EP1161442A2 - Exocytosis pathway proteins and methods of use - Google Patents

Exocytosis pathway proteins and methods of use

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Publication number
EP1161442A2
EP1161442A2 EP00902456A EP00902456A EP1161442A2 EP 1161442 A2 EP1161442 A2 EP 1161442A2 EP 00902456 A EP00902456 A EP 00902456A EP 00902456 A EP00902456 A EP 00902456A EP 1161442 A2 EP1161442 A2 EP 1161442A2
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Prior art keywords
seq
protein
exo
nucleic acid
acid sequence
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German (de)
French (fr)
Inventor
Ying Luo
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Rigel Pharmaceuticals Inc
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Rigel Pharmaceuticals Inc
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to moleculesinvolvedin the exocytosispathway and more particularly to novel polypeptideswhich associatewith exocytoticproteins, nucleicacids and antibodies.
  • the invention further relates to the use of proteins associated with the exocytosis pathway in methods for identifying candidate agents which modulate exocytosis.
  • proteins destined forthe plasma membrane or the extracellular space are delivered along the secretory pathway. This comprises a series of sequential, vesicle- mediated transport steps, each of which requires the specific targeting of transportvesicles to the appropriate acceptor membrane and the subsequent fusion of vesicle and acceptor membranes.
  • proteins to be secreted by the cell are translocated into the endoplasmic reticulum and then travel through the Golgi complex.
  • the proteins are sorted into secretory vesicles in the frans-Golgi network and these vesicles then fuse with the plasma membrane. This final membrane fusion event is known as exocytosis and results in the discharge of vesicle contents into the extracellular space as well as the incorporation of vesicle membrane lipids and proteins into the plasma membrane.
  • Exocytosis can be divided into two classes: constitutive and regulated. In constitutive exocytosis, secretor vesicles fuse with the plasma membrane immediately after formation; in regulated exocytosis, secretory vesicles accumulate in the cytoplasm and only undergo fusion upon receipt of an appropriate signal. All eukaryotic cells exhibit constitutive Although the fundamental purpose of exocytosis is to deliver iipids and proteins to the plasma membrane and to release vesicle contents from the cell, different cell types utilize this mechanism to fulfill their own particular physiological role. Some examples of the various functions of exocytosis in different cell types are listed in Table 1.
  • exocytosis i.e., the fusion of vesicles with the plasma membrane
  • exocytosis is not only the end point of the secretory pathway, but can also involve vesicles which did not originate fromthe endoplasmic reticulum.
  • transcytosis occurs in polarized cells and involves endocytic vesicle budding from one pole ofthe cell, transport to the other pole (often via endosomes) and subsequent exocytic fusion.
  • transcytosis is used in the uptake of antibodies from the blood and their subsequent secretion in milk.
  • some vesicles undergo cycles of exo/endocytic fusion via endosomes without returning to the Golgi.
  • Exocytosis of recycling vesicles may be either constitutive (e.g., transferrin receptor-containing vesicles) or regulated (e.g., synaptic vesicles).
  • regulated secretory cells must possess two types of secretory vesicle: one constitutive and on regulated. Morphological studies indicate this to be the case, since constitutive secretory vesicles appear small and clear in the electron microscope, whereas regulated secretory vesicles typically appear larger and opaque. Furthermore, the two types of vesicle usually contain different substances (an exception is the mammary cell, where casein secretion occurs by both constitutive and regulated exocytosis).
  • cells may contain more than one type of regulated secretory vesicle.
  • the best example of this is seen in neurons, which may possess synaptic vesicles and large dense-core vesicles in addition to constitutive secretory vesicles.
  • Some properties ofthe two type of neuronal regulated secretory vesicle are listed in Table 2.
  • Large dense- core vesicles contain peptide neurotransmitters and these are very similar to regulated secretory vesicles in endocrine cells. Indeed, much ofthe information on large dense-core vesicle biogenesis and exocytosis has come from studies of adrenal chromaffin cells and theirtumor cell derivatives, PC12 cells, both popular neuronal cell models.
  • Synaptic vesicles appear clear in the electron microscope, are much smaller than large dense-core vesicles and contain fast neurotransmitters. Synaptic vesicles have evolved in animals to allow the extremely rapid point-to-point communication required for brain function. Recently synaptic- like vesicles have been found in endocrine cells, such as adrenal chromaffin cells and pancreatic ⁇ -cells. These vesicles also appearto contain fast neurotransmitters, although their physiological role is unclear.
  • GABA ⁇ -aminobutyric acid
  • ACh acetylcholine
  • VIP vasoactive intestinal peptide
  • exocytosis The more information that is gathered regarding exocytosis, the easier it will be to manipulate exocytosis. Moreover, the more information which is gathered, the easier it will be to diagnosis and treat disorders involving exocytosis.
  • inflammatory mediator release from mast cells leads to a variety of disorders, including asthma.
  • Therapy for allergy remains limited to blocking the individual mediators released from mast cells (anti- histamines), non-specificanti-inflammatoryagentssuch as steroidsand mast cell stabilizers which are only marginally effective at limiting the symptomatology of allergies
  • Chediak-Higashi Syndrome is a rare autosomal recessive disease in which neutrophils, monocytes and lymphocytes contain giant cytoplasmic granules
  • rab GTPases represent a diverse family of homologous proteins that are generally associated with the membrane of organelles in a wide variety of cells, where they regulatedefined steps of intracellular membrane traffic (Ze ⁇ al, M and Stenmark, H , Curr Opin Cell Biol 5 613 (1993))
  • An example of this are the rab3 subfamily proteins which have been found to have limited expression in regulated secretion-competent cells, and to be associated with synaptic or secretory granules, suggesting that they are involved in stimulus-secretion coupling (Lledo, et al , Trends Neurobiol Sci 17426 (1994))
  • overexpression of rab3d or its GTP binding mutant form (N 1351) in the rat basophil line RBL leads to significantinhibit
  • Rab3a, Rab3b, Rab3c, and Rab3d constitute a subgroup of the rab family implicated in regulated exocytosis
  • Rab3a has been detected in regulated secretory cells such as neurons, endocrine cells, and exocnne cells but not in constitutive secretory cells such as hepatocytes and lymphocytes (Fischer von Mollard, Proc Natl Acad Sci 87 1988-92 (1990), Takai, et al , Int Rev Cytol 133 187-230 (1992))
  • rab3a has been localized at the synaptic vesicles (Mizoguchi, et al , Biochem Biophys Res Commun 202 1235-43 (1994))
  • Rab3a has also been detected at the secretory granules of chromaffin cells and at the zymogen granules in the exocnne cells of pancreat ⁇ cac ⁇ n ⁇ (Padfield,
  • Rab3a is cloned and known in the art (see, i.e., Genbank accession number (no.) M28210).
  • Rab3d is thought to be involved in the modulation of regulated secretion in a number of celltypes. Rab3d is predominantly expressed in fat tissue but can also be found expressed, at lower levels, in a wide range of tissue types including lung, spleen, heart, and brain. Baldini, G., et al., (1992) Proc. Natl. Acad. Sci. USA 89: 5049-52. Rab3d has also been implicated in the translocation of the Giut4 glucose transporter in adipocytes. Rab3d has been cloned and is known in the art, i.e., Genbank accession no.: AF081353.
  • rabs particularly, tissue /cell specific isoforms of rabs and the proteins which they interact with are of great pharmaceutical interest.
  • Rab7 is cloned and known in the art, (see, i.e., Genbank accession no. U44104).
  • Rab9 is localized to the surface of late endosomes where it appears to act to stimulate the transportof mannose ⁇ -phosphate receptors between late endosomes and the trans-Golgi network, both in vitro and in vivo. Recent studies suggest that this GTPase is a rate-limiting component for transport between late endosomes and the trans-Golgi network. Rab9 has been cloned and is known in the art, (see i.e., U44103).
  • Rab11 was identified by screening a Madin-Darby canine kidney cell cDNA library using degenerate oligonucleotides derived from conserved sequences of the Rab superfamily. Chavrier, P., et al., (1990), Mol. Cell. Biol. 10:6578-85. The predicted amino acid sequences ofthe canine, human, rat and rabbit Rab11 are 100% identical. This high level of conservation between species might reflect a particular importance of this member of the Rab family. Rab11 has been localized to both the constitutive and regulated secretory pathway in PC12 cells.
  • Rab11 a homolog of Rab11 (91% identity at the amino acid level) has been found to be associated with cholinergicsynapticvesiclesderived from the electric organ of the marine ray.
  • the function of Rab11 has yet to be definitively determined, a number of lines of evidence suggest that it may play a role in the targeting of transport vesicles of different origin to a common destination, the plasma membrane.
  • Northern blot analysis has shown that Rab11 is ubiquitously expressed but is generally more abundant in tissues with a high level of secretion. Rab11 has been cloned and is known in the art, i.e., Genbank accession no. X56740.
  • Rab5 (a, b, and c) make up a subgroup of the Rab protein family. They are located at the cytoplasmic surface of the plasma membrane, on early endosomes and on plasma- membrane derived clathrin coated vesicles. Antibodies directed against Rab5a inhibit the fusion of early endosomes in vitro suggesting that its activity is required in this process. In vivo, overexpression of wild type and mutant Rab ⁇ a leads to changes in the rate of internalizationofendocyticmarkersand in morphologicalalterations ofthe early endosomes.
  • Rab ⁇ a is a rate-limiting factor that regulates the kinetics of both lateral fusion of early endosomesand fusion of plasma membranederivedendocyticvesicles with early endosomes.
  • Some proteins have been identified to associate with Rab5. For example, a 62 kDa coiled-coil protein that specifically interacts with the GTP-bound form of Rab5 has been identified. This protein shares 42% sequence identity with Rabaptin-5, a previously identified effector of Rab5, and has been named it Rabaptin-5beta.
  • Rabaptin- ⁇ beta displays heptad repeats characteristic of coiled-coil proteins and is recruited on the endosomal membrane by Rab5 in a GTP-dependent manner.
  • Rabaptin- ⁇ beta has features that distinguish it from Rabaptin-5.
  • the relative expression levels of the two proteins varies in different cell types. Rabaptin- ⁇ beta does not heterodimerize with Rabaptin- ⁇ , and forms a distinct complex with Rabex- ⁇ , the GDP/GTP exchange factor for Rab ⁇ .
  • Immunodepletion of the Rabaptin- ⁇ beta complex from cytosol only partially inhibits early endosome fusion in vitro, whereas the additional depletion of the Rabaptin- ⁇ complex has a stronger inhibitory effect. Fusion activity can mostly be recovered by addition of the Rabaptin- ⁇ complex alone, but maximal fusion efficiency requires the presence of both Rabaptin- ⁇ and Rabaptin-5beta complexes.
  • Rab ⁇ is cloned and known in the art (see, i.e., Genbank accession no. M28216).
  • key proteins that act in Ca 2+ -regulated exocytosis in neurons and endocrine cells includethevesicleproteinssynaptotagmin.VAMP/synaptobrevin, the target membrane protein SYNTAXINs and SNAP-23/26 and, in addition, the soluble N-ethylmaleimide- sensitive fusion protein (NSF) and soluble NSF-attachment proteins ( ⁇ -, ⁇ -, ⁇ -SNAPs).
  • NSF soluble N-ethylmaleimide- sensitive fusion protein
  • ⁇ -, ⁇ -, ⁇ -SNAPs soluble NSF-attachment proteins
  • GS27 is associatedwith the Golgi apparatusand is believed to behave like a SNARE.
  • SNAP-23 (used interchangeablywith snap-23) was first identified in a human B lymphocyte cDNA library (Ravichandran, V., et al., (1996), J. Biol. Chem. 271:13300-03). Subsequently, others have independently reported the identification of SNAP-23 in several celltypesincludingmastcells.
  • the primary structure of SNAP-23 is 69% identical to SNAP- 2 ⁇ ; it contains a central cluster of cysteine residues that is a site of palmitoylation in SNAP- 0 2 ⁇ and predicted coiled-coil that are thought to serve in binding other SNAREs, especially syntaxins 1 ,3, and 4.
  • SNAP-23 like SNAP-26, has been localized mainly to the plasma membrane.
  • SNAP-23 translocates to the surface of secretory granules upon cellular activation and forms a complex with SYNTAXIN-3 and VAMP-2 (Guo. Z., etal., (1998),Cell. 94:637-48).
  • Thissame study suggests thatSNAP-23 6 functioniscrucialforcompound exocytosis in mast cells in thatSNAP-23specificantibodies can completely block secretion in permeabilized cells.
  • SNAP-23 is cloned and known in the art, (see, i.e., Genbank accession no. U65936).
  • NSF and alpha-snap were originally detected as factors required for transport through the Golgi in in vitro assays and yeast homologues of these proteins, sec18 and sec17, are 0 essential for secretion in vivo.
  • ⁇ and ⁇ -SNAP appear to be functionally redundant.
  • ⁇ and ⁇ -SNAP havedistinctfunctionsin regulated exocytosis based on the ability of alpha-snap to displace synaptotagmin from the SNARE complex. Sollner, T., et al., Cell, 75: 409-18 (1993).
  • Alpha-snap has been cloned and is known in the art, i.e., Genbank accession number (no.) U39412.
  • Sec1 is a hydrophilic protein that plays an essential role in exocytosis from the yeast Saccharomyces cerevisiae.
  • Syntaxin a T-SNARE
  • SNAP-25 a T- and a V-SNARE, respectively
  • Proteins that exhibit similarity to Sed were identified in the nervous system of Drosophila melanogaster (Rop) and Caenorhabditiselegans(UNC18).Munc-18/n-Sec1/rbSec1 ,a brain homoiogueoftheyeast Sedp protein, is thought to participate in regulating the docking and fusion of synaptic vesicles. Munc-18/n-Sec1 /rbSed expression has been reported to be neural-specific and a number of non-neural isoforms have been identified which are more ubiquitously expressed. Shaywitz, D. A., et al., J. Cell. Biol. 128:769-777 (199 ⁇ ).
  • Munc18c previously identified as an n-Sec1/Munc18 homolog in 3T3-L1 adipocytes, in insulin-regulated GLUT4 trafficking has been investigated in 3T3-L1 adipocytes. Lowe, S. L., et al, Nature. 389:881-884 (1997). In these cells, Munc18c predominantly associated with syntaxin4, although it bound both syntaxin2 and syntaxin4 to similar extents in vitro. In addition, SNAP-23, an adipocyte homolog of SNAP-25, associated with both syntaxins 2 and 4 in 3T3-L1 adipocytes.
  • Tetanus toxin inhibits neurotransmitter release by selectively blocking fusion of synaptic vesicles.
  • Tetanus toxin has been shown to proteolytically degrade synaptobrevin II (also named VAMP-2), a synaptic vesicle-specific protein, in vitro and in nerve terminals.
  • synaptobrevin II also named VAMP-2
  • Asynaptobrevinhomologue,cellubrevin (VAMP-3) present in all cells and tissues tested, is a membrane trafficking protein of a constitutively recycling pathway. McMahon H.T., et al, Nature.
  • Vamp3 has been localizedto mast cell granules and may play a critical role in mast cell exocytosis (Guo, Z., etal., Cell, 94:537-48 (1998)).
  • Vamp3 has been cloned and is known in the art, i.e., Genbank accession no.: AF26007.
  • Exo proteins proteins involved in exocytosis, termed Exo proteins herein, particularly those associated with GS27, Rab3a, Rab7, Rab9, Rab11 , Rab3d, Rab5, alphasnap, und ⁇ -
  • vamp3, and snap-23 are of interest, and it is desirable to providesuch proteinsand related molecules. It is a further aspect of the invention to provide recombinant nucleic acids encoding Exo proteins and expression vectors and host cells containing the nucleic acid encoding the Exo protein. A further aspect of the invention is to provide methods for screening for antagonists and agonists of Exo proteins, particularly those which modulate exocytosis, secretion and/or vesicular transport.
  • the present invention provides recombinant nucleic acids encoding an Exo protein that has at least about 8 ⁇ % sequence identity, and more preferably at least about 90% sequence identity, and most preferably about 95% sequence identity with an amino acid sequence encoded by a nucleic acid comprising the first 100 nucleic acid residues of a sequence selected from the group consisting of SEQ ID NOS:15, 17, 19, 21 , 23, 2 ⁇ , 27, 29, 31 , 33, 3 ⁇ , 37, 39, 41 , 43, 4 ⁇ , 47, 49, 51 , 53, 63, 64, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124,
  • the Exo proteins bind to a protein selected from the group consisting of GS27, rab7, rab9, snap-23, rab3a, rabl 1 , rab3d, rab ⁇ , alpha-snap, und 8-1 , and vamp3.
  • nucleic acids which have at least about 7 ⁇ % sequence identity, more preferably, at least 8 ⁇ % sequence identity and most preferably at least about 9 ⁇ % sequence identity with a nucleicacid sequencecomprisingthe first 100 nucleic acid residues of a sequenceselected from the group consisting of SEQ ID NOS: 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 36, 37, 39, 41 , 43, 45, 47, 49, 51 , 63, 63, 64, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129,
  • the invention provides recombinant Exo proteinsExo3, Exo4, Exo ⁇ , Exo6, Exo7, Exo ⁇ , Exo9, Exo10, Exo11 , Exo12, Exo13, Exo14, Exol ⁇ , Exo16, Exo17a, 0 Exo17b, Exo18, Exo19, Exo20, Exo21 , Exo22, Exo23, Exo24, Exo2 ⁇ , Exo26, Exo27, Exo28,
  • the invention provides methods of making Exo proteins, comprising providing a cell comprising an Exo nucleic acid and subjecting the cell to conditions which allow the expression of Exo proteins.
  • the present invention provides methods for screening for a bioactive agent capable of binding to an Exo protein.
  • the method comprises combining an Exo ⁇ protein and a candidate bioactive agent, and determining the binding of the candidateagent to the Exo protein.
  • the present invention provides methods for screening for agents capable of interfering with the binding of an Exo protein and GS27.
  • the methods comprise combining an Exo protein, a candidate bioactiveagentand a GS27 protein, and determining 0 the binding of the Exo protein and the GS27 protein.
  • the present invention provides methods forscreeningforagents capable of interferingwith the binding of an Exo protein and rab7.
  • the methods comprise combining an Exo protein, a candidatebioactive agent and a rab7 protein, and determining the binding of the Exo protein and the rab7 protein
  • the presentinvention provides methods for screening for agents capable of interfering with the binding of an Exo protein and rab9
  • the methods comprise combining an Exo protein, a candidate bioactiveagentand a rab9 protein, and determining the binding of the Exo protein and the rab9 protein
  • the present invention provides methods for screening for agents capable of interfering with the binding of an Exo protein and snap-23
  • the methods comprise combining an Exo protein, a candidate bioactive agent and a snap-23 protein, and determining the binding of the Exo protein and the snap-23 protein
  • the present invention provides methods for screening for agents capable of interfering with the binding of an Exo protein and rab3a
  • the methods comprise combining an Exo protein, a candidatebioactiveagentand a rab3a protein, and determining the binding of the Exo protein and the rab3a protein
  • the presentinvention providesmethodsforscreening for agents capable of interfering with the binding of an Exo protein and rab11
  • the methods comprise combiningan Exo protein, a candidate bioactive agent and a rabl 1 protein, and determining the binding of the Exo protein and the rabl 1 protein
  • the present invention providesmethodsforscreeningfor agents capable of interfering with the binding of an Exo protein and rab3d
  • the methods comprise combiningan Exo protein, a candidatebioactiveagentand a rab3d protein, and determining the binding of the Exo protein and the rab3d protein
  • the present invention provides methodsforscreeningforagents capable of interfering with the binding of an Exo protein and rab ⁇
  • the methods comprise combining an Exo protein, a candidate bioactive agent and a rab ⁇ protein, and determining the binding of the Exo protein and the rab ⁇ protein
  • the presentinvention provides methodsforscreeningforagents capable of interfering with the binding of an Exo protein and alpha-snap
  • the methods comprise combining an Exo protein, a candidate bioactive agent and an alpha-snap protein, and determining the binding of the Exo protein and the alpha-snap protein
  • the present invention provides methods for screening for agents capable of interfering with the binding of an Exo protein and und ⁇ -1
  • the methods comprise combining an Exo protein, a candidate bioactive agent and an und ⁇ -1 protein, and determining the binding of the Exo protein and the und 8-1 protein
  • the present invention provides methods for screening for agents capable of interfering with the binding of an Exo protein and vamp3
  • the methods comprise combining an Exo protein, a candidatebioactiveagentand a vamp3 protein, and determining the binding of the Exo protein and the vamp3 protein
  • the invention provides methods for screening for an bioactive agent 0 capable of modulating the activity of an Exo protein
  • the method comprises the steps of adding a candidatebioactiveagentto a cell comprising a recombinant nucleic acid encoding an Exo protein, and determining the effect of the candidate bioactive agent on cellular activity
  • the cellular activity is exocytosis or vesicular transport
  • the invention provides a method of treating an exocytosisrelateddisorder ⁇ comprising administering an agent that interferes with specific binding of a protein selected from those shown in the Sequence Listing with a protein selected from the group consisting of GS27, Rab3a, Rab7, Rab9, Rab11 , Rab3d, Rab ⁇ , alpha snap, und 8-1 , vamp3, and snap-23, expressed in a tissue such that said disorder is ameolerated
  • Also provided herein is a method of treating an exocytosis related disorder comprising 0 administering to a patient an agent that binds to a protein encoded by a sequence selected from the group consisting of those set forth in the Sequence Listing, such that exocytosis is altered
  • a method of reducing or inhibiting exocytosis in a cell comprising administering an agent that interferes with specific binding of a protein selected from those 6 encoded by a sequence selected from the group consisting of SEQ ID NOS 1-61 (odd numbers) and 53-211w ⁇ th a protein selected from the group consisting of GS27, Rab3a, Rab7, Rab9, Rabl 1 , Rab3d, Rab ⁇ , alpha snap, und 8-1 , vamp3, and snap-23, expressed in said cell such that exocytosis is inhibited
  • the invention provides a method of neutralizing the effect of a protein 0 encoded by a sequence selected from the group consisting of SEQ ID NOS 1-61 (odd numbers) and 63-211 comprising contacting an agent specific for said protein with said protein in an amount sufficient to effect neutralization.
  • a sequence selected from the group consisting of SEQ ID NOS 1-61 (odd numbers) and 63-211 comprising contacting an agent specific for said protein with said protein in an amount sufficient to effect neutralization.
  • Figures 1 A and I B show the underlying mechanism of yeasttwo-hybridandyeastone-hybrid ⁇ systems.
  • Figure 1 A shows the two-hybrid system: GAL4A represent GAL4 transcription activation domain.
  • cDNA represents cDNA library inserts.
  • X represents any bait gene.
  • GAL4B represents GAL4 DNA binding domain.
  • HIS/lacZ indicates that the reporter gene is either HIS or lacZ.
  • Figure 2 shows the outline of yeast two-hybrid screening. Solid black dots represent 0 colonies on plates. Transformation steps of both bait plasmid and cDNA library plasmids are indicated.
  • Figure 3 shows the outline of yeast one-hybrid screening. Solid black dots represent colonies on plates.
  • Figures 4A-4D show vectors used in the yeast two-hybrid and one-hybrid screening, 5 respectively.
  • Figures4A-4B showthetwo-hybridvectors.
  • Bait vectors can be pHybLex/Zeo
  • the binding domain can be either GAL4 or LexA.
  • MCS underlined represents multiple cloning sites, where either bait gene or cDNA fragments should be cloned.
  • 2 ⁇ 0 Ori represents yeast2 micron replicationorigin.cDNAvectorscan be pYESTrp2(lnvitrogen), pAD-GAL4 (Stratagene), pACT2 (Clontech), pGADGH (Clontech), pGAD424 (Clontech), or pJG4-5 (Origene).
  • Activationdomain can be GAL4NP16, or other transcription activator.
  • Figures 4C-4D show the one-hybrid reporter vectors. D ⁇ A sequences of interest should be inserted into the multiple cloning sites (MCS) underlined.
  • MCS multiple cloning sites
  • the enzyme used to Linearize ⁇ reporter vector for integration is shown by solid arrow. The dashed arrow indicates the transcription of either HIS or lacZ gene.
  • the present invention provides Exo proteins and nucleic acids involved in the exocytotic pathway.
  • the Exo proteins are from vertebrates and more 0 preferably from mammals, including rodents (rats, mice, hamsters, guinea pigs, etc.), primates, farm animals (including sheep, goats, pigs, cows, horses, etc) and in the most preferred embodiment, from humans.
  • Exo protein of the present invention may be identified in several ways. "Protein” in this sense includes proteins, polypeptides, and peptides.
  • the Exo proteins of the invention fall into two general classes: proteins that are completely novel, i.e. are not part of a public database as of the time of discovery, although they may have homology to either known proteins or expressed sequence tags (ESTs).
  • ESTs expressed sequence tags
  • the Exo proteins are known proteins, but that were not known to be involved in exocytosis; i.e. they are identified herein as having a novel biological function. Accordingly, an Exo protein may be initially identified by its association with a protein known to be involved in exocytosis or vesicular transport.
  • Exo proteins bind to a protein selected from the group consisting of GS27, rab7, rab9, snap-23, rab3a, rab11, rab3d, rab ⁇ , alpha-snap, unc18-1, and vamp3.
  • Exo proteins may be novel or may have been known in the art to exist, but not known to bind to GS27, rab7, rab9, snap-23, rab3a, rab11, rab3d, rab ⁇ , alpha-snap, und ⁇ -1 , or vamp3.
  • the Exo proteins and nucleic acids are novel, compositions and methodsof useare provided herein.
  • Exo proteins and nucleic acids were known but not known to bind to GS27, rab7, rab9, snap-23, rab3a, rab11 , rab3d, rab ⁇ , alpha-snap, und ⁇ -1 , or vamp3, methods of use, i.e. functional screens, are provided.
  • Exo nucleic acids or Exo proteins are initially identified by substantial nucleic acid and/or amino acid sequence identity or similarity to the sequences provided herein.
  • ExonucleicacidsorExoproteins havesequenceidentity orsimilaritytothesequencesprovided herein as described below and bind to an exocytosis orvesiculartransportprotein.
