CG	−22.331	8.872	19.483
2937	TRP375	CD1	−21.851	9.306	20.704
2938	TRP375	CD2	−23.628	9.464	19.323
2939	TRP375	NE1	−22.769	10.151	21.262
2940	TRP375	CE2	−23.867	10.263	20.444
2941	TRP375	CE3	−24.638	9.38	18.341
2942	TRP375	CZ2	−25.054	10.984	20.627
2943	TRP375	CZ3	−25.831	10.092	18.509
2944	TRP375	CH2	−26.035	10.881	19.638
2945	LYS376	N	−21.516	10.383	15.928
2946	LYS376	CA	−22.219	11.011	14.814
2947	LYS376	C	−21.41	10.936	13.501
2948	LYS376	O	−21.746	11.557	12.494
2949	LYS376	CB	−22.584	12.47	15.123
2950	LYS376	CG	−23.353	12.625	16.44
2951	LYS376	CD	−23.905	14.043	16.608
2952	LYS376	CE	−24.52	14.269	17.983
2953	LYS376	NZ	−23.482	14.625	18.973
2954	ASN377	N	−20.364	10.027	13.505
2955	ASN377	CA	−19.79	9.578	12.237
2956	ASN377	C	−20.838	8.658	11.589
2957	ASN377	O	−21.536	7.917	12.289
2958	ASN377	CB	−18.536	8.733	12.44
2959	ASN377	CG	−17.282	9.556	12.579
2960	ASN377	OD1	−16.574	9.835	11.614
2961	ASN377	ND2	−16.991	9.935	13.859
2962	PRO378	N	−20.903	8.629	10.213
2963	PRO378	CA	−21.896	7.798	9.54
2964	PRO378	C	−21.427	6.334	9.541
2965	PRO378	O	−20.324	5.978	9.129
2966	PRO378	CB	−21.965	8.358	8.118
2967	PRO378	CG	−20.59	8.984	7.909
2968	PRO378	CD	−20.246	9.536	9.283
2969	GLN379	N	−22.355	5.455	10.063
2970	GLN379	CA	−22.13	4.02	10.099
2971	GLN379	C	−22.575	3.441	8.742
2972	GLN379	O	−23.457	3.955	8.053
2973	GLN379	CB	−22.913	3.342	11.236
2974	GLN379	CG	−22.593	3.905	12.627
2975	GLN379	CD	−23.27	5.236	12.913
2976	GLN379	OE1	−24.062	5.774	12.145
2977	GLN379	NE2	−22.924	5.779	14.122
2978	PHE380	N	−21.936	2.265	8.383
2979	PHE380	CA	−22.185	1.634	7.09
2980	PHE380	C	−22.63	0.18	7.295
2981	PHE380	O	−22.198	−0.546	8.188
2982	PHE380	CB	−20.949	1.658	6.181
2983	PHE380	CG	−20.569	3.043	5.716
2984	PHE380	CD1	−19.357	3.616	6.122
2985	PHE380	CD2	−21.416	3.779	4.873
2986	PHE380	CE1	−19.001	4.896	5.693
2987	PHE380	CE2	−21.056	5.057	4.443
2988	PHE380	CZ	−19.848	5.615	4.853
2989	LEU381	N	−23.534	−0.249	6.338
2990	LEU381	CA	−24.25	−1.514	6.46
2991	LEU381	C	−23.534	−2.548	5.567
2992	LEU381	O	−23.838	−2.741	4.388
2993	LEU381	CB	−25.714	−1.298	6.037
2994	LEU381	CG	−26.653	−2.459	6.414
2995	LEU381	CD1	−26.929	−2.494	7.92
2996	LEU381	CD2	−27.978	−2.326	5.66
2997	LEU382	N	−22.46	−3.169	6.173
2998	LEU382	CA	−21.835	−4.382	5.63
2999	LEU382	C	−22.284	−5.561	6.522
3000	LEU382	O	−22.899	−5.375	7.577
3001	LEU382	CB	−20.31	−4.248	5.591
3002	LEU382	CG	−19.805	−3.112	4.675
3003	LEU382	CD1	−18.293	−2.95	4.834
3004	LEU382	CD2	−20.143	−3.357	3.203
3005	SER383	N	−21.988	−6.824	6.048
3006	SER383	CA	−22.588	−8.052	6.617
3007	SER383	C	−21.629	−9.25	6.398
3008	SER383	O	−20.671	−9.146	5.623
3009	SER383	CB	−23.949	−8.315	5.955
3010	SER383	OG	−24.973	−7.4	6.396
3011	VAL384	N	−21.83	−10.37	7.195
3012	VAL384	CA	−21.372	−11.751	6.842
3013	VAL384	C	−22.079	−12.761	7.821
3014	VAL384	O	−22.425	−12.38	8.942
3015	VAL384	CB	−19.837	−11.888	6.772
3016	VAL384	CG1	−19.229	−12.214	8.117
3017	VAL384	CG2	−19.373	−12.917	5.744
3018	TRP385	N	−22.278	−14.06	7.353
3019	TRP385	CA	−22.723	−15.189	8.229
3020	TRP385	C	−21.53	−16.149	8.431
3021	TRP385	O	−20.479	−15.995	7.81
3022	TRP385	CB	−24.005	−15.93	7.763
3023	TRP385	CG	−23.947	−16.867	6.581
3024	TRP385	CD1	−23.168	−18.003	6.451
3025	TRP385	CD2	−24.804	−16.854	5.419
3026	TRP385	NE1	−23.438	−18.598	5.247
3027	TRP385	CE2	−24.405	−17.891	4.578
3028	TRP385	CE3	−25.913	−16.085	5.006
3029	TRP385	CZ2	−24.963	−18.119	3.313
3030	TRP385	CZ3	−26.5	−16.301	3.754
3031	TRP385	CH2	−26.014	−17.294	2.91
3032	ARG386	N	−21.73	−17.184	9.335
3033	ARG386	CA	−20.636	−18.069	9.763
3034	ARG386	C	−21.064	−19.578	9.786
3035	ARG386	O	−21.971	−19.986	10.516
3036	ARG386	CB	−20.162	−17.568	11.128
3037	ARG386	CG	−19.024	−18.381	11.725
3038	ARG386	CD	−18.422	−17.636	12.911
3039	ARG386	NE	−17.243	−16.862	12.502
3040	ARG386	CZ	−17.023	−15.559	12.755
3041	ARG386	NH1	−15.887	−14.992	12.315
3042	ARG386	NH2	−17.891	−14.792	13.442
3043	PRO387	N	−20.383	−20.443	8.926
3044	PRO387	CA	−20.892	−21.788	8.578
3045	PRO387	C	−20.298	−22.971	9.395
3046	PRO387	O	−19.435	−22.831	10.267
3047	PRO387	CB	−20.461	−21.931	7.113
3048	PRO387	CG	−19.113	−21.221	7.091
3049	PRO387	CD	−19.383	−20.011	7.958
3050	GLU388	N	−20.764	−24.221	9.005
3051	GLU388	CA	−20.389	−25.505	9.623
3052	GLU388	C	−19.082	−26.127	9.012
3053	GLU388	O	−18.926	−27.349	8.891
3054	GLU388	CB	−21.524	−26.554	9.497
3055	GLU388	CG	−22.909	−26.148	10.003
3056	GLU388	CD	−23.108	−26.087	11.515
3057	GLU388	OE1	−22.765	−27.093	12.197
3058	GLU388	OE2	−23.571	−24.979	11.934
3059	GLU389	N	−18.033	−25.254	8.789
3060	GLU389	CA	−16.664	−25.697	8.397
3061	GLU389	C	−15.987	−26.349	9.647
3062	GLU389	O	−16.605	−26.566	10.696
3063	GLU389	CB	−15.93	−24.498	7.763
3064	GLU389	CG	−14.713	−24.819	6.884
3065	GLU389	CD	−13.399	−24.46	7.563
3066	GLU389	OE1	−12.701	−23.533	7.064
3067	GLU389	OE2	−13.124	−25.121	8.61
3068	GLY390	N	−14.681	−26.751	9.516
3069	GLY390	CA	−14.027	−27.553	10.53
3070	GLY390	C	−13.423	−26.714	11.662
3071	GLY390	O	−14.015	−25.771	12.195
3072	ARG391	N	−12.162	−27.162	12.036
3073	ARG391	CA	−11.427	−26.628	13.18
3074	ARG391	C	−10.954	−25.21	12.832
3075	ARG391	O	−10.327	−24.927	11.809
3076	ARG391	CB	−10.224	−27.531	13.497
3077	ARG391	CG	−9.386	−27.126	14.715
3078	ARG391	CD	−10.134	−27.236	16.041
3079	ARG391	NE	−9.223	−26.995	17.173
3080	ARG391	CZ	−9.596	−27.072	18.477
3081	ARG391	NH1	−8.666	−26.907	19.442
3082	ARG391	NH2	−10.872	−27.303	18.843
3083	ARG392	N	−11.331	−24.267	13.785
3084	ARG392	CA	−11.305	−22.854	13.436
3085	ARG392	C	−10.846	−21.965	14.609
3086	ARG392	O	−11.058	−20.753	14.675
3087	ARG392	CB	−12.647	−22.412	12.835
3088	ARG392	CG	−12.793	−22.794	11.36
3089	ARG392	CD	−14.19	−22.585	10.807
3090	ARG392	NE	−15.126	−23.597	11.318
3091	ARG392	CZ	−16.472	−23.486	11.233
3092	ARG392	NH1	−17.268	−24.443	11.732
3093	ARG392	NH2	−17.067	−22.445	10.617
3094	SER393	N	−9.984	−22.596	15.478
3095	SER393	CA	−9.333	−21.934	16.611
3096	SER393	C	−7.956	−21.336	16.252
3097	SER393	O	−7.059	−21.28	17.093
3098	SER393	CB	−9.151	−22.928	17.767
3099	SER393	OG	−8.707	−22.282	18.955
3100	LEU394	N	−7.851	−20.775	14.996
3101	LEU394	CA	−6.568	−20.364	14.404
3102	LEU394	C	−6.794	−19.764	12.993
3103	LEU394	O	−6.362	−20.323	11.985
3104	LEU394	CB	−5.522	−21.52	14.394
3105	LEU394	CG	−5.762	−22.791	13.539
3106	LEU394	CD1	−4.561	−23.734	13.699
3107	LEU394	CD2	−7.04	−23.555	13.868
3108	ARG395	N	−7.593	−18.622	12.89
3109	ARG395	CA	−8.21	−18.3	11.586
3110	ARG395	C	−8.701	−16.782	11.178
3111	ARG395	O	−9.874	−16.525	10.892
3112	ARG395	CB	−9.39	−19.275	11.375
3113	ARG395	CG	−9.286	−20.806	11.46
