WO2002016600A2 - Nouvelles proteines et acides nucleiques les codant - Google Patents

Nouvelles proteines et acides nucleiques les codant Download PDF

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WO2002016600A2
WO2002016600A2 PCT/US2001/026518 US0126518W WO0216600A2 WO 2002016600 A2 WO2002016600 A2 WO 2002016600A2 US 0126518 W US0126518 W US 0126518W WO 0216600 A2 WO0216600 A2 WO 0216600A2
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amino acid
polypeptide
nucleic acid
ofthe
seq
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PCT/US2001/026518
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English (en)
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WO2002016600A3 (fr
Inventor
Valerie Gerlach
John R. Macdougall
Glennda Smithson
David J. Stone
Karen Ellerman
Kimberly A. Spytek
Bryan D. Zerhusen
Luca Rastelli
Corine A. M. Vernet
Meera Patturajan
Velizar T. Tchernev
Muralidhara Padigaru
Raymond J. Taupier, Jr.
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Curagen Corporation
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Priority to CA002420538A priority Critical patent/CA2420538A1/fr
Priority to JP2002522273A priority patent/JP2004516820A/ja
Priority to EP01964417A priority patent/EP1332212A2/fr
Priority to AU2001285271A priority patent/AU2001285271A1/en
Publication of WO2002016600A2 publication Critical patent/WO2002016600A2/fr
Publication of WO2002016600A3 publication Critical patent/WO2002016600A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the invention generally relates to nucleic acids and polypeptides encoded thereby. 5
  • the invention generally relates to nucleic acids and polypeptides encoded therefrom. More specifically, the invention relates to nucleic acids encodmg cytoplasmic, nuclear, membrane bound, and secreted polypeptides, as well as vectors, host cells, antibodies, and 10 recombinant methods for producing these nucleic acids and polypeptides.
  • the invention is based in part upon the discovery of nucleic acid sequences encoding novel polypeptides.
  • novel nucleic acids and polypeptides are referred to herein as NOVX, i 5 or NOV1 , NOV2, NOV3 , NOV4, NOV5, NOV6, NOV7, and NOV8 nucleic acids and? polypeptides.
  • NOVX nucleic acid or polypeptide sequences.
  • the invention provides an isolated NOVX nucleic acid molecule
  • NOVX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a NOVX nucleic acid sequence.
  • .5 includes an isolated nucleic acid that encodes a NOVX polypeptide, or a fragment, homolog, analog or derivative thereof.
  • the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37.
  • the nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid
  • oligonucleotide e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of aNOVX nucleic acid (e.g., SEQ JD NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36) or a complement of said oligonucleotide.
  • substantially purified NOVX polypeptides SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36.
  • the NOVX polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human NOVX polypeptide.
  • the invention also features antibodies that immunoselectively bind to NOVX polypeptides, or fragments, homologs, analogs or derivatives thereof.
  • the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically- acceptable carrier.
  • the therapeutic can be, e.g., a NOVX nucleic acid, a NOVX polypeptide, or an antibody specific for a NOVX polypeptide.
  • the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.
  • the invention includes a method of producing a polypeptide by culturing a cell that includes a NOVX nucleic acid, under conditions allowing for expression ofthe NOVX polypeptide encoded by the DNA. If desired, the NOVX polypeptide can then be recovered.
  • the invention includes a method of detecting the presence of a NOVX polypeptide in a sample.
  • a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound.
  • the complex is detected, if present, thereby identifying the NOVX polypeptide within the sample.
  • the invention also includes methods to identify specific cell or tissue types based on their expression of a NOVX.
  • Also included in the invention is a method of detecting the presence of a NOVX nucleic acid molecule in a sample by contacting the sample with a NOVX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a NOVX nucleic acid molecule in the sample.
  • the invention provides a method for modulating the activity of a NOVX polypeptide by contacting a cell sample that includes the NOVX polypeptide with a compound that binds to the NOVX polypeptide in an amount sufficient to modulate the activity of said polypeptide.
  • the compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
  • a small molecule such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
  • a therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g., Inflamation, Autoimmune disorders, scleroderma, transplantation, autoimmune disease, allergies, immunodeficiencies, systemic lupus erythematosus, lymphedema, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, graft versus host disease (GVHD), endometriosis, cystic fibrosis, pathological disorders involving spleen, thymus, lung, and peritoneal macrophages, Aging, Alzheimer's disease, stroke, Cancer, abnormal angiogenesis, like cancer and more specifically aggressive, metastatic cancer, more specifically tumor ofthe lung, kidney, brain, liver and colon, mastocytomas, prostate cancer, modulation of apoptosis, Oncogenesis such as lung, liver and ovarian cancers; cell growth and possibly differentiation, non-Hodgkin lympho
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like.
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding NOVX may be useful in gene therapy, and NOVX may be useful when administered to a subject in need thereof.
  • the compositions ofthe present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like.
  • the invention further includes a method for screening for a modulator of disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like.
  • the method includes contacting a test compound with a NOVX polypeptide and determining if the test compound binds to said NOVX polypeptide. Binding ofthe test compound to the NOVX polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.
  • Also within the scope ofthe invention is a method for screening for a modulator of activity, or of latency or predisposition to an disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes.
  • the test animal expresses a recombinant polypeptide encoded by a NOVX nucleic acid. Expression or activity of NOVX polypeptide is then measured in the test animal, as is expression or activity ofthe protein in a control animal which recombinantly- expresses NOVX polypeptide and is not at increased risk for the disorder or syndrome.
  • the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a NOVX polypeptide, a NOVX nucleic acid, or both, in a subject (e.g., a human subject). The method includes measuring the amount ofthe NOVX polypeptide in a test sample from the subject and comparing the amount ofthe polypeptide in the test sample to the amount ofthe NOVX polypeptide present in a control sample.
  • an alteration in the level ofthe NOVX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject.
  • the predisposition includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like.
  • the expression levels ofthe new polypeptides ofthe invention can be used in a method to screen for various cancers as well as to determine the stage of cancers.
  • the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subj ect a NOVX polypeptide, a NOVX nucleic acid, or a NOVX-specific antibody to a subject (e.g., a human subject), in an amount sufficient to alleviate or prevent the pathological condition.
  • the disorder includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like.
  • the invention can be used in a method to identity the cellular receptors and downstream effectors ofthe invention by any one of a number of techniques commonly employed in the art.
  • the present invention provides novel nucleotides and polypeptides encoded thereby. Included in the invention are the novel nucleic acid sequences and their encoded polypeptides. The sequences are collectively referred to herein as “NOVX nucleic acids” or “NOVX polynucleotides” and the corresponding encoded polypeptides are referred to as “NOVX polypeptides” or “NOVX proteins.” Unless indicated otherwise, “NOVX” is meant to refer to any ofthe novel sequences disclosed herein. Table A provides a summary ofthe NOVX nucleic acids and their encoded polypeptides.
  • NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts.
  • the various NOVX nucleic acids and polypeptides according to the invention are useful as novel members ofthe protein families according to the presence of domains and sequence relatedness to previously described proteins. Additionally, NOVX nucleic acids and polypeptides can also be used to identify proteins that are members ofthe family to which the NOVX polypeptides belong. NO VI is homologous to a thioredoxin -like family of proteins.
  • NO VI nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; Inflamation, Autoimmune disorders, Aging and Cancer, and/or other pathologies/disorders.
  • NOV2a and 2b are homologous to the Retinoic acid responsive/STRA-6-like family of proteins.
  • NOV2 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, scleroderma, transplantation, autoimmune disease, allergies, immunodeficiencies, renal artery stenosis, intersti
  • NOV3a and 3b are homologous to a family of LRR/GPCR-like proteins.
  • the NOV3 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; abnormal angiogenesis, like cancer and more specifically aggressive, metastatic cancer, more specifically rumor ofthe lung, kidney, brain, liver and colon and/or other pathologies/disorders.
  • NOV4 is homologous to the Trypsin inhibitor-like family of proteins.
  • NOV4 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; chronic lung diseases, periodontitis, pancreatitis and/or other pathologies/disorders.
  • NOV5a, b, c, d, e, f, g, h and i are homologous to the apolipoprotein e-like family of proteins.
  • NOV5 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; atherosclerosis, dysbetalipoproteinemia, hyperlipoproteinemia type ILT, xanthomatosis, coronary and/or peripheral vascular disease, hypothyroidism, systemic lupus erythematosus, diabetic acidosis, hypercholesterolemia, planar and tendon xanthomas, dysbetalipoproteinemia, hypercholesterolemia, accelerated vascular disease, Alzheimer disease, familial amyloidotic polyneuropathy and/or other pathologies/disorders.
  • NOV6 is homologous to the mastocytoma protease precursor -like family of proteins.
  • NOV6 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; allergies, asthma and cystic fibrosis, cancers such as mastocytomas, vascular diseases and/or other pathologies/disorders.
  • NOV7 is homologous to members ofthe thymosin beta-4-like family of proteins.
  • the NOV7 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; prostate cancer, immunological and autoimmune disorders (ie hyperthyroidism), angiogenesis and wound healing, modulation of apoptosis, neurodegenerative and neuropsychiatric disorders, age-related disorders, pathological disorders involving spleen, thymus, lung, and peritoneal macrophages and Cancer and/or other pathologies/disorders.
  • NOV8 is homologous to the Protein-tyrosine-phosphatase -like family of proteins.
  • NOV8 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; hematopoiesic disorders; leukemogenesis; polycystic kidney disease; Oncogenesis such as lung, liver and ovarian cancers; apoptosis; type 2 diabetes and obesity; cell growth and possibly differentiation; non-Hodgkin lymphomas; musculoskeletal disorders; and CNS development disorders and/or other pathologies/disorders.
  • the NOVX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVX activity or function.
  • the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., neurogenesis, cell differentiation, cell proliferation, hematopoiesis, wound healing and angiogenesis.
  • NOVX nucleic acids and polypeptides according to the invention are disclosed herein.
  • a disclosed NO VI nucleic acid of 620 nucleotides (also referred to as AC013554_dal) encoding a novel thioredoxin-like protein is shown in Table 1 A.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 282-284 and ending with a TAA codon at nucleotides 618-620.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 1 A. The start and stop codons are in bold letters.
  • Table 1A NOV1 nucleotide sequence (SEQ ID NO:l).
  • the NO VI nucleic acid sequence, ocated on the p31 region of chromsome 9 has 232 of 331 bases (70%) identical to a thioredoxin mRNA from Ovis aries (GENBANK-ID: OATHIORD
  • Public nucleotide databases include all GenBank databases and the GeneSeq patent database.
  • the "E-value” or “Expect” value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched.
  • the probability that the subject (“Sbjct”) retrieved from the NOV1 BLAST analysis, e.g., thioredoxin mRNA from Ovis aries, matched the Query NOV1 sequence purely by chance is 608e- .
  • the Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences. Essentially, the E value describes the random background noise that exists for matches between sequences.
  • the Expect value is used as a convenient way to create a significance threshold for reporting results.
  • the default value used for blasting is typically set to 0.0001.
  • the Expect value is also used instead ofthe P value (probability) to report the significance of matches.
  • an E value of one assigned to a hit can be interpreted as meaning that in a database ofthe current size one might expect to see one match with a similar score simply by chance.
  • An E value of zero means that one would not expect to see any matches with a similar score simply by chance. See, e.g., http://www.ncbi.nlm.nih.gov/Education BLASTinfo/. Occasionally, a string of X's or N's will result from a BLAST search.
  • the disclosed NOVl polypeptide (SEQ ID NO:2) encoded by SEQ ID NO:l has 112 amino acid residues and is presented in Table IB using the one-letter amino acid code.
  • Signal P, Psort and/or Hydropathy results predict that NOVl has a signal peptide and is likely to be localized in the cytoplasm with a certainty of 0. 6500.
  • NOVl may also be localized to the mitochondrial matrix space with acertainty of 0.1000, or the lysosome (lumen) with a certainty of 0.1000.
  • the NOVl peptide is not likely to have a signal peptide.
  • Table IB Encoded NOVl protein sequence (SEQ ID NO:2).
  • the disclosed NOVl polypeptide has homology to the amino acid sequences shown in the BLASTP data listed in Table lC.
  • the "strong” group of conserved amino acid residues may be any one ofthe following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
  • Table IE lists the domain description from DOMAIN analysis results against NOVl. This indicates that the NOVl sequence has properties similar to those of other proteins known to contain this domain.
  • NOVl also has high homology to members ofthe patp database shown in Table IF.
  • Thioredoxin is an oxidoreductase enzyme of 12,000 daltons, containing a dithiol- disulfide active site. It is ubiquitous and found in many organisms from plants and bacteria to mammals. Multiple in vitro substrates for thioredoxin have been identified, including ribonuclease, choriogonadotropins, coagulation factors, glucocorticoid receptor, and insulin. Reduction of insulin is classically used as an activity test. Wollman et al. (1988) identified a full-length cDNA clone coding for human thioredoxin. The open reading frame coded for a protein of 104 amino acids (excluding the initial methionine).
  • This protein possessed the highly conserved enzymatic active site common to plant and bacterial thioredoxins: trp-cys- gly-pro-cys (amino acids 30-34). Tonissen and Wells (1991) determined that the TRX gene extends over 13 kb and consists of 5 exons encoding a 12-kD protein. Southern analysis demonstrated the presence of several TXN genes in the human genome. At least one of these is inactive, i.e., apseudogene. Kaghad et al. (1994) also cloned the TXN gene and demonstrated that the 5 exons are separated by 4 introns. They determined the +1 transcription start point by primer extension. The +1 site is located 22 bp downstream from a TATAA box and defines a 5-prime untranslated region of 74 bp. Southern hybridization of genomic DNAs from several donors detected only 1 active TXN gene.
  • Proteins internalized into the endocytic pathway are usually degraded. Efficient proteolysis requires denaturation, induced by acidic conditions within lysosomes, and reduction of inter- and intrachain disulfide bonds. Cytosolic reduction is mediated enzymatically by thioredoxin (TXN; 187700). (Arunachalam et al.; Nat. Acad. Sci. 97: 745- 750, 2000) described a lysosomal thiol reductase, which they called GILT, that was optimally active at low pH and capable of catalyzing disulfide bond reduction both in vivo and in vitro.
  • TXN thioredoxin
  • the GILT gene encodes a predicted 261-amino acid protein composed of a 37- amino acid signal peptide and a 224-amino acid proform.
  • the enzyme was expressed constitutively in antigen-presenting cells and was induced by gamma-interferon (147570) in other cell types, suggesting a potentially important role in antigenprocessing.