  • Preferred exocytosis and vesiculartransportproteins include GS27, rab7, rab9, snap-23, rab3a, rab11 , rab3d, rab ⁇ , alpha-snap, und ⁇ -1 , and vamp3 (these proteins are known in the art are considered the same whether "-" is used within the name or capitals are used).
  • sequence identity or similarity can be based upon the overall nucleic acid or amino acid sequence.
  • SEQ ID NO: 1 shows nucleic acid sequence encoding at least a portionof mouse syntaxin4, Genbankaccessionno.: U76 ⁇ 32,described in HayJC, etal., J Cell Biol 199 ⁇ , 141 (7):14 ⁇ 9- 1502.
  • SEQ ID NO:3 shows nucleic acid sequence encoding at least a portion of mouse LZIP-1 and LZIP-2, Genbank accessionno.:AC003675,describedin BurbeloPD, etal. Gene 1994, 139(2):241-245.
  • SEQ ID NO:5 shows the nucleic acid sequence encoding at least a portion of mouse IL-3 receptor, Genbank accession no.:M29855, described in TabiraT, etal., Ann N Y Acad Sci. 1998, 640: 107-116.
  • SEQ ID NO:7 shows nucleic acid sequence encoding at least a portion of mouse IL-4 receptor, accession no.:M27969,describedin Ryan JJ, etal. , J Immunol.1998, 161 (4):1 ⁇ 11- 1621.
  • SEQ ID NO:9 shows a second nucleic acid sequence encoding at least a portion of mouse IL-4 receptor, accession no.:M27959, described in Ryan JJ, et al., J Immunol. 1996, 161(4):1 ⁇ 11-1 ⁇ 21.
  • SEQ ID NO:11 shows nucleic acid sequence encoding at least a portion of mouse LDL receptor-related protein 6 (Lrp6), accession no.:AF074265, described in Brown, SD, etal., Biochem. Biophys. Res. Commun. 248 (3):879-8 ⁇ (1996).
  • Lrp6 mouse LDL receptor-related protein 6
  • SEQ ID NO: 13 shows nucleic acid sequence encoding at least a portion of mouse abc2, accession no.:X76927, described in tiling M, et al., J Biol Chem 1997,11 ;272(15):10303-
  • SEQ ID NOS:1 ⁇ , 17, 19, 21 , 23, and 26 shownucleic acid sequences which encode Exo3-8, respectively.
  • SEQ ID NO:27 shows the nucleic acid sequence encoding Exo9, which may share some characteristics with human syntaxin16A, accession no.:AF00 ⁇ 937, described in Hay JC, et al., J Cell Biol 199 ⁇ , 141(7):14 ⁇ 9-1502.
  • SEQ ID NO:29 shows the nucleic acid sequence encoding Exo10, which which may share some characteristics with human putative RNA binding protein (RBP66), accession no.:U61334, described in Genomics 33:61-67 (1996).
  • SEQ ID NO:31 showsthenucleicacidsequenceencodingExo11 , which has some similarity with GenBank accession no.: M144063.
  • SEQ ID NO:33 shows the nucleicacid sequenceencoding Exo12, which has some similarity with GenBank accession no.: AA1031 ⁇ 5.
  • SEQ ID NO:3 ⁇ shows the nucleicacidsequenceencodingExol 3, which hassomesimilarity with GenBank accession no.: AA919222.
  • SEQ ID NO:37 shows the nucleic acid sequenceencodingExo14, which hassomesimilarity with GenBank accession no.:AA276016 and human (xs99).
  • SEQ ID NO:39 shows the nucleicacidsequenceencoding Exol ⁇ , which hassomesimilarity with GenBank accession no.:AA617266, and CREB-RP (creb-rp), Genebank accession no.: U31903.
  • SEQ ID NO:41 shows the nucleicacidsequenceencoding Exo16, which hassomesimilarity with GenBank accession no.:AA221293 and rat lamina-assocated peptide.
  • SEQ ID NO:43 shows the nucleic acid sequence encoding Exo17a, which has some similarity with GenBank accession no. : AA166109 and rat syntaxin ⁇ , Genebank accession no.:L20322, described in Rowe T, et al., Science 1998 279(6361 ):696-700.
  • SEQ ID NO:45 shows the nucleic acid sequence encoding Exo17b, which has some similarity with GenBank accession no. :AA166109 and rat syntaxin ⁇ , Genebank accession no.:L20822, described in Rowe T, et al., Science 199 ⁇ 279(5361 ):696-700.
  • SEQ ID NO:47 shows the nucleicacidsequenceencodingExol ⁇ , which has somesimilarity with GenBankaccession no. : AA166109and has some similarity to rat syntaxin ⁇ , Genebank accession no.:L20 ⁇ 22, described in Rowe T, et al., Science 1998 279(6361 ):696-700.
  • SEQ ID NO:49 shows the nucleicacidsequenceencodingExol 9, which hassomesimilarity with GenBank accession no.:U76832 and mouse syntaxin4, described in Hay JC, J Cell Biol 199 ⁇ , 141(7):1489-1502.
  • SEQ ID NO: ⁇ 1 shows the nucleic acid sequence which encodes Exo20.
  • SEQ ID NO:53 shows the nucleic acid sequence which encodes Exo21.
  • SEQ ID NO: ⁇ 4 shows the nucleic acid sequence encoding a portion of human axonal transporter of synaptic vesicles, Genbank accession no.: X90840.
  • SEQ ID NO: ⁇ shows the nucleic acid sequence encoding a portion of human cargo selection protein TIP47 (TIP47), Genbank accession no.: AF067140.
  • SEQ ID NO:56 shows the nucleic acid sequence encoding a portion of human cargo selection protein TIP47 (TIP47), Genbank accession no.: AF057140.
  • SEQ ID NO:57 shows the nucleic acid sequence encoding a portion of human cargo selection protein TIP47 (TIP47), Genbank accession no.: AF057140.
  • SEQ ID NO: ⁇ shows the nucleic acid sequence encoding a portion of human cargo selection protein TIP47 (TIP47), Genbank accession no.: AF057140.
  • SEQ ID NO:59 shows the nucleicacidsequenceencodinga portionof human tax interaction protein, Genbank accession no.: AF02 ⁇ 24.
  • SEQ ID NO:60 shows the nucleic acid sequenceencodinga portion of human tax interaction protein, Genbank accession no.: AF028824.
  • SEQ ID NO:61 shows the nucleic acid sequenceencodinga portionofhumantaxinteraction protein, Genbank accession no.: AF026824.
  • SEQ ID NO:62 shows the nucleicacid sequenceencodinga portionof human human inositol polyphosphate ⁇ -phosphatase, Genbank accession no.: M74161.
  • SEQ ID NO:63 shows the nucleic acid sequence encoding Exo22.
  • SEQ ID NO:64 shows the nucleic acid sequence encoding Exo23.
  • SEQ ID NO:65 shows the nucleic acid sequence encoding a portion of mouse C67BL/6J Sec61 protein complex gamma subunit; Genbank accession no.: U11027.
  • SEQ ID NO:66 shows the nucleic acid sequence encoding a portion of mouse HMG-1; GenBank accession no.: U00431.
  • SEQ ID NO:67 shows the nucleic acid sequence encoding a portion of mouse cyclin B2; GenBank accession no.: X66032.
  • SEQ ID NO:68 shows the nucleic acid sequence encoding a portion of mouse cyclin B2; GenBank accession no.: X66032.
  • SEQ ID NO:69 shows the nucleic acid sequence encoding a portion of mouse pancreatic beta-cell kinesin heavy chain; GenBank accession no.: U36090.
  • SEQ ID NO:70 shows the nucleic acid sequence encoding a portion of mouse pancreatic beta-cell kinesin heavy chain; GenBank accession no.: U66090.
  • SEQ ID NO:71 shows the nucleic acid sequence encoding a portion of mouse syntaxin4; GenBank accession no.:U76832.
  • SEQ ID NO:72 shows the nucleic acid sequence encoding a portion of mouse syntaxin4; GenBank accession no.: U76632.
  • SEQ ID NO:73 shows the nucleic acid sequence encoding a portion of mouse syntaxin4; GenBank accession no.:U76 ⁇ 32.
  • SEQ ID NO:74 shows the nucleicacid sequenceencodinga portion of mouse stearoyl-CoA desaturase (SCD2); GenBank accession no.: M26270.
  • SEQ ID NO:7 ⁇ shows the nucleic acid sequence encoding a portion of mouse spermidine aminopropyltransferase (Mspmsy); GenBank accession no.: AF031466.
  • SEQ ID NO:76 shows the nucleic acid sequence encoding a portion of mouse prothymosin alpha; GenBank accession no.: X66135.
  • SEQ ID NO:77 shows the nucleic acid sequence encoding a portion of mouse protein cofactor; GenBank accession no.: U74079.
  • SEQ ID NO:7 ⁇ shows the nucleic acid sequence encoding a portion of mouse outer dense fiber protein 2 (Odf2); GenBank accession no.: AF000966.
  • SEQ ID NO:79 shows the nucleic acid sequence encoding a portion of mouse protein expressed in E12 brain (clone C2); GenBank accession no.: X83569.
  • SEQ ID NO ⁇ O shows the nucleic acid sequence encoding a portion of mouse hnRNP K homologue,GenBank accession no L29769
  • SEQ ID NO 61 shows the nucleic acid sequence encoding a portion of mouse NRF1 (NFE2-related factor 1), GenBank accession no X78709
  • SEQ ID NO 82 shows the nucleic acid sequenceencodinga portion of mouse RNA-binding protein, GenBank accession no L17076
  • SEQ ID NO ⁇ 3 shows the nucleic acid sequence encoding a portion of mouse dynactinl , GenBank accession no U60312
  • SEQ ID NO 64 shows the nucleic acid sequence encoding a portion of mouse hormone-sensitive lipase, GenBank accession no UO ⁇ l ⁇
  • SEQ ID NO ⁇ 5 shows the nucleic acid sequence encoding a portion of mouse mtprda (human TPRD homologue), GenBank accession no AB00 ⁇ 516
  • SEQ ID NO 66 shows the nucleic acid sequence encoding Exo24, having some similarity with GenBank accession no AA266661
  • SEQ ID NO 67 shows the nucleic acid sequence encoding Exo25, having some similarity with GenBank accession no AA097037
  • SEQ ID NO ⁇ shows the nucleic acid sequence encoding Exo26, having some similarity with GenBank accession no AA097037
  • SEQ ID NO 89 shows the nucleic acid sequence encoding Exo27, having some similarity with GenBank accession no AA269474
  • SEQ ID NO 90 shows the nucleic acid sequence encoding Exo28, having some similarity with GenBank accession no AA55 ⁇ 6
  • SEQ ID NO 91 shows the nucleic acid sequence encoding Exo29, having some similarity with GenBank accession no AA770 ⁇ 39
  • SEQ ID NO:92 shows the nucleic acid sequence encoding Exo30, having some similarity with GenBank accession no.: AA770639.
  • SEQ ID NO:93 shows the nucleic acid sequence encoding Exo31 , having some similarity with GenBank accession no.: AA416604.
  • ⁇ SEQ ID NO:94 shows the nucleic acid sequence encoding Exo32, having some similarity with GenBank accession no.: AA416604.
  • SEQ ID NO:9 ⁇ shows the nucleic acid sequence encoding Exo33, having some similarity with GenBank accession no.: AA416504.
  • SEQ ID NO:96 shows the nucleic acid sequence encoding Exo34, having some similarity 0 with GenBank accession no.: AA416604.
  • SEQ ID NO:97 shows the nucleic acid sequence encoding Exo35, having some similarity with GenBank accession no.: AA415604.
  • SEQ ID NO:98 shows the nucleic acid sequence encoding Exo36, having some similarity with GenBank accession no.: AA416604.
  • ⁇ SEQ ID NO:99 shows the nucleic acid sequence encoding Exo37, having some similarity with GenBank accession no.: AA416604.
  • SEQ I D NO: 100 shows the nucleic acid sequence encoding Exo38, having some similarity with GenBank accession no.: AA416604.
  • SEQ ID NO: 101 shows the nucleic acid sequence encoding Exo39, having some similarity 0 with GenBank accession no.: AA416604.
  • SEQ ID NO: 102 shows the nucleic acid sequence encoding Exo40, having some similarity with GenBank accession no.: AA416604.
  • SEQ ID NO: 103 shows the nucleic acid sequence encoding Exo41 , having some similarity with GenBank accession no.: AA172926.
  • SEQ ID NO: 104 shows the nucleic acid sequence encoding Exo42, having some similarity with GenBank accession no.: AA2 ⁇ 130.
  • SEQ I D NO: 106 shows the nucleic acid sequence encoding Exo43, having some similarity with GenBank accession no.: AI131639.
  • SEQ ID NO: 106 shows the nucleic acid sequence encoding Exo44, having some similarity with GenBank accession no.: AA164709.
  • SEQ ID NO: 107 shows the nucleic acid sequence encoding Exo4 ⁇ , having some similarity with GenBank accession no.: AA266406.
  • SEQ I D NO: 10 ⁇ shows the nucleic acid sequence encoding Exo46, having some similarity with GenBank accession no.: AA5631 ⁇ .
  • SEQ ID NO: 109 shows the nucleic acid sequence encoding Exo47, having some similarity with GenBank accession no.: AA619170.
  • SEQ ID NO: 110 shows the nucleic acid sequence encoding Exo4 ⁇ , having some similarity with GenBank accession no.: AA519170.
  • SEQ ID NO:111 shows the nucleic acid sequence encoding Exo49, having some similarity with yeast ORMI, GenBank accession no.: AA175196.
  • SEQ ID NO: 112 shows the nucleic acid sequence encoding Exo ⁇ O, having some similarity with rat mt-GrpE no.1 precursor, GenBank accession no.: AA060 ⁇ 61.
  • SEQ ID NO: 113 shows the nucleic acid sequence encoding Exo ⁇ l , having some similarity with human CENP-F kinetochore protein, GenBank accession no.: AI034171.
  • SEQ ID NO: 114 shows the nucleic acid sequence encoding Exo ⁇ 2, having some similarity with human arfaptin 2 (putative target of ADP-ribosylation factor), GenBank accession no.:AA6439 ⁇ .
  • SEQ I D NO: 11 ⁇ shows the nucleic acid sequence encoding Exo ⁇ 3, having some similarity with human brain and reproductive organ-expressed protein (BRE); GenBank accession no.: AA200603.
  • SEQ ID NO: 116 shows the nucleic acid sequence encoding Exo ⁇ 4, having some similarity with human brain and reproductive organ-expressed protein (BRE); GenBank accession no.: AA200608.
  • SEQ ID NO: 117 shows the nucleic acid sequence encoding Exo ⁇ , having some similarity with human cell cycle progression 2 protein (CPR2); GenBank accession no.: W67077.
  • SEQ I D NO: 11 ⁇ shows the nucleic acid sequence encoding Exo ⁇ 6, having some similarity with human spliceosomeassociatedprotein(SAP14 ⁇ ); GenBankaccession no.: AH 19401.
  • SEQ ID NO: 119 shows the nucleic acid sequence encoding Exo ⁇ 7, having some similarity with mesocricetus auratus stearyl-CoA desaturase (FAR-17c); GenBank accession no.: AA337696.
  • SEQ ID NO: 120 shows the nucleic acid sequence encoding Exo ⁇ , having some similarity with rat inositol trisphosphate receptor subtype 3 (IP3R-3); GenBank accession no.: AA ⁇ 23026.
  • SEQ ID NO: 121 shows the nucleic acid sequence encoding Exo ⁇ 9, having some similarity with L-Asparaginase; GenBank accession no.: AH 13730.
  • SEQ ID NO: 122 shows the nucleic acid sequence encoding Exo60, having some similarity with L-Asparaginase; GenBank accession no.: AH 16730.
  • SEQ ID NO: 123 shows the nucleic acid sequence encoding Exo61 , having some similarity with human RB-binding protein 2 (RBBP-2); GenBank accession no.: AA756316.
  • SEQ I D NO: 124 shows the nucleic acid sequence encoding Exo62, having some similarity with human secreted apoptosis related protein 3 (SARP3); GenBank accession no.: AU01 ⁇ 90.
  • SEQ ID NO: 126 shows the nucleic acid sequence encoding Exo63, having some similarity with myosin heavy chain; GenBank accession no.: AA237764.
  • SEQ ID NO: 126 shows the nucleic acid sequence encoding Exo64, having some similarity with myosin heavy chain; GenBank accession no.: AA237764.
  • SEQ ID NO: 127 shows the nucleic acid sequence encoding Exo ⁇ , having some similarity with myosin heavy chain; GenBank accession no.: AA237764.
  • SEQ ID NO: 123 shows the nucleic acid sequence encoding Exo66, having some similarity with myosin heavy chain; GenBank accession no.: AA237764.
  • SEQ ID NO:129 shows the nucleic acid sequence encoding Exo67, having some similarity with rat tomosyn; GenBank accession no.: AA437465.
  • SEQ ID NO: 130 shows the nucleic acid sequence encoding Exo6 ⁇ , having some similarity with rat tomosyn; GenBank accession no.: AA437465.
  • SEQ ID NO: 131 shows the nucleic acid sequence encoding Exo69, having some similarity with rat tomosyn; GenBank accession no.: AA437465.
  • SEQ ID NO: 132 shows the nucleic acid sequence encoding Exo70, having some similarity with rat tomosyn; GenBank accession no.: AA437466.
  • SEQ ID NO: 133 shows the nucleic acid sequence encoding Exo71 , having some similarity with human mcag29 CTG repeat region; GenBank accession no.: AA039340.
  • SEQ ID NO: 134 shows the nucleic acid sequence encoding Exo72, having some similarity with rat G protein gamma-5 subunit; GenBank accession no.: AA021679.
  • SEQ ID NO: 136 shows the nucleic acid sequence encoding Exo73, having some similarity with rat G protein gamma-5 subunit; GenBank accession no.: AA021679.
  • SEQ ID NO: 136 shows the nucleic acid sequence encoding Exo74, having some similarity with rat G protein gamma-5 subunit; GenBank accession no.: AA021879.
  • SEQ ID NO: 137 shows the nucleic acid sequence encoding Exo75, having some similarity with rat G protein gamma- ⁇ subunit; GenBank accession no.: AA021879.
  • SEQ ID NO: 136 shows the nucleic acid sequence encoding Exo76, having some similarity with rat G protein gamma-5 subunit; GenBank accession no.: AA021679.
  • SEQ ID NO: 139 shows the nucleic acid sequence encoding Exo77, having some similarity with rat G protein gamma- ⁇ subunit; GenBank accession no.: AA021879.
  • SEQ ID NO: 140 shows the nucleic acid sequence encoding Exo78.
  • SEQ ID NO:141 shows the nucleic acid sequence encoding Exo79.
  • SEQ ID NO: 142 shows the nucleic acid sequence encoding Exo ⁇ O.
  • SEQ ID NO:143 shows the nucleic acid sequence encoding Exo81.
  • SEQ ID NO: 144 shows the nucleic acid sequence encoding a portion of human HLA-B-associated transcript 3; GenBank accession no.: M33619.
  • SEQ ID NO:14 ⁇ shows the nucleic acid sequence encoding a portion of human 0 HLA-B-associated transcript 3; GenBank accession no.: M33619.
  • SEQ ID NO:146 shows the nucleic acid sequence encoding a portion of human T cell leukemia/lymphoma 1 ; GenBank accession no.: X82240.
  • SEQ ID NO: 147 shows the nucleic acid sequence encoding Exo82.
  • SEQ ID NO:148 shows the nucleic acid sequence encoding Exo83; may have some homology with rat rabin3.
  • SEQ ID NO:149 shows the nucleic acid sequence encoding at least a portion of human KIAA0666; GenBank accession no.: AB014665.
  • SEQ ID NO: 160 shows the nucleic acid sequence encoding Exo84 which may have some 0 similarity with Rabin 3, GenBank accession no.: AA346676.
  • SEQ ID NO:161 shows the nucleic acid sequence encoding Exo ⁇ which may have some similarity with Rabin 3, GenBank accession no.: AA346676.
  • SEQ ID NO: 162 shows the nucleic acid sequence encoding Exo ⁇ which may have some ⁇ similarity with human KI 0665, GenBank accession no.: AA757034.
  • SEQ ID NO: 153 shows the nucleic acid sequence encoding a portion of mouse protein cofactor; GenBank accession no.: U74079.
  • SEQ ID N0.154 shows the nucleic acid sequence encoding a portion of mouse protein cofactor; GenBank accession no.: U74079.
  • SEQ ID NO:1 ⁇ 5 shows the nucleic acid sequence encoding Exo37, may have some similarity with calcium-dependent protein kinase, Genbank accession no.: AA770736.
  • SEQ ID NO: 156 shows the nucleic acid sequence encoding Exo88, may have some similarity with calcium-dependent protein kinase, Genbank accession no.: AA770736.
  • SEQ ID NO: 157 shows the nucleic acid sequence encoding Exo ⁇ 9, may have some similarity with calcium-dependent protein kinase, Genbank accession no.: AA770736.
  • SEQ ID NO: 153 shows the nucleic acid sequence encoding Exo90, may have some similarity with calcium-dependent protein kinase, Genbank accession no.: AA770736.
  • SEQ ID NO: 159 shows the nucleic acid sequence encoding Exo91 , may have some similarity with human mcag29 CTG repeat region, Genbank accession no.: AA473325.
  • SEQ ID NO: 160 shows the nucleic acid sequence encoding Exo92, may have some similarity with human mcag29 CTG repeat region, Genbank accession no.: AA473325.
  • SEQ ID NO:161 shows the nucleic acid sequence encoding Exo93, may have some similarity with Genbank accession no.: AA136122.
  • SEQ ID NO: 162 shows the nucleic acid sequence encoding Exo94, may have some similarity with Genbank accession no.: AA136122.
  • SEQ ID NO:163 shows the nucleic acid sequence encoding Exo9 ⁇ , may have some similarity with Genbank accession no.: AA133122.
  • SEQ ID NO: 164 shows the nucleic acid sequence encoding Exo96, may have some similarity with Genbank accession no.: AA133122.
  • SEQ ID NO: 165 shows the nucleic acid sequence encoding Exo97, may have some similarity with Genbank accession no.: AA136122.
  • SEQ ID NO: 166 shows the nucleic acid sequence encoding Exo98, may have some similarity with Genbank accession no.: AA060976.
  • SEQ ID NO: 167 shows the nucleic acid sequence encoding Exo99, may have some similarity with Genbank accession no.: AA277208.
  • SEQ ID NO: 168 shows the nucleic acid sequence encoding ExolOO, may have some similarity with Genbank accession no.: AA277208.
  • SEQ ID NO: 169 shows the nucleic acid sequence encoding Exo101 , may have some similarity with Genbank accession no.: AA467477.
  • SEQ ID NO: 170 shows the nucleic acid sequence encoding Exo102, may have some similarity with Genbank accession no.: AA467477.
  • SEQ ID NO:171 shows the nucleic acid sequence encoding Exo103, may have some similarity with Genbank accession no.: AA833213.
  • SEQ ID NO: 172 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403.
  • SEQ ID NO: 173 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
  • SEQ ID NO: 174 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403.
  • SEQ ID NO:175 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403.
  • SEQ ID NO: 176 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403.
  • SEQ ID NO: 177 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403.
  • SEQ ID NO:17 ⁇ shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403.
  • SEQ ID NO: 179 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
  • SEQ ID NO: 160 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
  • SEQ ID NO:1 ⁇ 1 shows the nucleic acid sequence encoding a portion of Rab2; GenBank 0 accession no.: X96403.
  • SEQ ID NO: 1 ⁇ 2 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
  • SEQ ID NO: 163 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
  • ⁇ SEQ ID NO: 164 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
  • SEQ ID NO:1 ⁇ shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
  • SEQ ID NO: 166 shows the nucleic acid sequence encoding a portion of Rab2; GenBank 0 accession no.: X96403.
  • SEQ ID NO:187 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
  • SEQ ID NO:188 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
  • SEQ ID NO: 189 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
  • SEQ ID NO: 190 shows the nucleic acid sequence encoding a portion of Rab5C; GenBank accession no.: AA230407.
  • SEQ ID NO: 191 shows the nucleic acid sequence encoding a portion of Rab ⁇ C; GenBank accession no.: AA230407.
  • SEQ ID NO: 192 shows the nucleic acid sequence encoding a portion of Rab ⁇ C; GenBank accession no.: AA230407.
  • SEQ ID NO: 193 shows the nucleic acid sequence encoding Exo104.
  • SEQ ID NO: 194 shows the nucleicacid sequence encoding Exol O ⁇ , a human gene similar to mouse testis-specific protein PBS13; may have some similarity to GenBank Accession no.: AA184366.
  • ⁇ SEQ ID NO: 196 shows the nucleic acid sequence encoding Exo106; may have some similarity to GenBank Accession no.: AA604490.
  • SEQ ID NO: 196 shows the nucleic acid sequence encoding Exo107; may have some similarity to GenBank Accession no.: AA504490.
  • SEQ ID NO: 197 shows the nucleic acid sequence encoding Exo108; may have some similarity to GenBank Accession no.: AI181750.
  • SEQ ID NO: 198 shows the nucleic acid sequence encoding Exo109; may have some similarity to GenBank Accession no.: AI181760.
  • ⁇ SEQ ID NO: 199 shows the nucleic acid sequence encoding Exo110; may have some similarity to GenBank Accession no.: AU043111.
  • SEQ ID NO:200 shows the nucleic acid sequence encoding Exol 11 , a human gene similar to rat alpha-soluble NSF attachment protein (SNAP).