3114	ARG395	CD	−8.562	−21.618	10.391
3115	ARG395	NE	−9.305	−21.658	9.128
3116	ARG395	CZ	−10.205	−22.574	8.688
3117	ARG395	NH1	−10.593	−23.658	9.402
3118	ARG395	NH2	−10.761	−22.39	7.476
3119	PRO396	N	−7.74	−15.79	11.005
3120	PRO396	CA	−7.983	−14.407	10.506
3121	PRO396	C	−8.425	−14.105	9.049
3122	PRO396	O	−8.923	−14.954	8.312
3123	PRO396	CB	−6.609	−13.747	10.679
3124	PRO396	CG	−5.993	−14.423	11.868
3125	PRO396	CD	−6.711	−15.746	11.993
3126	CYS397	N	−8.274	−12.755	8.692
3127	CYS397	CA	−8.72	−12.15	7.421
3128	CYS397	C	−8.328	−10.621	7.371
3129	CYS397	O	−7.924	−10.054	8.394
3130	CYS397	CB	−10.208	−12.41	7.221
3131	CYS397	SG	−11.279	−11.876	8.582
3132	SER398	N	−8.423	−9.94	6.157
3133	SER398	CA	−8.182	−8.484	6.024
3134	SER398	C	−9.232	−7.657	5.24
3135	SER398	O	−9.673	−7.98	4.142
3136	SER398	CB	−6.83	−8.139	5.399
3137	SER398	OG	−6.54	−6.747	5.627
3138	VAL399	N	−9.546	−6.441	5.84
3139	VAL399	CA	−10.484	−5.489	5.229
3140	VAL399	C	−9.877	−4.905	3.935
3141	VAL399	O	−8.669	−4.858	3.714
3142	VAL399	CB	−10.915	−4.399	6.256
3143	VAL399	CG1	−12.368	−4.572	6.703
3144	VAL399	CG2	−10.719	−2.942	5.827
3145	LEU400	N	−10.855	−4.406	3.088
3146	LEU400	CA	−10.577	−3.844	1.768
3147	LEU400	C	−11.708	−2.838	1.468
3148	LEU400	O	−12.448	−2.909	0.491
3149	LEU400	CB	−10.524	−4.964	0.726
3150	LEU400	CG	−10.008	−4.493	−0.644
3151	LEU400	CD1	−8.493	−4.301	−0.627
3152	LEU400	CD2	−10.408	−5.497	−1.721
3153	VAL401	N	−11.801	−1.835	2.407
3154	VAL401	CA	−12.723	−0.702	2.301
3155	VAL401	C	−11.844	0.483	2.736
3156	VAL401	O	−10.856	0.309	3.454
3157	VAL401	CB	−13.959	−0.896	3.208
3158	VAL401	CG1	−14.994	0.218	3.018
3159	VAL401	CG2	−14.649	−2.245	2.967
3160	SER402	N	−12.241	1.72	2.284
3161	SER402	CA	−11.36	2.883	2.401
3162	SER402	C	−12.157	4.151	2.745
3163	SER402	O	−13.38	4.229	2.642
3164	SER402	CB	−10.56	3.073	1.105
3165	SER402	OG	−11.443	3.091	−0.023
3166	LEU403	N	−11.356	5.198	3.161
3167	LEU403	CA	−11.854	6.504	3.597
3168	LEU403	C	−10.857	7.561	3.072
3169	LEU403	O	−10.423	8.519	3.706
3170	LEU403	CB	−12.133	6.514	5.096
3171	LEU403	CG	−12.702	7.82	5.681
3172	LEU403	CD1	−13.726	8.51	4.783
3173	LEU403	CD2	−13.325	7.552	7.053
3174	LEU404	N	−10.668	7.373	1.714
3175	LEU404	CA	−9.941	8.264	0.812
3176	LEU404	C	−10.818	9.529	0.723
3177	LEU404	O	−12.024	9.486	0.474
3178	LEU404	CB	−9.801	7.527	−0.53
3179	LEU404	CG	−8.834	8.136	−1.553
3180	LEU404	CD1	−8.581	7.127	−2.68
3181	LEU404	CD2	−9.37	9.432	−2.147
3182	GLN405	N	−10.159	10.714	1.016
3183	GLN405	CA	−10.916	11.95	1.155
3184	GLN405	C	−11.398	12.498	−0.194
3185	GLN405	O	−10.848	12.242	−1.26
3186	GLN405	CB	−10.152	13.044	1.92
3187	GLN405	CG	−8.982	13.679	1.17
3188	GLN405	CD	−8.409	14.848	1.941
3189	GLN405	OE1	−8.517	16.01	1.558
3190	GLN405	NE2	−7.772	14.524	3.104
3191	LYS406	N	−12.457	13.383	−0.088
3192	LYS406	CA	−12.778	14.302	−1.177
3193	LYS406	C	−12.141	15.635	−0.743
3194	LYS406	O	−12.518	16.183	0.306
3195	LYS406	CB	−14.292	14.496	−1.302
3196	LYS406	CG	−15.033	13.227	−1.744
3197	LYS406	CD	−16.543	13.452	−1.659
3198	LYS406	CE	−17.366	12.198	−1.9
3199	LYS406	NZ	−17.498	11.909	−3.337
3200	PRO407	N	−11.126	16.161	−1.511
3201	PRO407	CA	−10.416	17.373	−1.111
3202	PRO407	C	−11.214	18.633	−1.495
3203	PRO407	O	−12.201	18.619	−2.226
3204	PRO407	CB	−9.092	17.307	−1.875
3205	PRO407	CG	−9.469	16.556	−3.147
3206	PRO407	CD	−10.478	15.53	−2.653
3207	ARG408	N	−10.711	19.793	−0.93
3208	ARG408	CA	−11.226	21.121	−1.276
3209	ARG408	C	−10.01	21.966	−1.669
3210	ARG408	O	−8.867	21.71	−1.273
3211	ARG408	CB	−11.946	21.751	−0.075
3212	ARG408	CG	−13.319	21.114	0.167
3213	ARG408	CD	−13.694	21.058	1.65
3214	ARG408	NE	−14.581	19.919	1.947
3215	ARG408	CZ	−14.228	18.617	1.777
3216	ARG408	NH1	−12.974	18.264	1.444
3217	ARG408	NH2	−15.149	17.647	1.94
3218	HIS409	N	−10.29	23.114	−2.386
3219	HIS409	CA	−9.225	23.894	−3.039
3220	HIS409	C	−8.423	24.827	−2.092
3221	HIS409	O	−7.786	25.794	−2.516
3222	HIS409	CB	−9.749	24.675	−4.261
3223	HIS409	CG	−10.004	23.779	−5.431
3224	HIS409	ND1	−11.239	23.595	−6
3225	HIS409	CD2	−9.152	23.013	−6.2
3226	HIS409	CE1	−11.089	22.715	−7.037
3227	HIS409	NE2	−9.837	22.33	−7.181
3228	ARG410	N	−8.27	24.332	−0.808
3229	ARG410	CA	−7.107	24.629	0.028
3230	ARG410	C	−5.953	23.707	−0.422
3231	ARG410	O	−4.786	24.104	−0.504
3232	ARG410	CB	−7.462	24.385	1.5
3233	ARG410	CG	−6.329	24.746	2.464
3234	ARG410	CD	−6.767	24.581	3.916
3235	ARG410	NE	−5.679	24.889	4.856
3236	ARG410	CZ	−4.671	24.047	5.193
3237	ARG410	NH1	−4.554	22.815	4.655
3238	ARG410	NH2	−3.751	24.446	6.098
3239	CYS411	N	−6.287	22.387	−0.683
3240	CYS411	CA	−5.272	21.47	−1.206
3241	CYS411	C	−4.915	21.959	−2.62
3242	CYS411	O	−5.71	22.596	−3.317
3243	CYS411	CB	−5.753	20.019	−1.281
3244	CYS411	SG	−6.127	19.345	0.368
3245	ARG412	N	−3.614	21.698	−3.013
3246	ARG412	CA	−3.076	22.247	−4.266
3247	ARG412	C	−3.132	21.185	−5.377
3248	ARG412	O	−3.331	21.488	−6.55
3249	ARG412	CB	−1.624	22.739	−4.093
3250	ARG412	CG	−1.472	24.264	−3.936
3251	ARG412	CD	−1.97	24.831	−2.609
3252	ARG412	NE	−3.426	24.817	−2.508
3253	ARG412	CZ	−4.289	25.563	−3.227
3254	ARG412	NH1	−3.925	26.701	−3.841
3255	ARG412	NH2	−5.553	25.134	−3.334
3256	LYS413	N	−2.775	19.912	−4.979
3257	LYS413	CA	−2.423	18.864	−5.94
3258	LYS413	C	−3.566	17.87	−6.208
3259	LYS413	O	−3.417	16.89	−6.934
3260	LYS413	CB	−1.2	18.077	−5.447
3261	LYS413	CG	0.061	18.939	−5.336
3262	LYS413	CD	1.276	18.072	−5.005
3263	LYS413	CE	2.534	18.909	−4.838
3264	LYS413	NZ	3.687	18.012	−4.683
3265	ARG414	N	−4.745	18.116	−5.519
3266	ARG414	CA	−5.96	17.328	−5.776
3267	ARG414	C	−5.757	15.849	−5.368
3268	ARG414	O	−6.423	14.935	−5.854
3269	ARG414	CB	−6.46	17.417	−7.226
3270	ARG414	CG	−6.939	18.805	−7.644
3271	ARG414	CD	−8.386	19.082	−7.238
3272	ARG414	NE	−8.988	20.13	−8.069
3273	ARG414	CZ	−9.199	20.038	−9.405
3274	ARG414	NH1	−9.55	21.142	−10.09
3275	ARG414	NH2	−9.091	18.866	−10.056
3276	LYS415	N	−4.876	15.685	−4.308
3277	LYS415	CA	−4.204	14.401	−4.077
3278	LYS415	C	−5.175	13.385	−3.436
3279	LYS415	O	−5.764	13.646	−2.38
3280	LYS415	CB	−3.002	14.629	−3.139
3281	LYS415	CG	−2.124	13.386	−2.95
3282	LYS415	CD	−0.881	13.715	−2.115
3283	LYS415	CE	−0.054	12.499	−1.707
3284	LYS415	NZ	0.445	11.776	−2.873
3285	PRO416	N	−5.289	12.137	−4.035
3286	PRO416	CA	−6.208	11.119	−3.493
3287	PRO416	C	−5.699	10.44	−2.2
3288	PRO416	O	−5.502	9.229	−2.116
3289	PRO416	CB	−6.379	10.111	−4.639
3290	PRO416	CG	−6.094	10.922	−5.889
3291	PRO416	CD	−4.993	11.863	−5.44
3292	LEU417	N	−5.558	11.296	−1.119
3293	LEU417	CA	−5.151	10.828	0.207
3294	LEU417	C	−6.354	10.546	1.155
3295	LEU417	O	−7.531	10.596	0.797
3296	LEU417	CB	−4.029	11.711	0.793