  • T-cell thioredoxin reductase is a selenoenzyme that contains selenium in a conserved C-terminal region provides another example ofthe role of selenium in the major antioxidant enzyme system (i.e., thioredoxin-thioredoxin reductase), in addition to the well- known glutathione peroxidase enzyme system.
  • thioredoxin-thioredoxin reductase the major antioxidant enzyme system
  • glutathione peroxidase enzyme system i.e., glutathione peroxidase enzyme system.
  • the disclosed NOVl nucleic acid ofthe invention encoding a thioreductase-like protein includes the nucleic acid whose sequence is provided in Table 1 A, or a fragment thereof.
  • the invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 1 A while still encoding a protein that maintains its thioreductase-like activities and physiological functions, or a fragment of such a nucleic acid.
  • the invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any ofthe nucleic acids just described.
  • the invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications.
  • modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 30 % percent ofthe bases may be so changed.
  • the disclosed NOVl protein ofthe invention includes the thioreductase-like protein whose sequence is provided in Table IB.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue.shown in Table 2 while still encoding a protein that maintains its thioreductase-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 38 % percent ofthe residues may be so changed.
  • the invention further encompasses antibodies and antibody fragments, such as F a b or (F a b)2, that bind immunospecifically to any ofthe proteins ofthe invention.
  • This thioreductase-like protein may function as a member of a "Thioredoxin family”. Therefore, the NOVl nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below.
  • the potential therapeutic applications for this invention include, but are not limited to: protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vitro of all tissues and cell types composing (but not limited to) those defined here.
  • NOVl nucleic acids and proteins ofthe invention are useful in potential therapeutic applications implicated in cancer including but not limited to Inflamation,
  • a cDNA encoding the thioreductase- like protein (NOVl) may be useful in gene therapy, and the thioreductase-like protein (NOVl) may be useful when administered to a subject in need thereof.
  • the compositions ofthe present invention will have efficacy for treatment of patients suffering from Inflamation, Autoimmune disorders, Aging and Cancer.
  • the NOVl nucleic acid encoding thioreductase-like protein, and the thioreductase-like protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.
  • NOVl nucleic acids and polypeptides are further useful in the generation of antibodies that bind immuno-specifically to the novel NOVl substances for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOVl protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOVl epitope is from about amino acids 1 to 10. In another embodiment, a NOVl epitope is from about amino acids 20 to 35.
  • NOVl epitopes are from about amino acids 45 to 50, from about amino acids 55 to 60, from about amino acid 65 to 75, and from about amino acids 90 to 115.
  • NOV2 NOV2 includes two novel retinoic acid/SRA6-like proteins disclosed below. The disclosed proteins have been named NOV2a and NOV2b. NOV2a
  • NOV2a nucleic acid of 2632 nucleotides also referred to as 3277789.0.83_dal
  • Table 2A An open reading frame was identified beginning with an ATG initiation codon at nucleotides 31-33 and ending with a TGA codon at nucleotides 2011-2013.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 2A, and the start and stop codons are in bold letters.
  • Table 2A NOV2a nucleotide sequence (SEQ ID NO:3).
  • the NOV2a nucleic acid was identified by TblastN using CuraGen Corporation's sequence file for Retinoic acid responsive/STRA-6 or homolog as run against the Genomic Daily Files made available by GenBank or from files downloaded from the individual sequencing centers.
  • the nucleic acid sequence was predicted from the genomic file Genbank by homology to a known Retinoic acid responsive/STRA-6 or homolog. Exons were predicted by homology and the intron/exon boundaries were determined using standard genetic rules. Exons were further selected and refined by means of similarity determination using multiple BLAST (for example, tBlastN, BlastX, and BlastN) searches, and, in some instances, GeneScan and Grail. Expressed sequences from both public and proprietary databases were also added when available to further define and complete the gene sequence. The DNA sequence was then manually corrected for apparent inconsistencies thereby obtaining the sequences encoding the full-length protein.
  • the disclosed NO V2a nucleic acid sequence, localized to chromsome 15, has 1511 of 2207 bases (68%) identical to a retinoic acid-responsive protein (Stra6) mRNA from Mus musculus (GENBANK-ID: AF062476
  • acc: AF062476 (GENBANK-ID: M77668)(E 1.2e " 189 ).
  • a NOV2a polypeptide (SEQ ID NO:4) encoded by SEQ ID NO:3 has 660 amino acid residues and is presented using the one-letter code in Table 2B.
  • Signal P, Psort and/or Hydropathy results predict that NOV2A contain a signal peptide and is likely to be localized in the plasmam membrane with a certainty of 0.6000.
  • NOV2a may also be localized to the Golgi body with acertainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000.
  • the most likely cleavage site for a NOV2A peptide is between amino acids 8 and 9, at: AGN-QT.
  • Table 2B Encoded NOV2a protein sequence (SEQ ID NO:4).
  • NOV2a also has high homology to members ofthe patp database shown in Table 2C.
  • a disclosedNOV2b nucleic acid of2663 nucleotides (also referred to as CG52276-07) encoding a novel STRA6-like protein is shown in Table 2D.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 1-3 and ending with a TGA codon at nucleotides 2068-2070.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 2D, and the start and stop codons are in bold letters.
  • a disclosed NOV2b polypeptide (SEQ ID NO:6) encoded by SEQ ID NO:5 has 689 amino acid residues and is presented using the one-letter code in Table 2E.
  • Signal P, Psort and/or Hydropathy results predict that NOV2b has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • NOV2b could be localized at the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or a microbody (peroxisome) with a certainty of 0.3000.
  • the most likely cleavage site for a NOV2b peptide is between amino acids 8 and 9, at AGN-QT.
  • NOV2b maps to chromosome 15. This assignment was made using mapping information associated with genomic clones, public genes and ESTs sharing sequence identity with the disclosed sequence and CuraGen Corporation's Electronic Northern bioinformatic tool.
  • NOV2b is expressed in at least the following tissues: Brain, Cervix, Heart, Kidney, Lymph node, Lymphoid tissue, Ovary, Pituitary Gland, Placenta, Retina, Temporal Lobe, Thyroid, Uterus. Expression information was derived from the tissue sources ofthe sequences that were included in the derivation ofthe sequence of NOV2b. NOV2a also homology to the amino acid sequences shown in the BLASTP data listed in Table 2F.
  • Carcinogenesis involves inactivation or subversion ofthe normal controls of proliferation, differentiation, and apoptosis (Hurst RE, et.al.; Adv Exp Med Biol
  • hRAR alpha nuclear receptor occupies a central position with respect to induction of gene transcription in that when bound to appropriate retinoid ligands, its homodimers and heterodimers with hRXR alpha regulate the transcription of a number of retinoid-responsive genes. These include genes in other signaling pathways, so that the whole forms a complex network.
  • a set of cultured bladder-derived cells representing different stages of bladder rumorigenesis formed a model system. It consisted of 2 immortalized bladder cell lines (HUC-BC and HUC-PC), one squamous cell carcinoma cell line (SCaBER), one papilloma line (RT4), and 4 transitional cell carcinomas (TCC-Sup, 5637, T24, J82) of varying stages and grades. This set of cells were used to model the range of behaviors of bladder cancers.
  • aTRA all-trans-retinoic acid
  • CRABP I was not expressed by J82, T24, 5637 and RT4, but was expressed at low levels that did not change in SCaBER and at moderate levels that decreased, increased, or decreased sharply in HUC-BC, TCC-Sup and HUC-PC, respectively.
  • telomeres The phenotypes for inhibition of proliferation showed no obvious relationship to the expression of any single gene, but cell lines that were inhibited by aTRA (HUC-BC and TCC-Sup) were not sensitive to 4-HPR, and vice versa.
  • One line (RT4) was insensitive to either retinoid.
  • Transfection showed very little retinoid- stimulated transfection ofthe CAT reporter gene with RT4 or HUC-PC.
  • About 2-fold enhancement transactivation was observed with SCaBER, HUC-BC, J82 and T24 cells and 3- 8 fold with 5637, TCC-Sup cells.
  • HUC-BC a G to T point mutation was found at position 606 ofthe hRAR alpha gene. This mutation would substitute tyrosine for asparagine in a highly conserved domain.
  • Relative RT-PCR analysis of a panel of retinoid-responsive and inducible genes demonstrated changes in expression levels of all the genes in response to retinoic acid treatment together with numerous aberrations dysregulations. They concluded that retinoid signaling may be a target for inactivation during tumorigenesis by uncoupling gene expression, proliferation and differentiation. Therefore retinoids are more likely to be effective for chemoprevention than for treatment of bladder carcinomas.
  • the proliferative effects of retinoids were examined in the MC-26 and LoVo colon adenocarcinoma cell lines (Stewart LV, et.al.; Exp Cell Res 1997 Jun 15;233(2):321-9).
  • the proliferation ofthe LoVo cell line was not altered in the presence ofthe retinoids all trans- retinoic acid (atRA) and 9-cis-retinoic acid (9-cis-RA). Both retinoids, however, stimulated the growth, as measured by cell proliferation, of MC-26 cells. atRA and 9-cis-RA were equipotent in increasing MC-26 cell proliferation, suggesting that the growth stimulation is mediated by one or more retinoic acid receptors (RARs). To determine the RAR which might be responsible for this growth stimulatory effect, the authors characterized the RAR subtypes which were present in both cell lines. mRNA for the RAR alpha, RAR beta, and RAR gamma were detected in the MC-26 cell.
  • the RAR alpha does not mediate the growth stimulatory effects of retinoids, for a selective RAR alpha antagonist was unable to prevent the retinoid-induced increase in MC-26 cell growth.
  • RAR alpha, RAR beta, and RAR gamma mRNA are also expressed in the LoVo cell line; the lack of growth- stimulation by retinoids in LoVo cells, therefore, does not seem to be due to the absence of RARs.
  • the results obtained in these experiments demonstrate that the growth response elicited by retinoids can vary between colon cancer cells and that the differences in response may not be solely determined by the RAR subtypes which are expressed in a colon cancer cell line.
  • Retinoic acids well characterized regulators of proliferation and differentiation, partly re-differentiate follicular thyroid carcinoma cell lines (FTC-133, FTC-238, andHTC- TSHr) as well as SV40-transfected immortalized thyroid cell lines (ori3 and 7751) (Schmutzler C, et.al.;Exp Clin Endocrinol Diabetes 1996;104 Suppl 4:16-9). This is indicated by the stimulation of type 15'-deiodinase and other differentiation markers.
  • thyroid carcinoma cell lines express RA receptor rnRNAs and functional ligand- and DNA-binding receptor proteins able to mediate RA-dependent signal transduction. Together, these properties make these thyroid-derived cell lines useful in vitro models for studying the effects of an RA re-differentiation therapy of thyroid cancer.
  • RA chemotherapeutic agent retinoic acid
  • SCC head and neck squamous cell carcinoma
  • RAR retinoic acid receptors
  • RXR retinoid X receptors
  • CRABP cellular retinoic acid binding proteins I and II
  • clones expressing the antisense CRABP construct were less sensitive to RA mediated inhibition of proliferation. These clones were also less invasive in an in vitro invasion assay, likely due to downregulation of matrix metalloproteinase activity.
  • CRABP II affects the transcription of RA responsive genes which regulate proliferation and invasion of head and neck SCCs.
  • trans-RA trans-retinoic acid
  • RARs RA receptors
  • RXRs retinoid X receptors
  • Retinoic acid plays important roles in development, growth and differentiation by regulating the expression of target genes.
  • a new retinoic acid-inducible gene, Stra6 has been identified in PI 9 embryonal carcinoma cells using a subtractive hybridization cDNA cloning technique by Bouillet and coworkers (Mech Dev 1997;63:173-86).
  • Stra6 codes for a very hydrophobic membrane protein of a new type, which does not display similarities with previously characterized integral membrane proteins. Stra6, which exhibits a specific pattern of expression during development and in the adult, is strongly expressed at the level of blood- organ barriers.
  • Stra6 has a spermatogenic cycle-dependent expression which is lost in testes of RAR alpha null mutants where Stra6 is expressed in all tubules. This finding suggests that the Stra6 protein may be a component of an as yet unidentified transport machinery.
  • VHL Von Hippel-Lindau
  • Alzheimer's disease stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, scleroderma, transplantation, autoimmune disease, allergies, immunodeficiencies, renal artery stenosis, interstitial nephritis, glomeruloneph
  • VHL Von Hippel-Lindau
  • a cDNA encoding the Retinoic acid responsive/STRA-6-like protein may be useful in gene therapy, and the Retinoic acid responsive/STRA-6-like protein (NOV2) may be useful when administered to a subject in need thereof.
  • the compositions ofthe present invention will have efficacy for treatment of patients suffering from Inflamation, Autoimmune disorders, Aging or Cancer.
  • the NOV2A nucleic acid encoding Retinoic acid responsive/STRA-6-like protein, and the Retinoic acid responsive/STRA-6-like protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
  • NOV2 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOV2 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOV2a epitope is from about amino acids 20 to 30.
  • a NOV2a epitope is from about amino acids 40 to 60.
  • NOV2a epitopes are from about amino acids 75 to 80, from about 100 to 110, from about amino acids 180 to 190, from about amino acids 220 to 260, from about amino acids 310 to 330, from about amino acids 350 to 370, from about amino acids 380 to 400, from about amino acids 470 to 510, from about amino acids 550 to 570, and from about amino acids 590 to 650.
  • novel proteins can be used in assay systems for functional analysis of various human disorders, which are useful in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • a contemplated NOV2b epitope is from about amino acids 5 to 50. In another embodiment, a NOV2b epitope is from about amino acids 80 to 90. In additional embodiments, NOV2b epitopes are from about amino acids 140 to 160, from about 190 to 210, from about amino acids 230 to 290, from about amino acids 360 to 365, from about amino acids 390 to 440, from about amino acids 550 to 590, and from about amino acids 610 to 670. These novel proteins can be used in assay systems for functional analysis of various human disorders, which are useful in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • NOV3 NOV3 includes two novel LRR/GPCR-like proteins disclosed below. The disclosed proteins have been named NOV3a and NOV3b. NOV3a
  • a disclosed NOV3a nucleic acid of 7892 nucleotides (also referred to as 128900861ext) encoding a novel LRR/GPCR -like protein is shown in Table 3A.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 1-3 and ending with a TAA codon at nucleotides 5728-5730.
  • a putative untranslated region downstream from the termination codon is underlined in Table 3A, and the start and stop codons are in bold letters.
  • a NOV3a polypeptide (SEQ ID NO:8) encoded by SEQ ID NO:7 is 1542 amino acid residues and is presented using the one-letter code in Table 3C.