  • SEQ ID NO 201 shows the nucleic acid sequence encoding Exo112 shows the nucleic acid sequence encoding Exo105, a human gene similar to mouse testis-spe fic protein PBS13, may have some similarity to GenBank Accession no AA184366
  • SEQ ID NO 202 shows the nucleic acid sequence encoding Exol 13 which may be similar to a mouse zinc finger protein
  • SEQ ID NO 203 shows the nucleic acid sequence encoding Exol 14 which may be similar to a mouse zinc finger protein
  • SEQ ID NO 204 shows the nucleic acid sequence encoding Exol 15 which may be similar to chicken c-hairy 1 , may have some similarity to GenBank Accession no AA116067
  • SEQ ID NO 205 shows the nucleic acid sequence encoding Exol 16 which may be similar to chicken c-hairy 1 , may have some similarity to GenBank Accession no AA116067
  • SEQ ID NO 206 shows the nucleic acid sequence encoding a portion of mouse syntax ⁇ n4, GenBank accession no U76332
  • SEQ ID NO 207 shows the nucleic acid sequence encoding a portion of mouse interleukin (IL) -3 receptor, GenBank accession no M29356
  • SEQ ID NO 20 ⁇ shows the nucleic acid sequence encoding a portion of mouse interleukin (IL) -3 receptor, GenBank accession no M29866
  • SEQ ID NO 209 shows the nucleic acid sequence encoding a portion of mouse low density lipoprotem (LDL) receptor-related protein, GenBank accession no AF074266
  • SEQ ID NO 210 shows the nucleic acid sequence encoding Exo117, similar to a human ANF126 zinc protein
  • SEQ ID NO 211 shows the nucleic acid sequence encoding Exo118, similar to a rat isoprenylated 67 kDa protein
  • Exo3-Exo11 ⁇ are novel
  • the Exoproteinsencodedby SEQ ID NOS 1- 61 (odd numbers) and Sequence ID NOS 63-211 are each novel in the aspect that they are shown herein to bind to an exocytosisorvesiculartransport protein, or fragment thereof, for the first time.
  • the proteins encoded by SEQ ID NOS: 1-51 (odd numbers) bind to GS27; the proteins encoded by SEQ ID NO:53 bind to rab7; the proteins encoded by SEQ ID NOS: ⁇ 4-64 bind to rab9; the proteins encoded by SEQ ID NOS:6 ⁇ -143 bind to snap-23; the proteins encoded by SEQ ID NOS: 144-148 bind to rab3a; ⁇ the proteins encoded by SEQ ID NOS:149-162 bind to rab11 ; the proteinsencoded by SEQ
  • a protein is a "Exo protein" if the overall sequence identity of the protein sequence to any one ofthe amino acid sequences encoded by SEQ ID NOS: 1- 61 , odd numbers, and SEQ ID NOS:53-211 , preferably those sequences encoding Exo3- 11 ⁇ , is preferably greater than about 76%, more preferably greater than about 60%, even more preferably greater than about ⁇ % and most preferably greater than 90%. In some ⁇ embodiments the sequence identity will be as high as about 93 to 96 or 93%. As is known in the art, a number of different programs can be used to identify whether a nucleic acid has sequence identity or similarity to a known gene or expression sequence tag (EST).
  • EST gene or expression sequence tag
  • Sequence identity will be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. 0 Math.2:4 ⁇ 2 (19 ⁇ 1), by the sequence identity alignmentalgorithmofNeedleman&Wunsch,
  • percent identity is calculated by FastDB based upon the following parameters: mismatch penalty of 1 ; gap penalty of 1 ; gap size penalty of 0.33; and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis," MacromoleculeSequencingand Synthesis, Selected Methods and Applications, pp 127-149 0 (19 ⁇ ), Alan R. Liss, Inc.
  • PILEUP createsa multiplesequencealignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment.
  • PILEUP uses a simplificationof the progressivealignmentmethod of Feng & Doolittle,J. Mol. Evol.35:351- 5 360 ( 1937); the method is similar to that described by Higgins & Sharp CABIOS 5: 151 -163 (1989).
  • Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
  • BLAST algorithm described in Altschul et al., J. Mol. Biol. 275, 403-410, (1990) and Karlin et al., PNAS USA 90:6873-6737 (1993).
  • WU-BLAST-2 program is the WU-BLAST-2 program which was obtained from
  • WU-BLAST-2 uses several search parameters, most of which are set to the default values.
  • the HSP S and HSP S2 0 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
  • a % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of 5 the "longer" sequence in the aligned region.
  • the "longer” sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
  • percent (%) nucleic acid sequence identity with respect to the coding sequence of the polypeptides identified herein is defined as the percentage of nucleotide 0 residuesin a candidatesequencethatare identicalwith the nucleotideresiduesin the coding sequence of the Exo protein.
  • a preferred method utilizes the BLASTN module of WU- BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
  • the alignment may include the introduction of gaps in the sequences to be aligned.
  • sequences which contain either more or fewer amino acids than the protein encoded by the sequences in the Sequence Listing it is understood that the percentage of sequence identity will be determined based on the number of identical amino acids in relation to the total number of amino acids.
  • sequence identity of sequences shorter than that shown in the Sequence Listing will be determined using the number of amino acids in the shorter sequence.
  • SEQ ID NOS: 1-51 (odd numbers)and SEQ ID NOS:53-211 were identified as follows.
  • a basic Blast search has been performed using program "Blastn” and database "nr".
  • Blastn is a NCBI BLAST family of program used to compared a nucleotide query sequence against a nucleotide sequence database
  • nr is a nucleotide sequence d a ta ba s e t h a t i n c l u d es a l l n o n - re d u n d a n t G e n B a n k C D S translations+PDB+SwissProt+PIR+PRF.
  • the nucleic acid sequences ofthe invention can be used to generate protein sequences. There are a variety of ways to do this, including cloning the entire gene and verifying its frame and amino acid sequence, or by comparing it to known sequences to search for homology to provide a frame, assuming the novel Exo protein has homology to some protein in the database being used.
  • the nucleic acid sequences are input into a program that will search all three frames for homology. This is done in a preferred embodiment using the following NCBI Advanced BLAST parameters.
  • the program is blastx or blastn.
  • the database is nr.
  • the input data is as "Sequence in FASTA format".
  • the organism list is "none".
  • the "expect” is 10; the filter is default.
  • the “descriptions” is 600, the “alignments” is 600, and the “alignment view” is pairwise.
  • the "Query Genetic Codes” is standard (1).
  • the matrix is BLOSUM62; gap existence cost is 11 , per residue gap cost is 1 ; and the lambda ratio is .65 default. This results in the generation of a putative protein sequence. While this program can be used to generatea preferredproteinsequence, it is understood thatthepresentinventionprovides polypeptides encoded by each of the three frames of each nucleic acid provided herein. Thus, when a protein encoded by the nucleic acid herein is described, the skilled artisan understands that the protein begins with the first amino acid encoded by the first codon of the coding region, which is not necessarily the first nucleotide in the sequence listing.
  • the sequences of the present invention may contain sequencing errors. That is, there may be incorrect nucleosides, frameshifts, unknownnucleosides.orothertypesofsequencingerrorsin any ofthe sequences; however, the correct sequences will fall within the homology and stringency definitions herein.
  • the first 200 bases or so of sequence contains the fewest errors.
  • the Exo proteins are encoded by a nucleic acid comprising the first 100 nucleotides of the sequences set forth in the Sequence Listing and bind to an exocytosis or vesicular transport proteinorfragment thereof.
  • Exo proteins of the present invention may be shorter or longer than the amino acid sequences encoded by the nucleic acids shown in the Sequence Listing.
  • Exo proteins can be portions or fragments of the amino acid sequences encoded by the nucleic acid sequences provided herein.
  • fragmentsof Exo proteins are considered Exo proteins if a) they share at leastoneantigenic epitope; b) have at least the indicated sequence identity; c) and preferably have Exo biologicalactivity, including binding to an exocytosis or vesicular transport protein.
  • the sequence is used diagnostically, that is, when the presence or absence of Exo protein nucleic acid is determined, only the indicated sequence identity is required.
  • nucleic acids ofthe present invention may also be shorteror longerthan the sequences in the Sequence Listing.
  • Exo proteins can be made that are longer than those depicted in the Sequence Listings; for example, by the addition of epitope or purification tags, the addition of other fusion sequences, or the elucidation of additional coding and non-coding sequences.
  • a fluorescent peptide such as Green Fluorescent Peptide (GFP)
  • GFP Green Fluorescent Peptide
  • Exo proteins may also be identified as encoded by Exo nucleic acids which hybridize to any one of the sequences depicted in SEQ ID NOS: 1-51 , odd numbers, and SEQ ID NOS:53-211 , preferably those encoding Exo3-11 ⁇ . Hybridization conditions are further described below.
  • an Exo protein when an Exo protein is to be used to generate antibodies, an Exo protein must share at least one epitope or determinant with the full length protein.
  • epitope or “determinant” herein is meant a portion of a protein which will generate and/or bind an antibody. Thus, in most instances, antibodies made to a smaller Exo protein will be able to bind to the full length protein. In a preferred embodiment, the epitope is unique; that is, antibodies generated to a unique epitope show little or no cross-reactivity.
  • antibody includes antibody fragments, as are known in the art, including Fab Fab 2 , single chain antibodies (Fv for example), chimeric antibodies, etc., either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies.
  • the antibodies to Exo are capable of reducing or eliminating the biological function of Exo, as is described below. That is, the addition of anti-Exo antibodies(eitherpolyclonal or preferably monoclonal) to Exo (or cells containing Exo) may reduce or eliminate the Exo activity. Generally, at least a 2 ⁇ % decrease in activity is preferred, with at least about 50% being particularly preferred and about a 95-100% decrease being especially preferred.
  • the Exo antibodies of the invention specifically bind to Exo proteins.
  • the antibodies specifically bind to Exo proteins.
  • “specifically bind” herein is meant that the antibodies bind to the protein with a binding constant in the range of at least 10 "4 - 10 "6 M " ⁇ with a preferred range being 10 "7 - 10 "9 M “1 .
  • Antibodies are further described below.
  • the overall sequence identity of the nucleic acid sequence is commensurate with amino acid sequence identity but takes into account the degeneracy in the genetic code and codon bias of different organisms. Accordingly, the nucleic acid sequence identity may be either lower or higher than that of the protein sequence.
  • the sequence identity of the nucleic acid sequence as compared to the nucleic acid sequences ofthe Sequence Listing is preferablygreaterthan 75%, more preferablygreater thanabout30%, particularly greaterthan about ⁇ % and mostpreferablygreaterthan90%. In some embodiments the sequence identity will be as high as about 93 to 95 or 9 ⁇ %.
  • an Exo nucleic acid encodes an Exo protein.
  • an extremely large number of nucleic acids may be made, all of which encode the Exo proteins of the present invention.
  • those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the Exo.
  • the nucleic acid is determined through hybridization studies
  • nucleic acids which hybridize under high stringency to the nucleic acid sequences shown in the sequence listing, or its complement is considered an Exo gene High stringency conditions are known in the art, see for example Maniatis etal , Molecular Cloning A Laboratory Manual, 2d Edition, 1969, and Short Protocols in Molecular Biology, ed Ausubel.
  • stringent conditions are selected to be about ⁇ -10°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH
  • T m is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 60% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium)
  • Stringent conditions will be those in which the salt concentration is less than about 1 0 sodium ion, typically about 0 01 to 1 0
  • less stringent hybridization conditions are used, for example, moderate or low stringency conditions may be used, as are known in the art, see Maniatis and Ausubel, supra, and Tijssen, supra
  • Exo proteins and nucleic acids of the present invention are preferably recombinant
  • nucleic acid may refer to either DNA or RNA, or molecules which contain both deoxy- and nbonucleotides
  • the nucleic acids include genomic DNA, cDNA and oligonucleotides including sense and anti-sense nucleic acids
  • Such nucleic acids may also contain modifications in the nbose-phosphate backbone to increase stability and half life of such molecules in physiological environments
  • the nucleicacid may be doublestranded.singlestranded, or contain portionsof both double stranded or single stranded sequence
  • the depiction of a single strand also defines the sequence of the other strand (“Crick"), thus the sequences depicted in the Sequence Listing also includethe complement of the sequence.
  • recombinant nucleic acid herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleicacid by endonucleases, in a form not normally found in nature.
  • Exo nucleic acid in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e. using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention.
  • a "recombinant protein” is a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid as depicted above.
  • a recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics.
  • the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host, and thus may be substantially pure.
  • an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight ofthe total protein in a given sample.
  • a substantially pure protein comprises at least about 76% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred.
  • the definition includes the production of an Exo protein from one organism in a different organism or host cell.
  • the protein may be made at a significantly higher concentration than is normally seen, through the use of a inducible promoter or high expression promoter, such that the protein is made at increased concentration levels.
  • the protein may be in a form not normally found in nature, as in the addition of an epitope tag or amino acid substitutions, insertions and deletions, as discussed below.
  • Exo proteins of the present invention are amino acid sequence variants. These variants fall into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding an Exo protein, using cassette or PCR mutagenesisorothertechniqueswell known in the art, to produceDNAencodingthe variant, and thereafterexpressingthe DNA in recombinant cell culture as outlined above. However, variant Exo protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques.
  • Amino acid sequence variants are characte ⁇ zed by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the Exo protein ammo acid sequence
  • the variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below
  • the mutation per se need not be predetermined
  • random mutagenesis may be conducted at the targetcodon or region and theexpressedExovariantsscreenedfortheoptimalcombination of desired activity
  • Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis Screening of the mutants is done using assays of Exo protein activities
  • Ammo acid substitutions are typically of single residues, insertions usually will be on the order of from about 1 to 20 ammo acids, although considerably larger insertions may be tolerated Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger
  • Substantialchangesin functionor immunological identity are made by selectmgsubstitutions that are less conservative than those shown in Chart I
  • substitutions may be made which more significantly affect the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure, the charge or hydrophobicity of the molecule at the target site, or the bulk of the side chain
  • substitutions which in general are expected to produce the greatest changes in the poly peptide's properties are those in which (a) a hydrophi c residue, e g sery I or threonyl, is substituted for (or by) a hydrophobic residue, e g leucyl, isoleucyl, phenylalanyl, valyl or alanyl, (b) a cysteine or prolme is substituted for (or by) any other residue, (c) a residue having an electropositive side chain, e g lysyl, argmyl, or histid
  • variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurrmganalogue, although variants also are selected to modify the characteristics ofthe Exo proteins as needed Alternatively, the variant may be designed such that the biological activity of the Exo protein is altered For example, glycosylationsites may be alteredor removed Similarly, mutationswithin the kinasedomain and/or the cell death domain may be made
  • Covalent modifications of Exo polypeptides are included within the scope of this invention
  • One type of covalent modification includes reacting targeted aminoacid residues of an Exo polypeptide with an organic derivatizmg agent that is capable of reacting with selected side chamsorthe N-orC-terminalresiduesof an Exo polypeptide Denvatizationwith bifunctional agents is useful, for instance, for crosslmking Exo to a water-insoluble support matrix or surface for use in the method for purifying anti-Exo antibodies or screening assays, as is more fully described below
  • Commonly used crosslink gagents include, e g , 1 , 1 -b ⁇ s(d ⁇ azo- acetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimideesters, for example, esters with 4-az ⁇ dosal ⁇ cyl ⁇ c acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'
  • Addition of glycosylation sites to Exo polypeptides may be accomplished by altering the am o acid sequence thereof
  • the alteration may be made, for example, by the addition of, or substitution by, one or more serine orthreonine residues to the native sequence Exo polypeptide (for O-linked glycosylation sites)
  • the Exo aminoacid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the Exo polypeptide at preselected bases such that codons are generated thatwill translate into the desired ammo acids
  • Exo polypeptide Removal of carbohydrate moieties present on the Exo polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for ammo acid residues thatserveas targets for glycosylation
  • Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al , Arch Biochem Biophvs , 259 52 (1987) and by Edge et al , Anal Biochem , 118 131 (1931)
  • Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo-and exo-glycosidases as described by Thotakura et al , Meth Enzymol , 138 350 (1987)
  • Exo polypeptide comprises linking the Exo polypeptide to one of a variety of nonproteinaceous polymers, e g , polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U S Patent Nos 4,640,836, 4,496,689, 4,301 ,144, 4,670,417, 4,791 ,192 or 4,179,337
  • Exo polypeptides of the present invention may also be modified in a way to form chimeric molecules comprising an Exo polypeptide fused to another, heterologous polypeptide or am o acid sequence
  • a chimeric molecule comprises a fusion of an Exo polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind
  • the epitope tag is generally placed at the ammo-orcarboxy I- terminus of the Exo polypeptide
  • the presence of such epitope-tagged forms of an Exo polypeptide can be detected using an antibody against the tag polypeptide
  • provision of the epitope tag enables the Exo polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag
  • the chimeric molecule may comprise a fusion of an Exo polypeptide with an immunoglobulin or a particular region of an immunoglobu n
  • a fusion could be to the Fc region of an IgG molecule as discussed further below
  • Vanoustag polypeptidesandtheirrespectiveantibodies are well known in the art Examples include poly-histidme (poly-his) or poly-histidme-glycine (poly-his-gly) tags, the flu HA tag polypeptide and its antibody 12CA5 [Field et al , Mol Cell Biol . 8 2169-2165 (1988)], the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al , Molecular and Cellular Biology, 5 3610-3616 (1935)], and the Herpes Simplex virus glycoprote ⁇ n D (gD) tag and its antibody [Paborsky etal . Protein Engineering.
  • tag polypeptides include the Flag-peptide [Hopp et al , BioTechnology. 6 1204-1210 (1988)], the KT3 epitope peptide [Martin etal . Science, 255 192-194(1992)], tubulm epitope peptide [Skinner et al , J Biol Chem . 266 15163-15166 (1991)], and the T7 gene 10 protein pept ⁇ detag [Lutz-Freyermuthetal . Proc Natl Acad Sci USA, 87 6393-
  • Exo proteins of the Exo family and Exo proteins from other organisms are cloned and expressed as outlined below
  • probe or degenerate poly merase chain reaction (PCR) primer sequences may be used to find other related Exo proteins from humans or other organisms
  • particularly useful probe and/or PCR primer sequences include the unique areas of the Exo nucleic acid sequence
  • preferred PCR primers are from about 16 to about 36 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosine as needed
  • the conditions for the PCR reaction are well known in the art It is thereforealso understood that provided along with the sequences in the sequences listed herein are portions of those sequences, wherein unique portions of 16 nucleotides or more are particularly preferred
  • the skilled artisan can routinely synthesize or cut a nucleotide sequence to the desired length
  • Exo nucleic acid Once the Exo nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombmedtoform the entire Exo nucleic acid Once isolated from its natural source, e g , contained within a plasmid or other vector or excised therefrom as a linear nucleic acid segment, the recombinant Exo nucleic acid can be further-used as a probe to identify and isolate other Exo nucleic acids It can also be used as a "precursor" nucleic acid to make modified or variant Exo nucleic acids and proteins The skilled artisan understands that wherein two or more nucleic acids overlap, the overlapping port ⁇ on(s) of one of the overlapping nucleic acids can be omitted and the nucleic acids combined for example, by gation, to form a longer linear Exo nucleic acid so as to, for example, encode the full length or mature peptide The same applies to the ammo acid sequences of Exo polypeptides in that
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ⁇ bosome binding site Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleicacid sequence
  • a promoterorenhance ⁇ s operably Imkedto a coding sequence if it affects the transcription of the sequence, or a ⁇ bosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase However, enhancers do not have to be contiguous Linking is accomplished by ligation at convenient restriction sites If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice The transcnptional and translational regulatory nucleic acid will
  • the transcnptional and translational regulatory sequences may include, but are not limited to, promoter sequences, nbosomal binding sites, transcnptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences
  • the regulatory sequences include a promoterand transcnptional start and stop sequences
  • Promoter sequences encode either constitutive or mducible promoters
  • the promoters may be either naturally occurring promoters or hybrid promoters Hy rid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention
  • the expression vector may comprise additional elements
  • the expression vector may have two replication systems, thus allowing it to be maintained in twoorganisms, for example in mammalian or insect cells forexpressionand in a procaryotic host for cloning and amplification
  • the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct
  • the integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector Constructs for integrating vectors are well known in the art
  • the expression vector contains a selectable marker gene to allow the selection of transformed host cells Selection genes are well known in the art and will vary with the host cell used
  • a preferred expression vector system is a retroviral vector system such as is generally described in PCT/US97/01019 and PCT/US97/01048, both of which are hereby expressly incorporated by reference
  • Exo proteins ofthe present invention are produced by culturing a host cell transformed with an expressionvectorcontainmgnucleicacidencodingan Exo protein, undertheappropnate conditions to induce or cause expression of the Exo protein
  • the conditions appropriate for Exo protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation
  • the use of constitutive promoters in the expression vector will require optimizmgthegrowthand proliferationof the host cell, while the use of an mducible promoter requires the appropriate growth conditionsfor induction
  • the timing ofthe harvest is important
  • the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield
  • Appropriate host cells include yeast, bacteria, archebacte ⁇ a, fungi, and insect and animal cells, including mammalian cells Of particular interest are Drosophila melangaster cells, Saccharomycescerevisiae and otheryeasts, E coli, Bacillussubtilis, SF9cells, C129cells,
  • 293 cells Neurospora, BHK, CHO, COS, and HeLa cells, fibroblasts, Schwanoma cell lines, immortalized mammalian myeloid and lymphoidcell lines, Jurkatcells, livercells, mammary cells, sperm, egg, ad ⁇ pocytes,granulocytes,adrenalchromaffin cells, mast cells, basophils, endocrine and exocnne cells, muscle cells, eosinophils and neuronal cells
  • mammalian expressionsystems are also known in the art, and include retroviralsystems
  • a mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequencefor Exo protein into mRNA
  • a promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the transcription initiation site The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site
  • a mammalian promoter will also contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation
  • an upstream promoter element determines the rate at which transcription is initiated and can act in either orientation
  • Of particular use as mammalian promoters are the promoters from mamma
  • transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence The 3' terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation
  • transcription terminator and polyadenlytion signals includethosede ⁇ ved form SV40
  • Exo proteins are expressed in bacterial systems Bacterial expression systems are well known in the art
  • a suitable bacterial promote ⁇ any nucleicacid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription ofthe coding sequence of Exo protein into mRNA
  • a bacterial promoter has a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site
  • Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactoseand maltose, and sequencesderivedfrom biosynthetic enzymes such as tryptophan Promoters from bacte ⁇ ophage may also be used and are known in the art
  • synthetic promoters and hybrid promoters are also useful, for example, the fac promoter is a hybrid of the trp and lac promoter sequences
  • abacterialpromotercan include natu rally occurring promotersof non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription ln addition to a functioning promoter sequence, an efficient ribosome binding site is desirable In E coli, the ribosome binding site is called the Shme-Delgarno (SD) sequence and includes an initiation codon and a sequence 3-9 nucleotides in length located 3 - 11 nucleotides upstream of the initiation codon
  • SD Shme-Delgarno
  • the expression vector may also include a signal peptide sequence that provides for secretion of the Exo protein in bacteria
  • the signal sequence typically encodes a signal peptide comprised of hydrophobic am o acids which direct the secretion of the protein from the cell, as is well known in the art
  • the protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria)
  • the bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol.erythromycin, kanamycm, neomycinandtetracyc ne Selectablemarkers also include biosynthetic genes, such as those in the histidme, tryptophan and leucine biosynthetic pathways
  • Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E coli, Streptococcus cremons, and Streptococcus lividans, among others
  • the bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others
  • Exo proteins are produced in insect cells
  • Expression vectors for the transformation of insect cells and in particular, baculovirus-based expression vectors, are well known in the art
  • Exo protein is produced in yeast cells
  • Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candidaalbicansand C maltosa, Hansenula polymorpha, Kluyveromycesfragilis and K lactis, Pichia guillenmondii and P pastons, Schizosaccharomyces pombe, and Yarrowia lipolytica
  • Preferred promoter sequences for expression in yeast include the mducible GAL1.10 promoter, the promoters from alcohol dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase, hexokmase, phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and the acid phosphatase gene
  • Yeast selectable markers include ADE2, HIS
  • the Exo protein may also be made as a fusion protein, using techniques well known in the art Thus, for example, for the creation of monoclonal antibodies, if the desired epitope is small, the Exo protein may be fused to a carrier protein to form an immunogen Alternatively, the Exo protein may be made as a fusion protein to increase expression, or for other reasons For example, when the Exo protein is an Exo peptide, the nucleic acid encoding the peptide may be linked to othernucleicacid for expression purposes Similarly, Exo proteins of the invention can be linked to protein labels, such as green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), etc
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • BFP blue fluorescent protein
  • YFP yellow fluorescent protein
  • the Exo nucleic acids, proteins and antibodies of the invention are labeled
  • labeled herein is meant that a compound has at least one element, isotope or chemical compound attached to enablethe detectionof the compound
  • labels fall into three classes a) isotopic labels, which may be radioactive or heavy isotopes, b) immune labels, which may be antibodies or antigens, and c) colored or fluorescent dyes
  • isotopic labels which may be radioactive or heavy isotopes
  • immune labels which may be antibodies or antigens
  • colored or fluorescent dyes The labels may be incorporated into the compound at any position
  • the Exo protein is purified or isolated after expression
  • Exo proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample Standard purification methods includeelectrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusmg
  • the Exo protein may be purified using a standard anti-Exo antibody column Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful For general guidance in suitable purification techniques, see Scopes, R , Protein Purification, Sp ⁇ nger-Verlag, NY (1982) The degree of purification necessary will vary depending on the use of the Exo protein In some instances no purification will be necessary
  • Exo proteins and nucleic acids are useful in a number of applications
  • the nucleotide sequences (or their complement) encoding Exo proteins have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA Exo protein nucleic acid will also be useful for the preparation of Exo protein polypeptides by the recombinant techniques described herein
  • the full-length native sequence Exo protein gene, or portions thereof, may be used as hyb ⁇ dizationprobesfora cDNAiibrary to isolate the full-length Exo protemgeneorto isolate still other genes (for instance, those encoding naturally-occurring variants of Exo protein or Exo protein from other species) which have a desired sequenceidentity to the Exo protein coding sequence
  • the length ofthe probes will be about 20 to about 50 bases
  • hybridization probes may be derived from the nucleotide sequences herein or from genomic sequences including promoters, enhancer elements and mtrons of native sequences as provided herein
  • a screening method will comprise isolating the coding region of the Exo protein gene using the known DNA sequence to synthesize a selected probe of about 40 bases
  • Hybridization probes may be labeled by a variety of labels, including radionucleotides such as 32 P or 35 S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotm coupling systems
  • Labeled probes having a sequence complementary to that of the Exo protein gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes
  • the probes may also be employed in PCR techniques to generate a pool of sequences for identification of closely related Exo protein coding sequences