3297	LEU417	CG	−4.428	12.987	1.563
3298	LEU417	CD1	−3.186	13.579	2.241
3299	LEU417	CD2	−5.07	14.051	0.682
3300	LEU418	N	−5.985	10.162	2.433
3301	LEU418	CA	−6.93	9.736	3.481
3302	LEU418	C	−7.686	10.961	4.037
3303	LEU418	O	−7.214	12.1	4.002
3304	LEU418	CB	−6.102	9.081	4.61
3305	LEU418	CG	−6.88	8.332	5.706
3306	LEU418	CD1	−7.557	7.083	5.159
3307	LEU418	CD2	−5.933	7.937	6.844
3308	ALA419	N	−8.902	10.672	4.638
3309	ALA419	CA	−9.558	11.629	5.528
3310	ALA419	C	−9.125	11.34	6.98
3311	ALA419	O	−8.25	12	7.538
3312	ALA419	CB	−11.072	11.637	5.361
3313	ILE420	N	−9.751	10.262	7.572
3314	ILE420	CA	−9.394	9.73	8.889
3315	ILE420	C	−9.594	8.204	8.777
3316	ILE420	O	−10.033	7.702	7.74
3317	ILE420	CB	−10.195	10.35	10.062
3318	ILE420	CG1	−11.716	10.074	10.052
3319	ILE420	CG2	−9.889	11.838	10.256
3320	ILE420	CD1	−12.536	10.817	9.009
3321	GLY421	N	−9.242	7.453	9.882
3322	GLY421	CA	−9.545	6.029	9.923
3323	GLY421	C	−10.876	5.785	10.639
3324	GLY421	O	−11.293	6.56	11.506
3325	PHE422	N	−11.516	4.611	10.28
3326	PHE422	CA	−12.881	4.271	10.723
3327	PHE422	C	−12.978	2.908	11.459
3328	PHE422	O	−12.03	2.131	11.58
3329	PHE422	CB	−13.905	4.426	9.586
3330	PHE422	CG	−13.725	3.469	8.435
3331	PHE422	CD1	−14.505	2.311	8.335
3332	PHE422	CD2	−12.772	3.732	7.443
3333	PHE422	CE1	−14.332	1.436	7.261
3334	PHE422	CE2	−12.596	2.855	6.375
3335	PHE422	CZ	−13.376	1.708	6.286
3336	TYR423	N	−14.215	2.649	12.042
3337	TYR423	CA	−14.36	1.647	13.112
3338	TYR423	C	−15.679	0.909	12.861
3339	TYR423	O	−16.769	1.43	13.102
3340	TYR423	CB	−14.392	2.292	14.525
3341	TYR423	CG	−13.436	3.454	14.663
3342	TYR423	CD1	−13.908	4.752	14.408
3343	TYR423	CD2	−12.065	3.241	14.853
3344	TYR423	CE1	−13.014	5.801	14.239
3345	TYR423	CE2	−11.166	4.292	14.683
3346	TYR423	CZ	−11.652	5.542	14.327
3347	TYR423	OH	−10.774	6.499	13.905
3348	LEU424	N	−15.555	−0.326	12.252
3349	LEU424	CA	−16.735	−1.139	11.976
3350	LEU424	C	−17.057	−2.062	13.172
3351	LEU424	O	−16.209	−2.733	13.761
3352	LEU424	CB	−16.547	−2.012	10.728
3353	LEU424	CG	−16.532	−1.242	9.395
3354	LEU424	CD1	−16.122	−2.192	8.266
3355	LEU424	CD2	−17.888	−0.613	9.07
3356	TYR425	N	−18.407	−2.132	13.465
3357	TYR425	CA	−18.935	−2.946	14.562
3358	TYR425	C	−20.066	−3.78	13.949
3359	TYR425	O	−21.154	−3.292	13.65
3360	TYR425	CB	−19.461	−2.075	15.711
3361	TYR425	CG	−18.348	−1.351	16.436
3362	TYR425	CD1	−18.103	0.007	16.184
3363	TYR425	CD2	−17.52	−2.037	17.337
3364	TYR425	CE1	−17.053	0.67	16.823
3365	TYR425	CE2	−16.465	−1.376	17.97
3366	TYR425	CZ	−16.239	−0.029	17.709
3367	TYR425	OH	−15.195	0.579	18.343
3368	ARG426	N	−19.7	−5.072	13.62
3369	ARG426	CA	−20.619	−5.985	12.935
3370	ARG426	C	−21.328	−6.886	13.962
3371	ARG426	O	−20.791	−7.22	15.016
3372	ARG426	CB	−19.831	−6.833	11.928
3373	ARG426	CG	−19.498	−6.044	10.651
3374	ARG426	CD	−18.185	−6.492	10.014
3375	ARG426	NE	−18.271	−6.602	8.552
3376	ARG426	CZ	−18.851	−7.644	7.904
3377	ARG426	NH1	−18.874	−7.652	6.555
3378	ARG426	NH2	−19.409	−8.672	8.574
3379	MET427	N	−22.589	−7.326	13.584
3380	MET427	CA	−23.458	−8.081	14.497
3381	MET427	C	−24.524	−8.836	13.668
3382	MET427	O	−25.435	−8.232	13.105
3383	MET427	CB	−24.21	−7.149	15.474
3384	MET427	CG	−23.321	−6.538	16.555
3385	MET427	SD	−24.334	−5.591	17.739
3386	MET427	CE	−23.003	−4.993	18.809
3387	ASN428	N	−24.345	−10.21	13.56
3388	ASN428	CA	−25.495	−11.121	13.342
3389	ASN428	C	−26.198	−10.914	11.983
3390	ASN428	O	−27.267	−10.315	11.873
3391	ASN428	CB	−26.504	−11.046	14.496
3392	ASN428	CG	−25.841	−11.268	15.835
3393	ASN428	OD1	−25.592	−10.354	16.617
3394	ASN428	ND2	−25.496	−12.557	16.116
3395	LYS429	N	−25.502	−11.41	10.89
3396	LYS429	CA	−25.762	−10.926	9.529
3397	LYS429	C	−25.484	−12.015	8.456
3398	LYS429	O	−25.267	−13.184	8.767
3399	LYS429	CB	−24.914	−9.658	9.352
3400	LYS429	CG	−25.731	−8.384	9.563
3401	LYS429	CD	−24.844	−7.196	9.938
3402	LYS429	CE	−25.496	−5.884	9.55
3403	LYS429	NZ	−25.222	−5.639	8.124
3404	GLU430	N	−25.568	−11.583	7.132
3405	GLU430	CA	−25.522	−12.497	5.971
3406	GLU430	C	−24.303	−12.271	5.033
3407	GLU430	O	−23.738	−11.184	4.938
3408	GLU430	CB	−26.789	−12.36	5.12
3409	GLU430	CG	−28.058	−12.739	5.875
3410	GLU430	CD	−29.177	−12.747	4.838
3411	GLU430	OE1	−29.853	−11.683	4.78
3412	GLU430	OE2	−29.261	−13.809	4.158
3413	MET431	N	−23.937	−13.375	4.265
3414	MET431	CA	−22.64	−13.472	3.578
3415	MET431	C	−22.448	−12.481	2.41
3416	MET431	O	−23.38	−11.916	1.837
3417	MET431	CB	−22.355	−14.908	3.081
3418	MET431	CG	−21.332	−15.608	3.982
3419	MET431	SD	−21.075	−17.319	3.462
3420	MET431	CE	−19.889	−17.834	4.72
3421	THR432	N	−21.117	−12.353	2.026
3422	THR432	CA	−20.633	−11.428	1
3423	THR432	C	−20.416	−12.185	−0.332
3424	THR432	O	−21.316	−12.297	−1.171
3425	THR432	CB	−19.345	−10.708	1.456
3426	THR432	OG1	−18.337	−11.689	1.747
3427	THR432	CG2	−19.559	−9.846	2.692
3428	TRP433	N	−19.203	−12.821	−0.466
3429	TRP433	CA	−18.684	−13.334	−1.741
3430	TRP433	C	−17.643	−14.447	−1.47
3431	TRP433	O	−17.445	−14.869	−0.33
3432	TRP433	CB	−18.203	−12.196	−2.66
3433	TRP433	CG	−17.287	−11.209	−2.005
3434	TRP433	CD1	−16.045	−11.47	−1.461
3435	TRP433	CD2	−17.527	−9.805	−1.836
3436	TRP433	NE1	−15.553	−10.308	−0.933
3437	TRP433	CE2	−16.424	−9.273	−1.171
3438	TRP433	CE3	−18.578	−8.933	−2.188
3439	TRP433	CZ2	−16.306	−7.916	−0.848
3440	TRP433	CZ3	−18.473	−7.572	−1.876
3441	TRP433	CH2	−17.352	−7.072	−1.22
3442	SER434	N	−17.074	−14.988	−2.607
3443	SER434	CA	−16.302	−16.245	−2.675
3444	SER434	C	−14.816	−16.052	−2.31
3445	SER434	O	−14.388	−15.017	−1.798
3446	SER434	CB	−16.474	−16.814	−4.092
3447	SER434	OG	−16.026	−15.858	−5.058
3448	SER435	N	−13.978	−17.125	−2.611
3449	SER435	CA	−12.701	−17.345	−1.906
3450	SER435	C	−11.638	−16.241	−1.942
3451	SER435	O	−10.671	−16.273	−1.174
3452	SER435	CB	−12.027	−18.653	−2.353
3453	SER435	OG	−11.566	−18.579	−3.706
3454	LEU436	N	−11.87	−15.187	−2.797
3455	LEU436	CA	−11.111	−13.952	−2.65
3456	LEU436	C	−11.359	−13.328	−1.26
3457	LEU436	O	−10.531	−12.606	−0.705
3458	LEU436	CB	−11.517	−12.903	−3.684
3459	LEU436	CG	−11.315	−13.279	−5.154
3460	LEU436	CD1	−11.864	−12.154	−6.032
3461	LEU436	CD2	−9.846	−13.513	−5.489
3462	GLY437	N	−12.62	−13.556	−0.747
3463	GLY437	CA	−13.039	−13.084	0.547
3464	GLY437	C	−12.719	−14.037	1.689
3465	GLY437	O	−13.079	−13.791	2.842
3466	SER438	N	−11.88	−15.091	1.39
3467	SER438	CA	−11.446	−16.058	2.416
3468	SER438	C	−10.602	−15.312	3.458
3469	SER438	O	−10.428	−15.716	4.604
3470	SER438	CB	−10.628	−17.205	1.821
3471	SER438	OG	−9.526	−16.709	1.058
3472	ARG439	N	−10.038	−14.168	2.939
3473	ARG439	CA	−9.286	−13.194	3.686
3474	ARG439	C	−10.124	−11.915	3.874
3475	ARG439	O	−9.563	−10.825	3.969