  • Signal P, Psort and/or Hydropathy results predict that NOV3a contains a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.6400.
  • NOV3a may also be localized to the Golgi body with acertainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3700, or the endoplasmic reticulum (lumen) with a certainty of 0.1000.
  • the most likely cleavage site for a NOV3a peptide is between amino acids 26 and 27, at: ARG-AP.
  • Table 3C Encoded NOV3a protein sequence (SEQ ID NO:8).
  • VFDELPAL WDLGTEFLTCDCHLR LLP AQNRSLQLSEHTLCAYPSALHAHALGSLQEAQLCCEGALEL HTHHLIPSLRQWFQGDRLPFQCSASYLGNDTRIR YHNRAPVEGDEQAGILLAESLIHDCTFITSELTLS HIGVWASGE ECTVSMAQGNASKKVEIWLETSASYCPAERVANNRGDFRWPRTLAGITAYQSCLQYPFTS
  • VPLGGGAPGTRASRRCDRAGR EPGDYSHCLYTNDITRVLYTFVLMPINASNALTLAHQLRVYTAEAASFS DMMD WAQMIQKFLGYvDQIKELVEVMVDMPSNLMLVDEHLLWLAQREDKACSRIVGAIERIGGAALSP HAQHISVNARNVALEAYLIKPHSYVGLTCTAFQRREGGVPGTRPGSPGQNPPPEPEPPADQQLRFRCTTGR PNVSLSSFHIKNSVALASIQLPPSLFSSLPAALAPPVPPDCTLQLLVFRNGRLFHSHSNTSRPGAAGPGKR
  • ptnr SPTREMBL-ACC : 094898 KIAA0806 PROTEIN - Homo sapiens . . . 240 1.4e-15 2 ptnr : SPTREMBL-ACC : P70193 MEMBRANE GLYCOPROTEIN - Mus muse . . . 234 1.4e-15 3 ptnr : SPTREMBL-ACC :088278 MEGF2 - Rattus norvegicus (Rat) , . . . 190 1.6e-15 6 ptnr : TREMBLNE -ACC :AAF61929 PROTOCADHERIN FLAMINGO 1 - Ho . . . 214 8 .2e-15 4
  • NOV3a also has high homology to members ofthe patp database shown in Table 3E.
  • NOV3a is expressed in at least the following tissues: Blood, Brain, Breast, Colon, Ear, Germ Cell, Heart, Kidney, Lung, Muscle, Pancreas, Placenta, Prostate, Skin, Stomach, Testis, Uterus, Whole embryo, colon, lung, ovary, uterus Eye, Prostate, Tonsil, placenta, and thymus..
  • sequence is predicted to be expressed in the following tissues based on the expression pattern of its homolog, SeqCalling assembly 128900861: fetal heart, fetal lung fetal retina fetal_liver_spleen, total fetal tissue and multiple tumor from colon, lung, geerm cells, kidney prostate.
  • NOV3b The cDNA coding for the a domain of NOV3a from residue 444 to 937 was targeted for "in-frame" cloning by PCR.
  • the PCR template is based on human cDNA(s).
  • oligonucleotide primers were used to clone the target cDNA sequence:
  • the forward primer includes an in-frame BamHI restriction site and the reverse primer contains an in-frame Xhol restriction site.
  • the pool is composed of 5 micrograms of each ofthe following human tissue cDNAs: adrenal gland, whole brain, amygdala, cerebellum, thalamus, bone marrow, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, liver, lymphoma, Burkitt's Raji cell line, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small Intestine, spleen, stomach, thyroid, trachea, uterus.
  • human tissue cDNAs adrenal gland, whole brain, amygdala, cerebellum, thalamus, bone marrow, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, liver, lymphoma, Burkitt's Raji cell line, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small Intestine
  • the second PCR was performed using the above primers and 0.5ng-l .0 ng of one of the following human tissue cDNAs: skeleton muscle, testis, mammary gland, adrenal gland, ovary, colon, normal cerebellum, normal adipose, normal skin, bone marrow, brain amygdala, brain hippocampus, brain substantia nigra, brain thalamus, thyroid, fetal lung, fetal liver, fetal brain, kidney, heart, spleen, uterus, pituitary gland, lymph node, salivary gland, small intestine, prostate, placenta, spinal cord, peripheral blood, trachea, stomach, pancreas, hypothalamus.
  • the reaction mixtures contained 2 microliters of each ofthe primers (original concentration: 5 pmol/ul), 1 microliter of lOmM dNTP (Clontech Laboratories, Palo Alto CA) and 1 microliter of 50xAdvantage-HF 2 polymerase (Clontech Laboratories) in 50 microliter- reaction volume.
  • PCR condition 1 a) 96°C 3 minutes b) 96°C 30 seconds denaturation c) 60°C 30 seconds, primer annealing d) 72°C 6 minutes extension Repeat steps b-d 15 times e) 96°C 15 seconds denaturation f) 60°C 30 seconds, primer annealing g) 72°C 6 minutes extension Repeat steps e-g 29 times e) 72°C 10 minutes final extension
  • PCR condition 2 a) 96°C 3 minutes b) 96°C 15 seconds denaturation c) 76°C 30 seconds, primer annealing, reducing the temperature by 1 °C per cycle d) 72°C 4 minutes extension Repeat steps b-d 34 times e) 72°C 10 minutes final extension
  • SF2 GTGCTGATGCCCATCAATGCCT (SEQ ID NO:94 SF3: CGCATCGTGGGTGCCATAG (SEQ ID NO:95)
  • SF4 ACCGGGAGGCCCAATGTTTCTCT (SEQ ID NO:96)
  • SR2 ACGAAGGTGTACAGCACCCTG (SEQ ID NO:99)
  • SR3 GGCTGCAGGCCTTGTCCTC (SEQ ID NO:100)
  • the insert assembly NOV3b was found to encode an open reading frame between residues 444 and 937 ofthe target sequence NOV3a.
  • the cloned insert differs from the original sequence at 8 nucleotide positions and 4 amino acid positions. It also differs by a 55 amino acid deletion between amino acid residues 491 and 546 ofthe original sequence.
  • the alignment with NOV3a is displayed in a ClustalW below. Note that differing amino acids have a white or grey background, and deleted/inserted amino acids can be detected by a dashed line in the sequence that does not code at that position.
  • a disclosed NOV3b nucleic acid of 1329 nucleotides (also referred to as 188822778) encoding a novel LRR/GPCR -like protein is shown in Table 3F.
  • An open reading frame was identified beginning with an GGA initiation codon at nucleotides 1-3 and ending with a GAG codon at nucleotides 1327-1329.
  • a putative untranslated region upstream from the initiation codon is underlined in Table 3F, and the start and stop codons are in bold letters.
  • This reading frame may be a portion of a larger reading frame, since it does not posess traditional start and stop codons.
  • the coding region of this nucleic acid may extend in both the 5' and 3' directions, and the sequence of its expressed protein may extend in the N- or C terminal directions.
  • a NOV3b polypeptide (SEQ ID NO:l 1 encoded by SEQ ID NO:9 is 443 amino acid residues and is presented using the one-letter code in Table 3H.
  • NOV3 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 31.
  • Novel NOV3 (SEQ ID NO: 8) 2) Novel NOV4 (SEQ ID N ⁇ :ll)
  • NOV3a APRL RLHSKCTLSMAGNQKNQGGSSSQEAASGLMPSRRLQRGEVEGLCTCLCTDLRNNI 300 NOV3b 1 Q9P1Z7 I Q9BYB8
  • NOV3a ISTVQPGAFLGLGELKRLDLSNNRIGCLTSETFQGLPRLLRLNISGNIFSSLQPGVFDEL 360 NOV3b X Q9P1Z7 Q9BYB8 I
  • NOV3a PALKVVDLGTEFLTCDCHLR LLP AQNRSLQLSEHTLCAYPSALHAHALGSLQEAQLCC 20 NOV3b 1 Q9P1Z7 Q9BYB8 MARPVRGGLGAPRRS- 15
  • NOV3 AV ⁇ QRWLPRaVCSCl ⁇ GVAAS LGLF(3FTHHCARRRDVRfi ⁇ RgCCPPA PAAPHAPPRA 1302 NOV3b :' 443 Q9P1Z7 AvSQRWLPR ⁇ CSCJ4gGVAAS ⁇ LGLF * FTHHCARRRDVRA[pRlCCPPA ⁇ PAAPHAPPRA 820 Q9BYB8 Vs lVLTSL3wVCl(rgQTR-0 ⁇ EEYs2 ⁇ NTDETvVPPDVp
  • Tables 3K through 3Q list the domain description from DOMAIN analysis results against NOV3. This indicates that the NOV3a sequence has properties similar to those of other proteins known to contain these domains.
  • ID NO: 75 51 residues, 96.1% aligned
  • CD-Length 51 residues, only 68.6% aligned
  • CD-Length 70 residues, only 67.1% aligned
  • CD-Length 49 residues, 93.9% aligned
  • CD-Length 49 residues, 93.9% aligned
  • tumor endothelial markers were expressed in a wide range of tumor types, as well as in normal vessels associated with wound healing and corpus luteum formation. These studies demonstrate that tumor and normal endothelium are distinct at the molecular level, a finding that may have significant implications for the development of antiangiogenic therapies.
  • Small leucine-rich proteoglycans belong to an expanding gene class whose distinctive feature is a structural motif, called the trypsin inhibitor, found in an increasing number of intracellular and extracellular proteins with diverse biological attributes (Iozzo RV Crit Rev Biochem Mol Biol 1997;32(2): 141-74).
  • Three-dimensional modeling of their prototype protein core proposes a flexible, arch-shaped binding surface suitable for strong and distinctive interactions with ligand proteins. Changes in the properties of individual proteoglycans derive from amino acid substitutions in the less conserved surface residues, changes in the number and length ofthe trypsin inhibitors, and/or variation in glycosylation.
  • proteoglycans are tissue organizers, orienting and ordering collagen fibrils during ontogeny and in pathological processes such as wound healing, tissue repair, and tumor stroma formation. These properties are rooted in their bifunctional character: the protein moiety binding collagen fibrils at strategic loci, the microscopic gaps between staggered fibrils, and the highly charged glycosaminoglycans extending out to regulate interfibrillar distances and thereby establishing the exact topology of fibrillar collagens in tissues. These proteoglycans also interact with soluble growth factors, modulate their functional activity, and bind to cell surface receptors.
  • Trypsin inhibitors are short sequence motifs present in a number of proteins with diverse functions and cellular locations (Kobe B et al., Trends Biochem Sci 1994 Oct;19(10):415-21). All proteins containing these repeats are thought to be involved in protein-protein interactions.
  • the crystal structure of ribonuclease inhibitor protein has revealed that trypsin inhibitors correspond to beta-alpha structural units. These units are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protein acquires an unusual, nonglobular shape.
  • Latrophilin is a brain-specific Ca2+-independent receptor of alpha-latrotoxin, a potent presynaptic neurotoxin. Matsushita H. et al. report the finding of two novel latrophilin homologues (Matsushita H. et al., FEBS Lett 1999 Jan 29;443(3):348-52). All three latrophilins are unusual G protein-coupled receptors. They exhibit strong similarities within their lectin, olfactomedin and transmembrane domains but possess variable C-termini.
  • Latrophilins have up to seven sites of alternative splicing; some splice variants contain an altered third cytoplasmic loop or a truncated cytoplasmic tail. Only latrophilin-1 binds alpha- latrotoxin; it is abundant in brain and is present in endocrine cells. Latrophilin-3 is also brain- specific, whereas latrophilin-2 is ubiquitous. Together, latrophilins form a novel family of heterogeneous G protein-coupled receptors with distinct tissue distribution and functions.
  • the protein similarity information, expression pattern, and map location for the LRR/GPCR-like protein and nucleic acid (NOV3) disclosed herein suggest that this LRR/GPCR-like protein may have important structural and/or physiological functions characteristic ofthe transient receptor potential-related protein family. Therefore, the NOV3 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo.
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from disorder linked to abnormal angiogenesis, like cancer and more specifically aggressive, metastatic cancer, more specifically tumor ofthe lung, kidney, brain, liver and colon.
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from disorder linked to abnormal angiogenesis, like cancer and more specifically aggressive, metastatic cancer, more specifically tumor ofthe lung, kidney, brain, liver and colon.
  • These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods, specifically for the treatment of tumors, more specifically tumor ofthe lung, kidney, brain, liver and colon.
  • NOV3 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOV3 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • the disclosed NOV3 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOV3 epitope is from about amino acids 1 to 300. In another embodiment, a NOV3 epitope is from about amino acids 420 to 450.
  • NOV3 epitopes are from about amino acids 650 to 800, from about 700 to 800, from about amino acids 820 to 900, from about amino acids 1050 to 1150, from about amino acids 1200 to 1250, and from about amino acids 1300 to 1550.
  • NOV3 proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • NOV4 A disclosed NOV4 nucleic acid of 1123 nucleotides also referred to as
  • the disclosed NOV4 nucleic acid sequence has 1063 of its 1123 bases (95%) identical to an alpha- 1 -micro globulin mRNA from Homo sapiens (GENBANK-ID: HSHCR
  • acc:X04225) (E 3.3e -227 ).
  • a NOV4 polypeptide (SEQ ID NO: 13) encoded by SEQ ID NO: 12 is 372 amino acid residues and is presented using the one-letter amino acid code in Table 4B. Signal P, Psort and/or Hydropathy results predict that NOV4 has a signal peptide and is likely to be localized extracellularly with a certainty of 0.8200. In other embodiments, NOV4 may also be localized to the lysosome (lumen) with acertainty of 0.1900, the microbody (peroxisome) with a certainty of 0.1456, or the endoplasmic reticulum (membrane) with a certainty of 0.1000. The most likely cleavage site for a NOV4 peptide is between amino acids 19 and 20, at: VSA-GP.
  • the disclosed NOV4 amino acid sequence has
  • the disclosed NOV4 protein maps to chromosome 16 and is expressed in at least the following tissues: HEPG2 untreated (a hepatocellular carcinoma cell line); Fetal Liver; Pituitary Gland; pool of: Hypothalamus, Lymph Node, Fetal Liver, fetal_lung.
  • NOV4 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 4C.