  • Nucleotide sequences encoding a Exo protein can also be used to construct hybridization probes for mapping the gene which encodes that Exo protein and forthe genetic analysis of individuals with genetic disorders
  • the nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries
  • Nucleic acids which encode Exo protein or its modified forms can also be used to generate e ⁇ thertransgen ⁇ can ⁇ malsor"knockout” animalswhich, in turn, are useful in the development and screening of therapeutically useful reagents
  • a transgenic animal e g , a mouse or rat
  • a transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops
  • cDNA encoding an Exo protein can be used to clone genomic DNA encoding an Exo protein in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express the desired DNA Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U S Patent Nos 4,736,866 and 4,870,009 Typically, particular cells would be
  • non-human homologues of the Exo protein can be used to construct a Exo protein "knock out" animal which has a defective or altered gene encoding an Exo protein as a result of homologous recombination between the endogenous gene encoding an Exo protein and altered genomic DNA encoding an Exo protein introduced into an embryonic cell of the animal
  • cDNA encoding an Exo protein can be used to clone genomicDNAencodingan Exo protein in accordancewith established techniques
  • a portion of thegenomicDNAencodmgan Exo protein can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration
  • flanking DNA typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [seee g , Thomasand Capecchi, Cell, ⁇ . 603 (1987) for a description of homologous recombination vectors]
  • the vector is introduced into an embryonic stem cell line (e g , by electroporation) and cells in which the introduced DNA has homologously recombmed with the endogenous DNA are selected [see e g , Li etal , Cell, 69 915 (1992)]
  • the selected cells are then injected into a blastocyst of an animal (e g , a mouse or rat) to form aggregation chimeras [see e g , Bradley, in Teratocarcmomasand Embryonic Stem Cells A Practical Approach, E J Robertson, ed (IRL, Oxford, 1987), pp 113-152]
  • a chimeric embryocanthen be implantedintoa suitablepseudopregnantfemalefosteranimal and the embryo brought to term to create a "knock out" animal
  • Progeny harboring the homologously recombmed DNA in theirgermcellscan be identifiedbystandardtechniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the Exo protein polypeptide It is understood that cell based knock-out or "kno
  • Nucleic acid encoding the Exo polypeptides, antagonists or agonists may also be used in gene therapy
  • Gene therapy includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA
  • Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane (Zamecnik etal , Proc Natl Acad Sci USA 83.4143-4146 [1986]) The oligonucleotides can be modified to enhance their uptake, e g by substituting their negatively charged phosphodiester groups by un
  • nucleic acids there are a variety of techniques available for introducing nucleic acids into viable cells
  • the techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells ofthe intended host
  • Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphateprecipitationmethod, etc
  • the currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al , Trends in Biotechnology 11. 206-210 [1993])
  • an agent that targets the target cells such as an antibodyspecificfora cell surface membraneproteinorthetargetcell, a Iigandfora receptor on the target cell, etc
  • proteins which bind to a cell surface membrane protein associated with endocytosis may be used fortargetingand/orto facilitate uptake, e g capsid proteins orfragments thereof tropicfor a particular cell type, antibodies for proteins which undergo mternalization in cycling, proteins that target intracellular loca zation and enhance intracellular half-life
  • receptor-mediated endocytosis is described, for example, by Wu etal , J Biol Chem 262, 4429-4432 (1987), and Wagner et al , Proc Natl
  • Exo proteins, nucleic acids, modified proteins and cells containing the native or modified Exo proteins are used in screening assays Identification of this important exocytosis protein permits the design of drug screening assays for compounds that modulate Exo activity
  • Screens may be designed to first find candidate agents that can bind to Exo proteins, and then these agents may be used in assays that evaluate the ability of the candidate agent to modulate Exo activity
  • assays that evaluate the ability of the candidate agent to modulate Exo activity
  • the methods comprise combining an Exo protein and a candidate bioactive agent, and determining the binding ofthe candidate agent to the Exo protein
  • Preferred embodiments utilize the human Exo protein, although other mammalian proteins may also be used, including rodents (mice, rats, hamsters, guinea pigs, etc ), farm animals (cows, sheep, pigs, horses, etc ) and primates
  • rodents mice, rats, hamsters, guinea pigs, etc
  • farm animals cows, sheep, pigs, horses, etc
  • primates primates
  • variant or derivative Exo proteins may be used, including deletion Exo proteins as outlined above
  • candidatebioactiveagent or "exogeneous compound” as used herein describes any molecule, e g , protein, oligopeptide, small organic molecule, polysaccha ⁇ de, polynucleotide, etc , with the capability of directly or indirectly altering the bioactivity of Exo Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations Typically, one of these concentrations serves as a negative control, i e , at zero concentration or below the level of detection
  • Candidateagents encompass numerouschemicalclasses, though typically they areorganic molecules, preferably small organic compounds having a molecular weight of more than
  • Candidate agents comprise functional groups necessaryforstructural teractionwith proteins, particularlyhydrogen bonding, and typically include at least an amme, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more ofthe above functionalgroups
  • Candidateagents are also found among biomolecules including peptides, saccha des, fatty acids, steroids, pu ⁇ nes, pynmidines, derivatives, structural analogs or combinations thereof Particularly preferred are peptides
  • Candidate agents are obtained from a wide variety of sources including brariesof synthetic ornaturalcompounds forexample.numerousmeansare available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extractsareavailableor readily produced Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, estenfication, amidification to produce structural analogs
  • thecand ⁇ date bioactive agents are proteins
  • protein herein is meantatleasttwocovalentlyattachedaminoacids, which includesprotems, polypeptides, o gopeptides and peptides
  • the protem may be madeupofnaturallyoccurnngamino acids and peptide bonds, or synthetic peptidomimetic structures
  • “ammo acid”, or “peptide residue”, as used herein means both naturally occurring and synthetic ammo acids
  • homo-phenylalamne, citrullme and noreleucme are considered ammo acids for the purposes of the invention
  • Ammo acid also includes imino acid residues such as prolme and hydroxypro ne
  • the side chains may be in either the (R) or the (S) configuration lnthepreferredembod ⁇ ment,theam ⁇ noac ⁇ dsare ⁇ nthe(S)orL-configurat ⁇ on If non-naturally occurring side chains are used, non-a
  • the candidate bioactive agents are naturally occurring proteins or fragments of naturally occunng proteins
  • cellular extracts containing proteins, or random or directed digests of protemaceous cellular extracts may be used in this way libraries of procaryotic and eukaryotic proteins may be made for screening agamstExo
  • the candidate bioactive agents are peptides of from about ⁇ to about 30 ammo acids, with from about ⁇ to about 20 ammo acids being preferred, and from about 7 to about 16 being particularly preferred
  • the peptides may be digests of naturally occunng proteins as is outlined above, random peptides, or “biased” random peptides By "randomized” or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotidesandaminoacid
  • the library is fully randomized, with no sequence preferences or constants at any position
  • the library is biased That is, some positions within the sequenceareeitherheldconstant.orare selected from a limited number of possibilities
  • the nucleotides or am o acid residues are randomized within a defined class, for example, of hydrophobic am o acids, hydrophi c residues, stencally biased (either small or large) residues, towards the creation of cystemes, for cross-linking, prolines for SH-3 domains, sennes, threon es, tyrosmes or histidmes for phosphorylation sites, etc , or to purmes, etc
  • the candidate bioactive agents are nucleic acids
  • nucleic ac ⁇ d or "ol ⁇ gonucleot ⁇ de”or grammatical equivalents herein means at leasttwo nucleotides covalently linked together
  • a nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al , Tetrahedron 49(10) 1925 (1993) and references therein, Letsmger, J
  • nucleic acid candidate bioactive agents may be naturally occunng nucleicacids, randomnucle ⁇ cac ⁇ ds,or"b ⁇ ased”random nucleic acids
  • nucleic acid candidate bioactive agents may be naturally occunng nucleicacids, randomnucle ⁇ cac ⁇ ds,or"b ⁇ ased”random nucleic acids
  • digestsofprocaryoticoreucaryoticgenomes may beusedasisoutlinedabove for proteins
  • the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature
  • the assays provided utilize Exo proteins as defined herein
  • portions of Exo proteins are utilized, in a preferred embodiment, portions having Exo activity are used Exo activity is described further below and includes binding activity to GS27, rab7, rab9, snap-23, rab3a, rab11 , rab3d, rab ⁇ , alpha-snap, und 8-1 , vamp3 or Exo protein modulatorsasfurtherdescribed below
  • the assays described herein may utilize either isolated Exo proteins or cells comprising the Exo proteins
  • the Exo protein or the candidate agent is non-diffusibly bound to an insoluble support having isolated sample receiving areas (e g a microtiterplate, an array, etc )
  • the insolublesupports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening
  • the surface of such supports may be solid or porous and of any convenient shape
  • suitable insoluble supports include microtiter plates, arrays, membranes and beads These are typically made of glass, plastic (e g , polystyrene), polysacchandes, nylon or nitrocellulose, teflonTM, etc Microtiterplatesandarraysareespe ⁇ allyconvenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples In some cases magnetic beads and the like are included
  • the particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods
  • the Exo protein is bound to the support, and a candidate bioactive agent is added to the assay
  • the candidate agent is bound to the support and the Exo protein is added
  • Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc Of particular interest are screening assays for agents that have a low toxicity for human cells
  • assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc ) and the like
  • the determination of the binding of the candidate bioactive agent to the Exo protein may be done in a number of ways
  • the candidate bioactive agent is labelled, and binding determined directly For example, this may be done by attaching all or a portion ofthe Exo protein to a solid support, adding a labelled candidate agent (for example a fluorescent label) washing off excess reagent, and determining whether the
  • label herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e g radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc
  • Specific binding molecules include pairs, such as biotm and streptavidm, digoxin and antidigoxm etc
  • the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above
  • the label can directly or indirectly provide a detectable signal
  • the proteins may be labeled at tyrosine positions using 125 l, or with fluorophores Alternatively, more than one component may be labeled with different labels, using 125 l for the proteins, for example, and a fluorophor for the candidate agents
  • the binding of the candidate bioactive agent is determined through the use of competitive binding assays
  • the competitor is a binding moiety known to bind to the targetmolecule( ⁇ e Exo), such as an antibody, peptide, binding partner, ligand, etc
  • This assay can be used to determinecandidateagents which interfere with binding between Exo proteins and GS27, rab7, rab9, snap-23, rab3a, rab11 , rab3d, rab ⁇ , alpha-snap, und ⁇ -1 , or vamp3
  • the candidatebioactiveagent is labeled Eitherthe candidate bioactive agent, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present Incubations may be performed at any temperature which facilitates optimalactivity, typically between 4 and 40°C Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening Typically between 0 1 and 1 hour will be sufficient Excess reagent is generally removed or washed away The second component is then added, and the presence or absence ofthe labeled component is followed, to indicate binding
  • the competito ⁇ s added first, followed by the candidate bioactive agent Displacement of the competitor is an indication that the candidate bioactive agent is binding to the Exo protein and thus is capable of binding to, and potentially modulating, the activity ofthe Exo protein
  • either component can be labeled
  • the presence of label in the wash solution indicates displacement by the agent
  • the candidate bioactive agent is labeled
  • the presence of the label on the support indicates displacement
  • the candidate bioactive agent is added first, with incubation and washing, followed by the competitor
  • the absence of binding by the competitor may indicate that the bioactive agent is bound to the Exo protein with a higher affinity
  • the presenceof the label on the support, coupled with a lack of competitorbinding may indicate that the candidate agent is capable of binding to the Exo protein
  • the methods comprisedifferential screening to identity bioactive agents that are capable of modulating the activity ofthe Exo proteins
  • the methods comprise combining an Exo protein and a competitor in a first sample
  • a second sample comprises a candidate bioactive agent, an Exo protein and a competitor
  • the binding ofthe competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the Exo protein and potentially modulating its activity That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the Exo protein
  • a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native Exo protein, but cannot bind to modified Exo proteins
  • the structure of the Exo protein may be modeled, and used in rational drug design to synthesize agents that interact with that site
  • Drug candidates that affect Exo bioactivity are also identified by screening drugs for the ability to either enhance or reduce the activity of the protein
  • Positive controls and negative controls may be used in the assays
  • Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results Incubation of all samples is for a time sufficient for the binding of the agent to the protein Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, generally labeled agentdetermmed For example, where a radiolabel is employed, the samples may be counted in a scintillation counterto determmethe amount of bound compound
  • reagents may be included in the screening assays These include reagents like salts, neutral proteins, e g albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions Also reagents that otherwise improve the efficiency of the assay, such as proteasemhibitors.nuclease inhibitors, anti-microbialagents, etc , may be used The mixture of components may be added in any order that provides for the requisite binding
  • kits can be based on the use ofthe protein and/orthe nucleicacid encoding the Exo proteins Assays regarding the use of nucleic acids are further described below
  • methods for screening for a bioactive agent capableofmodulatingthe activity of Exo comprise the steps of adding a candidate bioactive agent to a sample of Exo, as above, and determinmganalterationin the biologicalactivity of Exo "Modulating the activity of Exo" includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present
  • the candidate agent should both bind to Exo (although this may not be necessary), and alter its biological or biochemical activity as defined herein
  • the methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution, activity or amount of Exo
  • the methods comprise combiningan Exo sampleand a candidate bioactive agent, and evaluating the effect on exocytosis
  • Exo activity or grammatical equivalents herein is meant one of Exo's biological activities, including, but not limited to, its ability to affect exocytosis, secretion and/or vesicular transport Included within exocytosis, secretion and/or vesicular transport activitiesmclude regulating or involvement in steps therein such as docking, fusion and targeting activities of proteins involved in the entire pathway of exocytosis, secretion and/or vesicular transport
  • vesicular refers to any vesicle including synaptic or secretory granules and vesicles those involved in exocytosis, endocytosis or the trans-golgi network
  • exo activity includes GTPase activity or regulation thereof
  • One exo activity herein is binding to at least one protein selected from the group consist
  • the activity ofthe Exo protein is increased, in another preferred embodiment, the activity ofthe Exo protein is decreased
  • bioactive agents that are antagonists are preferred in some embodiments, and bioactive agents that are agonists may be preferred in other embodiments
  • the invention providesmethodsforscreenmgforbioactiveagents capable of modulating the activity of an Exo protein
  • the methods comprise adding a candidate bioactive agent, as defined above, to a cell comprising Exo proteins
  • Preferred cell types include almost any cell
  • a library of candidate agents are tested on a plurality of cells
  • the assays include exposing the cells to an exocytosis agent that will induce exocytosis in control cells, i e cells ofthe same type but that do not contain the exogeneous nucleic acid encoding an Exo Suitable exocytosis agents are known in the art and include but are not limited to lonomycin and CaN such as, but not limited to the Ca ++ ⁇ onophoreA23137 Alternatively, the cells may be exposedtoconditionsthatnormaliy result in exocytosis, and changes in the normal exocytosis progression are determined Alternatively, the cells into which the Exo nucleic acids are introduced normally under exocytosis, and thus changes (for example, inhibition of exocytosis) are determined
  • the cells normally do not undergo exocytosis, and the introduction of a candidate agent causes exocytosis
  • exocytosis labels include, but are not limited to, annexm Accordingly, these agents can be used as an affinity ligand, and attached to a solid support such as a bead, a surface, etc and used to pull out cells that are undergoing exocytosis Similarly, these agents can be coupled to a fluorescent dye such as PerCP, and then used as the basis of a fluorescent-activated cell sorting (FACS) separation Moreover, FACS or other optical methods can be used to detect exocytosis activity and the modulation thereof based on light scattering, light absorption, dye uptake and release, granule enzyme activity and quantification of granule specific proteins
  • the compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host, as previously described
  • the agents may be administered in a variety of ways, orally, parenterally e g , subcutaneously, mtraperitoneally, mtravascularly, etc Depending upon the manner of introduction, the compounds may be formulatedin a variety of ways
  • the concentration of therapeutically active compound in the formulation may vary from about 0 1-100 wt %
  • compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like
  • Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds
  • Diluents known to the art include aqueous media, vegetable and animal oils and fats Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents
  • the invention provides methods for identifying cells containing variant Exo genes comprising determining all or part ofthe sequence of at least one endogeneous Exo genes in a cell As will be appreciated by those in the art, this may be done using any number of sequencing techniques
  • the invention provides methods of identifying the Exo genotype of an individual comprising determining all or part ofthe sequence of at least one Exo gene ofthe individual This is generally done in at least one tissue of the individual, and may include the evaluation of a number of tissues or different samples of the same tissue The method may include comparing the sequence of the sequenced Exo gene to a known Exo gene, i e a wild-type gene The sequence of all or part of the Exo gene can then be compared to the sequence of a known Exo gene to determine if any differences exist This can be done using any number of known sequence identity programs, such as Bestfit
  • Exo proteins and particularly Exo fragments, are useful in the study or treatment of conditions which are mediated by exocytosis, i e to diagnose, treatorpreventexocytosis-mediateddisorders
  • exocytosis mediated disorders or “disease state” include conditions involving both insufficient or excessive exocytosis, vesicular transport, and/or secretion via the secretory pathway, including inflammatory mediator release from mast cells including asthma, allergies, and Chediak-Higashi Syndrome (CHS) Additionally, control of neurotransmitter release can be used to treat Alzheimer's disease, Parkinson's and Huntmgton's disease states as well as some skitzophrenia, thus these can also be included in exocytosis mediated disorders in some cases In other cases, fertilization and lactation disorders can be included as disease states which can be treated with the compositions provided and/or identified herein Additionally, some diabetes, digestion and wound healing disorders can be exocytosis mediated disorders
  • the methods comprise administering to a cell an anti-Exo antibody that reduces or eliminates the biological activity ofthe endogeneous Exo protein
  • the methods comprise administering to a cell or organism a recombinant nucleic acid encoding an Exo protein
  • the activity of Exo is increased by increasing the amount of Exo in the cell, for example by overexpressmg the endogeneous Exo or by administering a gene encoding an Exo, using known gene- therapy techniques, for example
  • the gene therapy techniques include the incorporation of the exogeneous gene using enhanced homologous recombination (EHR), for example as described in PCT/US93/03868, hereby incorporated by reference in its entireity
  • EHR enhanced homologous recombination
  • the invention provides methods for diagnosing an exocytosis related condition in an individual
  • the methods comprise measuring the activity of Exo in a tissue from the individual or patient, which may include a measurement ofthe amount or specific activity of Exo This activity is compared to the activity of Exo from either a unaffected second individual orfroman unaffectedtissuefromthefirstmdividual When these activities are different, the first individual may be at risk for an exocytosis mediated disorder
  • the proteins and nucleic acids provided herein can also be used for screening purposes wherein the protein-protein interactions of the Exo proteins can be identified Genetic systems have been described to detect protein-protein interactions The first work was done in yeast systems, namely the "yeast two-hybrid" system The basic system requires a protein-protein interaction in order to turn on transcnptionof a reportergene Subsequent work was done in mammalian cells See Fields et al , Nature 340 245 (1989), Vasavada et al , PNAS USA ⁇ 10666 (1991), Fearon et al , PNAS USA 69 7958 (1992), Dang et al .
  • two nucleic acids are transformed into a cell, where one is a "bait" such as the geneencod ⁇ ngGS27, rab7, rab9, snap-23, rab3a, rabl 1 , rab3d, rab ⁇ , alpha-snap, und 8-1 , vamp3 or a portion thereof, and the other encodes a test candidate Only if the two expression products bind to one another will an indicator, such as a fluorescent protein, be expressed Expression of the indicator indicates when a test candidate binds to the GS27, rab7, rab9, snap-23, rab3a, rab11 , rab3d, rab ⁇ , alpha-snap, und 8-1 orvamp3and can be identified as an Exo protein Using the same system and the identified Exo proteins the reverse can be performed Namely, the Exo proteins provided herein can be used to identify new baits, or agents which interact with Exo proteins Additionally, the two-hybrid system can be used where
  • a mammalian two-hybrid system provides post-translational modifications of proteins which may contribute significantly to their ability to interact
  • a mammalian two-hybrid system can be used in a wide variety of mammaliancell types to mimic the regulation, induction, processing, etc of specific proteins within a particularcell type
  • proteins involved in a disease state such as those descnbedabove could be tested in the relevant disease cells
  • assaying them underthe relevant cellular conditions will give the highest positive results
  • the mammalian cells can be tested under a variety of experimental conditions that may affect intracellular protein-protein interactions, such as in the presence of hormones, drugs, growth factors and cytokines, cellular and chemical stimuli, etc , that may contribute to conditions which can effect protein-protein interactions, particularlythose involved in exocytosis, the secretory pathway, and/or vesiculartransport
  • the activity assays such as having an effect on exocytosis, secretion and/or vesiculartransport can be performed to confirm the activity of Exo proteins which have already been identified by their sequence identity/similarity or binding to GS27, rab7, rab9, snap-23, rab3a, rabl 1 , rab3d, rab ⁇ , alpha-snap, und 8-1 or vamp3 as well as to further confirm the activity of lead compounds identified as modulators of exocytosis, secretion and/or vesicular transport
  • the Exo proteins of the present invention may be used to generate polyclonal and monoclonalantibodiesto Exo proteins, which are useful as described herein Similarly, the Exo proteins can be coupled, using standard technology, to affinity chromatography columns These columns may then be used to purify Exo antibodies In a preferred embodiment, the antibodies are generated to epitopes unique to the Exo protein, that is, the antibodies show little or no cross-reactivity to other proteins These antibodies find use in a number of applications For example, the Exo antibodies may be coupled to standard affinity chromatography columns and used to purify Exo proteins as further described below The antibodies may also be used as blocking polypeptides, as outlined above, since they will specifically bind to the Exo protein
  • the anti-Exo protein antibodies may comprise polyclonal antibodies
  • Methods of preparing polyclonal antibodies are known to the skilled artisan Polyclonal antibodies can be raised in a mammal, forexample, by one or more injections of an immunizing agent and, if desired, an adjuvant Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intrapentoneal injections
  • the immunizing agent may include the Exo protein polypeptide or a fusion protein thereof It may be useful to conjugate the immunizing agent to a 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, bovinethyroglobu n, and soybean trypsin inhibitor Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant(monophosphorylL ⁇ p ⁇ d a, synthetic trehalosedicorynomy
  • the anti-Exo protein antibodies may, alternatively, be monoclonal antibodies
  • Monoclonal antibodies may be prepared using hybndoma methods, such as those described by Kohler and Milstem, Nature, 256 495 (1975)
  • a hybndoma 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 may be immunized in vitro
  • the immunizing agent will typically include the Exo protein polypeptide or a fusion protein thereof Generally, either peripheral blood lymphocytes ("PBLs") are used if cellsof human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired
  • PBLs peripheral blood lymphocytes
  • 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 hybndoma cell [Godmg, Monoclonal Antibodies Principles and Practice, Academic Press, (1986) pp 59-103]
  • Immortalized cell lines are usually transformed mammaliancells, particularlymyeloma cells of rodent, bovmeand human origin Usually, rator mouse myeloma cell lines are employed
  • the hybndoma cells may be cultured in a
  • 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 immortalizedcell 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, Rockville, Maryland
  • the culture medium in which the hybndoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against Exo protein
  • the binding specificity of monoclonal antibodies produced by the hybndoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the clones may be subcloned by limiting dilution proceduresand grown by standard methods[God ⁇ ng, supra] Suitableculturemedia forthis purpose include, forexample, Dulbecco'sModified Eagle's Medium and RPMI-1640 medium Alternatively, the hybndoma cells may be grown in vivo as ascites in a mammal
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein a-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography
  • the monoclonalantibodies may also be made by recombinantDNA methods, such as those described in U S Patent No 4,616,567
  • DNA encoding the monoclonal antibodies ofthe 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 lightchamsof mu ⁇ neantibodies)
  • the hybndoma cells ofthe invention serve as a preferred source of such DNA
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cellsthatdo not otherwiseproduceimmunoglobulm protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells
  • the DNA alsomay be modified, forexample, by substituting the coding sequencefor human heavy and light chain constant domains in placeofthehomologousmur ⁇ nese
  • the antibodies may be monovalent antibodies
  • Methods for preparing monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain.
  • the heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking.
  • the relevantcysteineresidues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in the art.
  • the anti-Exo protein antibodies ofthe invention may furthercomprise humanizedantibodies or human antibodies.
  • Humanizedforms of non-human(e.g., murine)antibodies are chimeric immunoglobulins, immunoglobulin chains orfragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins(recipientantibody)in which residues from a complementary determining region (CDR) ofthe recipientare replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanizedantibodies mayalsocomprise 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 of the
  • CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321 :522-626 (1936); Riechmann et al., Nature. 332:323-329 (19 ⁇ ); and Presta, Curr. Op.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typicallytakenfroman “import” variabledomain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature. 321:522-626 (1966); 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.
  • humanized antibodies are chimeric antibodies (U S Patent No 4,816,567), wherein substantially less thanan mtacthumanvariabledomain has been substituted by the correspondmgsequence from a non-human species
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies
  • Human antibodies can also be produced using vanoustechniquesknown in the art, including phage display libraries [Hoogenboom and Winter, J Mol Biol , 227 381 (1991), Marks et al , J Mol Biol , 222 581 (1991)1
  • the techniques of Cole etal and Boerner etal are also available for the preparation of human monoclonal antibodies (Cole et al , Monoclonal Antibodies and Cancer Therapy, Alan R Liss, p 77 (1985) and Boerner etal . J Immunol ,
  • human antibodies can be made by introducing of 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
  • transgenic animals e g , mice in which the endogenous immunoglobulin genes have been partially or completely inactivated
  • 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 5,545,607, 5,545,806, 5,669,625, 5,625, 126, 5,633,425, 5,661 ,016, and in the following scientific publications Marks et al , Bio/Technology 10.779-783 (1992).
  • Lonberg etal Nature 368856-869 (1994), Morrison, Nature 368, 812-13 (1994),
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodiesthat have bindmgspecificitiesforatleasttwodifferentantigens
  • one ofthe binding specificities is for the Exo protein, the other one is for any other antigen, and preferably for 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 [Milstem and Cuello, Nature, 305 537-539 (1983)] Because of the random assortment of immunoglobulin heavy and light chains, these hyb ⁇ domas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure The purification of the correct molecule is usually accomplished by affinitychromatographysteps Similarproceduresare disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al , EMBO J .