3476	ARG439	CB	−7.99	−12.932	2.92
3477	ARG439	CG	−6.867	−12.299	3.739
3478	ARG439	CD	−5.585	−12.165	2.925
3479	ARG439	NE	−5.069	−13.475	2.519
3480	ARG439	CZ	−5.407	−14.133	1.391
3481	ARG439	NH1	−5.1	−15.423	1.213
3482	ARG439	NH2	−6.106	−13.565	0.399
3483	GLN440	N	−11.463	−12.096	4.166
3484	GLN440	CA	−12.465	−11.035	4.403
3485	GLN440	C	−12.178	−10.022	5.552
3486	GLN440	O	−11.043	−9.693	5.896
3487	GLN440	CB	−12.943	−10.352	3.108
3488	GLN440	CG	−11.864	−9.689	2.259
3489	GLN440	CD	−12.414	−9.178	0.948
3490	GLN440	OE1	−13.474	−9.548	0.455
3491	GLN440	NE2	−11.597	−8.285	0.317
3492	PRO441	N	−13.239	−9.428	6.202
3493	PRO441	CA	−13.036	−8.136	6.862
3494	PRO441	C	−12.416	−8.117	8.287
3495	PRO441	O	−13.114	−7.991	9.295
3496	PRO441	CB	−14.447	−7.52	6.857
3497	PRO441	CG	−15.365	−8.731	6.917
3498	PRO441	CD	−14.652	−9.738	6.038
3499	PHE442	N	−11.029	−8.153	8.366
3500	PHE442	CA	−10.332	−7.805	9.62
3501	PHE442	C	−9.083	−6.896	9.496
3502	PHE442	O	−9.206	−5.679	9.672
3503	PHE442	CB	−10.116	−8.98	10.59
3504	PHE442	CG	−9.39	−8.563	11.855
3505	PHE442	CD1	−9.915	−7.563	12.689
3506	PHE442	CD2	−8.13	−9.099	12.155
3507	PHE442	CE1	−9.175	−7.076	13.767
3508	PHE442	CE2	−7.399	−8.621	13.245
3509	PHE442	CZ	−7.914	−7.601	14.042
3510	PHE443	N	−7.864	−7.47	9.167
3511	PHE443	CA	−6.592	−6.816	9.559
3512	PHE443	C	−6.519	−5.329	9.168
3513	PHE443	O	−6.065	−4.502	9.956
3514	PHE443	CB	−5.35	−7.501	8.958
3515	PHE443	CG	−4.915	−8.78	9.632
3516	PHE443	CD1	−4.499	−8.779	10.97
3517	PHE443	CD2	−4.845	−9.977	8.906
3518	PHE443	CE1	−4.053	−9.956	11.575
3519	PHE443	CE2	−4.392	−11.152	9.509
3520	PHE443	CZ	−4.002	−11.141	10.845
3521	SER444	N	−6.944	−5.024	7.889
3522	SER444	CA	−7.273	−3.663	7.462
3523	SER444	C	−6.103	−2.826	6.927
3524	SER444	O	−4.948	−2.922	7.329
3525	SER444	CB	−8.006	−2.808	8.523
3526	SER444	OG	−9.277	−3.34	8.873
3527	LEU445	N	−6.519	−1.872	6.004
3528	LEU445	CA	−5.909	−0.544	5.962
3529	LEU445	C	−7.064	0.398	6.361
3530	LEU445	O	−8.241	0.056	6.242
3531	LEU445	CB	−5.414	−0.178	4.561
3532	LEU445	CG	−4.232	−1.026	4.061
3533	LEU445	CD1	−3.967	−0.717	2.587
3534	LEU445	CD2	−2.959	−0.783	4.872
3535	GLU446	N	−6.68	1.619	6.875
3536	GLU446	CA	−7.618	2.736	7.041
3537	GLU446	C	−8.712	2.552	8.119
3538	GLU446	O	−9.611	3.382	8.283
3539	GLU446	CB	−8.281	3.187	5.724
3540	GLU446	CG	−7.293	3.445	4.588
3541	GLU446	CD	−7.919	4.079	3.345
3542	GLU446	OE1	−7.238	3.967	2.289
3543	GLU446	OE2	−9.047	4.637	3.496
3544	ALA447	N	−8.607	1.443	8.935
3545	ALA447	CA	−9.769	1.048	9.716
3546	ALA447	C	−9.445	0.063	10.841
3547	ALA447	O	−8.421	−0.612	10.864
3548	ALA447	CB	−10.827	0.409	8.815
3549	CYS448	N	−10.472	−0.034	11.766
3550	CYS448	CA	−10.613	−1.163	12.675
3551	CYS448	C	−11.992	−1.8	12.41
3552	CYS448	O	−12.953	−1.175	11.954
3553	CYS448	CB	−10.567	−0.751	14.15
3554	CYS448	SG	−8.969	−0.041	14.652
3555	GLN449	N	−12.101	−3.113	12.828
3556	GLN449	CA	−13.353	−3.852	12.697
3557	GLN449	C	−13.478	−4.789	13.901
3558	GLN449	O	−12.505	−5.372	14.383
3559	GLN449	CB	−13.392	−4.607	11.36
3560	GLN449	CG	−14.686	−5.391	11.114
3561	GLN449	CD	−14.77	−6.726	11.827
3562	GLN449	OE1	−15.787	−7.134	12.383
3563	GLN449	NE2	−13.64	−7.486	11.733
3564	GLY450	N	−14.782	−4.982	14.336
3565	GLY450	CA	−15.039	−5.922	15.4
3566	GLY450	C	−16.488	−6.393	15.59
3567	GLY450	O	−17.472	−5.878	15.069
3568	ILE451	N	−16.545	−7.431	16.511
3569	ILE451	CA	−17.782	−7.967	17.084
3570	ILE451	C	−17.402	−8.498	18.495
3571	ILE451	O	−16.283	−8.969	18.747
3572	ILE451	CB	−18.431	−9.052	16.18
3573	ILE451	CG1	−19.753	−9.581	16.776
3574	ILE451	CG2	−17.474	−10.212	15.914
3575	ILE451	CD1	−20.564	−10.47	15.844
3576	LEU452	N	−18.429	−8.406	19.42
3577	LEU452	CA	−18.457	−9.171	20.67
3578	LEU452	C	−19.38	−10.365	20.366
3579	LEU452	O	−20.564	−10.223	20.049
3580	LEU452	CB	−19.03	−8.361	21.838
3581	LEU452	CG	−17.969	−7.553	22.614
3582	LEU452	CD1	−17.285	−6.489	21.757
3583	LEU452	CD2	−18.613	−6.897	23.838
3584	ALA453	N	−18.72	−11.575	20.334
3585	ALA453	CA	−19.368	−12.823	19.955
3586	ALA453	C	−19.845	−13.573	21.207
3587	ALA453	O	−19.208	−13.543	22.263
3588	ALA453	CB	−18.397	−13.666	19.146
3589	LEU454	N	−20.992	−14.335	21.039
3590	LEU454	CA	−21.678	−14.884	22.218
3591	LEU454	C	−22.532	−16.117	21.838
3592	LEU454	O	−23.352	−16.077	20.924
3593	LEU454	CB	−22.578	−13.791	22.84
3594	LEU454	CG	−22.179	−13.341	24.254
3595	LEU454	CD1	−23.059	−12.174	24.702
3596	LEU454	CD2	−22.264	−14.459	25.287
3597	LEU455	N	−22.303	−17.232	22.626
3598	LEU455	CA	−23.037	−18.504	22.562
3599	LEU455	C	−22.923	−19.278	21.221
3600	LEU455	O	−21.809	−19.515	20.749
3601	LEU455	CB	−24.366	−18.564	23.336
3602	LEU455	CG	−25.471	−17.604	22.881
3603	LEU455	CD1	−26.844	−18.245	23.061
3604	LEU455	CD2	−25.453	−16.307	23.685
3605	ASP456	N	−24.08	−19.73	20.622
3606	ASP456	CA	−24.079	−20.807	19.612
3607	ASP456	C	−25.142	−20.552	18.52
3608	ASP456	O	−25.977	−21.383	18.16
3609	ASP456	CB	−24.277	−22.157	20.297
3610	ASP456	CG	−25.561	−22.276	21.117
3611	ASP456	OD1	−25.678	−23.357	21.757
3612	ASP456	OD2	−26.351	−21.282	21.082
3613	LEU457	N	−25.039	−19.315	17.918
3614	LEU457	CA	−26.163	−18.744	17.205
3615	LEU457	C	−26.23	−19.102	15.711
3616	LEU457	O	−25.275	−18.996	14.942
3617	LEU457	CB	−26.152	−17.21	17.303
3618	LEU457	CG	−26.172	−16.655	18.737
3619	LEU457	CD1	−26.215	−15.13	18.687
3620	LEU457	CD2	−27.342	−17.195	19.553
3621	ASN458	N	−27.517	−19.404	15.283
3622	ASN458	CA	−27.869	−19.553	13.868
3623	ASN458	C	−28.119	−18.161	13.271
3624	ASN458	O	−28.082	−17.963	12.057
3625	ASN458	CB	−29.094	−20.438	13.704
3626	ASN458	CG	−29.345	−20.88	12.281
3627	ASN458	OD1	−30.369	−20.599	11.654
3628	ASN458	ND2	−28.395	−21.678	11.711
3629	ALA459	N	−28.339	−17.149	14.183
3630	ALA459	CA	−28.623	−15.775	13.76
3631	ALA459	C	−27.383	−15.129	13.104
3632	ALA459	O	−27.454	−14.087	12.457
3633	ALA459	CB	−29.048	−14.929	14.952
3634	SER460	N	−26.191	−15.771	13.377
3635	SER460	CA	−24.923	−15.423	12.737
3636	SER460	C	−24.465	−16.523	11.762
3637	SER460	O	−23.307	−16.571	11.336
3638	SER460	CB	−23.826	−15.211	13.796
3639	SER460	OG	−24.097	−14.046	14.573
3640	GLY461	N	−25.421	−17.425	11.328
3641	GLY461	CA	−24.972	−18.751	10.942
3642	GLY461	C	−26.015	−19.652	10.301
3643	GLY461	O	−26.07	−20.852	10.568
3644	THR462	N	−26.747	−19.036	9.319
3645	THR462	CA	−27.6	−19.729	8.332
3646	THR462	C	−27.351	−18.909	7.069
3647	THR462	O	−27.307	−17.676	7.15
3648	THR462	CB	−29.076	−19.719	8.781
3649	THR462	OG1	−29.416	−21.025	9.273
3650	THR462	CG2	−30.111	−19.384	7.717
3651	MET463	N	−27.18	−19.512	5.856
3652	MET463	CA	−27.327	−20.912	5.478
3653	MET463	C	−26.033	−21.723	5.745
3654	MET463	O	−25.904	−22.367	6.789