  • AMBP_HUMAN h ambp protein precursor [contains: alpha-1-microglobulin (protein he) (complex-forming glycoprotein heterogeneous in charge) ; inter-alpha- trypsin inhibitor light chai (SEQ ID NO: 49)
  • AMBP_PIG sus scrofa (pig), ambp protein precursor [contains: alpha-1- microglobulin; inter-alpha- trypsin inhibitor light chain (iti-lc) (bikunin) (hi-30) (ei-14)] (fragment) (SEQ ID N ⁇ :50)
  • AMBP_MESAU mesocricetus auratus golden hamster
  • ambp protein precursor [contains: alpha-1-microglobulin; inter-alpha- trypsin inhibitor light chain (iti- lc) (bikunin) (hi-30)] (SEQ ID NO: 51)
  • AMBP_RAT rattus norvegicus rat
  • ambp protein precursor [contains: alpha-1- microglobulin; inter-alpha- trypsin inhibitor light chain (iti-lc) (bikunin) (hi- 30)]. 7/1999and (Pasted_No.1.18 :22-343) (SEQ ID N0:52)
  • AMBP_BOVIN b ambp protein precursor [contains: alpha-1-microglobulin; inter- alpha- trypsin inhibitor light chain (iti-lc) (bikunin) (hi-30) (bi-14) (cumulus extracellular matrix s (SEQ ID NO: 53)
  • Tables 4E-I list the domain description from DOMAIN analysis results against NOV4. This indicates that the NOV4 sequence has properties similar to those of other proteins known to contain this domain.
  • Table 4F Domain Analysis of NOV4 gnl I Smart
  • One member of the family is encoded by an alternatively-spliced form of Alzheimer's amyloid beta- protein (SEQ ID NO: 81)
  • NOV4 also has high homology to members ofthe patp database shown in Table 4J.
  • NOV4 is homologous in various ways to the following molecules whose functions are described below.
  • An imbalance between increased neutrophil elastase and a decreased antiprotease shield has been suggested as a factor contributing to the development of chronic lung disease (CLD).
  • CLD chronic lung disease
  • Stiskal JA et al. hypothesized that administration of alpha 1-proteinase inhibitor (A1PI), also known as alphal-antitrypsin, to premature neonates would prevent CLD.
  • A1PI alpha 1-proteinase inhibitor
  • alpha 1-PI alpha 1-proteinase inhibitor
  • the serum protein, alpha 1-proteinase inhibitor (alpha 1-PI) defends the host against serine proteinases, e.g. PMN elastase.
  • this protein in GCF was quantified from a standard curve constructed from dot-blot analysis and characterized by Western blot (Lee HM et al., J Periodontal Res 1997 Jan;32(l Pt 1):9-19).
  • GCF was collected on filter paper strips from healthy (H), gingivitis (G) and adult periodontitis (AP) patients, then extracted with Tris/NaCl/CaC12 buffer, pH 7.6. alpha 1-PI concentration increased with G and was highest in AP subjects.
  • alpha 1-PI degradation fragments were seen in 17% and 71% of GCF samples, respectively.
  • Both collagenase and alpha 1-PI-degrading activities in GCF increased with severity of inflammation (GCF flow).
  • the alpha 1-PI degrading (or serpinolytic) activity was characterized as a matrix metalloproteinase, probably collagenase, based on its in vitro response to a panel of different proteinase inhibitors including doxycycline.
  • protease- antiprotease balance was studied by measuring immunoreactive anionic trypsin (irAT), cationic trypsin (irCT), complexes between cationic trypsin and alpha 1 -protease inhibitor (irCT-alpha 1 PI), leukocyte elastase and neutrophil proteinase 4 (NP4), as well as the endogenous protease inhibitors, pancreatic secretory trypsin inhibitor (PSTI), alpha 2- macro globulin (alpha 2M), alpha 1 -protease inhibitor (alpha 1 PI), antichymotrypsin (ACHY), and secretory leukocyte protease inhibitor (SLPI).
  • irAT immunoreactive anionic trypsin
  • irCT cationic trypsin
  • irCT-alpha 1 PI complexes between cationic trypsin and alpha 1 -protease inhibitor
  • NP4 neutrophil proteinase 4
  • Intraperitoneal levels were studied before and after the lavage procedure, and plasma levels were followed for 21 d.
  • the control group had lower plasma levels of SLPI and analysis of peritoneal fluid showed the reduction of irCT- alpha 1 PI to be more pronounced in the aprotinin group. None ofthe other variables measured differed significantly between the two groups. All patients had very high levels of leukocyte elastase and NP4 both in peritoneal exudate and in plasma. Peritoneal levels of PSTI were higher after the lavage procedure in contrast to the other measured variables that all showed lower peritoneal levels after the lavage.
  • the protein similarity information, expression pattern, and map location for the trypsin inhibitor-like protein and nucleic acid (NOV4) disclosed herein suggest that this NOV4 protein may have important structural and/or physiological functions characteristic ofthe trypsin inhibitor family. Therefore, the NOV4 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo.
  • the NOV4 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies.
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from chronic lung diseases, periodontitis, pancreatitis.
  • the NOV4 nucleic acids, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.
  • NOV4 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOV4 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOV4 epitope is from about amino acids 20 to 50.
  • a NOV4 epitope is from about amino acids 75 to 170.
  • NOV4 epitopes are from about amino acids 180 to 270, and from about amino acids 320 to 370.
  • NOV5 includes three novel apolipoprotein e-like proteins disclosed below.
  • the disclosed proteins have been named NOV5a, NOV5b, NOV5c, NOV5d, NOV5e, NOV5f, NOV5g, NOV5h, andNOV5i.
  • NOV5a A disclosed NOV5a nucleic acid of 2249 nucleotides (also referred to as sggc_draft_dj784al6_20000723_dal) encoding a novel apolipoprotein e-like protein is shown in Table 5A.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 30-32 and ending with a TGA codon at nucleotides 2217-2219.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 5A, and the start and stop codons are in bold letters.
  • the NOV5a sequence was identified by subjecting Ace. No. RP4-784A16 to an exon linking process.
  • PCR primers were designed to Ace. No. RP4-784A16 by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case ofthe reverse primer, until the stop codon was reached.
  • suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species.
  • the resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5a sequence.
  • NOV5a nucleic acid was identified on chromosome 1 and has 2099 of 2118 bases (99%) identical to a 2549 bp apolipoprotein E receptor 2 mRNA from Homo sapiens (GENBANK-ID: HSZ75190
  • acc:Z75190 (E 0.0)
  • a disclosed NOV5a polypeptide (SEQ ID NO: 15) encoded by SEQ ID NO: 14 is 729 amino acid residues and is presented using the one-letter code in Table 5B.
  • Signal P, Psort and/or Hydropathy results predict that NOV5a has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6000.
  • NOV5a may also be localized to the Golgi body with acertainty of 0.4000, the endoplasmic reticulum
  • the most likely cleavage site for a NOV5a peptide is between amino acids 59 and 60, at: AAA-AA.
  • NOV5b nucleic acid of 2348 nucleotides also referred to as sggc_draft_dj784al6_20000723_da2 encoding a novel apolipoprotein e-like protein is shown in Table 5C.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 30-32 and ending with a TGA codon at nucleotides 2316-2318.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 5C, and the start and stop codons are in bold letters.
  • Table 5C NOV5b Nucleotide Sequence (SEQ ID NO:16)
  • the NOV5b sequence was identified through an exon linking process.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library contaimng a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5b sequence.
  • NOV5b nucleic acid was identified on chromosome 1 and has 1151 of 1206 bases (95%) identical to a a 2549 bp apolipoprotein E receptor 2 mRNA from Homo sapiens (GENBANK-ID: HSZ75190
  • acc:Z75190 (E 0.0)
  • a disclosed NOV5b polypeptide (SEQ ID NO: 17) encoded by SEQ ID NO: 16 is 762 amino acid residues and is presented using the one-letter code in Table 5D.
  • Signal P, Psort and/or Hydropathy results predict that NOV5b has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6000.
  • NOV5b may also be localized to the Golgi body with acertainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000.
  • the most likely cleavage site for a NOV5b peptide is between amino acids 59 and 60, at: AAA-AA.
  • NOV5c nucleic acid of 2537 nucleotides also referred to as sggc_draft_dj784al6_20000723_da3 encoding a novel apolipoprotein e-like protein is shown in Table 5E.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 30-32 and ending with a TGA codon at nucleotides 2505-2507.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 5E, and the start and stop codons are in bold letters.
  • the NOV5c sequence was identified through an exon linking process.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5c sequence.
  • NOV5c nucleic acid was identified on chromosome 1 and has 1626 of 1922 bases (84%) identical to a 4468 bp apolipoprotein E receptor 2 mRNA from Homo sapiens (GENBANK-ID:D50678
  • acc:D50678 ) (E ie '127 )
  • a disclosed NOV5c polypeptide (SEQ ID NO: 19) encoded by SEQ ID NO: 18 is 825 amino acid residues and is presented using the one-letter code in Table 5F.
  • Signal P, Psort and/or Hydropathy results predict that NOV5c has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6000.
  • NOV5c may also be localized to the Golgi body with acertainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000.
  • the most likely cleavage site for a NOV5c peptide is between amino acids 44 and 45, at: AAA-DP.
  • a disclosed NOV5d nucleic acid was isolated by subjecting NOV5c to ⁇ & exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case ofthe reverse primer, until the stop codon was reached.
  • Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) ofthe DNA or protein sequence ofthe target sequence, or by translated homology ofthe predicted exons to closely related human sequences sequences from other species.
  • primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone manow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • the NOV5d sequence was identified through an exon linking process.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5d sequence.
  • the NOV5d nucleic acid was identified to the p34 region of human chromosome 1 and has 2508 of 2995 bases (83%) identical to a gb:GENBANK-ID:D50678
  • acc:D50678.1 mRNA from Homo sapiens (Human mRNA for apolipoprotein E receptor 2, complete eds) (E 0.0)
  • a disclosed NOV5d polypeptide (SEQ ID NO:21) encoded by SEQ ID NO:20 is 1012 amino acid residues and is presented using the one-letter code in Table 5H. Signal P, Psort and/or Hydropathy results predict that NOV5d has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6760.
  • NOV5d may also be localized to the endoplasmic reticulum (membrane) with acertainty of 0.1000, the endoplasmic reticulum (lumen) with a certainty of 0.1000, or the exfracellularly with a certainty of 0.1000.
  • the most likely cleavage site for a NOV5d peptide is between amino acids 32 and 33, at: AAA-AA.
  • the disclosed NOV5d amino acid sequence has 742 of 931 amino acid residues (79%) identical to, and 777 of 931 amino acid residues (83%) similar to, the 963 amino acid residue ptnr:SPTREMBL-ACC:Q14114 protein from Homo sapiens (Human) (APOLIPOPROTEIN E
  • NOV5d is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • This information was derived by determining the tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
  • sequence is predicted to be expressed in the following tissues because ofthe expression pattern of (GENBANK-ID: gb:GENBANK-ID:D50678
  • a disclosed NOV5e nucleic acid was isolated by subjecting NOV5c to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case ofthe reverse primer, until the stop codon was reached.
  • Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) ofthe DNA or protein sequence ofthe target sequence, or by translated homology ofthe predicted exons to closely related human sequences sequences from other species.
  • primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • the NOV5e sequence was identified through an exon linking process.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5e sequence.
  • the NOV5e nucleic acid was identified to the p34 region of human chromosome 1 and has 1359 of 1359 bases (100%) identical to a gb:GENBANK-ID:HSZ75190
  • acc:Z75190.1 mRNA from Homo sapiens (H.sapiens mRNA for apolipoprotein E receptor 2) (E 0.0)
  • a disclosed NOV5e polypeptide (SEQ ID NO:23) encoded by SEQ ID NO:22 is 695 amino acid residues and is presented using the one-letter code in Table 5 J. Signal P, Psort and/or Hydropathy results predict that NOV5e has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6760. In other embodiments, NOV5e may also be localized to the endoplasmic reticulum (membrane) with acertainty of 0.1000, the endoplasmic reticulum (lumen) with a certainty of 0.1000, or the extracellularly with a certainty of 0.1000. The most likely cleavage site for a NOV5e peptide is between amino acids 32 and 33, at: AAA-AA.
  • NOV5e is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, and/or RACE sources.
  • sequence is predicted to be expressed in the following tissues because ofthe expression pattern of (GENBANK-ID: gb:GENBANK-JD:HSZ75190
  • this protein contains the following protein domains (as defined by Interpro) at the indicated positions: low density lipoprotein receptor domains at amino acid positions 45 to 83, 84 to 165, 168 to 206; low density receptor repeat at amino acid ⁇ ositions333 to 378, 380 to 416, 418 to 460, 462 to 546; EGF-like domains at amino acid positions 86 tol22, 170 to 204, 211 to 245, 251 to 285, 556 to 600, 739 to 783; and trypsin inhibitor like cysteine rich domain at amino acid positions 195 to 251.
  • sequence ofthe invention has properties similar to those of other proteins known to contain this/these domain(s) and similar to the properties of these domains.
  • a disclosed NOV5f nucleic acid was isolated by subjecting NOV5c to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case ofthe reverse primer, until the stop codon was reached.
  • Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) ofthe DNA or protein sequence ofthe target sequence, or by translated homology ofthe predicted exons to closely related human sequences sequences from other species.
  • primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was. encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5f sequence.
  • the NOV5f nucleic acid was identified to the p34 region of human chromosome 1 and has 2047 of 2341 bases (87%) identical to a gb:GENBANK-JX>:D50678
  • acc:D50678.1 mRNA from Homo sapiens (Human mRNA for apolipoprotein E receptor 2, complete eds) (E 0.0)
  • a disclosed NOV5f polypeptide (SEQ ID NO:25) encoded by SEQ ID NO:24 is 804 amino acid residues and is presented using the one-letter code in Table 5L. Signal P, Psort and/or Hydropathy results predict that NOV5f has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6000.
  • NOV5f may also be localized to the Golgi body with acertainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000.
  • the most likely cleavage site for a NOV5f peptide is between amino acids 44 and 45, at: AAA-DP.
  • NOV5f is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • This information was derived by determining the tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
  • sequence is predicted to be expressed in the following tissues because ofthe expression pattern of (GENBANK-ID: gb:GENBANK-ID:D50678
  • the presence of identifiable domains in NOV5f was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/).
  • this protein contains the following protein domains (as defined by Interpro) at the indicated positions: domain name low density lipoprotein receptor domains at amino acid positions 55 to 93, 96 to 177, 180 to 218; low density receptor repeat domains at amino acid positions 303 to 348, 350 to 391, 393 to 435, 437 to 521; EGF-like domains at amino acid positions 182 to 216, 223 to 257, 531 to 575; and fusion glycoprotein FO domain at amino acid positions 650 to 683.