  • Antibodyvariabledomainswiththedesiredbindingspecificities(antibody-antigencombining sites) can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are cotransfected into a suitable host organism.
  • DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are cotransfected into a suitable host organism.
  • Heteroconjugate antibodies are also within the scope of the present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089].
  • the antibodies may be prepared/ ⁇ vitro using known methods in syntheticprotein chemistry, includingthoseinvolvingcrosslinkingagents.
  • immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolateand methyl-4-mercaptobutyrimidateand those disclosed, forexample, in U.S. Patent No. 4,676,980.
  • anti-Exoprotein antibodies ofthe invention have various utilities.
  • anti-Exo protein antibodies may be used in diagnostic assays for an Exo protein, e.g. , detecting its expression in specif iccells, tissues, orserum.
  • diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in eitherheterogeneousor homogeneous phases [Zola, Monoclonal Antibodies: a Manual of Technigues, CRC Press, Inc. (1987) pp. 147-158].
  • the antibodiesused in the diagnosticassays can be labeled with a detectable moiety.
  • the detectable moiety should be capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety may be a radioisotope, such as
  • a fluorescent or chemiluminescent compound such asfluorescein isothiocyanate.rhodamine, orluciferin
  • an enzyme such as alkaline phosphatase, beta- galactosidase or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter etal.. Nature. 144:946 (1962): David et al.. Biochemistry. 13:1014 (1974): Pain et al., J. Immunol. Meth., 40:219 (1981 ); and Nygren.J. Histochem.
  • Anti-Exo protein antibodies also are useful for the affinity purification of Exo protein from recombinant cell culture or natural sources
  • the antibodies against Exo protein are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art
  • the immobilized antibody then is contacted with a sample containmgthe Exo protemto be purified, and thereafterthesupportis washed with a suitable solvent that will remove substantially all the material in the sample except the Exo protein, which is bound to the immobilized antibody
  • the support is washed with another suitable solvent that will release the Exo protein from the antibody
  • the anti-Exo protein antibodies may also be used in treatment
  • the genes encoding the antibodies are provided, such that the antibodies bind to and modulate the Exo protein within the cell
  • a therapeuticallyeffectivedose of an Exo protein, agonist orantagonist is administered to a patient
  • therapeutically effective dose herein is meant a dose that produces theeffectsforwhich it is administered The exact dose will depend on the purpose ofthe treatment, and will be ascertainabie by one skilled in the art using known techniques
  • a "patient” for the purposes of the present invention includes both humans and other animals, particularly mammals, and organisms Thus the methods are applicable to both human therapy and veterinary applications
  • the patient is a mammal, and in the most preferred embodiment the patient is human
  • Exo protein, agonist or antagonist of the present invention can be done in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, ⁇ ntranasally,transdermally, ⁇ ntrape ⁇ toneally, ⁇ ntramuscularly, intrapulmonary, vaginally, rectally, or intraocularly
  • the Exo may be directly applied as a solution or spray
  • compositionsof the present invention comprise an Exo protein, agonist orantagonist in a form suitable for administration to a patient
  • the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, 5 sulfuric acid, nitric acid, phosphoricacid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, 5 sulfuric acid, nitric acid, phosphoricacid and the like
  • organic acids such as acetic acid, propionic acid, glycolic acid, pyru
  • compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring 0 agents; and polyethylene glycol.
  • carrier proteins such as serum albumin
  • buffers such as buffers
  • fillers such as microcrystalline cellulose, lactose, corn and other starches
  • binding agents such as microcrystalline cellulose, lactose, corn and other starches
  • sweeteners and other flavoring agents such as sweeteners and other flavoring agents
  • coloring 0 agents such as polyethylene glycol.
  • additives are well known in the art, and are used in a variety of formulations.
  • yeast two-hybrid cDNA cloning technology is a powerful in vivo protein-protein interaction assay first introduced in Fields S, Song O (1989) Nature 340:246-247 (Chevray PM, Nathans D (1992) Proc Natl Acad Sci USA 89:5789-6793; Chien CT, Bartel PL, Stemglanz R, Fields S (1991 ) Proc Natl Acad Sci USA 88:9578-9682; Durfee T, Becherer K, Chen PL, Yeh SH, Yang Y, Kilburn AE, Lee WH, Elledge S (1993) Genes Dev 7:566-669; 0 Fields S, Song O (1989) Nature 340:245-247; Mendelsohn AR, Brent R (1994)
  • the yeast one-hybrid system is widely used in cloning new transcription factors (Lehming N, Thanos D, Brickman JM, Ma J, Maniatis T, Ptashne M (1994) Nature 371:175-179; Li JJ, Herskowitz I (1993) Science 262:1870-1873; Luo Y, Stile J, Zhu L (1996) BioTechniques 20:564-568; Shang J, Luo Y, Clayton D (1997) Developmental Dynamics 209:242-253; Strubin M., Newell JW, Matthias P (1995) Cell
  • the experimental flow-chart of a yeast two-hybrid cDNA screening experiment is outlined in Figure 2.
  • the experimental flow-chart of a yeast one-hybrid cDNA screening experiment is outlined in Figure 3.
  • yeast culture mediums including YPD, YPD Agar, DOB, DOBA, CSM-TRP,
  • CSM-LEU, CSM-HIS, CSM-URA, CSM-LYS, CSM-LEU-TRP, CSM-LEU-HIS, and CSM-LEU-TRP-HIS are available from Bio101 , Inc.
  • 3AT (3-amino-1 ,2,4-triazol) is available from Sigma (Cat no.: A-8066, St. Louis, MO, USA).
  • Yeast two-hybrid system reporter strain Y190 (MATa, ura3-52, his3-200, Iys2-801 , ade2-101, trp1-901 , leu2-3, 112, gal4 ⁇ , gal ⁇ O ⁇ , cyhr2, LYS2::GAL1 UAS -HIS3 TATA -
  • HIS3, URA3::GAL1 UAS -GAL1 TATA -lacZ) and yeast one-hybrid system reporter strain YM4271 (MATa, ura3- ⁇ 2, his3-200, Iys2-801 , ade2-101 , trp1-903, leu2-3, 112, tyr1-601) are available from Clontech Laboratories, Inc. (Cat no.K1603-1, Clontech, Palo Alto, CA, USA). Plasmids and cDNA Libraries pAS2 and pACT2 series were originally constructed by Elledge lab (Durfee et al. 1993) and are available from Clontech laboratories (Cat no.K1604-A, K1604-B).
  • GAL4 based two-hybrid vectors such as pGBT9 and pGAD424 series, were originally published by Fields lab and are available from both Stratagene, Inc. (Cat no.235700,235722) and Clontech Laboratories, Inc. (Cat no.K1605-A, K1605-B).
  • LexA based two-hybrid vectors are available from Origene Technologies, Inc. (Cat no.DPL-100, DPL-102).
  • cDNA libraries for two-hybrid and one-hybrid screening are available from Origene, Stratagene, Clontech, and Invitrogen.
  • Rigel also makes its own two-hybrid cDNA libraries from various tissues. All of these two- hybrid vectors share basic structures as shown in Figure 4A.
  • the cDNA library is to be amplified before screening. It is recommended that at least 200 15cm-plates should be used to grow up 10 million independent cDNA clones. High quality plasmid can be obtained with Qiagene DNA preparation kits.
  • Single-strand carrier DNA for yeast transformation is available from Origene or Clontech.
  • Carrier DNA can also be made according to protocol by Ito et al. (Ito H,
  • Nylon membrane and Whatman filter for lacZ color assay are available from Fisher Scientific.
  • Gal is from either Promega (Cat no.V3941 , Madison, Wl, USA) or Denville Scientific (Cat no.CX-3000-3, Metuchen, NJ, USA). All plastic wares are from Fisher Scientific or VWR.
  • PROCEDURE Yeast Two-Hybrid System Screening Grow up yeast reporter strains on YPD plates from frozen stock.
  • yeast reporter strain be streaked on SD-W, SD-L, SD-H, SD-U, and SD-K plates to test other markers of the yeast before cDNA library screening. Reporter strain such as Y190 should be able to grow up on SD-K, SD-U and SD-H plates, but not on SD-
  • Y190 consistently showed higher sensitivity than other yeast strains such as HF7c.
  • Yeast reporter strains with both lacZ reporter gene and HIS3 reporter gene are strongly recommended. HIS selection will ensure that only interaction positive clones will grow, which makes colony picking much easier later.
  • pGBT9 series plasmids (Estojak J, Brent R, Golemis EA (1995) Molecular and Cellular Biology 15:6820-6829; Legrain P, Dokhelar MC, Transy C (1994) Nucleic Acids Research 22:3241-3242).
  • the disadvantage of using pAS2 is the large size of this plasmid (8 kb), which may present a challenge to cloning large cDNA fragments into the plasmid.
  • cDNA fragments should fused to the C-terminal of Gal4 binding domain in frame ( Figure 4A).
  • the junction sequence between GAL4 and cDNA should have a GGG amino acid sequence to avoid any interruption of domain structure. Either full-length cDNA or partial fragments can used to generate bait plasmid. Transform bait into yeast: 1st round.
  • 1 ⁇ g of bait plasmid is transformed into Y190 with small-scale yeast transformation protocol (see SUBPROTOCOL section).
  • Transformants should be plated on SD- W, SD-WH, and SD-WH+3AT(5-40mM) plates.
  • LacZ color assay can also be done after colonies grow to a diameter of 1 mm. If colonies grow up on SD- WH+40mM3AT plates after 3 days incubation and/or LacZ color assay of these colonies show positive result after only 30 minutes incubation with X-Gal, the bait gene should be determined not suitable for two-hybrid screening without further modification.
  • the bait gene itself may be able to activate transcription of reporter genes HIS/lacZ.
  • co-transformation of bait plasmid and cDNA library can be done in a single step, co-transformation efficiency is at least 10 fold lower than single plasmid transformation.
  • Mating approach may also be used to introduce cDNA library into yeast cells containing the bait vector. Please refer to protocol published by Finley and Brent (Finley R, Brent R (1994) Proc Natl Acad Sci USA 91:12980- 12984).
  • Transform cDNA library 2nd round. Y190 containing bait plasmid is grown up for second round of transformation by cDNA library plasmid (see SUBPROTOCOL section) Incubation time after transformation varies significantly from 4 days to 11 days Identify positive clones Identification of positive clones needs experience It should also be pointed out that background colonies at lightly populated area of the plate tend to grow bigger, occasionally reaching the size of a positive colony in a dense area on the same plate The size of the positive colony should at least 4 times bigger than the neighboring background colonies Positive colonies may also turn red faster Perform lacZ color assay
  • Electroporation method is by far the most efficient method to transform plasmids from yeast mmiprep into E coli Bait and cDNA plasmid may carry different antibiotic selection markers to facilitate separation in E coli
  • Rigel's bait plasmid carries Kan r gene and the cDNA plasmid carries Amp r gene Verify positive clones cDNA clones recovered from positive HIS/lacZ positive colonies should be re- transformed into yeast with other non-specific bait control to rule non-specific binding
  • In vitro protein binding assays and function assays should also be done to rule out false positive clones
  • PROCEDURE Yeast One-Hybrid System Screening Construct HIS and lacZ reporter plasmids
  • yeast reporter strain be streaked on SD-W, SD-L, SD-H, SD-U, and SD-K plates to test other markers of the yeast before cDNA library screening.
  • Reporter strain such as YM4271 should be able to grow up on SD-K, SD-U SD-H, SD-W, and SD-L plates.
  • pHISi-1 should be linearized at Xho I site. Since pHISi-1 has no yeast replication origin and can not survive in yeast without integration, no gel purification of digested plasmid is required. Transform 1 ⁇ g of digested plasmid into YM4271 using the small-scale yeast transformation protocol (see SUBPROTOCOL section). Use more plasmids if integration efficiency is low. Transformants should be plated on SD-H, and SD-H plates with different concentration of 3AT( ⁇ -40 mM) and incubated at 30°C for at least 4 days. Determine optimal 3AT concentration.
  • step 6 Same as step 6 in two-hybrid screening procedure. Perform lacZ color assays.
  • step 7 Same as step 7 in two-hybrid screening procedure.
  • step 8 Same as step 8 in two-hybrid screening procedure.
  • DNA gel retardation assay and other function assays are required to verify one- hybrid screening results.
  • Competent cells can be kept on at room temperature for several hours without significant reduction of transformation efficiency, or at 4°C overnight with a slight reduction of transformation efficiency.
  • a clean eppendorf mix 1 ⁇ g of plasmid with 10 ⁇ l 10 mg/ml carrier DNA.
  • PEG/LiAc should be freshly made. Pre-mixed PEG/LiAc of up to 2 weeks old can also be used if transformation efficiency is not critical. Incubate at 30°C for 30 minutes with or without shaking. Add 70 ⁇ l DMSO and mix well. Incubate in 42°C water bath for 15 minutes.
  • cDNA library transformation (1-10X10 6 transformants/100 ⁇ g cDNA) Inoculate one colony of yeast in 200 ml YPD (one-hybrid screening) or corresponding selection medium (SD-W for two-hybrid screening) at 240 rpm in a 30°C shaker overnight, ⁇ 1.
  • Check OD 600 the next day. If OD 600 is between 0.8 and 1.0, the yeast can be used to prepare competent cells. Otherwise, dilute to OD 600 0.6 and grow another 3 to 4 hours.
  • PEG/LiAc should be freshly made. Pre-mixed PEG/LiAc of up to 2 weeks old can also be used if transformation efficiency is not critical. Incubate at 30°C for 30 minutes with shaking. Either an orbital shaker or rotator can be used. 5 Add 140 ⁇ l DMSO and mix well.
  • SD-L for cDNA library plasmid pACT with a yeast colony. Incubate in a 30°C shaker or rotator overnight or until confluent. Spin down yeast in a bench-top centrifuge at 3000 rpm at room temperature. Remove medium and re-suspend pellet in 200 ⁇ l lysis buffer. Transfer to an eppendorf. Add 200 ⁇ l volume glass beads.
  • the lid of eppendorf can be used as scoop to collect 200 ⁇ l glass beads. Add 200 ⁇ l phenol/chloroform/isoamyl alcohol (25:24:1). Vortex at the highest speed for 3 minutes. Spin in micro-centrifuge at 14000 rpm for 10 minutes. Transfer top water layer to another eppendorf, add 20 ⁇ l 3M NaAc and 500 ⁇ l ethanol.
  • Precipitate should be visible immediately. Put the eppendorf into a dry ice bath for 15 minutes or until frozen. Spin in a micro-centrifuge at 14000 rpm for 10 minutes. Remove supernatant and dry pellet. Wash pellet by 100 ⁇ l of 80% ethanol, and dry the pellet in air.
  • Bait peptide was cloned into pAS2-1 to screen for proteins that can bind thereto. Results are listed below.
  • PCNA is a published binding protein of the bait peptide. Yeast one-hybrid screening results were previously published ( Luo Y, Stile J, Zhu L (1996) BioTechniques 20:564- 568).

Abstract

The present invention is directed to novel polypeptides such as the Exo protein and related molecules which have an effect on or are related to exocytosis and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention. Further provided by the present invention are method for identifying novel compositions which mediated Exo bioactivity, and the use of such compositions in diagnosis and treatment of disease.

Description

EXOCYTOSIS PATHWAY PROTEINS AND METHODS OF USE
FIELD OF THE INVENTION
The invention relates to moleculesinvolvedin the exocytosispathway and more particularly to novel polypeptideswhich associatewith exocytoticproteins, nucleicacids and antibodies. The invention further relates to the use of proteins associated with the exocytosis pathway in methods for identifying candidate agents which modulate exocytosis.
BACKGROUND OF THE INVENTION
In eukaryotic cells, proteins destined forthe plasma membrane or the extracellular space are delivered along the secretory pathway. This comprises a series of sequential, vesicle- mediated transport steps, each of which requires the specific targeting of transportvesicles to the appropriate acceptor membrane and the subsequent fusion of vesicle and acceptor membranes. In this way, proteins to be secreted by the cell are translocated into the endoplasmic reticulum and then travel through the Golgi complex. The proteins are sorted into secretory vesicles in the frans-Golgi network and these vesicles then fuse with the plasma membrane. This final membrane fusion event is known as exocytosis and results in the discharge of vesicle contents into the extracellular space as well as the incorporation of vesicle membrane lipids and proteins into the plasma membrane.
Exocytosis can be divided into two classes: constitutive and regulated. In constitutive exocytosis, secretor vesicles fuse with the plasma membrane immediately after formation; in regulated exocytosis, secretory vesicles accumulate in the cytoplasm and only undergo fusion upon receipt of an appropriate signal. All eukaryotic cells exhibit constitutive Although the fundamental purpose of exocytosis is to deliver iipids and proteins to the plasma membrane and to release vesicle contents from the cell, different cell types utilize this mechanism to fulfill their own particular physiological role. Some examples of the various functions of exocytosis in different cell types are listed in Table 1.
Table 1. Functions of exocytosis
It should be noted that exocytosis (i.e., the fusion of vesicles with the plasma membrane) is not only the end point of the secretory pathway, but can also involve vesicles which did not originate fromthe endoplasmic reticulum. For instance, transcytosis occurs in polarized cells and involves endocytic vesicle budding from one pole ofthe cell, transport to the other pole (often via endosomes) and subsequent exocytic fusion. In mammary cells, transcytosis is used in the uptake of antibodies from the blood and their subsequent secretion in milk. Similarly, some vesicles undergo cycles of exo/endocytic fusion via endosomes without returning to the Golgi. Exocytosis of recycling vesicles may be either constitutive (e.g., transferrin receptor-containing vesicles) or regulated (e.g., synaptic vesicles).
Since all cells exhibit constitutive exocytosis, it follows that regulated secretory cells must possess two types of secretory vesicle: one constitutive and on regulated. Morphological studies indicate this to be the case, since constitutive secretory vesicles appear small and clear in the electron microscope, whereas regulated secretory vesicles typically appear larger and opaque. Furthermore, the two types of vesicle usually contain different substances (an exception is the mammary cell, where casein secretion occurs by both constitutive and regulated exocytosis).
It should be noted that cells may contain more than one type of regulated secretory vesicle. The best example of this is seen in neurons, which may possess synaptic vesicles and large dense-core vesicles in addition to constitutive secretory vesicles. Some properties ofthe two type of neuronal regulated secretory vesicle are listed in Table 2. Large dense- core vesicles contain peptide neurotransmitters and these are very similar to regulated secretory vesicles in endocrine cells. Indeed, much ofthe information on large dense-core vesicle biogenesis and exocytosis has come from studies of adrenal chromaffin cells and theirtumor cell derivatives, PC12 cells, both popular neuronal cell models. Synaptic vesicles appear clear in the electron microscope, are much smaller than large dense-core vesicles and contain fast neurotransmitters. Synaptic vesicles have evolved in animals to allow the extremely rapid point-to-point communication required for brain function. Recently synaptic- like vesicles have been found in endocrine cells, such as adrenal chromaffin cells and pancreatic β-cells. These vesicles also appearto contain fast neurotransmitters, although their physiological role is unclear.
Table 2. Characteristics of regulated secretory vesicles in neurons
Abbreviations used: GABA, λ-aminobutyric acid; ACh, acetylcholine; VIP, vasoactive intestinal peptide.
The more information that is gathered regarding exocytosis, the easier it will be to manipulate exocytosis. Moreover, the more information which is gathered, the easier it will be to diagnosis and treat disorders involving exocytosis. For example, inflammatory mediator release from mast cells leads to a variety of disorders, including asthma. Therapy for allergy remains limited to blocking the individual mediators released from mast cells (anti- histamines), non-specificanti-inflammatoryagentssuch as steroidsand mast cell stabilizers which are only marginally effective at limiting the symptomatology of allergies
Similarly, the Chediak-Higashi Syndrome (CHS) is a rare autosomal recessive disease in which neutrophils, monocytes and lymphocytes contain giant cytoplasmic granules
Similar disorders have been described in mice, mink, cattle, cats and killer whales, with the murine homolog of CHS (designated beige or bg) being the best characterized See Perouetal , J Biol Chem 272(47) 29790(1997)and Barbosaetal , Nature382 262(1996), both of which are hereby incorporated by reference
There is a therefore a need to determine the proteins involved in exocytosis Some insights into the process of regulated secretion at the molecular level have allowed the definition of G proteins as important regulators Early experiments showed that non-hydrolyzable analogues of GTP could inducesecretiomn peritoneal mast cells (Fernandes, etal . Nature 312 453 (1984)) More recently, a large body of evidence has been accumulating implicating smallG proteinsoftherabfamilyasregulatorsinthefusionof secretory granules with plasma membranes during exocytosis The rab GTPases represent a diverse family of homologous proteins that are generally associated with the membrane of organelles in a wide variety of cells, where they regulatedefined steps of intracellular membrane traffic (Zeπal, M and Stenmark, H , Curr Opin Cell Biol 5 613 (1993)) An example of this are the rab3 subfamily proteins which have been found to have limited expression in regulated secretion-competent cells, and to be associated with synaptic or secretory granules, suggesting that they are involved in stimulus-secretion coupling (Lledo, et al , Trends Neurobiol Sci 17426 (1994)) Furthermore, overexpression of rab3d or its GTP binding mutant form (N 1351) in the rat basophil line RBL leads to significantinhibitionoflgE mediated exocytosis (Roa, J Immunol , 1592815 (1997))
Rab3a, Rab3b, Rab3c, and Rab3d constitute a subgroup of the rab family implicated in regulated exocytosis Rab3a has been detected in regulated secretory cells such as neurons, endocrine cells, and exocnne cells but not in constitutive secretory cells such as hepatocytes and lymphocytes (Fischer von Mollard, Proc Natl Acad Sci 87 1988-92 (1990), Takai, et al , Int Rev Cytol 133 187-230 (1992)) In neuromuscular synapses, rab3a has been localized at the synaptic vesicles (Mizoguchi, et al , Biochem Biophys Res Commun 202 1235-43 (1994)) Furthermore, Rab3a has also been detected at the secretory granules of chromaffin cells and at the zymogen granules in the exocnne cells of pancreatιcacιnι(Padfield,etal , Proc Natl Acad Sci , USA 89 1656-60 (1992)) Rab3a has been shown to interact with a numberof other proteins including the exchange proteins GDI and GRF (Matsui, etal., Mol. Cell. Biol. 10:4116-22(1990), Burstein, E.S. and Macara I.G., Proc. Natl. acad. Sci., USA 89:1154-8 (1992)) as well as GAP (Burstein, J. Biol. Chem. 266: 2689-92 (1991)) and Rabphilin (Shirataki, et al., Mol. Cell Biol. 13: 2061-8 (1993)). ltisbelievedthatRab3amodulatesexocytosisin regulatorysecretorycells. Rab3a is cloned and known in the art (see, i.e., Genbank accession number (no.) M28210).
Rab3d is thought to be involved in the modulation of regulated secretion in a number of celltypes. Rab3d is predominantly expressed in fat tissue but can also be found expressed, at lower levels, in a wide range of tissue types including lung, spleen, heart, and brain. Baldini, G., et al., (1992) Proc. Natl. Acad. Sci. USA 89: 5049-52. Rab3d has also been implicated in the translocation of the Giut4 glucose transporter in adipocytes. Rab3d has been cloned and is known in the art, i.e., Genbank accession no.: AF081353.
Thus, rabs, particularly, tissue /cell specific isoforms of rabs and the proteins which they interact with are of great pharmaceutical interest. Rab7 is cloned and known in the art, (see, i.e., Genbank accession no. U44104).
Rab9 is localized to the surface of late endosomes where it appears to act to stimulate the transportof mannoseδ-phosphate receptors between late endosomes and the trans-Golgi network, both in vitro and in vivo. Recent studies suggest that this GTPase is a rate-limiting component for transport between late endosomes and the trans-Golgi network. Rab9 has been cloned and is known in the art, (see i.e., U44103).
Rab11 was identified by screening a Madin-Darby canine kidney cell cDNA library using degenerate oligonucleotides derived from conserved sequences of the Rab superfamily. Chavrier, P., et al., (1990), Mol. Cell. Biol. 10:6578-85. The predicted amino acid sequences ofthe canine, human, rat and rabbit Rab11 are 100% identical. This high level of conservation between species might reflect a particular importance of this member of the Rab family. Rab11 has been localized to both the constitutive and regulated secretory pathway in PC12 cells. Ora3, a homolog of Rab11 (91% identity at the amino acid level) has been found to be associated with cholinergicsynapticvesiclesderived from the electric organ of the marine ray. Although the function of Rab11 has yet to be definitively determined, a number of lines of evidence suggest that it may play a role in the targeting of transport vesicles of different origin to a common destination, the plasma membrane. Northern blot analysis has shown that Rab11 is ubiquitously expressed but is generally more abundant in tissues with a high level of secretion. Rab11 has been cloned and is known in the art, i.e., Genbank accession no. X56740.
Rab5 (a, b, and c) make up a subgroup of the Rab protein family. They are located at the cytoplasmic surface of the plasma membrane, on early endosomes and on plasma- membrane derived clathrin coated vesicles. Antibodies directed against Rab5a inhibit the fusion of early endosomes in vitro suggesting that its activity is required in this process. In vivo, overexpression of wild type and mutant Rabδa leads to changes in the rate of internalizationofendocyticmarkersand in morphologicalalterations ofthe early endosomes. These data suggest that Rabδa is a rate-limiting factor that regulates the kinetics of both lateral fusion of early endosomesand fusion of plasma membranederivedendocyticvesicles with early endosomes. Some proteins have been identified to associate with Rab5. For example, a 62 kDa coiled-coil protein that specifically interacts with the GTP-bound form of Rab5 has been identified. This protein shares 42% sequence identity with Rabaptin-5, a previously identified effector of Rab5, and has been named it Rabaptin-5beta. Like Rabaptin-5, Rabaptin-δbeta displays heptad repeats characteristic of coiled-coil proteins and is recruited on the endosomal membrane by Rab5 in a GTP-dependent manner. However, Rabaptin-δbeta has features that distinguish it from Rabaptin-5. The relative expression levels of the two proteins varies in different cell types. Rabaptin-δbeta does not heterodimerize with Rabaptin-δ, and forms a distinct complex with Rabex-δ, the GDP/GTP exchange factor for Rabδ. Immunodepletion of the Rabaptin-δbeta complex from cytosol only partially inhibits early endosome fusion in vitro, whereas the additional depletion of the Rabaptin-δ complex has a stronger inhibitory effect. Fusion activity can mostly be recovered by addition of the Rabaptin-δ complex alone, but maximal fusion efficiency requires the presence of both Rabaptin-δ and Rabaptin-5beta complexes. Gournier H., et al., (1998), EMBO J. 17(7): 1930-1940. Rabδ is cloned and known in the art (see, i.e., Genbank accession no. M28216).