3655	MET463	CB	−27.787	−21.064	4.019
3656	MET463	CG	−29.23	−20.624	3.748
3657	MET463	SD	−29.329	−19.017	2.886
3658	MET463	CE	−29.982	−17.973	4.209
3659	SER464	N	−25.026	−21.675	4.795
3660	SER464	CA	−23.925	−22.653	4.837
3661	SER464	C	−22.64	−22.062	4.223
3662	SER464	O	−22.496	−20.845	4.081
3663	SER464	CB	−24.372	−23.959	4.164
3664	SER464	OG	−24.026	−24.012	2.783
3665	ILE465	N	−21.628	−22.979	3.953
3666	ILE465	CA	−20.496	−22.62	3.087
3667	ILE465	C	−20.695	−23.153	1.652
3668	ILE465	O	−19.948	−22.821	0.73
3669	ILE465	CB	−19.145	−23.079	3.699
3670	ILE465	CG1	−17.945	−22.428	2.976
3671	ILE465	CG2	−19	−24.602	3.768
3672	ILE465	CD1	−16.65	−22.488	3.777
3673	GLN466	N	−21.729	−24.05	1.464
3674	GLN466	CA	−21.953	−24.678	0.166
3675	GLN466	C	−22.583	−23.712	−0.858
3676	GLN466	O	−22.796	−24.041	−2.023
3677	GLN466	CB	−22.784	−25.961	0.289
3678	GLN466	CG	−22.141	−26.997	1.216
3679	GLN466	CD	−22.575	−26.832	2.658
3680	GLN466	OE1	−21.911	−26.247	3.509
3681	GLN466	NE2	−23.792	−27.386	2.948
3682	GLU467	N	−22.787	−22.432	−0.379
3683	GLU467	CA	−23.107	−21.313	−1.25
3684	GLU467	C	−21.82	−20.77	−1.92
3685	GLU467	O	−21.881	−19.958	−2.844
3686	GLU467	CB	−23.776	−20.18	−0.456
3687	GLU467	CG	−25.224	−20.493	−0.071
3688	GLU467	CD	−25.479	−21.591	0.957
3689	GLU467	OE1	−26.685	−21.909	1.125
3690	GLU467	OE2	−24.462	−22.076	1.549
3691	PHE468	N	−20.625	−21.177	−1.354
3692	PHE468	CA	−19.297	−20.857	−1.887
3693	PHE468	C	−18.973	−19.378	−1.635
3694	PHE468	O	−18.508	−18.641	−2.502
3695	PHE468	CB	−19.081	−21.242	−3.361
3696	PHE468	CG	−19.031	−22.735	−3.584
3697	PHE468	CD1	−20.188	−23.444	−3.925
3698	PHE468	CD2	−17.824	−23.433	−3.437
3699	PHE468	CE1	−20.141	−24.826	−4.118
3700	PHE468	CE2	−17.777	−24.814	−3.637
3701	PHE468	CZ	−18.935	−25.51	−3.977
3702	ARG469	N	−19.176	−18.978	−0.326
3703	ARG469	CA	−18.955	−17.606	0.138
3704	ARG469	C	−18.233	−17.68	1.516
3705	ARG469	O	−18.179	−18.736	2.152
3706	ARG469	CB	−20.289	−16.841	0.146
3707	ARG469	CG	−20.949	−16.705	−1.238
3708	ARG469	CD	−22.418	−16.286	−1.154
3709	ARG469	NE	−22.576	−14.835	−1.038
3710	ARG469	CZ	−23.571	−14.191	−0.388
3711	ARG469	NH1	−23.577	−12.85	−0.416
3712	ARG469	NH2	−24.531	−14.828	0.309
3713	ASP470	N	−17.645	−16.493	1.966
3714	ASP470	CA	−16.41	−16.571	2.797
3715	ASP470	C	−16.38	−15.736	4.16
3716	ASP470	O	−17.401	−15.304	4.688
3717	ASP470	CB	−15.244	−16.262	1.861
3718	ASP470	CG	−14.765	−17.549	1.208
3719	ASP470	OD1	−13.791	−18.098	1.798
3720	ASP470	OD2	−15.341	−17.873	0.133
3721	LEU471	N	−15.118	−15.643	4.759
3722	LEU471	CA	−14.762	−15.476	6.201
3723	LEU471	C	−15.071	−14.186	7.029
3724	LEU471	O	−15.89	−13.351	6.66
3725	LEU471	CB	−15.013	−16.798	6.953
3726	LEU471	CG	−15.999	−16.857	8.125
3727	LEU471	CD1	−17.41	−16.426	7.758
3728	LEU471	CD2	−16.021	−18.296	8.651
3729	TRP472	N	−14.336	−14.066	8.234
3730	TRP472	CA	−14.807	−13.29	9.424
3731	TRP472	C	−13.809	−13.453	10.645
3732	TRP472	O	−13.539	−14.592	11.04
3733	TRP472	CB	−15.274	−11.864	9.136
3734	TRP472	CG	−16.065	−11.203	10.232
3735	TRP472	CD1	−15.809	−9.925	10.672
3736	TRP472	CD2	−17.245	−11.631	10.929
3737	TRP472	NE1	−16.69	−9.598	11.66
3738	TRP472	CE2	−17.645	−10.577	11.759
3739	TRP472	CE3	−18.065	−12.779	10.891
3740	TRP472	CZ2	−18.831	−10.599	12.5
3741	TRP472	CZ3	−19.274	−12.8	11.599
3742	TRP472	CH2	−19.649	−11.725	12.395
3743	LYS473	N	−13.317	−12.328	11.277
3744	LYS473	CA	−12.528	−12.244	12.559
3745	LYS473	C	−12.555	−10.73	12.919
3746	LYS473	O	−13.222	−9.962	12.208
3747	LYS473	CB	−11.089	−12.779	12.405
3748	LYS473	CG	−10.658	−13.618	13.62
3749	LYS473	CD	−9.176	−13.615	14.033
3750	LYS473	CE	−8.406	−12.325	13.805
3751	LYS473	NZ	−7.253	−12.238	14.705
3752	GLN474	N	−11.902	−10.125	13.948
3753	GLN474	CA	−11.485	−10.565	15.29
3754	GLN474	C	−12.758	−10.505	16.136
3755	GLN474	O	−13.294	−9.445	16.479
3756	GLN474	CB	−10.453	−9.581	15.879
3757	GLN474	CG	−10.067	−9.819	17.345
3758	GLN474	CD	−8.796	−10.613	17.546
3759	GLN474	OE1	−8.217	−11.236	16.662
3760	GLN474	NE2	−8.339	−10.619	18.834
3761	LEU475	N	−13.276	−11.753	16.425
3762	LEU475	CA	−14.466	−11.861	17.241
3763	LEU475	C	−14.037	−12.156	18.68
3764	LEU475	O	−13.301	−13.098	18.97
3765	LEU475	CB	−15.503	−12.844	16.703
3766	LEU475	CG	−15.106	−14.314	16.529
3767	LEU475	CD1	−16.382	−15.157	16.439
3768	LEU475	CD2	−14.241	−14.554	15.294
3769	LYS476	N	−14.52	−11.229	19.594
3770	LYS476	CA	−14.296	−11.408	21.023
3771	LYS476	C	−15.403	−12.373	21.456
3772	LYS476	O	−16.557	−11.986	21.643
3773	LYS476	CB	−14.448	−10.084	21.787
3774	LYS476	CG	−13.182	−9.226	21.871
3775	LYS476	CD	−13.144	−8.045	20.896
3776	LYS476	CE	−12.76	−8.445	19.483
3777	LYS476	NZ	−13.864	−8.18	18.544
3778	LEU477	N	−15.034	−13.706	21.475
3779	LEU477	CA	−15.975	−14.735	21.911
3780	LEU477	C	−16.033	−14.625	23.426
3781	LEU477	O	−15.074	−14.88	24.151
3782	LEU477	CB	−15.534	−16.129	21.469
3783	LEU477	CG	−16.481	−17.3	21.82
3784	LEU477	CD1	−16.3	−17.827	23.24
3785	LEU477	CD2	−17.957	−17.001	21.566
3786	SER478	N	−17.236	−14.142	23.884
3787	SER478	CA	−17.475	−13.916	25.289
3788	SER478	C	−17.836	−15.275	25.896
3789	SER478	O	−18.871	−15.882	25.619
3790	SER478	CB	−18.596	−12.9	25.497
3791	SER478	OG	−18.322	−12.077	26.646
3792	GLN479	N	−16.886	−15.779	26.773
3793	GLN479	CA	−17.03	−17.108	27.39
3794	GLN479	C	−18.077	−16.997	28.527
3795	GLN479	O	−17.791	−17.049	29.722
3796	GLN479	CB	−15.688	−17.598	27.959
3797	GLN479	CG	−14.69	−18.011	26.879
3798	GLN479	CD	−14.908	−19.453	26.483
3799	GLN479	OE1	−15.734	−19.8	25.647
3800	GLN479	NE2	−14.156	−20.36	27.173
3801	LYS480	N	−19.356	−16.773	28.067
3802	LYS480	CA	−20.517	−16.487	28.906
3803	LYS480	C	−21.77	−16.665	28.014
3804	LYS480	O	−21.677	−17.136	26.877
3805	LYS480	CB	−20.356	−15.172	29.687
3806	LYS480	CG	−19.899	−13.945	28.877
3807	LYS480	CD	−18.895	−13.069	29.654
3808	LYS480	CE	−17.427	−13.47	29.478
3809	LYS480	NZ	−16.768	−12.661	28.431
3810	VAL481	N	−22.997	−16.427	28.604
3811	VAL481	CA	−24.229	−17.002	28.043
3812	VAL481	C	−25.429	−16.015	28.089
3813	VAL481	O	−25.351	−14.874	28.538
3814	VAL481	CB	−24.567	−18.365	28.717
3815	VAL481	CG1	−23.517	−19.439	28.415
3816	VAL481	CG2	−24.767	−18.237	30.229
3817	PHE482	N	−26.597	−16.56	27.571
3818	PHE482	CA	−27.802	−15.775	27.256
3819	PHE482	C	−28.301	−14.989	28.477
3820	PHE482	O	−28.668	−15.532	29.517
3821	PHE482	CB	−28.909	−16.748	26.797
3822	PHE482	CG	−30.248	−16.149	26.442
3823	PHE482	CD1	−30.36	−15.064	25.568
3824	PHE482	CD2	−31.424	−16.738	26.933
3825	PHE482	CE1	−31.614	−14.568	25.206
3826	PHE482	CE2	−32.678	−16.249	26.563
3827	PHE482	CZ	−32.773	−15.165	25.696
3828	HIS483	N	−28.23	−13.613	28.311