  • domain name low density lipoprotein receptor domains at amino acid positions 55 to 93, 96 to 177, 180 to 218
  • low density receptor repeat domains at amino acid positions 303 to 348, 350 to 391, 393 to 435, 437 to 521
  • EGF-like domains at amino acid positions 182 to 216, 223 to 257, 531 to 575
  • fusion glycoprotein FO domain at amino acid positions 650 to 683.
  • NOV5g nucleic acid was isolated by The sequence of NOV5g was derived by laboratory cloning of cDNA fragments, by in silico prediction ofthe sequence. cDNA fragments covering either the full length ofthe DNA sequence, or part ofthe sequence, or both, were cloned. In silico prediction was based on sequences available in Curagen's proprietary sequence databases or in the public human sequence databases, and provided either the full length DNA sequence, or some portion thereof.
  • the novel NOV5g sequence of 4150 nucleotides also referred to as CG55256-05
  • Table 5M The novel NOV5g sequence of 4150 nucleotides (also referred to as CG55256-05) encoding a novel apolipoprotein e-like protein is shown in Table 5M.
  • the NOV5g sequence was identified through an exon linking process.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library contaimng a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5g sequence.
  • the NOV5g nucleic acid was identified to the p34 region of human chromosome 1 and has 3358 of 3361 bases (99%) identical to a gb:GENBANK-ID:D50678
  • acc:D50678.1 mRNA from Homo sapiens (Human mRNA for apolipoprotein E receptor 2, complete eds) (E 0.0)
  • a disclosed NOV5g polypeptide (SEQ ID NO:27) encoded by SEQ ID NO:26 is 857 amino acid residues and is presented using the one-letter code in Table 5N. Signal P, Psort and/or Hydropathy results predict that NOV5g has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6760.
  • NOV5g may also be localized to the endoplasmic reticulum (membrane) with acertainty of 0.1000, the endoplasmic reticulum (lumen) with a certainty of 0.1000, or exfracellularly with a certainty of 0.1000.
  • the most likely cleavage site for a NOV5g peptide is between amino acids 32 and 33, at: AAA-DP.
  • NOV5g is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus, Cerebral Medulla/Cerebral white matter, Colon, Lung, Lymphoid tissue, Parietal Lobe, Tonsils, Whole Organism. Expression information was derived from the tissue sources ofthe sequences that were included in the derivation ofthe sequence of NOV5g.
  • IPR000033 YWTD repeat, from amino acid 356 to amino acid 401, from amino acid 403 to amino acid 444, from amino acid 446 to amino acid 488, from amino acid 490 to amino acid 533, from amino acid 534 to amino acid 574.
  • This domain is also known as the YWTD motif after the most conserved region ofthe repeat.
  • the YWTD repeat is found in multiple tandem repeats and has been predicted to form a beta-propeller structure. It is found in a variety of proteins that include, vitellogenin receptor from Drosophila, low-density lipoprotein (LDL) receptor, preproepidermal growth factor, nidogen (entactin) and others.
  • LDL Low density lipoprotein
  • LDLRA Low density lipoprotein-receptor class A domain
  • the receptor protein binds LDL and transports it into cells by endocytosis. In order to be internalised, the receptor-ligand complex must first cluster into clathrin-coated pits. Seven successive cysteine-rich repeats of about 40 amino acids are present in the N-terminal of this multidomain membrane protein.
  • the LDL- receptor class A domain contains 6 disulfide-bound cysteines and a highly conserved cluster of negatively charged amino acids, of which many are clustered on one face ofthe module.
  • the class A domains form the binding site for LDL and calcium.
  • the acidic residues between the fourth and sixth cysteines are important for high-affinity binding of positively charged sequences in LDLR's ligands.
  • the repeat has been shown to consist of a beta-hairpin structure followed by a series of beta turns. The binding of calcium seems to induce no significant conformational change. Following these repeats is a 350 residue domain that resembles part ofthe epidermal growth factor (EGF) precursor. Similar domains have been found in several extracellular and membrane proteins.
  • EGF epidermal growth factor
  • EGF-like domain from amino acid 234 to amino acid 268, from amino acid 274 to amino acid 308
  • a sequence of about thirty to forty amino-acid residues long found in the sequence of epidermal growth factor (EGF) has been shown to be present, in a more or less conserved form, in a large number of other, mostly animal proteins.
  • the list of proteins currently known to contain one or more copies of an EGF-like pattern is large and varied.
  • the functional significance of EGF domains in what appear to be unrelated proteins is not yet clear. However, a common feature is that these repeats are found in the extracellular domain of membrane-bound proteins or in proteins known to be secreted (exception: prostaglandin G H synthase).
  • the EGF domain includes six cysteine residues which have been shown (in EGF) to be involved in disulfide bonds.
  • the main structure is a two-stranded beta-sheet followed by a loop to a C-terminal short two-stranded sheet. Subdomains between the conserved cysteines vary in length.
  • NOV5h A disclosed NOV5h nucleic acid was isolated by The sequence of NOV5h was derived by laboratory cloning of cDNA fragments, by in silico prediction ofthe sequence. cDNA fragments covering either the full length ofthe DNA sequence, or part ofthe sequence, or both, were cloned.
  • the NOV5h'sequence was identified through. an exon linking process.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5h sequence.
  • the NOV5h nucleic acid was identified to the p34 region of human chromosome 1 and has 3634 of 3851 bases (94%) identical to a gb:GENBANK-ID:D50678
  • acc:D50678.1 mRNA from Homo sapiens (Human mRNA for apolipoprotein E receptor 2, complete eds) (E 0.0)
  • a disclosed NOV5h polypeptide (SEQ ID NO:29) encoded by SEQ ID NO:28 is 847 amino acid residues and is presented using the one-letter code in Table 5P. Signal P, Psort and/or Hydropathy results predict that NOV5h has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6760.
  • NOV5h may also be localized to the endoplasmic reticulum (membrane) with acertainty of 0.1000, the endoplasmic reticulum (lumen) with a certainty of 0.1000, or exfracellularly with a certainty of 0.1000.
  • the most likely cleavage site for a NOV5h peptide is between amino acids 32 and 33, at: AAA-AA.
  • NOV5h is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus, Cerebral Medulla/Cerebral white matter, Colon, Lung, Lymphoid tissue, Parietal Lobe, Tonsils, Whole Organism. Expression information was derived from the tissue sources ofthe sequences that were included in the derivation ofthe sequence of NOV5h
  • the sequence is predicted to be expressed in placenta because ofthe expression pattern of (GENBANK-ID: gb:GENBANK-ID:D50678
  • LDL Low density lipoprotein
  • LDLRA Low density lipoprotein-receptor class A domain
  • the receptor-ligand complex In order to be internalised, the receptor-ligand complex must first cluster into clathrin-coated pits. Seven successive cysteine-rich repeats of about 40 amino acids are present in the N-terminal of this multidomain membrane protein.
  • the LDL- receptor class A domain contains 6 disulfide-bound cysteines and a highly conserved cluster of negatively charged amino acids, of which many are clustered on one face ofthe module. In LDL-receptors the class A domains form the binding site for LDL and calcium.
  • the acidic residues between the fourth and sixth cysteines are important for high-affinity binding of positively charged sequences in LDLR's ligands.
  • the repeat has been shown to consist of a beta-hairpin structure followed by a series of beta turns. The binding of calcium seems to induce no significant confonnational change. Following these repeats is a 350 residue domain that resembles part ofthe epidermal growth factor (EGF) precursor. Similar domains have been found in several extracellular and membrane
  • TPR000033 YWTD repeat, from amino acid 346 to amino acid 391, from amino acid 393 to amino acid 434, from amino acid 436 to amino acid 478, from amino acid 480 to amino acid 523, from amino acid 524 to amino acid 564.
  • This domain is also known as the YWTD motif after the most conserved region ofthe repeat.
  • the YWTD repeat is found in multiple tandem repeats and has been predicted to form a beta-propeller structure. It is found in a variety of proteins that include, vitellogenin receptor from Drosophila, low-density lipoprotein (LDL) receptor, preproepidermal growth factor, nidogen (entactin) and others.
  • EGF-like domain from amino acid 224 to amino acid 258, from amino acid 264 to amino acid 298, from amino acid 574 to amino acid 618.
  • a sequence of about thirty to forty amino-acid residues long found in the sequence of epidermal growth factor (EGF) has been shown to be present, in a more or less conserved form, in a large number of other, mostly animal proteins.
  • the list of proteins currently known to contain one or more copies of an EGF-like pattern is large and varied.
  • the functional significance of EGF domains in what appear to be unrelated proteins is not yet clear. However, a common feature is that these repeats are found in the extracellular domain of membrane-bound proteins or in proteins known to be secreted (exception: prostaglandin G/H synthase).
  • the EGF domain includes six cysteine residues which have been shown (in EGF) to be involved in disulfide bonds.
  • the main structure is a two-stranded beta-sheet followed by a loop to a C-terminal short two- stranded sheet.
  • Subdomains between the conserved cysteines vary in length.
  • NOV5i A disclosed NOV5i nucleic acid was isolated by The sequence of NOV5i was derived by laboratory cloning of cDNA fragments, by in silico prediction ofthe sequence. cDNA fragments covering either the full length ofthe DNA sequence, or part ofthe sequence, or both, were cloned. In silico prediction was based on sequences available in Curagen ' s proprietary sequence databases or in the public human sequence databases, and provided either the full length DNA sequence, or some portion thereof.
  • the novel NOV5i sequence of 4294 nucleotides (also referred to as CG55256-07) encoding a novel apolipoprotein e-like protein is shown in Table 5Q.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 103-105 and ending with a TGA codon at nucleotides 2818-2820.
  • a putative untranslated region upsfream from the initiation codon and downstream from the termination codon is underlined in Table 5Q, and the start and stop codons are in bold letters.
  • the NOV5i sequence was identified through an exon linking process.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5i sequence.
  • the NOV5i nucleic acid was identified to the p34 region of human chromosome 1 and has 3375 of 3389 bases (99%) identical to a gb:GENBANK-ID:D50678
  • acc:D50678.1 mRNA from Homo sapiens (Human mRNA for apolipoprotein E receptor 2, complete eds) (E 0.0)
  • a disclosed NOV5i polypeptide (SEQ ID NO:31) encoded by SEQ ID NO:30 is 905 amino acid residues and is presented using the one-letter code in Table 5R. Signal P, Psort and/or Hydropathy results predict that NOV5i has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6760.
  • NOV5i may also be localized to the endoplasmic reticulum (membrane) with acertainty of 0.1000, the endoplasmic reticulum (lumen) with a certainty of 0.1000, or extracellularly with a certainty of 0.1000.
  • the most likely cleavage site for a NOV5i peptide is between amino acids 32 and 33, at: AAA-AA. Table 5R. Encoded NOV5i protein sequence (SEQ ID NO:31)
  • NOV5i is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus, Cerebral Medulla/Cerebral white matter, Colon, Lung, Lymphoid tissue, Parietal Lobe, Tonsils, Whole Organism.
  • Expression information was derived from the tissue sources ofthe sequences that were included in the derivation ofthe sequence of NOV5L The sequence is predicted to be expressed in placenta because ofthe expression pattern of (GENBANK-ID: gb:GENBANK-ID:D50678
  • LDL Low density lipoprotein
  • LDLRA Low density lipoprotein-receptor class A domain
  • the receptor protein binds LDL and transports it into cells by endocytosis. In order to be internalised, the receptor-ligand complex must first cluster into clathrin-coated pits. Seven successive cysteine-rich repeats of about 40 amino acids are present in the N-terminal of this multidomain membrane protein.
  • the LDL- receptor class A domain contains 6 disulfide-bound cysteines and a highly conserved cluster of negatively charged amino acids, of which many are clustered on one face ofthe module.
  • the class A domains form the binding site for LDL and calcium.
  • the acidic residues between the fourth and sixth cysteines are important for high-affinity binding of positively charged sequences in LDLR's ligands.
  • the repeat has been shown to consist of a beta-hairpin structure followed by a series of beta turns. The binding of calcium seems to induce no significant conformational change. Following these repeats is a 350 residue domain that resembles part ofthe epidermal growth factor (EGF) precursor. Similar domains have been found in several extracellular and membrane proteins.
  • EGF epidermal growth factor
  • IPR000033 YWTD repeat, from amino acid 404 to amino acid 449, from amino acid 451 to amino acid 492, from amino acid 494 to amino acid 536, from amino acid 538 to amino acid 581, from amino acid 582 to amino acid 622.
  • This domain is also known as the YWTD motif after the most conserved region of the repeat.
  • the YWTD repeat is found in multiple tandem repeats and has been predicted to form a beta-propeller structure. It is found in a variety of proteins that include, vitellogenin receptor from Drosophila, low-density lipoprotein (LDL) receptor, preproepidermal growth factor, nidogen (entactin) and others.
  • EGF-like domain from amino acid 322 to amino acid 356, from amino acid 632 to amino acid 676.
  • a sequence of about thirty to forty amino-acid residues long found in the sequence of epidermal growth factor (EGF) has been shown to be present, in a more or less conserved form, in a large number of other, mostly animal proteins.
  • the list of proteins currently known to contain one or more copies of an EGF-like pattern is large and varied.
  • the functional significance of EGF domains in what appear to be unrelated proteins is not yet clear. However, a common feature is that these repeats are found in the extracellular domain of membrane-bound proteins or in proteins known to be secreted (exception: prostaglandin G/H synthase).
  • the EGF domain includes six cysteine residues which have been shown (in EGF) to be involved in disulfide bonds.
  • the main structure is a two-stranded beta- sheet followed by a loop to a C-terminal short two-stranded sheet. Subdomains between the conserved cysteines vary in length.
  • NOV5a also has homology to the amino acid sequences shown in the BLASTP data listed in Table 5S.
  • N0V5g SEQ ID NO: 7
  • NOV5h SEQ ID NO: 29
  • Tables 5U-Y list the domain description from DOMAIN analysis results against NOV5a. This indicates that the NOV5a sequence has properties similar to those of other proteins known to contain this domain.
  • Table 5U Domain Analysis of NOV5a qnl 1 Smart
  • LDL Low-density lipoprotein receptor domain class A
  • Cysteine-rich repeat in the low-density lipoprotein (LDL) receptor that plays a central role in mammalian cholesterol metabolism.
  • the N-terminal type A repeats in LDL receptor bind the lipoproteins.
  • Other homologous domains occur in related receptors, including the very low-density lipoprotein receptor and the LDL receptor-related protein/alpha 2-macroglobulin receptor, and in proteins which are functionally unrelated, such as the C9 component of complement . Mutations in the LDL receptor gene cause familial hypercholesterolemia. (SEQ ID NO:85)
  • NOV5a also has high homology to members ofthe patp database shown in Table 5Z.