Moreover, key proteins that act in Ca2+ -regulated exocytosis in neurons and endocrine cells includethevesicleproteinssynaptotagmin.VAMP/synaptobrevin, the target membrane protein SYNTAXINs and SNAP-23/26 and, in addition, the soluble N-ethylmaleimide- sensitive fusion protein (NSF) and soluble NSF-attachment proteins (α-, β-, γ-SNAPs).
Functional evidence for the importance of the membrane proteins has come from their sensitivitytothespecificproteolyticactionsofclostridialneurotoxinsand/orgeneticanalysis in mice and Drosophila. The soluble factors NSF and SNAP were found to interact, in a 20S complex, with the neurotoxin substrates leading to them being designated as SNAP- receptors (SNAREs). Many of the proteins that make up the SNARE complexes contain coiled coil domainswhich are thoughttointereact primarily th rough hydrophobicinteractions. These proteins described function at some point in the exocytic pathway either as central membersofthevesiclefusioncomplexoras accessory proteins involved in some regulatory step in the vesicle fusion cycle.
δ GS27 is associatedwith the Golgi apparatusand is believed to behave like a SNARE. GS27
(for GolgiSNAREof27K), is identicaltomembrin, a protein implicated earlier in ER-to-Golgi transport. Regarding SNAREs, these proteins are known to mediate vesicle transport by docking vesicles onto the target membrane. One study has have reported that the cytoplasmic domain of GS27 or antibodies raised against it quantitatively inhibit transport 0 in vitro from the ER to the trans-Golgi/TGN, acting at a stage between the cis/medial- and the trans-Golgi/TGN,indicatingthat protein movement from medial- to the trans-Golgi/TGN depends on SNARE-mediated vesicular transport and that GS27 plays a functional role (Lowe, S. L, et al., Nature, 389:881-884 (1997)). Therefore, GS27 is implicated in exocytosis by its relation to vesicular transport.
δ SNAP-23 (used interchangeablywith snap-23) was first identified in a human B lymphocyte cDNA library (Ravichandran, V., et al., (1996), J. Biol. Chem. 271:13300-03). Subsequently, others have independently reported the identification of SNAP-23 in several celltypesincludingmastcells. The primary structure of SNAP-23 is 69% identical to SNAP- 2δ; it contains a central cluster of cysteine residues that is a site of palmitoylation in SNAP- 0 2δ and predicted coiled-coil that are thought to serve in binding other SNAREs, especially syntaxins 1 ,3, and 4. SNAP-23, like SNAP-26, has been localized mainly to the plasma membrane. However, recent evidence suggests that SNAP-23 translocates to the surface of secretory granules upon cellular activation and forms a complex with SYNTAXIN-3 and VAMP-2 (Guo. Z., etal., (1998),Cell. 94:637-48). Thissame study suggests thatSNAP-23 6 functioniscrucialforcompound exocytosis in mast cells in thatSNAP-23specificantibodies can completely block secretion in permeabilized cells. SNAP-23 is cloned and known in the art, (see, i.e., Genbank accession no. U65936).
NSF and alpha-snap were originally detected as factors required for transport through the Golgi in in vitro assays and yeast homologues of these proteins, sec18 and sec17, are 0 essential for secretion in vivo. In Golgi transport assays and in the formation of a Golgi membrane derived 20S complex, α and β-SNAP appear to be functionally redundant. In contrastmore recent results suggestthat α and β-SNAP havedistinctfunctionsin regulated exocytosis based on the ability of alpha-snap to displace synaptotagmin from the SNARE complex. Sollner, T., et al., Cell, 75: 409-18 (1993). Alpha-snap has been cloned and is known in the art, i.e., Genbank accession number (no.) U39412.
Sec1 is a hydrophilic protein that plays an essential role in exocytosis from the yeast Saccharomyces cerevisiae. Syntaxin (a T-SNARE), together with SNAP-25 and synaptobrevin/VAMP (a T- and a V-SNARE, respectively), is thought to form the core of the docking-fusion complex in synaptic vesicle exocytosis. Proteins that exhibit similarity to Sed were identified in the nervous system of Drosophila melanogaster (Rop) and Caenorhabditiselegans(UNC18).Munc-18/n-Sec1/rbSec1 ,a brain homoiogueoftheyeast Sedp protein, is thought to participate in regulating the docking and fusion of synaptic vesicles. Munc-18/n-Sec1 /rbSed expression has been reported to be neural-specific and a number of non-neural isoforms have been identified which are more ubiquitously expressed. Shaywitz, D. A., et al., J. Cell. Biol. 128:769-777 (199δ).
The role of Munc18c, previously identified as an n-Sec1/Munc18 homolog in 3T3-L1 adipocytes, in insulin-regulated GLUT4 trafficking has been investigated in 3T3-L1 adipocytes. Lowe, S. L., et al, Nature. 389:881-884 (1997). In these cells, Munc18c predominantly associated with syntaxin4, although it bound both syntaxin2 and syntaxin4 to similar extents in vitro. In addition, SNAP-23, an adipocyte homolog of SNAP-25, associated with both syntaxins 2 and 4 in 3T3-L1 adipocytes. Overexpression of Mund 8c in 3T3-L1 adipocytes by adenovirus-mediated gene transfer results in inhibition of insulin- stimulated glucose transport in a virus dose-dependent manner (maximal effect, approximately 60%) as well as in inhibition of sorbitol-induced glucose transport (by approximately 35%), which is mediated by a pathway different from that used by insulin. In contrast, Mund 8b, which is also expressed in adipocytes but which did not bind to syntaxin4, had no effect on glucose transport. These results suggest that Mund 8c is involved in the insulin-dependent trafficking of GLUT4 from the intracellular storage compartment to the plasma membrane in 3T3-L1 adipocytes by modulating the formation of a SNARE complex that includes syntaxin4. Unc18-1 has been cloned and is known in the art, i.e., Genbank accession number (no.) D63851.
Tetanus toxin inhibits neurotransmitter release by selectively blocking fusion of synaptic vesicles. Tetanus toxin has been shown to proteolytically degrade synaptobrevin II (also named VAMP-2), a synaptic vesicle-specific protein, in vitro and in nerve terminals. As targetsoftetanustoxin.synaptobrevinsprobablyfunctionintheexocytoticfusionof synaptic vesicles. Asynaptobrevinhomologue,cellubrevin (VAMP-3), present in all cells and tissues tested, is a membrane trafficking protein of a constitutively recycling pathway. McMahon H.T., et al, Nature. 364(6435):346-52 (1993). Like synaptobrevin II, cellubrevin is proteolysed by tetanus toxin light chain in vitro and aftertransfection. These results indicate that constitutive and regulated vesicularpathwaysusehomologousproteins for membrane trafficking, likely for membrane fusion at the plasma membrane, indicating a greater mechanistic and evolutionary similarity between these pathways than previously thought.
The homologueof Vamp3, vamp2 (synaptobrevin), has been localizedto mast cell granules and may play a critical role in mast cell exocytosis (Guo, Z., etal., Cell, 94:537-48 (1998)). Vamp3 has been cloned and is known in the art, i.e., Genbank accession no.: AF26007.
Accordingly, the proteins involved in exocytosis, termed Exo proteins herein, particularly those associated with GS27, Rab3a, Rab7, Rab9, Rab11 , Rab3d, Rab5, alphasnap, undδ-
1 , vamp3, and snap-23 are of interest, and it is desirable to providesuch proteinsand related molecules. It is a further aspect of the invention to provide recombinant nucleic acids encoding Exo proteins and expression vectors and host cells containing the nucleic acid encoding the Exo protein. A further aspect of the invention is to provide methods for screening for antagonists and agonists of Exo proteins, particularly those which modulate exocytosis, secretion and/or vesicular transport.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides recombinant nucleic acids encoding an Exo protein that has at least about 8δ% sequence identity, and more preferably at least about 90% sequence identity, and most preferably about 95% sequence identity with an amino acid sequence encoded by a nucleic acid comprising the first 100 nucleic acid residues of a sequence selected from the group consisting of SEQ ID NOS:15, 17, 19, 21 , 23, 2δ, 27, 29, 31 , 33, 3δ, 37, 39, 41 , 43, 4δ, 47, 49, 51 , 53, 63, 64, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130,
131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 147, 148, 150, 151 , 152, 155, 156, 157, 158, 1δ9, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 206, 210, 211. Preferably, the Exo proteins bind to a protein selected from the group consisting of GS27, rab7, rab9, snap-23, rab3a, rabl 1 , rab3d, rabδ, alpha-snap, und 8-1 , and vamp3. Also provided are recombinant nucleic acids which have at least about 7δ% sequence identity, more preferably, at least 8δ% sequence identity and most preferably at least about 9δ% sequence identity with a nucleicacid sequencecomprisingthe first 100 nucleic acid residues of a sequenceselected from the group consisting of SEQ ID NOS: 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 36, 37, 39, 41 , 43, 45, 47, 49, 51 , 63, 63, 64, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 147, 148, 150, 151 , 152, 156, 166, 157, 158, 159, 5 160, 161 , 162, 163, 164, 166, 166, 167, 168, 169, 170, 171 , 193, 194, 196, 196, 197, 198,
199, 200, 201 , 202, 203, 204, 20δ, 210, 211 and their complements. Recombinant Exo proteins, expression vectors and host cells comprising the nucleic acids are also included.
In an additionalaspect, the invention provides recombinant Exo proteinsExo3, Exo4, Exoδ, Exo6, Exo7, Exoδ, Exo9, Exo10, Exo11 , Exo12, Exo13, Exo14, Exolδ, Exo16, Exo17a, 0 Exo17b, Exo18, Exo19, Exo20, Exo21 , Exo22, Exo23, Exo24, Exo2δ, Exo26, Exo27, Exo28,
Exo29, Exo30, Exo31 , Exo32, Exo33, Exo34, Exo35, Exo36, Exo37, Exo38, Exo39, Exo40, Exo41 , Exo42, Exo43, Exo44, Exo45, Exo46, Exo47, Exo48, Exo49, Exo50, Exoδl , Exoδ2, Exoδ3, Exo54, Exoδδ, Exoδ6, Exoδ7, Exoδδ, Exoδ9, Exo60, Exo61 , Exo62, Exo63, Exo64, Exo6δ, Exo66, Exo67, Exo68, Exo69, Exo70, Exo71 , Exo72, Exo73, Exo74, Exo7δ, Exo76, δ Exo77, Exo78, Exo79, Exo80, Exo81 , Exo82, Exo83, Exo84, Exoδδ, Exo86, Exoδ7, Exoδδ,
Exoδ9, Exo90, Exo91 , Exo92, Exo93, Exo94, Exo9δ, Exo96, Exo97, Exo9δ, Exo99,Exo100, Exo101 , Exo102, Exo103, Exo104, Exo105, Exo106, Exo107, Exo10δ, Exo109, Exo110, Exo111 , Exo112, Exo113, Exo114, Exo11 δ, Exo116, Exo117, and Exo11δ, and the nucleic acids encoding said Exo proteins.
0 In a further aspect, the invention provides methods of making Exo proteins, comprising providing a cell comprising an Exo nucleic acid and subjecting the cell to conditions which allow the expression of Exo proteins.
In a further aspect, the present invention provides methods for screening for a bioactive agent capable of binding to an Exo protein. The method comprises combining an Exo δ protein and a candidate bioactive agent, and determining the binding of the candidateagent to the Exo protein.
In an additional aspect, the present invention provides methods for screening for agents capable of interfering with the binding of an Exo protein and GS27. The methods comprise combining an Exo protein, a candidate bioactiveagentand a GS27 protein, and determining 0 the binding of the Exo protein and the GS27 protein.
Inanotheraspect, the present invention provides methods forscreeningforagents capable of interferingwith the binding of an Exo protein and rab7. The methodscomprise combining an Exo protein, a candidatebioactive agent and a rab7 protein, and determining the binding of the Exo protein and the rab7 protein
In afurtheraspect the presentinvention provides methods for screening for agents capable of interfering with the binding of an Exo protein and rab9 The methodscomprise combining an Exo protein, a candidate bioactiveagentand a rab9 protein, and determining the binding of the Exo protein and the rab9 protein
In yet another aspect, the present invention provides methods for screening for agents capable of interfering with the binding of an Exo protein and snap-23 The methods comprise combining an Exo protein, a candidate bioactive agent and a snap-23 protein, and determining the binding of the Exo protein and the snap-23 protein
In an additional aspect, the present invention provides methods for screening for agents capable of interfering with the binding of an Exo protein and rab3a The methods comprise combining an Exo protein, a candidatebioactiveagentand a rab3a protein, and determining the binding of the Exo protein and the rab3a protein
In another aspect, the presentinventionprovidesmethodsforscreening for agents capable of interfering with the binding of an Exo protein and rab11 The methods comprise combiningan Exo protein, a candidate bioactive agent and a rabl 1 protein, and determining the binding of the Exo protein and the rabl 1 protein
In afurtheraspect, the present invention providesmethodsforscreeningfor agents capable of interfering with the binding of an Exo protein and rab3d The methods comprise combiningan Exo protein, a candidatebioactiveagentand a rab3d protein, and determining the binding of the Exo protein and the rab3d protein
In yet an additional aspect, the present invention provides methodsforscreeningforagents capable of interfering with the binding of an Exo protein and rabδ The methods comprise combining an Exo protein, a candidate bioactive agent and a rabδ protein, and determining the binding of the Exo protein and the rabδ protein
Inafurtheraspect, the presentinvention provides methodsforscreeningforagents capable of interfering with the binding of an Exo protein and alpha-snap The methods comprise combining an Exo protein, a candidate bioactive agent and an alpha-snap protein, and determining the binding of the Exo protein and the alpha-snap protein In yet another aspect, the present invention provides methods for screening for agents capable of interfering with the binding of an Exo protein and undδ-1 The methods comprise combining an Exo protein, a candidate bioactive agent and an und δ-1 protein, and determining the binding of the Exo protein and the und 8-1 protein
δ In an additional aspect, the present invention provides methods for screening for agents capable of interfering with the binding of an Exo protein and vamp3 The methods comprise combining an Exo protein, a candidatebioactiveagentand a vamp3 protein, and determining the binding of the Exo protein and the vamp3 protein
In another aspect, the invention provides methods for screening for an bioactive agent 0 capable of modulating the activity of an Exo protein The method comprises the steps of adding a candidatebioactiveagentto a cell comprising a recombinant nucleic acid encoding an Exo protein, and determining the effect of the candidate bioactive agent on cellular activity In a preferred embodiment the cellular activity is exocytosis or vesicular transport
In anotheraspect,the invention provides a method of treating an exocytosisrelateddisorder δ comprising administering an agent that interferes with specific binding of a protein selected from those shown in the Sequence Listing with a protein selected from the group consisting of GS27, Rab3a, Rab7, Rab9, Rab11 , Rab3d, Rabδ, alpha snap, und 8-1 , vamp3, and snap-23, expressed in a tissue such that said disorder is ameolerated
Also provided herein is a method of treating an exocytosis related disorder comprising 0 administering to a patient an agent that binds to a protein encoded by a sequence selected from the group consisting of those set forth in the Sequence Listing, such that exocytosis is altered
Further provided herein is a method of reducing or inhibiting exocytosis in a cell comprising administering an agent that interferes with specific binding of a protein selected from those 6 encoded by a sequence selected from the group consisting of SEQ ID NOS 1-61 (odd numbers) and 53-211wιth a protein selected from the group consisting of GS27, Rab3a, Rab7, Rab9, Rabl 1 , Rab3d, Rabδ, alpha snap, und 8-1 , vamp3, and snap-23, expressed in said cell such that exocytosis is inhibited
In yet another aspect, the invention provides a method of neutralizing the effect of a protein 0 encoded by a sequence selected from the group consisting of SEQ ID NOS 1-61 (odd numbers) and 63-211 comprising contacting an agent specific for said protein with said protein in an amount sufficient to effect neutralization. Other aspects of the invention are set forth as described below.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1 A and I Bshowthe underlying mechanism of yeasttwo-hybridandyeastone-hybrid δ systems. Figure 1 A shows the two-hybrid system: GAL4A represent GAL4 transcription activation domain. cDNA represents cDNA library inserts. X represents any bait gene. GAL4B represents GAL4 DNA binding domain. HIS/lacZ indicates that the reporter gene is either HIS or lacZ.
Figure 2 shows the outline of yeast two-hybrid screening. Solid black dots represent 0 colonies on plates. Transformation steps of both bait plasmid and cDNA library plasmids are indicated.
Figure 3 shows the outline of yeast one-hybrid screening. Solid black dots represent colonies on plates.
Figures 4A-4D show vectors used in the yeast two-hybrid and one-hybrid screening, 5 respectively. Figures4A-4Bshowthetwo-hybridvectors. Bait vectors can be pHybLex/Zeo
(Invitrogen), pBD-GAL4 (Stratagene), pAS2-1 (Clontech), pGBT9 (Clontech), or pGilda
(Origene, Clontech). Arrows indicate transcription of fusion proteins on either bait or cDNA vector. The binding domain can be either GAL4 or LexA. MCS underlined represents multiple cloning sites, where either bait gene or cDNA fragments should be cloned. 2 μ 0 Ori represents yeast2 micron replicationorigin.cDNAvectorscan be pYESTrp2(lnvitrogen), pAD-GAL4 (Stratagene), pACT2 (Clontech), pGADGH (Clontech), pGAD424 (Clontech), or pJG4-5 (Origene). Activationdomain can be GAL4NP16, or other transcription activator.
Figures 4C-4D show the one-hybrid reporter vectors. DΝA sequences of interest should be inserted into the multiple cloning sites (MCS) underlined. The enzyme used to Linearize δ reporter vector for integration is shown by solid arrow. The dashed arrow indicates the transcription of either HIS or lacZ gene.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides Exo proteins and nucleic acids involved in the exocytotic pathway. In a preferred embodiment, the Exo proteins are from vertebrates and more 0 preferably from mammals, including rodents (rats, mice, hamsters, guinea pigs, etc.), primates, farm animals (including sheep, goats, pigs, cows, horses, etc) and in the most preferred embodiment, from humans.
An Exo protein of the present invention may be identified in several ways. "Protein" in this sense includes proteins, polypeptides, and peptides. The Exo proteins of the invention fall into two general classes: proteins that are completely novel, i.e. are not part of a public database as of the time of discovery, although they may have homology to either known proteins or expressed sequence tags (ESTs). Alternatively, the Exo proteins are known proteins, but that were not known to be involved in exocytosis; i.e. they are identified herein as having a novel biological function. Accordingly, an Exo protein may be initially identified by its association with a protein known to be involved in exocytosis or vesicular transport.
In one embodiment provided herein, Exo proteins bind to a protein selected from the group consisting of GS27, rab7, rab9, snap-23, rab3a, rab11, rab3d, rabδ, alpha-snap, unc18-1, and vamp3. Exo proteins may be novel or may have been known in the art to exist, but not known to bind to GS27, rab7, rab9, snap-23, rab3a, rab11, rab3d, rabδ, alpha-snap, und δ-1 , or vamp3. Wherein the Exo proteins and nucleic acids are novel, compositions and methodsof useare provided herein. In the case that the Exo proteins and nucleic acids were known but not known to bind to GS27, rab7, rab9, snap-23, rab3a, rab11 , rab3d, rabδ, alpha-snap, und δ-1 , or vamp3, methods of use, i.e. functional screens, are provided.
In one embodiment, Exo nucleic acids or Exo proteins are initially identified by substantial nucleic acid and/or amino acid sequence identity or similarity to the sequences provided herein. Ina preferredembodiment.ExonucleicacidsorExoproteinshavesequenceidentity orsimilaritytothesequencesprovided herein as described below and bind to an exocytosis orvesiculartransportprotein. Preferred exocytosis and vesiculartransportproteinsinclude GS27, rab7, rab9, snap-23, rab3a, rab11 , rab3d, rabδ, alpha-snap, und δ-1 , and vamp3 (these proteins are known in the art are considered the same whether "-" is used within the name or capitals are used). Such sequence identity or similarity can be based upon the overall nucleic acid or amino acid sequence.
SEQ ID NO: 1 shows nucleic acid sequence encoding at least a portionof mouse syntaxin4, Genbankaccessionno.: U76δ32,described in HayJC, etal., J Cell Biol 199δ, 141 (7):14δ9- 1502.
SEQ ID NO:3 shows nucleic acid sequence encoding at least a portion of mouse LZIP-1 and LZIP-2, Genbank accessionno.:AC003675,describedin BurbeloPD, etal. Gene 1994, 139(2):241-245. SEQ ID NO:5 shows the nucleic acid sequence encoding at least a portion of mouse IL-3 receptor, Genbank accession no.:M29855, described in TabiraT, etal., Ann N Y Acad Sci. 1998, 640: 107-116.
SEQ ID NO:7 shows nucleic acid sequence encoding at least a portion of mouse IL-4 receptor, accession no.:M27969,describedin Ryan JJ, etal. , J Immunol.1998, 161 (4):1δ11- 1621.
SEQ ID NO:9 shows a second nucleic acid sequence encoding at least a portion of mouse IL-4 receptor, accession no.:M27959, described in Ryan JJ, et al., J Immunol. 1996, 161(4):1δ11-1δ21.
SEQ ID NO:11 shows nucleic acid sequence encoding at least a portion of mouse LDL receptor-related protein 6 (Lrp6), accession no.:AF074265, described in Brown, SD, etal., Biochem. Biophys. Res. Commun. 248 (3):879-8δδ (1996).
SEQ ID NO: 13 shows nucleic acid sequence encoding at least a portion of mouse abc2, accession no.:X76927, described in tiling M, et al., J Biol Chem 1997,11 ;272(15):10303-
10310.
SEQ ID NOS:1 δ, 17, 19, 21 , 23, and 26 shownucleic acid sequences which encode Exo3-8, respectively.
SEQ ID NO:27 shows the nucleic acid sequence encoding Exo9, which may share some characteristics with human syntaxin16A, accession no.:AF00δ937, described in Hay JC, et al., J Cell Biol 199δ, 141(7):14δ9-1502.
SEQ ID NO:29 shows the nucleic acid sequence encoding Exo10, which which may share some characteristics with human putative RNA binding protein (RBP66), accession no.:U61334, described in Genomics 33:61-67 (1996).
SEQ ID NO:31 showsthenucleicacidsequenceencodingExo11 , which has some similarity with GenBank accession no.: M144063.
SEQ ID NO:33 shows the nucleicacid sequenceencoding Exo12, which has some similarity with GenBank accession no.: AA1031δ5. SEQ ID NO:3δ shows the nucleicacidsequenceencodingExol 3, which hassomesimilarity with GenBank accession no.: AA919222.
SEQ ID NO:37 shows the nucleic acid sequenceencodingExo14, which hassomesimilarity with GenBank accession no.:AA276016 and human (xs99).
SEQ ID NO:39shows the nucleicacidsequenceencoding Exolδ, which hassomesimilarity with GenBank accession no.:AA617266, and CREB-RP (creb-rp), Genebank accession no.: U31903.
SEQ ID NO:41 shows the nucleicacidsequenceencoding Exo16, which hassomesimilarity with GenBank accession no.:AA221293 and rat lamina-assocated peptide.
SEQ ID NO:43 shows the nucleic acid sequence encoding Exo17a, which has some similarity with GenBank accession no. : AA166109 and rat syntaxinδ, Genebank accession no.:L20322, described in Rowe T, et al., Science 1998 279(6361 ):696-700.
SEQ ID NO:45 shows the nucleic acid sequence encoding Exo17b, which has some similarity with GenBank accession no. :AA166109 and rat syntaxinδ, Genebank accession no.:L20822, described in Rowe T, et al., Science 199δ 279(5361 ):696-700.
SEQ ID NO:47 shows the nucleicacidsequenceencodingExolδ, which has somesimilarity with GenBankaccession no. : AA166109and has some similarity to rat syntaxinδ, Genebank accession no.:L20δ22, described in Rowe T, et al., Science 1998 279(6361 ):696-700.
SEQ ID NO:49shows the nucleicacidsequenceencodingExol 9, which hassomesimilarity with GenBank accession no.:U76832 and mouse syntaxin4, described in Hay JC, J Cell Biol 199δ, 141(7):1489-1502.
SEQ ID NO:δ1 shows the nucleic acid sequence which encodes Exo20.
SEQ ID NO:53 shows the nucleic acid sequence which encodes Exo21.
SEQ ID NO:δ4 shows the nucleic acid sequence encoding a portion of human axonal transporter of synaptic vesicles, Genbank accession no.: X90840. SEQ ID NO:δδ shows the nucleic acid sequence encoding a portion of human cargo selection protein TIP47 (TIP47), Genbank accession no.: AF067140.
SEQ ID NO:56 shows the nucleic acid sequence encoding a portion of human cargo selection protein TIP47 (TIP47), Genbank accession no.: AF057140.
SEQ ID NO:57 shows the nucleic acid sequence encoding a portion of human cargo selection protein TIP47 (TIP47), Genbank accession no.: AF057140.
SEQ ID NO:δδ shows the nucleic acid sequence encoding a portion of human cargo selection protein TIP47 (TIP47), Genbank accession no.: AF057140.
SEQ ID NO:59 shows the nucleicacidsequenceencodinga portionof human tax interaction protein, Genbank accession no.: AF02δδ24.
SEQ ID NO:60shows the nucleic acid sequenceencodinga portion of human tax interaction protein, Genbank accession no.: AF028824.
SEQ ID NO:61 shows the nucleic acid sequenceencodinga portionofhumantaxinteraction protein, Genbank accession no.: AF026824.
SEQ ID NO:62 shows the nucleicacid sequenceencodinga portionof human human inositol polyphosphate δ-phosphatase, Genbank accession no.: M74161.
SEQ ID NO:63 shows the nucleic acid sequence encoding Exo22.