3829	HIS483	CA	−28.735	−12.668	29.304
3830	HIS483	C	−27.97	−12.722	30.637
3831	HIS483	O	−28.45	−12.283	31.683
3832	HIS483	CB	−30.254	−12.74	29.528
3833	HIS483	CG	−31.073	−12.26	28.373
3834	HIS483	ND1	−32.394	−12.607	28.232
3835	HIS483	CD2	−30.826	−11.421	27.306
3836	HIS483	CE1	−32.878	−11.963	27.13
3837	HIS483	NE2	−31.948	−11.251	26.535
3838	LYS484	N	−26.657	−13.133	30.533
3839	LYS484	CA	−25.685	−12.862	31.58
3840	LYS484	C	−24.653	−11.904	30.972
3841	LYS484	O	−24.407	−11.865	29.767
3842	LYS484	CB	−25.05	−14.143	32.123
3843	LYS484	CG	−26.115	−15.069	32.724
3844	LYS484	CD	−25.513	−16.271	33.448
3845	LYS484	CE	−26.619	−17.214	33.907
3846	LYS484	NZ	−26.018	−18.377	34.601
3847	GLN485	N	−24.023	−11.091	31.9
3848	GLN485	CA	−23.392	−9.862	31.435
3849	GLN485	C	−22.075	−10.171	30.7
3850	GLN485	O	−21.35	−11.127	30.976
3851	GLN485	CB	−23.103	−8.888	32.593
3852	GLN485	CG	−24.353	−8.174	33.122
3853	GLN485	CD	−25.272	−9.063	33.935
3854	GLN485	OE1	−25.095	−10.268	34.093
3855	GLN485	NE2	−26.344	−8.412	34.475
3856	ASP486	N	−21.755	−9.213	29.744
3857	ASP486	CA	−20.474	−9.264	29.055
3858	ASP486	C	−19.431	−8.63	29.985
3859	ASP486	O	−19.696	−7.757	30.808
3860	ASP486	CB	−20.494	−8.536	27.718
3861	ASP486	CG	−20.864	−9.533	26.626
3862	ASP486	OD1	−21.919	−9.27	25.974
3863	ASP486	OD2	−20.035	−10.491	26.497
3864	ARG487	N	−18.167	−9.147	29.798
3865	ARG487	CA	−17.035	−8.814	30.649
3866	ARG487	C	−15.801	−9.489	30.029
3867	ARG487	O	−15.896	−10.495	29.316
3868	ARG487	CB	−17.223	−9.265	32.114
3869	ARG487	CG	−17.552	−10.755	32.235
3870	ARG487	CD	−17.844	−11.202	33.664
3871	ARG487	NE	−18.195	−12.636	33.704
3872	ARG487	CZ	−17.357	−13.643	33.349
3873	ARG487	NH1	−17.852	−14.883	33.153
3874	ARG487	NH2	−16.047	−13.438	33.161
3875	GLY488	N	−14.596	−8.926	30.413
3876	GLY488	CA	−13.358	−9.294	29.739
3877	GLY488	C	−12.713	−10.564	30.276
3878	GLY488	O	−11.803	−11.146	29.685
3879	SER489	N	−13.197	−10.993	31.488
3880	SER489	CA	−12.728	−12.203	32.159
3881	SER489	C	−13.311	−13.449	31.456
3882	SER489	O	−14.069	−14.252	32.002
3883	SER489	CB	−13.068	−12.139	33.651
3884	SER489	OG	−14.433	−11.726	33.865
3885	GLY490	N	−12.87	−13.583	30.151
3886	GLY490	CA	−13.229	−14.693	29.3
3887	GLY490	C	−13.465	−14.203	27.871
3888	GLY490	O	−14.604	−13.942	27.472
3889	TYR491	N	−12.286	−14.06	27.158
3890	TYR491	CA	−12.146	−13.768	25.722
3891	TYR491	C	−10.925	−14.598	25.239
3892	TYR491	O	−10.081	−15.004	26.04
3893	TYR491	CB	−11.825	−12.287	25.44
3894	TYR491	CG	−12.903	−11.243	25.626
3895	TYR491	CD1	−12.514	−9.911	25.86
3896	TYR491	CD2	−14.265	−11.515	25.474
3897	TYR491	CE1	−13.461	−8.888	25.963
3898	TYR491	CE2	−15.218	−10.499	25.599
3899	TYR491	CZ	−14.811	−9.194	25.84
3900	TYR491	OH	−15.774	−8.234	25.938
3901	LEU492	N	−10.827	−14.786	23.871
3902	LEU492	CA	−9.715	−15.511	23.231
3903	LEU492	C	−9.789	−15.324	21.697
3904	LEU492	O	−10.873	−15.114	21.145
3905	LEU492	CB	−9.668	−17.007	23.602
3906	LEU492	CG	−10.781	−17.923	23.041
3907	LEU492	CD1	−10.571	−19.357	23.543
3908	LEU492	CD2	−12.197	−17.488	23.413
3909	ASN493	N	−8.603	−15.444	20.997
3910	ASN493	CA	−8.429	−15.381	19.514
3911	ASN493	C	−6.895	−15.31	19.247
3912	ASN493	O	−6.15	−14.752	20.061
3913	ASN493	CB	−9.05	−14.117	18.881
3914	ASN493	CG	−10.391	−14.229	18.165
3915	ASN493	OD1	−10.682	−13.55	17.18
3916	ASN493	ND2	−11.348	−15.029	18.73
3917	TRP494	N	−6.359	−15.716	18.044
3918	TRP494	CA	−6.597	−17.008	17.376
3919	TRP494	C	−6.2	−16.972	15.869
3920	TRP494	O	−7.085	−17.043	15.008
3921	TRP494	CB	−5.864	−18.162	18.077
3922	TRP494	CG	−6.493	−18.602	19.362
3923	TRP494	CD1	−7.744	−19.168	19.512
3924	TRP494	CD2	−5.893	−18.577	20.663
3925	TRP494	NE1	−7.895	−19.543	20.819
3926	TRP494	CE2	−6.784	−19.19	21.546
3927	TRP494	CE3	−4.659	−18.117	21.169
3928	TRP494	CZ2	−6.494	−19.381	22.902
3929	TRP494	CZ3	−4.361	−18.287	22.526
3930	TRP494	CH2	−5.266	−18.913	23.378
3931	GLU495	N	−4.845	−16.903	15.565
3932	GLU495	CA	−4.323	−16.498	14.235
3933	GLU495	C	−4.049	−17.662	13.21
3934	GLU495	O	−3.999	−18.834	13.572
3935	GLU495	CB	−3.105	−15.573	14.392
3936	GLU495	CG	−3.338	−14.37	15.316
3937	GLU495	CD	−4.475	−13.47	14.863
3938	GLU495	OE1	−5.652	−13.786	15.24
3939	GLU495	OE2	−4.185	−12.479	14.142
3940	GLN496	N	−3.91	−17.286	11.867
3941	GLN496	CA	−4.128	−18.198	10.691
3942	GLN496	C	−5.056	−17.459	9.649
3943	GLN496	O	−4.914	−16.249	9.455
3944	GLN496	CB	−2.773	−18.562	10.06
3945	GLN496	CG	−2.739	−19.971	9.465
3946	GLN496	CD	−3.606	−20.099	8.236
3947	GLN496	OE1	−4.785	−20.438	8.302
3948	GLN496	NE2	−3.013	−19.72	7.067
3949	LEU497	N	−6.036	−18.212	9
3950	LEU497	CA	−7.082	−17.633	8.107
3951	LEU497	C	−8.492	−18.334	8.207
3952	LEU497	O	−8.592	−19.564	8.188
3953	LEU497	CB	−6.683	−17.756	6.624
3954	LEU497	CG	−5.572	−16.816	6.133
3955	LEU497	CD1	−5.241	−17.167	4.678
3956	LEU497	CD2	−5.983	−15.347	6.229
3957	HIS498	N	−9.613	−17.501	8.234
3958	HIS498	CA	−11.051	−17.916	8.02
3959	HIS498	C	−11.841	−18.503	9.278
3960	HIS498	O	−12.191	−19.685	9.35
3961	HIS498	CB	−11.123	−18.798	6.747
3962	HIS498	CG	−12.453	−19.296	6.297
3963	HIS498	ND1	−13.171	−20.239	6.994
3964	HIS498	CD2	−13.177	−19.116	5.137
3965	HIS498	CE1	−14.275	−20.556	6.265
3966	HIS498	NE2	−14.325	−19.868	5.15
3967	ALA499	N	−12.13	−17.598	10.312
3968	ALA499	CA	−12.588	−18.068	11.649
3969	ALA499	C	−14.09	−18.292	11.984
3970	ALA499	O	−15.033	−17.654	11.517
3971	ALA499	CB	−12.103	−17.133	12.772
3972	ALA500	N	−14.242	−19.215	13.024
3973	ALA500	CA	−15.505	−19.589	13.67
3974	ALA500	C	−15.146	−20.461	14.896
3975	ALA500	O	−14.092	−20.275	15.507
3976	ALA500	CB	−16.39	−20.34	12.698
3977	MET501	N	−16.081	−21.397	15.283
3978	MET501	CA	−15.902	−22.298	16.434
3979	MET501	C	−15.979	−21.489	17.736
3980	MET501	O	−15.297	−21.742	18.725
3981	MET501	CB	−14.634	−23.153	16.353
3982	MET501	CG	−14.778	−24.434	17.177
3983	MET501	SD	−13.151	−25.163	17.543
3984	MET501	CE	−12.736	−24.159	18.999
3985	ARG502	N	−16.942	−20.505	17.697
3986	ARG502	CA	−17.038	−19.447	18.694
3987	ARG502	C	−18.557	−19.172	18.805
3988	ARG502	O	−19.316	−20.076	19.158
3989	ARG502	CB	−16.146	−18.28	18.257
3990	ARG502	CG	−14.626	−18.523	18.409
3991	ARG502	CD	−13.871	−17.382	17.745
3992	ARG502	NE	−12.409	−17.446	17.791
3993	ARG502	CZ	−11.63	−18.186	16.968
3994	ARG502	NH1	−10.307	−17.933	16.92
3995	ARG502	NH2	−12.109	−19.143	16.165
3996	GLU503	N	−19.034	−17.964	18.301
3997	GLU503	CA	−20.488	−17.651	18.368
3998	GLU503	C	−21.257	−18.617	17.459
3999	GLU503	O	−22.454	−18.869	17.584
4000	GLU503	CB	−20.749	−16.219	17.876
4001	GLU503	CG	−22.228	−15.886	17.704
4002	GLU503	CD	−22.439	−14.387	17.63