  • NOV5c also has high homology to members ofthe patp database shown in Table 5AA.
  • Apolipoprotein E is a 34-kDa lipophilic protein that circulates in the plasma primarily as a major component of various lipoproteins including chylomicron remnants, intermediate density lipoprotein, very low density lipoprotein (VLDL), ⁇ -migrating VLDL ( ⁇ - VLDL), and high density lipoprotein (with apoE). It is a key molecule responsible for the cellular recognition and internalization of these lipoproteins. Biochemical and genetic studies have demonstrated that apoE is involved in the hepatic clearance of chylomicron remnants and VLDL remnants from the plasma. Apolipoprotein E receptor 2 binds apoE-rich ⁇ -VLDL with high affinity and internalizes it into the cells.
  • Apolipoprotein E receptor 2 is a receptor belonging to a family of low and very low density lipoprotein receptors (LDL-R). This family consists of cell-surface receptors that recognize extracellular ligands and internalize them for degradation by lysosomes. The family members contain four major structural modules: the cysteine-rich complement-type repeats, epidermal growth factor precursor-like repeats, a transmembrane domain, and a cytoplasmic domain. Each structural module serves distinct and important functions. These receptors bind several structurally dissimilar ligands. It is proposed that instead of a primary sequence, positive electrostatic potential in different ligands constitutes a receptor bmding domain. This family of receptors plays crucial roles in various physiologic functions.
  • LDL-R low and very low density lipoprotein receptors
  • LDL-R plays an important role in cholesterol homeostasis. Mutations cause familial hypercholesterolemia and premature coronary artery disease. LDL-R-related protein plays an important role in the clearance of plasma-activated alpha 2-macroglobulin and apolipoprotein E-enriched lipoproteins. It is essential for fetal development and has been associated with Alzheimer's disease. The regulation of expression of these receptors occurs at the transcriptional level.
  • the NOV5 nucleic acids and proteins ofthe invention are useful in potential therapeutic applications implicated in atherosclerosis, dysbetalipoproteinemia, hyperlipoproteinemia type III, xanthomatosis, coronary and/or peripheral vascular disease, hypothyroidism, systemic lupus erythematosus, diabetic acidosis, hypercholesterolemia, planar and tendon xanthomas, dysbetalipoproteinemia, hypercholesterolemia, accelerated vascular disease, Alzheimer disease, familial amyloidotic polyneuropathy, as well as other diseases, disorders and conditions.
  • the LDL receptor related protein, the apolipoprotein E receptor 2 and the very low density lipoprotein (VLDL) receptor are preferentially expressed in the brain and they have been suspected to be involved in Alzheimer disease at various levels (Microsc Res Tech 2000 Aug 15;50(4):273-7)., and/or other pathologies and disorders.
  • a cDNA encoding the apolipoprotein e-like protein may be useful in gene therapy, and the apolipoprotein e-like protein may be useful when administered to a subject in need thereof.
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from atherosclerosis, dysbetalipoproteinemia, hyperlipoproteinemia type III, xanthomatosis, coronary and or peripheral vascular disease, hypothyroidism, systemic lupus erythematosus, diabetic acidosis, hypercholesterolemia, planar and tendon xanthomas, dysbetalipoproteinemia, hypercholesterolemia, accelerated vascular disease, Alzheimer disease, familial amyloidotic polyneuropathy, as well as other diseases, disorders and conditions.
  • NOV5 nucleic acid, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.
  • NOV5 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOV5a, e, and f proteins have multiple hydrophilic regions, each of which can be used as an immunogen.
  • contemplated NOV5a, e, and f epitopes are from about amino acids 80 to 220.
  • NOV5a, e, and f epitopes are from about amino acids 250 to 340, from about amino acids 370 to 540, from about amino acids 590 to 650, or from about amino acids 680 to 720.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • the disclosed NOV5b protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOV5b epitope is from about amino acids 80 to 280.
  • a NOV5b epitope is from about amino acids 290 to 350, from about amino acids 390 to 560, from about amino acids 600 to 670, or from about amino acids 690 to 750.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • the disclosed NOV5c protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOV5c epitope is from about amino acids 50 to 220. In other embodiments, a NOV5c epitope is from about amino acids
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • the disclosed NOV5d protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOV5d epitope is from about amino acids 50 to 180.
  • a NOV5d epitope is from about amino acids 190 to 230, from about amino acids 250 to 390, from about amino acids 420 to 550,from about amino acids 470 to 650, from about amino acids 780 to 850, from amino acids 870 to 900, from about amino acids 920 to 980, or from about amino acids 990 to 1050.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • contemplated NOV5g and h epitopes are from about amino acids 30 to 220. In other embodiments, NOV5g and h epitopes are from about amino acids 250 to 330, from about amino acids 350 to 540, from about amino acids 550 to 680, from about amino acids 690 to 720, from about amino acids 750 to 780, or from about amino acids 790 to 850.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • the disclosed NOV5i protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOV5i epitope is from about amino acids 5 to 150.
  • a NOV5i epitope is from about amino acids 190 to 230, from about amino acids 170 to 320, or from about amino acids 360 to 450.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • a disclosed NOV6 nucleic acid of 843 nucleotides (also referred to as SC_129296119- A) encoding a novel mastocytoma protease precursor -like protein is shown in Table 6A.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 26- 28 and ending with a TAG codon at nucleotides 836-838.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 6A, and the start and stop codons are in bold letters.
  • a disclosed NOV6 polypeptide (SEQ ID NO:33) encoded by SEQ ID NO:32 is 270 amino acid residues and is presented using the one-letter amino acid code in Table 6B.
  • Signal P, Psort and/or Hydropathy results predict that NOV6 contains a signal peptide and is likely to be localized in thecytosol with a certainty of 0.4500.
  • the most likely cleavage site for a NOV6 peptide is between amino acids 20 and 21, at: KVG-IT.
  • Table 6B Encoded NOV6 protein sequence (SEQ ID NO.33).
  • the global sequence homology (as defined by FASTA alignment with the full length sequence of NOV6) is 66.171 % amino acid homology and 61.338 % amino acid identity.
  • this protein contains the following protem domains (as defined by Interpro) at the indicated nucleotide positions: Trypsin domain (IPR001254) at amino acid positions 21 to 260.
  • NOV6 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 6F.
  • N0V6 SEQ ID NO: 33
  • TRYM_CANFA canis familiaris (dog), mastocytoma protease precursor (ec 3.4.21.-) (SEQ ID NO: 58)
  • MCT7_RAT rattus norvegicus rat
  • mast cell protease 7 precursor ec 3.4.21.-
  • rmcp-7 tryptase, skin
  • MCT6_MOUSE mus musculus (mouse), mast cell protease 6 precursor (ec 3.4.21.-) (mmcp-6) (tryptase) (SEQ ID NO: 61)
  • Table 6H lists the domain description from DOMAIN analysis results against NOV6.
  • Table 6H Domain Analysis of NOV6 gnl I Smart
  • CD-Length 217 residues, 100.0% aligned
  • Mast cell tryptase is a secretory granule associated serine protease with trypsin-like specificity released extracellularly during mast cell degranulation.
  • the tryptase sequence includes the essential residues ofthe catalytic triad and an aspartic acid at the base ofthe putative substrate binding pocket that confers PI Arg and Lys specificity on tryptic serine proteases.
  • the apparent N-terminal signal/activation peptide terminates in a glycine. A glycine in this position has not been observed previously in serine proteases and suggests a novel mode of activation. Mast cell activation in vivo is often associated with areas of oedema and connective- tissue degradation.
  • Tryptase and chymase are the major serine proteinases released by mast cells, but they appear to have little activity on most components ofthe extracellular matrix.
  • the matrix metalloproteinases (MMP) are purported to degrade almost all connective tissue elements and are secreted by cells in the form of inactive precursors. Since the mechanisms of MMP activation in vivo are poorly understood we have examined the potential of mast cell proteinases to activate the precursor forms of human collagenase (MMP-1), stromelysin (MMP-3), gelatinase A (MMP-2) and gelatinase B (MMP-9).
  • Mast cell proteinases prepared from purified dog mastocytoma cells were shown to process and activate purified precursor forms of both MMP-1 and MMP-3. Using antipain and chymostatin, inhibitors for tryptase and chymase, respectively, it was demonstrated that both pMMP-1 and pMMP-3 were effectively processed and activated by the chymase component. By contrast, tryptase activated only pMMP-3. The mast cell proteinases were unable to process or activate purified precursor forms of MMP-2 and MMP-9. However, MMP-3 previously activated by mast cell proteinases was shown to activate pMMP-9, but not pMMP-2. Since we have no evidence that mast cells express these four metalloenzymes, the release of mast cell serine proteinases following activation/degranulation could contribute to local metalloproteinase activation and subsequent matrix degradation.
  • the mast cell proteases tryptase and chymase have long been known to constitute one- fifth ofthe total protein in mast cells.
  • their biological functions have not been easy to study because ofthe difficulty in obtaining sufficient amounts ofthe enzymes to study their biological functions.
  • We have discovered unique and potent actions ofthe enzymes that may provide important insights into the pathogenesis of diseases such as asthma and cystic fibrosis. Important biological activities are also likely to exist in other tissues. Because of their structures, mast cell proteases are likely to act in proximity to their sites of release.
  • NOV6 nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below.
  • the potential therapeutic applications for this invention include, but are not limited to: protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vitro of all tissues and cell types composing (but not limited to) those defined here.
  • nucleic acids and proteins of NOV6 are useful in potential therapeutic applications implicated in immune disorders and airway pathologies such as allergies, asthma and cystic fibrosis, cancers such as mastocytomas, vascular diseases, and/or other pathologies and disorders
  • a cDNA encodingNOV6 may be useful in gene therapy, and NOV6 may be useful when administered to a subject in need thereof.
  • NOV6 will have efficacy for treatment of patients suffering from immune disorders and airway pathologies such as allergies, asthma and cystic fibrosis, cancers such as mastocytomas, and vascular diseases.
  • novel NOV6 nucleic acid encoding NOV6 protein, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods.
  • NOV6 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immuno-specifically to the novel NOV6 substances for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders. NOV7
  • a disclosed NOV7 nucleic acid of 239 nucleotides (also referred to GM10d7_A) encoding a novel thymosin beta-4-like receptor protein is shown in Table 7A.
  • An open reading frame was identified beginning with a TCA initiation codon at nucleotides 40-42 and ending with a TGA codon at nucleotides 190-092.
  • the start and stop codons are in bold letters.
  • a disclosed NOV7 polypeptide (SEQ ID NO:35) encoded by SEQ ID NO:34 is 50 amino acid residues and is presented using the one-letter amino acid code in Table 7B. Signal P, Psort and/or Hydropathy results predict that NOV7 has no signal peptide and is likely to be localized in the cytoplasm with a certainty of 0.4500.
  • NOV7 maps to chromosome 5p35 and was found to be expressed in at least the following tissues: mammary gland, small intestine, colon, testis and leukocytes.
  • the disclosed NOV7 amino acid sequence has 26 of 43 amino acid residues (83 %) identical to, and 37 of 43 residues (86 %) positive with, the 50 amino acid residue
  • THYMOSIN BETA-4 protein ftom Mus musculus (ptnr:SPTREMBL-ACC: P20065) ( 2.2e " 12 ).
  • the global sequence homology (as defined by FASTA alignment with the full length sequence of this protein) is 72.000% amino acid homology and 72.000% amino acid identity.
  • NOV7 contains the following protein domains (as defined by Interpro) at the indicated nucleotide positions: Thymosin domain(JPR001152) at amino acid positions 8 to 48 NOV7 also has homology to the amino acid sequence shown in the BLASTP data listed in Table 7C.
  • TYB4_HUMAN homo sapiens human
  • bos taurus bos taurus
  • rattus norvegicus rat
  • equus caballus horse
  • thymosin beta-4 [contains: hematopoietic system regulatory peptide] (SEQ ID NO: 66)
  • TYB4_RABIT oryctolagus cuniculus (rabbit) .
  • thymosin beta-4 (SEQ ID NO: 67)
  • NOV7 also has high homology to members ofthe patp database shown in Table 7E.
  • Thymosin beta-4 is a small polypeptide whose exact physiological role is not yet known . It was first isolated as a thymic hormone that induces terminal deoxynucleotidyltransferase. It is found in high quantity in thymus and spleen but is widely distributed in many tissues. It has also been shown to bind to actin monomers and thus to inhibit actin polymerization .It has inhibitory activity on the proliferation of hematopoeitic pluripotent stem cells, and is localized in the cytosol of cells. It is induced by alpha interferon, nerve and fibroblast growth factors. It belongs to the thymosin beta family.
  • thymosin beta 4 is expressed in ROS 17/2.8 osteosarcoma cells in a regulated manner.
  • the differential expression of rnRNAs between the closely related rat osteosarcoma cell lines ROS 17/2.8 and ROS 25/1 was used to identify genes whose expression is associated with the osteoblast phenotype.
  • Thymosin beta 4 cDNA was cloned from an ROS 17/2.8 complimentary DAN library on the basis of its differential hybridization with radiolabeled cDNA prepared from ROS 17/2.8 and ROS 25/1 cells.
  • Northern blot analysis confirmed that thymosin beta 4, hitherto a putative immunodulatory hormone, was indeed differentially expressed.
  • Thymosin-beta(4) binds actin monomers stoichiometrically and maintains the bulk ofthe actin monomer pool in metazoan cells. Tbeta(4) binding quenches the fluorescence of N-iodoacetyl-N'-(5-sulfo-l-naphthyl)ethylenediamine (AEDANS) conjugated to Cys(374) of actin monomers.
  • AEDANS N-iodoacetyl-N'-(5-sulfo-l-naphthyl)ethylenediamine
  • the K(d) ofthe actin-Tbeta(4) complex depends on the cation and nucleotide bound to actin but is not affected by the AEDANS probe. The different stabilities are determined primarily by the rates of dissociation.
  • Tbeta(4) binding MgATP-actin is primarily enthalpic in origin but entropic for CaATP-actin. Binding is coupled to the dissociation of bound water molecules, which is greater for CaATP-actin than MgATP-actin monomers. Proteolysis of MgATP-actin, but not CaATP-actin, at Gly(46) on subdomain 2 is >12 times faster when Tbeta(4) is bound. The C terminus of Tbeta(4) contacts actin near this cleavage site, at His(40). By tritium exchange, Tbeta(4) slows the exchange rate of approximately eight rapidly exchanging amide protons on actin. We conclude that Tbeta(4) changes the conformation and structural dynamics ("breathing") of actin monomers. The conformational change may reflect the unique ability of Tbeta(4) to sequester actin monomers and inhibit nucleotide exchange.