SEQ ID NO:64 shows the nucleic acid sequence encoding Exo23.
SEQ ID NO:65 shows the nucleic acid sequence encoding a portion of mouse C67BL/6J Sec61 protein complex gamma subunit; Genbank accession no.: U11027.
SEQ ID NO:66 shows the nucleic acid sequence encoding a portion of mouse HMG-1; GenBank accession no.: U00431.
SEQ ID NO:67 shows the nucleic acid sequence encoding a portion of mouse cyclin B2; GenBank accession no.: X66032. SEQ ID NO:68 shows the nucleic acid sequence encoding a portion of mouse cyclin B2; GenBank accession no.: X66032.
SEQ ID NO:69 shows the nucleic acid sequence encoding a portion of mouse pancreatic beta-cell kinesin heavy chain; GenBank accession no.: U36090.
SEQ ID NO:70 shows the nucleic acid sequence encoding a portion of mouse pancreatic beta-cell kinesin heavy chain; GenBank accession no.: U66090.
SEQ ID NO:71 shows the nucleic acid sequence encoding a portion of mouse syntaxin4; GenBank accession no.:U76832.
SEQ ID NO:72 shows the nucleic acid sequence encoding a portion of mouse syntaxin4; GenBank accession no.: U76632.
SEQ ID NO:73 shows the nucleic acid sequence encoding a portion of mouse syntaxin4; GenBank accession no.:U76δ32.
SEQ ID NO:74 shows the nucleicacid sequenceencodinga portion of mouse stearoyl-CoA desaturase (SCD2); GenBank accession no.: M26270.
SEQ ID NO:7δ shows the nucleic acid sequence encoding a portion of mouse spermidine aminopropyltransferase (Mspmsy); GenBank accession no.: AF031466.
SEQ ID NO:76 shows the nucleic acid sequence encoding a portion of mouse prothymosin alpha; GenBank accession no.: X66135.
SEQ ID NO:77 shows the nucleic acid sequence encoding a portion of mouse protein cofactor; GenBank accession no.: U74079.
SEQ ID NO:7δ shows the nucleic acid sequence encoding a portion of mouse outer dense fiber protein 2 (Odf2); GenBank accession no.: AF000966.
SEQ ID NO:79 shows the nucleic acid sequence encoding a portion of mouse protein expressed in E12 brain (clone C2); GenBank accession no.: X83569. SEQ ID NO δO shows the nucleic acid sequence encoding a portion of mouse hnRNP K homologue,GenBank accession no L29769
SEQ ID NO 61 shows the nucleic acid sequence encoding a portion of mouse NRF1 (NFE2-related factor 1), GenBank accession no X78709
SEQ ID NO 82 shows the nucleic acid sequenceencodinga portion of mouse RNA-binding protein, GenBank accession no L17076
SEQ ID NO δ3 shows the nucleic acid sequence encoding a portion of mouse dynactinl , GenBank accession no U60312
SEQ ID NO 64 shows the nucleic acid sequence encoding a portion of mouse hormone-sensitive lipase, GenBank accession no UOδlδδ
SEQ ID NO δ5 shows the nucleic acid sequence encoding a portion of mouse mtprda (human TPRD homologue), GenBank accession no AB00δ516
SEQ ID NO 66 shows the nucleic acid sequence encoding Exo24, having some similarity with GenBank accession no AA266661
SEQ ID NO 67 shows the nucleic acid sequence encoding Exo25, having some similarity with GenBank accession no AA097037
SEQ ID NO δδ shows the nucleic acid sequence encoding Exo26, having some similarity with GenBank accession no AA097037
SEQ ID NO 89 shows the nucleic acid sequence encoding Exo27, having some similarity with GenBank accession no AA269474
SEQ ID NO 90 shows the nucleic acid sequence encoding Exo28, having some similarity with GenBank accession no AA55δδδ6
SEQ ID NO 91 shows the nucleic acid sequence encoding Exo29, having some similarity with GenBank accession no AA770δ39 SEQ ID NO:92 shows the nucleic acid sequence encoding Exo30, having some similarity with GenBank accession no.: AA770639.
SEQ ID NO:93 shows the nucleic acid sequence encoding Exo31 , having some similarity with GenBank accession no.: AA416604.
δ SEQ ID NO:94 shows the nucleic acid sequence encoding Exo32, having some similarity with GenBank accession no.: AA416604.
SEQ ID NO:9δ shows the nucleic acid sequence encoding Exo33, having some similarity with GenBank accession no.: AA416504.
SEQ ID NO:96 shows the nucleic acid sequence encoding Exo34, having some similarity 0 with GenBank accession no.: AA416604.
SEQ ID NO:97 shows the nucleic acid sequence encoding Exo35, having some similarity with GenBank accession no.: AA415604.
SEQ ID NO:98 shows the nucleic acid sequence encoding Exo36, having some similarity with GenBank accession no.: AA416604.
δ SEQ ID NO:99 shows the nucleic acid sequence encoding Exo37, having some similarity with GenBank accession no.: AA416604.
SEQ I D NO: 100 shows the nucleic acid sequence encoding Exo38, having some similarity with GenBank accession no.: AA416604.
SEQ ID NO: 101 shows the nucleic acid sequence encoding Exo39, having some similarity 0 with GenBank accession no.: AA416604.
SEQ ID NO: 102 shows the nucleic acid sequence encoding Exo40, having some similarity with GenBank accession no.: AA416604.
SEQ ID NO: 103 shows the nucleic acid sequence encoding Exo41 , having some similarity with GenBank accession no.: AA172926. SEQ ID NO: 104 shows the nucleic acid sequence encoding Exo42, having some similarity with GenBank accession no.: AA2δδ130.
SEQ I D NO: 106 shows the nucleic acid sequence encoding Exo43, having some similarity with GenBank accession no.: AI131639.
SEQ ID NO: 106 shows the nucleic acid sequence encoding Exo44, having some similarity with GenBank accession no.: AA164709.
SEQ ID NO: 107 shows the nucleic acid sequence encoding Exo4δ, having some similarity with GenBank accession no.: AA266406.
SEQ I D NO: 10δ shows the nucleic acid sequence encoding Exo46, having some similarity with GenBank accession no.: AA5631δδ.
SEQ ID NO: 109 shows the nucleic acid sequence encoding Exo47, having some similarity with GenBank accession no.: AA619170.
SEQ ID NO: 110 shows the nucleic acid sequence encoding Exo4δ, having some similarity with GenBank accession no.: AA519170.
SEQ ID NO:111 shows the nucleic acid sequence encoding Exo49, having some similarity with yeast ORMI, GenBank accession no.: AA175196.
SEQ ID NO: 112 shows the nucleic acid sequence encoding ExoδO, having some similarity with rat mt-GrpE no.1 precursor, GenBank accession no.: AA060δ61.
SEQ ID NO: 113 shows the nucleic acid sequence encoding Exoδl , having some similarity with human CENP-F kinetochore protein, GenBank accession no.: AI034171.
SEQ ID NO: 114 shows the nucleic acid sequence encoding Exoδ2, having some similarity with human arfaptin 2 (putative target of ADP-ribosylation factor), GenBank accession no.:AA6439δδ.
SEQ I D NO: 11 δ shows the nucleic acid sequence encoding Exoδ3, having some similarity with human brain and reproductive organ-expressed protein (BRE); GenBank accession no.: AA200603. SEQ ID NO: 116 shows the nucleic acid sequence encoding Exoδ4, having some similarity with human brain and reproductive organ-expressed protein (BRE); GenBank accession no.: AA200608.
SEQ ID NO: 117 shows the nucleic acid sequence encoding Exoδδ, having some similarity with human cell cycle progression 2 protein (CPR2); GenBank accession no.: W67077.
SEQ I D NO: 11 δ shows the nucleic acid sequence encoding Exoδ6, having some similarity with human spliceosomeassociatedprotein(SAP14δ); GenBankaccession no.: AH 19401.
SEQ ID NO: 119 shows the nucleic acid sequence encoding Exoδ7, having some similarity with mesocricetus auratus stearyl-CoA desaturase (FAR-17c); GenBank accession no.: AA337696.
SEQ ID NO: 120 shows the nucleic acid sequence encoding Exoδδ, having some similarity with rat inositol trisphosphate receptor subtype 3 (IP3R-3); GenBank accession no.: AAδ23026.
SEQ ID NO: 121 shows the nucleic acid sequence encoding Exoδ9, having some similarity with L-Asparaginase; GenBank accession no.: AH 13730.
SEQ ID NO: 122 shows the nucleic acid sequence encoding Exo60, having some similarity with L-Asparaginase; GenBank accession no.: AH 16730.
SEQ ID NO: 123 shows the nucleic acid sequence encoding Exo61 , having some similarity with human RB-binding protein 2 (RBBP-2); GenBank accession no.: AA756316.
SEQ I D NO: 124 shows the nucleic acid sequence encoding Exo62, having some similarity with human secreted apoptosis related protein 3 (SARP3); GenBank accession no.: AU01δδ90.
SEQ ID NO: 126 shows the nucleic acid sequence encoding Exo63, having some similarity with myosin heavy chain; GenBank accession no.: AA237764.
SEQ ID NO: 126 shows the nucleic acid sequence encoding Exo64, having some similarity with myosin heavy chain; GenBank accession no.: AA237764. SEQ ID NO: 127 shows the nucleic acid sequence encoding Exoδδ, having some similarity with myosin heavy chain; GenBank accession no.: AA237764.
SEQ ID NO: 123 shows the nucleic acid sequence encoding Exo66, having some similarity with myosin heavy chain; GenBank accession no.: AA237764.
SEQ ID NO:129 shows the nucleic acid sequence encoding Exo67, having some similarity with rat tomosyn; GenBank accession no.: AA437465.
SEQ ID NO: 130 shows the nucleic acid sequence encoding Exo6δ, having some similarity with rat tomosyn; GenBank accession no.: AA437465.
SEQ ID NO: 131 shows the nucleic acid sequence encoding Exo69, having some similarity with rat tomosyn; GenBank accession no.: AA437465.
SEQ ID NO: 132 shows the nucleic acid sequence encoding Exo70, having some similarity with rat tomosyn; GenBank accession no.: AA437466.
SEQ ID NO: 133 shows the nucleic acid sequence encoding Exo71 , having some similarity with human mcag29 CTG repeat region; GenBank accession no.: AA039340.
SEQ ID NO: 134 shows the nucleic acid sequence encoding Exo72, having some similarity with rat G protein gamma-5 subunit; GenBank accession no.: AA021679.
SEQ ID NO: 136 shows the nucleic acid sequence encoding Exo73, having some similarity with rat G protein gamma-5 subunit; GenBank accession no.: AA021679.
SEQ ID NO: 136 shows the nucleic acid sequence encoding Exo74, having some similarity with rat G protein gamma-5 subunit; GenBank accession no.: AA021879.
SEQ ID NO: 137 shows the nucleic acid sequence encoding Exo75, having some similarity with rat G protein gamma-δ subunit; GenBank accession no.: AA021879.
SEQ ID NO: 136 shows the nucleic acid sequence encoding Exo76, having some similarity with rat G protein gamma-5 subunit; GenBank accession no.: AA021679. SEQ ID NO: 139 shows the nucleic acid sequence encoding Exo77, having some similarity with rat G protein gamma-δ subunit; GenBank accession no.: AA021879.
SEQ ID NO: 140 shows the nucleic acid sequence encoding Exo78.
SEQ ID NO:141 shows the nucleic acid sequence encoding Exo79.
δ SEQ ID NO: 142 shows the nucleic acid sequence encoding ExoδO.
SEQ ID NO:143 shows the nucleic acid sequence encoding Exo81.
SEQ ID NO: 144 shows the nucleic acid sequence encoding a portion of human HLA-B-associated transcript 3; GenBank accession no.: M33619.
SEQ ID NO:14δ shows the nucleic acid sequence encoding a portion of human 0 HLA-B-associated transcript 3; GenBank accession no.: M33619.
SEQ ID NO:146 shows the nucleic acid sequence encoding a portion of human T cell leukemia/lymphoma 1 ; GenBank accession no.: X82240.
SEQ ID NO: 147 shows the nucleic acid sequence encoding Exo82.
6 SEQ ID NO:148 shows the nucleic acid sequence encoding Exo83; may have some homology with rat rabin3.
SEQ ID NO:149 shows the nucleic acid sequence encoding at least a portion of human KIAA0666; GenBank accession no.: AB014665.
SEQ ID NO: 160 shows the nucleic acid sequence encoding Exo84 which may have some 0 similarity with Rabin 3, GenBank accession no.: AA346676.
SEQ ID NO:161 shows the nucleic acid sequence encoding Exoδδ which may have some similarity with Rabin 3, GenBank accession no.: AA346676.
SEQ ID NO: 162 shows the nucleic acid sequence encoding Exoδδ which may have some δ similarity with human KI 0665, GenBank accession no.: AA757034. SEQ ID NO: 153 shows the nucleic acid sequence encoding a portion of mouse protein cofactor; GenBank accession no.: U74079.
SEQ ID N0.154 shows the nucleic acid sequence encoding a portion of mouse protein cofactor; GenBank accession no.: U74079.
SEQ ID NO:1δ5 shows the nucleic acid sequence encoding Exo37, may have some similarity with calcium-dependent protein kinase, Genbank accession no.: AA770736.
SEQ ID NO: 156 shows the nucleic acid sequence encoding Exo88, may have some similarity with calcium-dependent protein kinase, Genbank accession no.: AA770736.
SEQ ID NO: 157 shows the nucleic acid sequence encoding Exoδ9, may have some similarity with calcium-dependent protein kinase, Genbank accession no.: AA770736.
SEQ ID NO: 153 shows the nucleic acid sequence encoding Exo90, may have some similarity with calcium-dependent protein kinase, Genbank accession no.: AA770736.
SEQ ID NO: 159 shows the nucleic acid sequence encoding Exo91 , may have some similarity with human mcag29 CTG repeat region, Genbank accession no.: AA473325.
SEQ ID NO: 160 shows the nucleic acid sequence encoding Exo92, may have some similarity with human mcag29 CTG repeat region, Genbank accession no.: AA473325.
SEQ ID NO:161 shows the nucleic acid sequence encoding Exo93, may have some similarity with Genbank accession no.: AA136122.
SEQ ID NO: 162 shows the nucleic acid sequence encoding Exo94, may have some similarity with Genbank accession no.: AA136122.
SEQ ID NO:163 shows the nucleic acid sequence encoding Exo9δ, may have some similarity with Genbank accession no.: AA133122.
SEQ ID NO: 164 shows the nucleic acid sequence encoding Exo96, may have some similarity with Genbank accession no.: AA133122. SEQ ID NO: 165 shows the nucleic acid sequence encoding Exo97, may have some similarity with Genbank accession no.: AA136122.
SEQ ID NO: 166 shows the nucleic acid sequence encoding Exo98, may have some similarity with Genbank accession no.: AA060976.
SEQ ID NO: 167 shows the nucleic acid sequence encoding Exo99, may have some similarity with Genbank accession no.: AA277208.
SEQ ID NO: 168 shows the nucleic acid sequence encoding ExolOO, may have some similarity with Genbank accession no.: AA277208.
SEQ ID NO: 169 shows the nucleic acid sequence encoding Exo101 , may have some similarity with Genbank accession no.: AA467477.
SEQ ID NO: 170 shows the nucleic acid sequence encoding Exo102, may have some similarity with Genbank accession no.: AA467477.
SEQ ID NO:171 shows the nucleic acid sequence encoding Exo103, may have some similarity with Genbank accession no.: AA833213.
SEQ ID NO: 172 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403.
SEQ ID NO: 173 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
SEQ ID NO: 174 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403.
SEQ ID NO:175 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403.
SEQ ID NO: 176 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403. SEQ ID NO: 177 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403.
SEQ ID NO:17δ shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X95403.
δ SEQ ID NO: 179 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
SEQ ID NO: 160 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
SEQ ID NO:1δ1 shows the nucleic acid sequence encoding a portion of Rab2; GenBank 0 accession no.: X96403.
SEQ ID NO: 1δ2 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
SEQ ID NO: 163 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
δ SEQ ID NO: 164 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
SEQ ID NO:1 δδ shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
SEQ ID NO: 166 shows the nucleic acid sequence encoding a portion of Rab2; GenBank 0 accession no.: X96403.
SEQ ID NO:187 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
SEQ ID NO:188 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403. SEQ ID NO: 189 shows the nucleic acid sequence encoding a portion of Rab2; GenBank accession no.: X96403.
SEQ ID NO: 190 shows the nucleic acid sequence encoding a portion of Rab5C; GenBank accession no.: AA230407.
5
SEQ ID NO: 191 shows the nucleic acid sequence encoding a portion of RabδC; GenBank accession no.: AA230407.
SEQ ID NO: 192 shows the nucleic acid sequence encoding a portion of RabδC; GenBank accession no.: AA230407.
0 SEQ ID NO: 193 shows the nucleic acid sequence encoding Exo104.
SEQ ID NO: 194 shows the nucleicacid sequence encoding Exol Oδ, a human gene similar to mouse testis-specific protein PBS13; may have some similarity to GenBank Accession no.: AA184366.
δ SEQ ID NO: 196 shows the nucleic acid sequence encoding Exo106; may have some similarity to GenBank Accession no.: AA604490.
SEQ ID NO: 196 shows the nucleic acid sequence encoding Exo107; may have some similarity to GenBank Accession no.: AA504490.
0 SEQ ID NO: 197 shows the nucleic acid sequence encoding Exo108; may have some similarity to GenBank Accession no.: AI181750.
SEQ ID NO: 198 shows the nucleic acid sequence encoding Exo109; may have some similarity to GenBank Accession no.: AI181760.
δ SEQ ID NO: 199 shows the nucleic acid sequence encoding Exo110; may have some similarity to GenBank Accession no.: AU043111.
SEQ ID NO:200 shows the nucleic acid sequence encoding Exol 11 , a human gene similar to rat alpha-soluble NSF attachment protein (SNAP). SEQ ID NO 201 shows the nucleic acid sequence encoding Exo112 shows the nucleic acid sequence encoding Exo105, a human gene similar to mouse testis-spe fic protein PBS13, may have some similarity to GenBank Accession no AA184366
SEQ ID NO 202 shows the nucleic acid sequence encoding Exol 13 which may be similar to a mouse zinc finger protein
SEQ ID NO 203 shows the nucleic acid sequence encoding Exol 14 which may be similar to a mouse zinc finger protein
SEQ ID NO 204 shows the nucleic acid sequence encoding Exol 15 which may be similar to chicken c-hairy 1 , may have some similarity to GenBank Accession no AA116067
SEQ ID NO 205 shows the nucleic acid sequence encoding Exol 16 which may be similar to chicken c-hairy 1 , may have some similarity to GenBank Accession no AA116067
SEQ ID NO 206 shows the nucleic acid sequence encoding a portion of mouse syntaxιn4, GenBank accession no U76332
SEQ ID NO 207 shows the nucleic acid sequence encoding a portion of mouse interleukin (IL) -3 receptor, GenBank accession no M29356
SEQ ID NO 20δ shows the nucleic acid sequence encoding a portion of mouse interleukin (IL) -3 receptor, GenBank accession no M29866
SEQ ID NO 209 shows the nucleic acid sequence encoding a portion of mouse low density lipoprotem (LDL) receptor-related protein, GenBank accession no AF074266
SEQ ID NO 210 shows the nucleic acid sequence encoding Exo117, similar to a human ANF126 zinc protein
SEQ ID NO 211 shows the nucleic acid sequence encoding Exo118, similar to a rat isoprenylated 67 kDa protein
As indicated above, Exo3-Exo11δ are novel The Exoproteinsencodedby SEQ ID NOS 1- 61 (odd numbers) and Sequence ID NOS 63-211 are each novel in the aspect that they are shown herein to bind to an exocytosisorvesiculartransport protein, or fragment thereof, for the first time. In preferred embodiments, the proteins encoded by SEQ ID NOS: 1-51 (odd numbers) bind to GS27; the proteins encoded by SEQ ID NO:53 bind to rab7; the proteins encoded by SEQ ID NOS:δ4-64 bind to rab9; the proteins encoded by SEQ ID NOS:6δ-143 bind to snap-23; the proteins encoded by SEQ ID NOS: 144-148 bind to rab3a; δ the proteins encoded by SEQ ID NOS:149-162 bind to rab11 ; the proteinsencoded by SEQ
ID NOS: 163-171 bind to rab3d; the proteinsencoded by SEQ ID NOS: 172-193 bind to rab5; the proteins encoded by SEQ ID NOS: 194-201 bind to alpha-snap; the proteins encoded by SEQ ID NOS:202-205 bind to und 8-1 ; and, the proteins encoded by SEQ ID NOS:206- 211 bind to vamp3.
0 In a preferred embodiment, a protein is a "Exo protein" if the overall sequence identity of the protein sequence to any one ofthe amino acid sequences encoded by SEQ ID NOS: 1- 61 , odd numbers, and SEQ ID NOS:53-211 , preferably those sequences encoding Exo3- 11 δ, is preferably greater than about 76%, more preferably greater than about 60%, even more preferably greater than about δδ% and most preferably greater than 90%. In some δ embodiments the sequence identity will be as high as about 93 to 96 or 93%. As is known in the art, a number of different programs can be used to identify whether a nucleic acid has sequence identity or similarity to a known gene or expression sequence tag (EST). Sequence identity will be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. 0 Math.2:4δ2 (19δ1), by the sequence identity alignmentalgorithmofNeedleman&Wunsch,
J. Mol. Biool.4δ:443(1970), by the search for similarity method of Pearson& Lipman. PNAS USA 85:2444 (193δ), by computerizedimplementationsof these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wl), the Best Fit sequence program described by 5 Devereux eta/., Nucl. Acid Res. 72:337-395 (1964), preferably using the default settings, or by inspection. Preferably, percent identity is calculated by FastDB based upon the following parameters: mismatch penalty of 1 ; gap penalty of 1 ; gap size penalty of 0.33; and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis," MacromoleculeSequencingand Synthesis, Selected Methods and Applications, pp 127-149 0 (19δδ), Alan R. Liss, Inc.
Anexampleofa usefulalgorithmis PILEUP. PILEUPcreatesa multiplesequencealignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplificationof the progressivealignmentmethod of Feng & Doolittle,J. Mol. Evol.35:351- 5 360 ( 1937); the method is similar to that described by Higgins & Sharp CABIOS 5: 151 -163 (1989). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 275, 403-410, (1990) and Karlin et al., PNAS USA 90:6873-6737 (1993). A δ particularly useful BLAST program is the WU-BLAST-2 program which was obtained from
Altschul et al., Methods in Enzymology, 266: 460-480 (1996); http://blast.wustl/edu/blast/ README.html]. WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span =1 , overlap fraction = 0.125, word threshold (T) = 11. The HSP S and HSP S2 0 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. A % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of 5 the "longer" sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
In a similar manner, "percent (%) nucleic acid sequence identity" with respect to the coding sequence of the polypeptides identified herein is defined as the percentage of nucleotide 0 residuesin a candidatesequencethatare identicalwith the nucleotideresiduesin the coding sequence of the Exo protein. A preferred method utilizes the BLASTN module of WU- BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
The alignment may include the introduction of gaps in the sequences to be aligned. In 5 addition, for sequences which contain either more or fewer amino acids than the protein encoded by the sequences in the Sequence Listing, it is understood that the percentage of sequence identity will be determined based on the number of identical amino acids in relation to the total number of amino acids. Thus, for example, sequence identity of sequences shorter than that shown in the Sequence Listing, as discussed below, will be determined using the number of amino acids in the shorter sequence.
As an example, SEQ ID NOS: 1-51 (odd numbers)and SEQ ID NOS:53-211 were identified as follows. A basic Blast search has been performed using program "Blastn" and database "nr". Blastn is a NCBI BLAST family of program used to compared a nucleotide query sequence against a nucleotide sequence database, and nr is a nucleotide sequence d a ta ba s e t h a t i n c l u d es a l l n o n - re d u n d a n t G e n B a n k C D S translations+PDB+SwissProt+PIR+PRF. Two numbers, Score (bits) and E values, will be returned after search querry is submitted. In general sequences considered known had a Score > 100 and E < 0.001. Using the same Blast search, the nucleic acid sequences encoding Exo3-118 had a Score < 100 or E > 0.001. These nucleic acid sequences encoding Exo3-11δ were then further searched using program "Blastn" and database "dbest". The dbest database is a nucleotidesequencedatabasethatincludesnon-redundant Database of GenBank+EMBL+DDBJEST Divisions. Using this criteria, some ofthe nucleic acid sequences had a Score > 100 and E < 0.001 , thus, these sequences are considered novel, yet have "somesimilarity"to a known sequenceas indicated by the accession number provided. The sequences ofthe accession numbers provided herein are readily available to the skilled artisan.
As will be appreciated by those in the art, the nucleic acid sequences ofthe invention can be used to generate protein sequences. There are a variety of ways to do this, including cloning the entire gene and verifying its frame and amino acid sequence, or by comparing it to known sequences to search for homology to provide a frame, assuming the novel Exo protein has homology to some protein in the database being used. Generally, the nucleic acid sequences are input into a program that will search all three frames for homology. This is done in a preferred embodiment using the following NCBI Advanced BLAST parameters. The program is blastx or blastn. The database is nr. The input data is as "Sequence in FASTA format". The organism list is "none". The "expect" is 10; the filter is default. The "descriptions" is 600, the "alignments" is 600, and the "alignment view" is pairwise. The "Query Genetic Codes" is standard (1). The matrix is BLOSUM62; gap existence cost is 11 , per residue gap cost is 1 ; and the lambda ratio is .65 default. This results in the generation of a putative protein sequence. While this program can be used to generatea preferredproteinsequence, it is understood thatthepresentinventionprovides polypeptides encoded by each of the three frames of each nucleic acid provided herein. Thus, when a protein encoded by the nucleic acid herein is described, the skilled artisan understands that the protein begins with the first amino acid encoded by the first codon of the coding region, which is not necessarily the first nucleotide in the sequence listing.