4003	GLU503	OE1	−22.748	−13.938	16.486
4004	GLU503	OE2	−22.316	−13.748	18.729
4005	ALA504	N	−20.506	−19.008	16.372
4006	ALA504	CA	−20.935	−20.061	15.492
4007	ALA504	C	−19.729	−20.986	15.274
4008	ALA504	O	−18.558	−20.607	15.372
4009	ALA504	CB	−21.442	−19.471	14.191
4010	GLY505	N	−20.091	−22.262	14.917
4011	GLY505	CA	−19.139	−23.332	14.703
4012	GLY505	C	−19.814	−24.4	13.856
4013	GLY505	O	−20.725	−24.12	13.072
4014	ARG506	N	−19.264	−25.662	14.004
4015	ARG506	CA	−19.955	−26.86	13.504
4016	ARG506	C	−20.693	−27.446	14.714
4017	ARG506	O	−20.065	−27.881	15.68
4018	ARG506	CB	−18.946	−27.862	12.938
4019	ARG506	CG	−19.611	−29.153	12.447
4020	ARG506	CD	−18.652	−30.027	11.642
4021	ARG506	NE	−18.39	−29.446	10.322
4022	ARG506	CZ	−17.684	−30.031	9.334
4023	ARG506	NH1	−17.591	−29.385	8.157
4024	ARG506	NH2	−17.083	−31.226	9.486
4025	HIS507	N	−22.069	−27.325	14.683
4026	HIS507	CA	−22.898	−27.667	15.848
4027	HIS507	C	−24.408	−27.458	15.594
4028	HIS507	O	−24.865	−26.798	14.664
4029	HIS507	CB	−22.481	−26.938	17.157
4030	HIS507	CG	−22.311	−25.457	17.051
4031	HIS507	ND1	−21.963	−24.674	18.126
4032	HIS507	CD2	−22.406	−24.57	16.004
4033	HIS507	CE1	−21.806	−23.393	17.674
4034	HIS507	NE2	−22.047	−23.299	16.384
4035	ARG508	N	−25.233	−28.045	16.547
4036	ARG508	CA	−26.681	−27.836	16.521
4037	ARG508	C	−26.953	−26.425	17.074
4038	ARG508	O	−27.094	−26.198	18.273
4039	ARG508	CB	−27.41	−28.873	17.387
4040	ARG508	CG	−27.241	−30.306	16.881
4041	ARG508	CD	−28.092	−31.173	17.698
4042	ARG508	NE	−27.858	−32.671	17.307
4043	ARG508	CZ	−26.814	−33.431	17.719
4044	ARG508	NH1	−26.724	−34.713	17.302
4045	ARG508	NH2	−25.855	−32.95	18.538
4046	LYS509	N	−26.933	−25.445	16.096
4047	LYS509	CA	−26.969	−24.029	16.467
4048	LYS509	C	−28.345	−23.634	17.061
4049	LYS509	O	−29.398	−24.176	16.727
4050	LYS509	CB	−26.716	−23.15	15.232
4051	LYS509	CG	−25.253	−23.169	14.785
4052	LYS509	CD	−25.024	−22.297	13.551
4053	LYS509	CE	−23.562	−21.957	13.328
4054	LYS509	NZ	−22.88	−22.921	12.47
4055	SER510	N	−28.295	−22.573	17.952
4056	SER510	CA	−29.498	−22.004	18.563
4057	SER510	C	−30.006	−20.796	17.746
4058	SER510	O	−29.321	−20.221	16.898
4059	SER510	CB	−29.268	−21.629	20.032
4060	SER510	OG	−28.392	−20.517	20.163
4061	TRP511	N	−31.309	−20.407	18.039
4062	TRP511	CA	−31.959	−19.289	17.34
4063	TRP511	C	−32.482	−18.271	18.358
4064	TRP511	O	−32.101	−17.099	18.366
4065	TRP511	CB	−33.115	−19.757	16.431
4066	TRP511	CG	−32.941	−19.317	15.011
4067	TRP511	CD1	−32.722	−20.154	13.938
4068	TRP511	CD2	−32.94	−17.976	14.5
4069	TRP511	NE1	−32.51	−19.383	12.827
4070	TRP511	CE2	−32.641	−18.051	13.138
4071	TRP511	CE3	−33.131	−16.699	15.07
4072	TRP511	CZ2	−32.5	−16.919	12.326
4073	TRP511	CZ3	−33.004	−15.557	14.271
4074	TRP511	CH2	−32.69	−15.668	12.919
4075	SER512	N	−33.435	−18.746	19.244
4076	SER512	CA	−34.294	−17.813	19.992
4077	SER512	C	−33.484	−16.939	20.958
4078	SER512	O	−33.897	−15.859	21.378
4079	SER512	CB	−35.369	−18.565	20.78
4080	SER512	OG	−34.785	−19.668	21.473
4081	CYS513	N	−32.281	−17.495	21.329
4082	CYS513	CA	−31.384	−16.86	22.278
4083	CYS513	C	−30.615	−15.692	21.628
4084	CYS513	O	−29.947	−14.898	22.298
4085	CYS513	CB	−30.354	−17.852	22.818
4086	CYS513	SG	−31.115	−19.286	23.643
4087	GLY514	N	−30.674	−15.588	20.256
4088	GLY514	CA	−29.821	−14.695	19.484
4089	GLY514	C	−30.244	−13.23	19.509
4090	GLY514	O	−30.297	−12.547	18.49
4091	HIS515	N	−30.4	−12.719	20.783
4092	HIS515	CA	−31.066	−11.438	21.038
4093	HIS515	C	−30.372	−10.761	22.233
4094	HIS515	O	−30.942	−9.978	22.991
4095	HIS515	CB	−32.567	−11.643	21.32
4096	HIS515	CG	−33.314	−12.194	20.15
4097	HIS515	ND1	−33.83	−13.466	20.107
4098	HIS515	CD2	−33.647	−11.648	18.927
4099	HIS515	CE1	−34.429	−13.627	18.887
4100	HIS515	NE2	−34.34	−12.543	18.154
4101	THR516	N	−29.009	−10.996	22.305
4102	THR516	CA	−28.221	−10.644	23.488
4103	THR516	C	−26.824	−10.167	23.058
4104	THR516	O	−26.179	−10.748	22.183
4105	THR516	CB	−28.127	−11.847	24.46
4106	THR516	OG1	−27.657	−11.48	25.761
4107	THR516	CG2	−27.272	−12.999	23.942
4108	ARG517	N	−26.341	−9.092	23.773
4109	ARG517	CA	−24.985	−8.515	23.652
4110	ARG517	C	−24.85	−7.641	24.913
4111	ARG517	O	−25.456	−6.566	24.999
4112	ARG517	CB	−24.819	−7.564	22.45
4113	ARG517	CG	−24.775	−8.14	21.035
4114	ARG517	CD	−23.657	−9.149	20.771
4115	ARG517	NE	−24.174	−10.51	20.9
4116	ARG517	CZ	−23.72	−11.583	20.231
4117	ARG517	NH1	−24.407	−12.739	20.281
4118	ARG517	NH2	−22.586	−11.579	19.529
4119	ALA518	N	−24.215	−8.205	25.999
4120	ALA518	CA	−24.204	−7.553	27.314
4121	ALA518	C	−25.608	−7.517	27.958
4122	ALA518	O	−25.869	−8.139	28.986
4123	ALA518	CB	−23.545	−6.175	27.309
4124	GLY519	N	−26.512	−6.701	27.309
4125	GLY519	CA	−27.93	−6.718	27.587
4126	GLY519	C	−28.688	−7.409	26.449
4127	GLY519	O	−28.14	−8.03	25.539
4128	CYS520	N	−30.062	−7.253	26.557
4129	CYS520	CA	−30.985	−7.773	25.539
4130	CYS520	C	−31.014	−6.765	24.375
4131	CYS520	O	−30.996	−5.539	24.549
4132	CYS520	CB	−32.391	−7.922	26.13
4133	CYS520	SG	−33.538	−8.817	25.043
4134	THR521	N	−31.107	−7.334	23.121
4135	THR521	CA	−31.076	−6.541	21.899
4136	THR521	C	−31.737	−7.262	20.707
4137	THR521	O	−32.218	−8.387	20.775
4138	THR521	CB	−29.655	−6.04	21.56
4139	THR521	OG1	−29.725	−5.151	20.433
4140	THR521	CG2	−28.67	−7.165	21.268
4141	LEU522	N	−31.81	−6.461	19.576
4142	LEU522	CA	−32.287	−6.938	18.274
4143	LEU522	C	−33.808	−7.211	18.26
4144	LEU522	O	−34.349	−7.886	17.388
4145	LEU522	CB	−31.478	−8.115	17.702
4146	LEU522	CG	−30	−7.79	17.401
4147	LEU522	CD1	−29.235	−9.081	17.093
4148	LEU522	CD2	−29.848	−6.812	16.235
4149	ILE523	N	−34.516	−6.48	19.199
4150	ILE523	CA	−35.973	−6.535	19.323
4151	ILE523	C	−36.545	−5.152	19.708
4152	ILE523	O	−37.73	−4.987	20
4153	ILE523	CB	−36.474	−7.617	20.317
4154	ILE523	CG1	−35.954	−7.381	21.752
4155	ILE523	CG2	−36.139	−9.03	19.829
4156	ILE523	CD1	−36.643	−8.264	22.785
4157	ARG524	N	−35.627	−4.116	19.641
4158	ARG524	CA	−36.041	−2.731	19.862
4159	ARG524	C	−36.617	−2.156	18.555
4160	ARG524	O	−36.56	−2.757	17.481
4161	ARG524	CB	−34.864	−1.887	20.374
4162	ARG524	CG	−34.488	−2.249	21.814
4163	ARG524	CD	−33.444	−1.292	22.384
4164	ARG524	NE	−33.203	−1.547	23.815
4165	ARG524	CZ	−32.502	−2.592	24.316
4166	ARG524	NH1	−32.437	−2.773	25.651
4167	ARG524	NH2	−31.859	−3.466	23.526
4168	GLN525	N	−37.257	−0.95	18.72
4169	GLN525	CA	−37.785	−0.124	17.626
4170	GLN525	CB	−39.314	−0.234	17.512
4171	GLN525	CG	−39.806	−1.609	17.056
4172	GLN525	CD	−39.88	−2.607	18.192
4173	GLN525	OE1	−40.549	−2.433	19.205
4174	GLN525	NE2	−39.152	−3.75	17.988
4175	GLN525	C	−37.468	1.347	17.904
4176	GLN525	O	−36.858	1.67	18.923
4177	GLN525	OXT	−37.884	2.231	16.996