  • glucocorticoids upregulate the expression of anti-inflammatory mediators is an exciting prospect for therapy in inflammatory diseases, because these molecules could give the therapeutic benefits of steroids without toxic side effects.
  • Supernatants from monocytes and macrophages cultured in the presence of glucocorticoids increase the dispersion of neutrophils from a cell pellet in the capillary tube migration assay. This supernatant factor, unlike other neutrophil agonists, promotes dispersive locomotion of neutrophils at uniform concentration, lowers their adhesion to endothelial cells, inhibits their chemotactic response to fMLP and induces distinctive morphological changes.
  • thymosin beta4 sulfoxide is generated by monocytes in the presence of glucocorticoids and acts as a signal to inhibit an inflammatory response.
  • thymosin beta4 sulfoxide inhibited neutrophil chemotaxis, and in vivo, the oxidized peptide, but not the native form, was a potent inhibitor of carrageenin-induced edema in the mouse paw.
  • Thymosin beta4 is unique, because oxidation attenuates its intracellular G-actin sequestering activity, but greatly enhances its extracellular signaling properties. This description of methionine oxidation conferring extracellular function on a cytosolic protein may have far-reaching implications for future strategies of ai ti-inflammatory therapy.
  • Angiogenesis is an essential step in the repair process that occurs after injury.
  • Tbeta4 angiogenic thymic peptide thymosin beta4
  • Tbeta4 stimulated keratinocyte migration in the Boyden chamber assay. After 4-5 h, migration was stimulated 2-3-fold over migration with medium alone when as little as 10 pg of Tbeta4 was added to the assay.
  • NOV7 thymosin beta-4-like protein and nucleic acid
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo.
  • the NOV7 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies.
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from prostate cancer, immunological and autoimmune disorders (ie hyperthyroidism), angiogenesis and wound healing, modulation of apoptosis, neurodegenerative and neuropsychiatric disorders, age-related disorders, pathological disorders involving spleen, thymus, lung, and peritoneal macrophages and/or other pathologies and disorders.
  • immunological and autoimmune disorders ie hyperthyroidism
  • angiogenesis ie hyperthyroidism
  • wound healing modulation of apoptosis
  • neurodegenerative and neuropsychiatric disorders ie hyperthyroidism
  • age-related disorders e.g., chronic myethelial fibrosis
  • pathological disorders involving spleen thymus, lung, and peritoneal macrophages and/or other pathologies and disorders.
  • the NOV7 nucleic acid, or fragments thereof may further be useful in diagnostic applications, wherein the presence or
  • NOV7 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods.
  • These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOV7 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • a disclosed NOV8 nucleic acid of 1348 nucleotides (also referred to as GMba550p23_A) encoding a novel Protein-tyrosine-phosphatase -like protein is shown in Table 8A.
  • An open reading frame was identified beginning with a ATG initiation codon at nucleotides 57-59 and ending with a TAA codon at nucleotides 1302-1304.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 8A, and the start and stop codons are in bold letters.
  • a disclosed NOV8 protein (SEQ ID NO:37) encoded by SEQ ID NO:36 has 415 amino acid residues, and is presented using the one-letter code in Table 8B.
  • Signal P, Psort and/or Hydropathy results predict that NOV8 is likely to be localized to the endoplasmic reticulum (membrane) with a certainty of 0.8500.
  • Table 8B Encoded NOV8 protein sequence (SEQ ID NO:37).
  • NOV8 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 8C.
  • PTN2_M0USE mus musculus (mouse) . protein-tyrosine phosphatase ptp-2 (ec 3.1.3.48) (mptp) (SEQ ID N ⁇ :69)
  • PTN2_RAT rattus norvegicus (rat) . protein-tyrosine phosphatase ptp-s (ec 3.1.3.48) (SEQ ID N ⁇ :70)
  • PTN1_CHICK gallus gallus (chicken) .
  • protein-tyrosine phosphatase lb (ec 3.1.3.48) (ptp-lb) (cptpl).
  • Pa (SEQ ID N0:71)
  • PTN1_M0USE mus musculus (mouse), protein-tyrosine phosphatase lb (ec 3.1.3.48) (ptp-lb) (protein- protein-tyrosine phosphatase ha2) (ptp-ha2) (SEQ ID NO: 72)
  • Tables 8E-8G list the domain description from DOMAIN analysis results against NOV8. This indicates that the NOV8 sequence has properties similar to those of other proteins known to contain this domain.
  • CD-Length 264 residues, 98.5% aligned
  • CD-Length 235 residues, 100.0% aligned
  • CD-Length 105 residues, 100.0% aligned
  • NOV8 also has high homology to members ofthe patp database shown in Table 8H.
  • T-cell PTPase was cloned by Cool et al. (1989) who showed that it shares 72% amino acid sequence identity with PTP1B in a 236- amino acid core region present in all PTPases.
  • Sakaguchi et al. (1991, 1992) used a human PTPT cDNA to assign the gene to human chromosome 18 and mouse chromosome 18 by Southern analysis of human/mouse and mouse/Chinese hamster somatic cell hybrids, respectively.
  • Johnson et al. (1993) mapped the PTPN2 gene to 18pll.3-pl 1.2 by fluorescence in situ hybridization.
  • PTPs Protein tyrosine phosphatases
  • NOV8 protein may have important structural and/or physiological functions characteristic of the protein-tyrosine-phosphatase F family. Therefore, the NOV8 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo.
  • the NOV8 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies.
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from hematopoiesic disorders; leukemogenesis; polycystic kidney disease; Oncogenesis such as lung, liver and ovarian cancers; apoptosis; type 2 diabetes and obesity; cell growth and possibly differentiation; non-Hodgkin lymphomas; musculoskeletal disorders; and CNS development disorders.
  • NOV8 nucleic acid, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.
  • NOV8 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOV8 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • nucleic acid molecules that encode NOVX polypeptides or biologically active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify NOVX-encoding nucleic acids (e.g., NOVX mRNAs) and fragments for use as PCR primers for the amplification and/or mutation of NOVX nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g. , mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • the nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double- stranded DNA.
  • an NOVX nucleic acid can encode a mature NOVX polypeptide.
  • a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein.
  • the naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein.
  • the product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises.
  • Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage ofthe N-terminal methionine residue encoded by the initiation codon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence.
  • a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine would have residues 2 through N remaining after removal ofthe N-terminal methionine.
  • a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved would have the residues from residue M+1 to residue N remaining.
  • a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event.
  • additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation.
  • a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
  • probes refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
  • isolated nucleic acid molecule is one, which is separated from other nucleic acid molecules which are present in the natural source ofthe nucleic acid.
  • an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5'- and 3 '-termini ofthe nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived.
  • the isolated NOVX nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA ofthe cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.).
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule ofthe invention e.g., a nucleic acid molecule having the nucleotide sequence SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et ⁇ l., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et ⁇ l., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
  • a nucleic acid ofthe invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g. , using an automated DNA synthesizer.
  • oligonucleotide refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.
  • a short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
  • Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length.
  • an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or a complement thereof. Oligonucleotides maybe chemically synthesized and may also be used as probes.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement ofthe nucleotide sequence shown in SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or a portion of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of an NOVX polypeptide).
  • a nucleic acid molecule that is complementary to the nucleotide sequence shown SEQ JD NOS:l, 3, 5, 7, 9, 11, 13, 15, 17 or 19 is one that is sufficiently complementary to the nucleotide sequence shown SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17 or 19 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, thereby forming a stable duplex.
  • binding means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like.
  • a physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
  • Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains.
  • Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.
  • Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.
  • Derivatives and analogs maybe full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
  • nucleic acids or proteins ofthe invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins ofthe invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below.
  • a “homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above.
  • Homologous nucleotide sequences encode those sequences coding for isoforms of NOVX polypeptides. Isoforms can be expressed in different tissues ofthe same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes.
  • homologous nucleotide sequences include nucleotide sequences encoding for an NOVX polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms.
  • homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations ofthe nucleotide sequences set forth herein.
  • a homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human NOVX protein.
  • Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, as well as a polypeptide possessing NOVX biological activity. Various biological activities ofthe NOVX proteins are described below.
  • An NOVX polypeptide is encoded by the open reading frame ("ORF") of an NOVX nucleic acid.
  • An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide.
  • a stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon.
  • An ORF that represents the coding sequence for a full protein begins with an ATG "start” codon and terminates with one ofthe three “stop” codons, namely, TAA, TAG, or TGA.
  • an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both.
  • a minimum size requirement is often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.
  • the nucleotide sequences determined from the cloning ofthe human NOVX genes allows for the generation of probes and primers designed for use in identifying and/or cloning NOVX homologues in other cell types, e.g. from other tissues, as well as NOVX homologues from other vertebrates.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17 or 19; or an anti-sense strand nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17 or 19; or of a naturally occurring mutant of SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36.
  • Probes based on the human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis- express an NOVX protein, such as by measuring a level of an NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX niRNA levels or determining whether a genomic NOVX gene has been mutated or deleted.
  • a polypeptide having a biologically-active portion of an NOVX polypeptide refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide ofthe invention, including mature forms, as measured in a particular biological assay, with or without dose dependency.
  • a nucleic acid fragment encoding a "biologically- active portion of NOVX” can be prepared by isolating a portion SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17 or 19, that encodes a polypeptide having an NOVX biological activity (the biological activities ofthe NOVX proteins are described below), expressing the encoded portion of NOVX protein (e.g. , by recombinant expression in vitro) and assessing the activity ofthe encoded portion of NOVX.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 due to degeneracy ofthe genetic code and thus encode the same NOVX proteins as that encoded by the nucleotide sequences shown in SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36.
  • an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37.
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of the NOVX polypeptides may exist within a population (e.g. , the human population).
  • Such genetic polymorphism in the NOVX genes may exist among individuals within a population due to natural allelic variation.
  • the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame (ORF) encoding an NOVX protein, preferably a vertebrate NOVX protein.
  • Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence ofthe NOVX genes. Any and all such nucleotide variations and resulting amino acid polymorphisms in the NOVX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity ofthe NOVX polypeptides, are intended to be within the scope ofthe invention.
  • nucleic acid molecules encoding NOVX proteins from other species and thus that have a nucleotide sequence that differs from the human SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 are intended to be within the scope ofthe invention.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the NOVX cDNAs ofthe invention can be isolated based on their homology to the human NOVX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36.
  • the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length.
  • an isolated nucleic acid molecule ofthe mvention hybridizes to the coding region.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
  • Homologs i.e., nucleic acids encoding NOVX proteins derived from species other than human
  • other related sequences e.g., paralogs
  • stringent hybridization conditions refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% ofthe probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% ofthe probes are occupied at equilibrium.
  • Tm thermal melting point
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides.
  • Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al, (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.
  • a non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C.
  • An isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequences SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or fragments, analogs or derivatives thereof, under conditions of moderate stringency.
  • moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5%) SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C.
  • Other conditions of moderate stringency that maybe used are well-known within the art.
  • nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided.
  • low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C.
  • Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations).
  • nucleotide sequences SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 can be introduced by mutation into the nucleotide sequences SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, thereby leading to changes in the amino acid sequences ofthe encoded NOVX proteins, without altering the functional ability of said NOVX proteins.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence SEQ ID NOS :2, 4, 6, 8, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31, 33, 35, or 37.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequences ofthe NOVX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity.
  • amino acid residues that are conserved among the NOVX proteins ofthe invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.
  • nucleic acid molecules encoding NOVX proteins that contain changes in amino acid residues that are not essential for activity.
  • NOVX proteins differ in amino acid sequence from SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 yet retain biological activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequences SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37.
  • the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37; more preferably at least about 70% homologous SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37; still more preferably at least about 80% homologous to SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37; even more preferably at least about 90% homologous to SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37; and most preferably at least about 95% homologous to SEQ ID NOS.2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37.
  • An isolated nucleic acid molecule encoding an NOVX protein homologous to the protein of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
  • Mutations can be introduced into SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted non-essential amino acid residue in the NOVX protein is replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of an NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity.
  • SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 the encoded protein can be expressed by any recombinant technology known in the art and the activity ofthe protein can be determined.
  • the relatedness of amino acid families may also be determined based on side chain interactions.
  • Substituted amino acids may be fully conserved "strong” residues or fully conserved “weak” residues.
  • the "strong” group of conserved amino acid residues may be any one ofthe following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other.
  • the "weak” group of conserved residues may be any one ofthe following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLJJVI, HFY, wherein the letters within each group represent the single letter amino acid code.
  • a mutant NOVX protein can be assayed for (i) the ability to form proteimprotein interactions with other NOVX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant NOVX protein and an NOVX ligand; or (iii) the ability of a mutant NOVX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g. avidin proteins).
  • a mutant NOVX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).
  • Another aspect ofthe invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or fragments, analogs or derivatives thereof.
  • An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence).
  • antisense nucleic acid molecules comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" ofthe coding strand of a nucleotide sequence encoding an NOVX protein.
  • coding region refers to the region ofthe nucleotide sequence comprising codons which are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" ofthe coding strand of a nucleotide sequence encoding the NOVX protein.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of NOVX mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid ofthe invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).
  • 2-methylthio-N6-isopentenyladenine 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-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules ofthe invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an NOVX protein to thereby inhibit expression of the protein (e.g. , by inhibiting transcription and/or translation).
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove ofthe double helix.
  • An example of a route of administration of antisense nucleic acid molecules ofthe invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens).
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule ofthe invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other. See, e.g., Gaultier, et al, 1987. Nucl. Acids Res. 15: 6625-6641.
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (See, e.g., ⁇ noue, et al. 1987. Nucl. Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (See, e.g., Inoue, et al, 1987. FEBS Lett. 215: 327-330.
  • Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
  • an antisense nucleic acid ofthe invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes as described in
  • Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave NOVX mRNA transcripts to thereby inhibit translation of NOVX mRNA.
  • a ribozyme having specificity for an NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of an NOVX cDNA disclosed herein (i.e., SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence ofthe active site is complementary to the nucleotide sequence to be cleaved in an NOVX-encoding mRNA.
  • NOVX-encoding mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Barrel et al, (1993) Science 261:1411-1418.
  • NOVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe NOVX nucleic acid (e.g., the NOVX promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe NOVX gene in target cells.
  • nucleotide sequences complementary to the regulatory region ofthe NOVX nucleic acid e.g., the NOVX promoter and/or enhancers
  • the NOVX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility ofthe molecule.
  • the deoxyribose phosphate backbone ofthe nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al, 1996. Bioorg Med Chem 4: 5-23.
  • peptide nucleic acids refer to nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al, 1996. supra; Perry-O'Keefe, et al, 1996. Proc. Natl Acad. Sci. USA 93: 14670-14675.
  • PNAs of NOVX can be used in therapeutic and diagnostic applications. For example,
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.
  • PNAs of NOVX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., Si nucleases (See, Hyrup, et al, 1996.supra); or as probes or primers for DNA sequence and hybridization (See, Hyrup, et al, 1996, supra; Perry-O'Keefe, et al, 1996. supra).
  • PNAs of NOVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of NOVX can be generated that may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (see, Hyrup, et al., 1996. supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al, 1996. supra and Finn, et al, 1996. Nucl Acids Res 24: 3357-3363.
  • aDNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5 , -(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA. See, e.g., Mag, et al, 1989. Nucl Acid Res 17: 5973-5988. PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment. See, e.g., Finn, et al, 1996. supra.
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, e.g., Petersen, et al, 1975. Bioorg. Med. Chem. Lett. 5: 1119-11124.
  • 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, 1989. Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556; Lemaitre, et al, 1987. Proc. Natl. Acad. Sci.
  • oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol, et al, 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988. Pharm. Res. 5: 539-549).
  • the oligonucleotide maybe conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.
  • a polypeptide according to the invention includes a polypeptide including the amino acid sequence of NOVX polypeptides whose sequences are provided in SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37 while still encoding a protein that maintains its NOVX activities and physiological functions, or a functional fragment thereof.
  • an NOVX variant that preserves NOVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues ofthe parent protein as well as the possibility of deleting one or more residues from the parent sequence.
  • Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
  • One aspect ofthe invention pertains to isolated NOVX proteins, and biologically- active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NOVX antibodies.
  • native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques, hi another embodiment, NOVX proteins are produced by recombinant DNA techniques.
  • an NOVX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NOVX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of NOVX proteins in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly-produced.
  • the language "substantially free of cellular material” includes preparations of NOVX proteins having less than about 30% (by dry weight) of non-NOVX proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-NOVX proteins, still more preferably less than about 10% of non-NOVX proteins, and most preferably less than about 5% of non-NOVX proteins.
  • non-NOVX proteins also referred to herein as a "contaminating protein”
  • contaminating protein also preferably substantially free of non-NOVX proteins
  • the NOVX protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% ofthe volume of the NOVX protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of NOVX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis ofthe protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of NOVX proteins having less than about 30% (by dry weight) of chemical precursors or non-NOVX chemicals, more preferably less than about 20% chemical precursors or non-NOVX chemicals, still more preferably less than about 10% chemical precursors or non-NOVX chemicals, and most preferably less than about 5% chemical precursors or non-NOVX chemicals.
  • Biologically-active portions of NOVX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences ofthe NOVX proteins (e.g., the amino acid sequence shown in SEQ JD NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37) that include fewer amino acids than the full-length NOVX proteins, and exhibit at least one activity of an NOVX protein.
  • biologically- active portions comprise a domain or motif with at least one activity ofthe NOVX protein.
  • a biologically-active portion of an NOVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.
  • biologically-active portions in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native NOVX protein.
  • the NOVX protein has an amino acid sequence shown SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37.
  • the NOVX protein is substantially homologous to SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37, and retains the functional activity ofthe protein of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below.
  • the NOVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37, and retains the functional activity of the NOVX proteins of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid "identity").
  • the nucleic acid sequence homology may be determined as the degree of identity between two sequences.
  • the homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. JMol Biol 48: 443-453.
  • sequence identity refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, U, or I, in the case of nucleic acids
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
  • the invention also provides NOVX chimeric or fusion proteins.
  • NOVX chimeric or fusion proteins.
  • NOVX "chimeric protein” or “fusion protein” comprises an NOVX polypeptide operatively- linked to a non-NOVX polypeptide.
  • An "NOVX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to an NOVX protein SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37), whereas a “non-NOVX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the NOVX protein, e.g., a protein that is different from the NOVX protein and that is derived from the same or a different organism.
  • an NOVX fusion protein comprises at least one biologically-active portion of an NOVX protein.
  • an NOVX fusion protein comprises at least two biologically- active portions of an NOVX protein.
  • an NOVX fusion protein comprises at least three biologically-active portions of an NOVX protein.
  • the term "operatively-linked" is intended to indicate that the NOVX polypeptide and the non-NOVX polypeptide are fused in-frame with one another.
  • the non-NOVX polypeptide can be fused to the N-terminus or C-terminus ofthe NOVX polypeptide.
  • the fusion protein is a GST-NO VX fusion protein in which the
  • NOVX sequences are fused to the C-terminus ofthe GST (glutathione S -transferase) sequences. Such fusion proteins can facilitate the purification of recombinant NOVX polypeptides.
  • the fusion protein is an NOVX protein containing a heterologous signal sequence at its N-terminus.
  • NOVX a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of NOVX can be increased through use of a heterologous signal sequence.
  • the fusion protein is an NOVX-immunoglobulin fusion protein in which the NOVX sequences are fused to sequences derived from a member ofthe immunoglobulin protein family.
  • the NOVX-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between an NOVX ligand and an NOVX protein on the surface of a cell, to thereby suppress NOVX-mediated signal transduction in vivo.
  • the NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of an NOVX cognate ligand.
  • NOVX-immunoglobulin fusion proteins ofthe invention can be used as immunogens to produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in screening assays to identify molecules that inhibit the interaction of NOVX with an NOVX ligand.
  • An NOVX chimeric or fusion protein ofthe invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).
  • anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • An NOVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.
  • the invention also pertains to variants ofthe NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists.
  • Variants ofthe NOVX protein can be generated by mutagenesis (e.g., discrete point mutation or truncation ofthe NOVX protein).
  • An agonist ofthe NOVX protein can retain substantially the same, or a subset of, the biological activities ofthe naturally occurring form ofthe NOVX protein.
  • An antagonist of the NOVX protein can inhibit one or more ofthe activities ofthe naturally occurring form of the NOVX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the NOVX protein.
  • treatment of a subject with a variant having a subset ofthe biological activities ofthe naturally occurring form ofthe protein has fewer side effects in a subject relative to treatment with the naturally occurring form ofthe NOVX proteins.
  • Variants ofthe NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists can be identified by screening combinatorial libraries of mutants (e.g. , truncation mutants) of the NOVX proteins for NOVX protein agonist or antagonist activity.
  • a variegated library of NOVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of NOVX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein.
  • a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein.
  • methods which can be used to produce libraries of potential NOVX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector.
  • degenerate set of genes allows for the provision, in one mixture, of all ofthe sequences encoding the desired set of potential NOVX sequences.
  • Methods for synthesizing degenerate oligonucleotides are well-known within the art. See, e.g., Narang, 1983. Tetrahedron 39: 3; Itakura, et al, 1984. Annu. Rev. Biochem. 53: 323; Itakura, et al, 1984. Science 198: 1056; Ike, et al, 1983. Nucl. Acids Res. 11: 477.
  • libraries of fragments ofthe NOVX protein coding sequences can be used to generate a variegated population of NOVX fragments for screening and subsequent selection of variants of an NOVX protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an NOVX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with Si nuclease, and ligating the resulting fragment library into an expression vector.
  • expression libraries can be derived which encodes N-terminal and internal fragments of various sizes ofthe NOVX proteins.
  • Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify NOVX variants. See, e.g., Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave, et al, 1993. Protein Engineering 6:327-331.
  • antibodies to NOVX proteins, or fragments of NOVX proteins.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • immunoglobulin (Ig) molecules i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F a , F ab * and F (a ')2 fragments, and an F ab expression library.
  • an antibody molecule obtained from humans relates to any ofthe classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature ofthe heavy chain present in the molecule.
  • Certain classes have subclasses as well, such as IgGi, IgG 2 , and others.
  • the light chain may be a kappa chain or a lambda chain.
  • Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
  • An isolated NOVX-related protein ofthe invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments ofthe antigen for use as immunogens.
  • An antigenic peptide fragment comprises at least 6 amino acid residues ofthe amino acid sequence ofthe full length protein and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.
  • the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions ofthe protein that are located on its surface; commonly these are hydrophilic regions.
  • At least one epitope encompassed by the antigenic peptide is a region of NOVX-related protein that is located on the surface ofthe protein, e.g., a hydrophilic region.
  • a hydrophobicity analysis ofthe human NOVX-related protein sequence will indicate which regions of a NOVX-related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.
  • hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78:
  • Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
  • a protein ofthe invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
  • polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative ofthe foregoing.
  • An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.
  • the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • the preparation can further include an adjuvant.
  • adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
  • Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • the polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target ofthe immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by inimunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
  • MAb monoclonal antibody
  • CDRs complementarity determining regions
  • Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse, 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.
  • the lymphocytes can be immunized in vitro.
  • the immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.
  • peripheral blood lymphocytes 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.
  • rat or mouse myeloma cell lines are employed.
  • the hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells.
  • 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, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J.
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
  • 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 immunoabsorbent assay (ELISA). Such techniques and assays are known in the art.
  • the binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
  • the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • 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).
  • the hybridoma cells ofthe invention serve as a preferred source of such DNA.
  • the DNA can be placed into expression vectors, which are then transfected 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 can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, N ⁇ twre 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody ofthe invention, or can be substituted for the variable domains of one antigen-combining site of an antibody ofthe invention to create a chimeric bivalent antibody.
  • the antibodies directed against the protein antigens ofthe invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.
  • Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen- binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.
  • Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann 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. (See also U.S. Patent No. 5,225,539.) In some instances, Fv framework residues ofthe human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin and all or substantially all ofthe framework 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., 1986; Riechmann et al, 1988; and Presta, Curr. Op. Struct. Biol, 2:593-596 (1992)).
  • Fc immunoglobulin constant region
  • Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or “fully human antibodies” herein.
  • Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 hnmunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, hie, pp. 77-96).
  • Human monoclonal antibodies may be utilized in the practice ofthe present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al., J. Mol. Biol, 222:581 (1991)).
  • 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 antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825;
  • Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.
  • WO94/02602 The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encodmg human heavy and light chain immunoglobulins are inserted into the host's genome.
  • the human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments.
  • An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement ofthe modifications.
  • the preferred embodiment of such a nonhuman animal is a mouse, and is termed the XenomouseTM as disclosed in PCT publications WO 96/33735 and WO 96/34096.
  • This animal produces B cells which secrete fully human immunoglobulins.
  • the antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
  • U.S. Patent No. 5,939,598 An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
  • a method for producing an antibody of interest such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
  • the hybrid cell expresses an antibody containing the heavy chain and the light chain.
  • F a b Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein ofthe invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of F ab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F a fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.
  • Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F( a ' )2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F( a b ' ) 2 fragment; (iii) an F a b fragment generated by the treatment ofthe antibody molecule with papain and a reducing agent and (iv) F v fragments.
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • one ofthe binding specificities is for an antigenic protein ofthe invention.
  • the second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
  • 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 ofthe random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the correct bispecific structure. The purification ofthe 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, 1991 EMBO J, 10:3655-3659.
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one ofthe fusions.
  • CHI first heavy-chain constant region
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part ofthe CH3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface ofthe second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield ofthe heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') 2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence ofthe dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • One ofthe Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount ofthe other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

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Abstract

L'invention concerne des séquences d'acides nucléiques qui codent de nouveaux polypeptides. Elle concerne également des polypeptides codés par ces séquences d'acides nucléiques, et des anticorps, qui se lient de manière immunospécifique au polypeptide, ainsi que des dérivés, des variants, des mutants ou des fragments du polypeptide, du polynucléotide ou de l'anticorps susmentionné. L'invention concerne en outre des procédés thérapeutiques, diagnostiques et de recherche destinés à diagnostiquer, traiter et prévenir des troubles impliquant l'un ou l'autre de ces nouveaux acides nucléiques et protéines.
PCT/US2001/026518 2000-08-25 2001-08-27 Nouvelles proteines et acides nucleiques les codant WO2002016600A2 (fr)

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CA002420538A CA2420538A1 (fr) 2000-08-25 2001-08-27 Nouvelles proteines et acides nucleiques les codant
JP2002522273A JP2004516820A (ja) 2000-08-25 2001-08-27 新規タンパク質およびそれらのタンパク質をコードする核酸
EP01964417A EP1332212A2 (fr) 2000-08-25 2001-08-27 Nouvelles proteines et acides nucleiques les codant
AU2001285271A AU2001285271A1 (en) 2000-08-25 2001-08-27 Novel proteins and nucleic acids encoding same

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US60/228,324 2000-08-25
US60/227,800 2000-08-25
US22918500P 2000-08-30 2000-08-30
US22899700P 2000-08-30 2000-08-30
US60/229,185 2000-08-30
US60/228,997 2000-08-30
US22978000P 2000-09-01 2000-09-01
US22984800P 2000-09-01 2000-09-01
US22985000P 2000-09-01 2000-09-01
US60/229,780 2000-09-01
US60/229,848 2000-09-01
US60/229,850 2000-09-01
US26333701P 2001-01-22 2001-01-22
US60/263,337 2001-01-22
US26551801P 2001-01-31 2001-01-31
US60/265,518 2001-01-31
US27645101P 2001-03-15 2001-03-15
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EP1776134A4 (fr) * 2004-06-17 2009-08-26 Amgen Mountain View Inc Proteines de liaison de kinase c-met
US9758554B2 (en) 2012-01-31 2017-09-12 Technische Universitaet Muenchen Muteins of α1m lipocalin and method of production therefor

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1776134A4 (fr) * 2004-06-17 2009-08-26 Amgen Mountain View Inc Proteines de liaison de kinase c-met
EP2151453A1 (fr) * 2004-06-17 2010-02-10 Amgen Mountain View Inc. Protéines de liaison de kinase c-met
US7803907B2 (en) 2004-06-17 2010-09-28 Amgen Mountain View, Inc. c-MET kinase binding proteins
US7968681B2 (en) 2004-06-17 2011-06-28 Amgen Mountain View, Inc. c-MET kinase binding proteins
AU2005265150B2 (en) * 2004-06-17 2012-04-12 Amgen Mountain View Inc. C-MET kinase binding proteins
US9758554B2 (en) 2012-01-31 2017-09-12 Technische Universitaet Muenchen Muteins of α1m lipocalin and method of production therefor
US10738092B2 (en) 2012-01-31 2020-08-11 Technische Universitaet Muenchen Muteins of a1m lipocalin and method of production therefor

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