Claims

1-23. (canceled)

24. An isolated polypeptide comprising a polypeptide sequence selected from the group consisting of:

(a) an isolated polypeptide comprising amino acids 1 to 697 of SEQ ID NO:56;

(b) an isolated polypeptide comprising amino acids 2 to 697 of SEQ ID NO:56;

(c) an isolated polypeptide comprising a polypeptide sequence encoded by nucleotides 9 to 2099 of SEQ ID NO:55; and

(d) an isolated polypeptide comprising a polypeptide sequence encoded by nucleotides 12 to 2099 of SEQ ID NO:55.

25. The isolated polypeptide of claim 24, wherein said polypeptide is (a).

26. The isolated polypeptide of claim 24, wherein said polypeptide is (b).

27. The isolated polypeptide of claim 24, wherein said polypeptide is (c).

28. The isolated polypeptide of claim 24, wherein said polypeptide is (d).

29. An isolated polypeptide produced by a method comprising:

(a) culturing an isolated recombinant host cell comprising a vector that comprises the coding region encoding the polypeptide of claim 24 under conditions such that the polypeptide of claim 24 is expressed; and

(b) recovering said polypeptide.

30. The isolated polypeptide of claim 24 further comprising a heterologous polypeptide sequence.

31. The isolated polypeptide of claim 28 wherein said heterologous polypeptide is the Fc domain of immunoglobulin.

32. An isolated polypeptide comprising a polypeptide sequence selected from the group consisting of:

(a) an isolated polypeptide comprising amino acids 1 to 694 of SEQ ID NO:54;

(b) an isolated polypeptide comprising amino acids 2 to 694 of SEQ ID NO:54;

(c) an isolated polypeptide comprising a polypeptide sequence encoded by nucleotides 9 to 2090 of SEQ ID NO:53; and

(d) an isolated polypeptide comprising a polypeptide sequence encoded by nucleotides 12 to 2090 of SEQ ID NO:53.

33. The isolated polypeptide of claim 32, wherein said polypeptide is (a).

34. The isolated polypeptide of claim 32, wherein said polypeptide is (b).

35. The isolated polypeptide of claim 32, wherein said polypeptide is (c).

36. The isolated polypeptide of claim 32,.wherein said polypeptide is (d).

37. An isolated polypeptide produced by a method comprising:

(a) culturing an isolated recombinant host cell comprising a vector that comprises the coding region encoding the polypeptide of claim 32 under conditions such that the polypeptide of claim 32 is expressed; and

(b) recovering said polypeptide..

38. The isolated polypeptide of claim 32 further comprising a heterologous polypeptide sequence.

39. The isolated polypeptide of claim 38 wherein said heterologous polypeptide is the Fc domain of immunoglobulin.

40. An isolated polypeptide comprising the polypeptide encoded by the CAN-12v1 or the CAN-12v2 cDNA clone contained in ATCC Deposit No: PTA-3434.

41. An isolated polypeptide comprising amino acids from position 2 through position 587 of SEQ ID NO:56, wherein said polypeptide has calcium-dependent cysteine protease activity.

42. An isolated nucleic acid comprising a polynucleotide encoding a polypeptide comprising the sequence of amino acids from position 90 through position 697 of SEQ ID NO:56, wherein said polypeptide has calcium-dependent cysteine protease activity.

43. An isolated polypeptide comprising amino acids from position 2 through position 584 of SEQ ID NO:54, wherein said polypeptide has calcium-dependent cysteine protease activity.

44. An isolated nucleic acid comprising a polynucleotide encoding a polypeptide comprising the sequence of amino acids from position 90 through position 694 of SEQ ID NO:54, wherein said polypeptide has calcium-dependent cysteine protease activity.

45. An isolated polypeptide having as least 95.0% identity to amino acids 2 to 697 of SEQ ID NO:56, wherein percent identity is calculated using a CLUSTALW global sequence alignment using default parameters, wherein said polypeptide has calcium-dependent cysteine protease activity.

46. An isolated polypeptide having as least 95.0% identity to amino acids 2 to 694 of SEQ ID NO:54, wherein percent identity is calculated using a CLUSTALW global sequence alignment using default parameters, wherein said polypeptide has calcium-dependent cysteine protease activity.