As will be appreciated by those skilled in the art, the sequences of the present invention may contain sequencing errors. That is, there may be incorrect nucleosides, frameshifts, unknownnucleosides.orothertypesofsequencingerrorsin any ofthe sequences; however, the correct sequences will fall within the homology and stringency definitions herein. In addition, as will be appreciated by those in the art, in general, the first 200 bases or so of sequence contains the fewest errors. In a preferred embodiment, the Exo proteins are encoded by a nucleic acid comprising the first 100 nucleotides of the sequences set forth in the Sequence Listing and bind to an exocytosis or vesicular transport proteinorfragment thereof.
Exo proteins of the present invention may be shorter or longer than the amino acid sequences encoded by the nucleic acids shown in the Sequence Listing. Thus, in one embodiment, Exo proteins can be portions or fragments of the amino acid sequences encoded by the nucleic acid sequences provided herein. In one embodiment herein, fragmentsof Exo proteinsare considered Exo proteins if a) they share at leastoneantigenic epitope; b) have at least the indicated sequence identity; c) and preferably have Exo biologicalactivity, including binding to an exocytosis or vesicular transport protein. In some cases, where the sequence is used diagnostically, that is, when the presence or absence of Exo protein nucleic acid is determined, only the indicated sequence identity is required. The nucleic acids ofthe present invention may also be shorteror longerthan the sequences in the Sequence Listing. The nucleic acid fragmentsincludeany portion of the nucleic acids provided herein which have a sequence not exactly previously identified; fragments having sequences with the indicated sequence identity to that portion not previously identified are provided in an embodiment herein.
In addition, as is more fully outlined below, Exo proteins can be made that are longer than those depicted in the Sequence Listings; for example, by the addition of epitope or purification tags, the addition of other fusion sequences, or the elucidation of additional coding and non-coding sequences. As described below, the fusion of an Exo peptide to a fluorescent peptide, such as Green Fluorescent Peptide (GFP), is particularly preferred.
Exo proteins may also be identified as encoded by Exo nucleic acids which hybridize to any one of the sequences depicted in SEQ ID NOS: 1-51 , odd numbers, and SEQ ID NOS:53-211 , preferably those encoding Exo3-11δ. Hybridization conditions are further described below.
In a preferred embodiment, when an Exo protein is to be used to generate antibodies, an Exo protein must share at least one epitope or determinant with the full length protein. By
"epitope" or "determinant" herein is meant a portion of a protein which will generate and/or bind an antibody. Thus, in most instances, antibodies made to a smaller Exo protein will be able to bind to the full length protein. In a preferred embodiment, the epitope is unique; that is, antibodies generated to a unique epitope show little or no cross-reactivity. The term "antibody" includes antibody fragments, as are known in the art, including Fab Fab2, single chain antibodies (Fv for example), chimeric antibodies, etc., either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies.
In a preferred embodiment, the antibodies to Exo are capable of reducing or eliminating the biological function of Exo, as is described below. That is, the addition of anti-Exo antibodies(eitherpolyclonal or preferably monoclonal) to Exo (or cells containing Exo) may reduce or eliminate the Exo activity. Generally, at least a 2δ% decrease in activity is preferred, with at least about 50% being particularly preferred and about a 95-100% decrease being especially preferred.
The Exo antibodies of the invention specifically bind to Exo proteins. In a preferred embodiment, the antibodies specifically bind to Exo proteins. By "specifically bind" herein is meant that the antibodies bind to the protein with a binding constant in the range of at least 10"4- 10"6 M"\ with a preferred range being 10"7 - 10"9 M"1. Antibodies are further described below.
in the case of the nucleic acid, the overall sequence identity of the nucleic acid sequence is commensurate with amino acid sequence identity but takes into account the degeneracy in the genetic code and codon bias of different organisms. Accordingly, the nucleic acid sequence identity may be either lower or higher than that of the protein sequence. Thus the sequence identity of the nucleic acid sequence as compared to the nucleic acid sequences ofthe Sequence Listing, is preferablygreaterthan 75%, more preferablygreater thanabout30%, particularly greaterthan about δδ% and mostpreferablygreaterthan90%. In some embodiments the sequence identity will be as high as about 93 to 95 or 9δ%.
In a preferred embodiment, an Exo nucleic acid encodes an Exo protein. As will be appreciated by those in the art, due to the degeneracy of the genetic code, an extremely large number of nucleic acids may be made, all of which encode the Exo proteins of the present invention. Thus, having identified a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the Exo. In one embodiment, the nucleic acid is determined through hybridization studies Thus, for example, nucleic acids which hybridize under high stringency to the nucleic acid sequences shown in the sequence listing, or its complement is considered an Exo gene High stringency conditions are known in the art, see for example Maniatis etal , Molecular Cloning A Laboratory Manual, 2d Edition, 1969, and Short Protocols in Molecular Biology, ed Ausubel. etal , both of which are hereby incorporatedby reference Stnngentconditions are sequence-dependentand will be differentin differentcircumstances Longersequences hybridize specifically at higher temperatures An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology- Hybridization with Nucleic Acid Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993) Generally, stringent conditions are selected to be about δ-10°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 60% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium) Stringent conditions will be those in which the salt concentration is less than about 1 0 sodium ion, typically about 0 01 to 1 0 M sodium ion concentration (or other salts) at pH 7 0 to 8 3 and the temperature is at least about 30°C for short probes (e g 10 to 60 nucleotides) and at least about 60°C for long probes (e g greater than 60 nucleotides) Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide
In another embodiment, less stringent hybridization conditions are used, for example, moderate or low stringency conditions may be used, as are known in the art, see Maniatis and Ausubel, supra, and Tijssen, supra
The Exo proteins and nucleic acids of the present invention are preferably recombinant
As used herein, "nucleic acid" may refer to either DNA or RNA, or molecules which contain both deoxy- and nbonucleotides The nucleic acids include genomic DNA, cDNA and oligonucleotides including sense and anti-sense nucleic acids Such nucleic acids may also contain modifications in the nbose-phosphate backbone to increase stability and half life of such molecules in physiological environments
The nucleicacid may be doublestranded.singlestranded, or contain portionsof both double stranded or single stranded sequence As will be appreciated by those in the art, the depiction of a single strand ("Watson") also defines the sequence of the other strand ("Crick"), thus the sequences depicted in the Sequence Listing also includethe complement of the sequence. By the term "recombinant nucleic acid" herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleicacid by endonucleases, in a form not normally found in nature. Thus an isolated Exo nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e. using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention.
Similarly, a "recombinant protein" is a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid as depicted above. A recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics. For example, the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host, and thus may be substantially pure. For example, an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight ofthe total protein in a given sample. A substantially pure protein comprises at least about 76% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred. The definition includes the production of an Exo protein from one organism in a different organism or host cell. Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of a inducible promoter or high expression promoter, such that the protein is made at increased concentration levels. Alternatively, the protein may be in a form not normally found in nature, as in the addition of an epitope tag or amino acid substitutions, insertions and deletions, as discussed below.
Also included within the definition of Exo proteins of the present invention are amino acid sequence variants. These variants fall into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding an Exo protein, using cassette or PCR mutagenesisorothertechniqueswell known in the art, to produceDNAencodingthe variant, and thereafterexpressingthe DNA in recombinant cell culture as outlined above. However, variant Exo protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques. Amino acid sequence variants are characteπzed by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the Exo protein ammo acid sequence The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below
While the site or region for introducing an ammo acid sequence variation is predetermined, the mutation per se need not be predetermined For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the targetcodon or region and theexpressedExovariantsscreenedfortheoptimalcombination of desired activity Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis Screening of the mutants is done using assays of Exo protein activities
Ammo acid substitutions are typically of single residues, insertions usually will be on the order of from about 1 to 20 ammo acids, although considerably larger insertions may be tolerated Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger
Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative Generally these changes are done on a few ammo acids to minimize the alteration of the molecule However, larger changes may be tolerated in certain circumstances When small alterations in the characteristics of the Exo protein are desired, substitutions are generally made in accordance with the following chart
Chart I Original Residue Exemplary Substitutions
Ala Ser
Arg Lys
Asn Gin, His
Asp Glu
Cys Ser Gin Asn
Glu Asp
Gly Pro lle Leu, Val
Leu lie, Val
Lys Arg, Gin, Glu
Met Leu, lie Phe Met, Leu, Tyr
Ser Thr
Thr Ser
Trp Tyr
Tyr Trp, Phe Val lie, Leu
Substantialchangesin functionor immunological identity are made by selectmgsubstitutions that are less conservative than those shown in Chart I For example, substitutions may be made which more significantly affect the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure, the charge or hydrophobicity of the molecule at the target site, or the bulk of the side chain The substitutions which in general are expected to produce the greatest changes in the poly peptide's properties are those in which (a) a hydrophi c residue, e g sery I or threonyl, is substituted for (or by) a hydrophobic residue, e g leucyl, isoleucyl, phenylalanyl, valyl or alanyl, (b) a cysteine or prolme is substituted for (or by) any other residue, (c) a residue having an electropositive side chain, e g lysyl, argmyl, or histidyl, is substituted for (or by) an electronegative residue, e g glutamyl or aspartyl, or (d) a residue having a bulky side chain, e g phenylalanine, is substituted for (or by) one not having a side chain, e g glycine
The variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurrmganalogue, although variants also are selected to modify the characteristics ofthe Exo proteins as needed Alternatively, the variant may be designed such that the biological activity of the Exo protein is altered For example, glycosylationsites may be alteredor removed Similarly, mutationswithin the kinasedomain and/or the cell death domain may be made
Covalent modifications of Exo polypeptides are included within the scope of this invention One type of covalent modification includes reacting targeted aminoacid residues of an Exo polypeptide with an organic derivatizmg agent that is capable of reacting with selected side chamsorthe N-orC-terminalresiduesof an Exo polypeptide Denvatizationwith bifunctional agents is useful, for instance, for crosslmking Exo to a water-insoluble support matrix or surface for use in the method for purifying anti-Exo antibodies or screening assays, as is more fully described below Commonly used crosslink gagents include, e g , 1 , 1 -bιs(dιazo- acetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimideesters, for example, esters with 4-azιdosalιcylιc acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dιthιobιs(succιnιmιdylpropιonate), bifunctional maleimides such as bis-N- maleιmιdo-1 ,δ-octane and agents such as methyl-3-[(p-azιdophenyl)dιthιo]propιoιmιdate
Other modifications include deamidation of glutaminyl and asparagmyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysme, phosphorylation of hydroxyl groups of seryl orthreonyl residues, methylation ofthe "-ammo groups of lysme, arginme, and histidme side chains [T E Creighton, Proteins
Structureand MolecularProperties, W H Freeman & Co , San Francisco, pp 79-36(1963)], acetylation of the N-terminal amme, and amidation of any C-terminal carboxyl group
Another type of covalent modification of the Exo polypeptide included within the scope of this inventioncompπsesalteringthenativeglycosylationpatternof the polypeptide "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence Exo polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence Exo polypeptide
Addition of glycosylation sites to Exo polypeptides may be accomplished by altering the am o acid sequence thereof The alteration may be made, for example, by the addition of, or substitution by, one or more serine orthreonine residues to the native sequence Exo polypeptide (for O-linked glycosylation sites) The Exo aminoacid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the Exo polypeptide at preselected bases such that codons are generated thatwill translate into the desired ammo acids
Another means of increasing the number of carbohydrate moieties on the Exo polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide Such methods are described in the art, e g , in WO 67/05330 published 11 September 1967, and in Aplm and Wriston, CRC Cnt Rev Biochem , pp 259-306 (1961)
Removal of carbohydrate moieties present on the Exo polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for ammo acid residues thatserveas targets for glycosylation Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al , Arch Biochem Biophvs , 259 52 (1987) and by Edge et al , Anal Biochem , 118 131 (1931) Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo-and exo-glycosidases as described by Thotakura et al , Meth Enzymol , 138 350 (1987)
Another type of covalent modification of Exo comprises linking the Exo polypeptide to one ofa variety of nonproteinaceous polymers, e g , polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U S Patent Nos 4,640,836, 4,496,689, 4,301 ,144, 4,670,417, 4,791 ,192 or 4,179,337
Exo polypeptides of the present invention may also be modified in a way to form chimeric molecules comprising an Exo polypeptide fused to another, heterologous polypeptide or am o acid sequence In one embodiment, such a chimeric molecule comprises a fusion of an Exo polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind The epitope tag is generally placed at the ammo-orcarboxy I- terminus of the Exo polypeptide The presence of such epitope-tagged forms of an Exo polypeptide can be detected using an antibody against the tag polypeptide Also, provision of the epitope tag enables the Exo polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag
In an alternative embodiment, the chimeric molecule may comprise a fusion of an Exo polypeptide with an immunoglobulin or a particular region of an immunoglobu n For a bivalent form of the chimeric molecule, such a fusion could be to the Fc region of an IgG molecule as discussed further below
Vanoustag polypeptidesandtheirrespectiveantibodiesare well known in the art Examples include poly-histidme (poly-his) or poly-histidme-glycine (poly-his-gly) tags, the flu HA tag polypeptide and its antibody 12CA5 [Field et al , Mol Cell Biol . 8 2169-2165 (1988)], the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al , Molecular and Cellular Biology, 5 3610-3616 (1935)], and the Herpes Simplex virus glycoproteιn D (gD) tag and its antibody [Paborsky etal . Protein Engineering. 3(6) 547-553 (1990)] Other tag polypeptides include the Flag-peptide [Hopp et al , BioTechnology. 6 1204-1210 (1988)], the KT3 epitope peptide [Martin etal . Science, 255 192-194(1992)], tubulm epitope peptide [Skinner et al , J Biol Chem . 266 15163-15166 (1991)], and the T7 gene 10 protein peptιdetag [Lutz-Freyermuthetal . Proc Natl Acad Sci USA, 87 6393-
6397 (1990)]
In an embodiment herein, Exo proteins of the Exo family and Exo proteins from other organisms are cloned and expressed as outlined below Thus, probe or degenerate poly merase chain reaction (PCR) primer sequences may be used to find other related Exo proteins from humans or other organisms As will be appreciated by those in the art, particularly useful probe and/or PCR primer sequences include the unique areas of the Exo nucleic acid sequence As is generally known in the art, preferred PCR primers are from about 16 to about 36 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosine as needed The conditions for the PCR reaction are well known in the art It is thereforealso understood that provided along with the sequences in the sequences listed herein are portions of those sequences, wherein unique portions of 16 nucleotides or more are particularly preferred The skilled artisan can routinely synthesize or cut a nucleotide sequence to the desired length
Once the Exo nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombmedtoform the entire Exo nucleic acid Once isolated from its natural source, e g , contained within a plasmid or other vector or excised therefrom as a linear nucleic acid segment, the recombinant Exo nucleic acid can be further-used as a probe to identify and isolate other Exo nucleic acids It can also be used as a "precursor" nucleic acid to make modified or variant Exo nucleic acids and proteins The skilled artisan understands that wherein two or more nucleic acids overlap, the overlapping portιon(s) of one of the overlapping nucleic acids can be omitted and the nucleic acids combined for example, by gation, to form a longer linear Exo nucleic acid so as to, for example, encode the full length or mature peptide The same applies to the ammo acid sequences of Exo polypeptides in that they can be combined so as to form one contiguous peptide
Using the nucleic acids of the present invention which encode an Exo protein, a variety of expression vectors are made The expression vectors may be either self-replicating extrachromosomalvectors or vectors which integrate into a hostgenome Generally, these expressionvectorsincludetranscπptional and translational regulatory nucleicacid operably linked to the nucleic acid encoding the Exo protein The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a πbosome binding site Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers
Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleicacid sequence Forexample. DNAfora presequenceorsecretory leadens operably linked to DNA for a polypeptide if it is expressed as a preprotem that participates in the secretionof the polypeptide.a promoterorenhanceπs operably Imkedto a coding sequence if it affects the transcription of the sequence, or a πbosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase However, enhancers do not have to be contiguous Linking is accomplished by ligation at convenient restriction sites If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice The transcnptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the Exo protein, for example, transcnptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the Exo protein in Bacillus Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells
In general, the transcnptional and translational regulatory sequences may include, but are not limited to, promoter sequences, nbosomal binding sites, transcnptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences In a preferredembodiment, the regulatory sequences include a promoterand transcnptional start and stop sequences
Promoter sequences encode either constitutive or mducible promoters The promoters may be either naturally occurring promoters or hybrid promoters Hy rid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention
In addition, the expression vector may comprise additional elements For example, the expression vector may have two replication systems, thus allowing it to be maintained in twoorganisms, for example in mammalian or insect cells forexpressionand in a procaryotic host for cloning and amplification Furthermore, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct The integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector Constructs for integrating vectors are well known in the art In addition, in a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells Selection genes are well known in the art and will vary with the host cell used
A preferred expression vector system is a retroviral vector system such as is generally described in PCT/US97/01019 and PCT/US97/01048, both of which are hereby expressly incorporated by reference
Exo proteins ofthe present invention are produced by culturing a host cell transformed with an expressionvectorcontainmgnucleicacidencodingan Exo protein, undertheappropnate conditions to induce or cause expression of the Exo protein The conditions appropriate for Exo protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation For example, the use of constitutive promoters in the expression vector will require optimizmgthegrowthand proliferationof the host cell, while the use of an mducible promoter requires the appropriate growth conditionsfor induction In addition, in someembodiments, the timing ofthe harvest is important For example, the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield
Appropriate host cells include yeast, bacteria, archebacteπa, fungi, and insect and animal cells, including mammalian cells Of particular interest are Drosophila melangaster cells, Saccharomycescerevisiae and otheryeasts, E coli, Bacillussubtilis, SF9cells, C129cells,
293 cells, Neurospora, BHK, CHO, COS, and HeLa cells, fibroblasts, Schwanoma cell lines, immortalized mammalian myeloid and lymphoidcell lines, Jurkatcells, livercells, mammary cells, sperm, egg, adιpocytes,granulocytes,adrenalchromaffin cells, mast cells, basophils, endocrine and exocnne cells, muscle cells, eosinophils and neuronal cells
In a preferredembodiment.theExoprotemsareexpressedin mammaliancells Mammalian expressionsystemsare also known in the art, and include retroviralsystems A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequencefor Exo protein into mRNA A promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the transcription initiation site The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site A mammalian promoter will also contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation Of particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter
Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence The 3' terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation
Examples of transcription terminator and polyadenlytion signalsincludethosedeπved form SV40
The methods of introducing exogenous nucleic acid into mammalian hosts, as well as other hosts, is well known in the art, and will vary with the host cell used Techniques include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of the polynucleotιde(s) in liposomes, and direct microinjection of the DNA into nuclei
In a preferred embodiment, Exo proteins are expressed in bacterial systems Bacterial expression systems are well known in the art
A suitable bacterial promoteπs any nucleicacid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription ofthe coding sequence of Exo protein into mRNA A bacterial promoter has a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactoseand maltose, and sequencesderivedfrom biosynthetic enzymes such as tryptophan Promoters from bacteπophage may also be used and are known in the art In addition, synthetic promoters and hybrid promoters are also useful, for example, the fac promoter is a hybrid of the trp and lac promoter sequences
Furthermore.abacterialpromotercan include natu rally occurring promotersof non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription ln addition to a functioning promoter sequence, an efficient ribosome binding site is desirable In E coli, the ribosome binding site is called the Shme-Delgarno (SD) sequence and includes an initiation codon and a sequence 3-9 nucleotides in length located 3 - 11 nucleotides upstream of the initiation codon
The expression vector may also include a signal peptide sequence that provides for secretion of the Exo protein in bacteria The signal sequence typically encodes a signal peptide comprised of hydrophobic am o acids which direct the secretion of the protein from the cell, as is well known in the art The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria)
The bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol.erythromycin, kanamycm, neomycinandtetracyc ne Selectablemarkers also include biosynthetic genes, such as those in the histidme, tryptophan and leucine biosynthetic pathways
These componentsareassembledintoexpressionvectors Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E coli, Streptococcus cremons, and Streptococcus lividans, among others
The bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others
In one embodiment, Exo proteins are produced in insect cells Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art
In a preferred embodiment, Exo protein is produced in yeast cells Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candidaalbicansand C maltosa, Hansenula polymorpha, Kluyveromycesfragilis and K lactis, Pichia guillenmondii and P pastons, Schizosaccharomyces pombe, and Yarrowia lipolytica Preferred promoter sequences for expression in yeast include the mducible GAL1.10 promoter, the promoters from alcohol dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase, hexokmase, phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and the acid phosphatase gene Yeast selectable markers include ADE2, HIS4, LEU2, TRP1 , and ALG7, which confers resistance to tunicamycin, the neomycin phosphotransferase gene, which confers resistance to G418, and the CUP1 gene, which allows yeast to grow in the presence of copper ions
The Exo protein may also be made as a fusion protein, using techniques well known in the art Thus, for example, for the creation of monoclonal antibodies, if the desired epitope is small, the Exo protein may be fused to a carrier protein to form an immunogen Alternatively, the Exo protein may be made as a fusion protein to increase expression, or for other reasons For example, when the Exo protein is an Exo peptide, the nucleic acid encoding the peptide may be linked to othernucleicacid for expression purposes Similarly, Exo proteins of the invention can be linked to protein labels, such as green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), etc
In one embodiment, the Exo nucleic acids, proteins and antibodies of the invention are labeled By "labeled" herein is meant that a compound has at least one element, isotope or chemical compound attached to enablethe detectionof the compound In general, labels fall into three classes a) isotopic labels, which may be radioactive or heavy isotopes, b) immune labels, which may be antibodies or antigens, and c) colored or fluorescent dyes The labels may be incorporated into the compound at any position
In a preferred embodiment, the Exo protein is purified or isolated after expression Exo proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample Standard purification methods includeelectrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusmg For example, the Exo protein may be purified using a standard anti-Exo antibody column Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful For general guidance in suitable purification techniques, see Scopes, R , Protein Purification, Spπnger-Verlag, NY (1982) The degree of purification necessary will vary depending on the use of the Exo protein In some instances no purification will be necessary
Once expressed and purified if necessary, the Exo proteins and nucleic acids are useful in a number of applications The nucleotide sequences (or their complement) encoding Exo proteins have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA Exo protein nucleic acid will also be useful for the preparation of Exo protein polypeptides by the recombinant techniques described herein
The full-length native sequence Exo protein gene, or portions thereof, may be used as hybπdizationprobesfora cDNAiibrary to isolate the full-length Exo protemgeneorto isolate still other genes (for instance, those encoding naturally-occurring variants of Exo protein or Exo protein from other species) which have a desired sequenceidentity to the Exo protein coding sequence Optionally, the length ofthe probes will be about 20 to about 50 bases
The hybridization probes may be derived from the nucleotide sequences herein or from genomic sequences including promoters, enhancer elements and mtrons of native sequences as provided herein By way of example, a screening method will comprise isolating the coding region of the Exo protein gene using the known DNA sequence to synthesize a selected probe of about 40 bases Hybridization probes may be labeled by a variety of labels, including radionucleotides such as 32P or 35S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotm coupling systems Labeled probes having a sequence complementary to that of the Exo protein gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes
The probes may also be employed in PCR techniques to generate a pool of sequences for identification of closely related Exo protein coding sequences
Nucleotide sequences encoding a Exo protein can also be used to construct hybridization probes for mapping the gene which encodes that Exo protein and forthe genetic analysis of individuals with genetic disorders The nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries
Nucleic acids which encode Exo protein or its modified forms can also be used to generate eιthertransgenιcanιmalsor"knockout" animalswhich, in turn, are useful in the development and screening of therapeutically useful reagents A transgenic animal (e g , a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e g , an embryonic stage A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops In one embodiment, cDNA encoding an Exo protein can be used to clone genomic DNA encoding an Exo protein in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express the desired DNA Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U S Patent Nos 4,736,866 and 4,870,009 Typically, particular cells would be targeted for the Exo protein transgene incorporation with tissue-specific enhancers Transgenic animals that include a copy of a transgene encoding an Exo protein introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of the desired nucleic acid Such animals can be used as tester animalsforreagentsthought to confer protection from, for example, pathologicalconditions associated with its overexpression In accordance with this facetofthe invention, an animal is treated with the reagent and a reduced incidence ofthe pathologicalcondition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition
Alternatively, non-human homologues of the Exo protein can be used to construct a Exo protein "knock out" animal which has a defective or altered gene encoding an Exo protein as a result of homologous recombination between the endogenous gene encoding an Exo protein and altered genomic DNA encoding an Exo protein introduced into an embryonic cell of the animal For example, cDNA encoding an Exo protein can be used to clone genomicDNAencodingan Exo protein in accordancewith established techniques A portion of thegenomicDNAencodmgan Exo protein can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration
Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [seee g , Thomasand Capecchi, Cell, δ .603 (1987) for a description of homologous recombination vectors] The vector is introduced into an embryonic stem cell line (e g , by electroporation) and cells in which the introduced DNA has homologously recombmed with the endogenous DNA are selected [see e g , Li etal , Cell, 69 915 (1992)]
The selected cells are then injected into a blastocyst of an animal (e g , a mouse or rat) to form aggregation chimeras [see e g , Bradley, in Teratocarcmomasand Embryonic Stem Cells A Practical Approach, E J Robertson, ed (IRL, Oxford, 1987), pp 113-152] A chimeric embryocanthen be implantedintoa suitablepseudopregnantfemalefosteranimal and the embryo brought to term to create a "knock out" animal Progeny harboring the homologously recombmed DNA in theirgermcellscan be identifiedbystandardtechniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the Exo protein polypeptide It is understood that cell based knock-out or "knock-in" systems can also be made and utilized in accordance with the present disclosure
It is understood that the models described herein can be varied For example, "knock-in" models can be formed, or the models can be cell-based rather than animal models
Nucleic acid encoding the Exo polypeptides, antagonists or agonists may also be used in gene therapy In gene therapy applications, genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene "Gene therapy" includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane (Zamecnik etal , Proc Natl Acad Sci USA 83.4143-4146 [1986]) The oligonucleotides can be modified to enhance their uptake, e g by substituting their negatively charged phosphodiester groups by uncharged groups
There are a variety of techniques available for introducing nucleic acids into viable cells The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells ofthe intended host Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphateprecipitationmethod, etc
The currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al , Trends in Biotechnology 11. 206-210 [1993]) In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibodyspecificfora cell surface membraneproteinorthetargetcell, a Iigandfora receptor on the target cell, etc Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosismay be used fortargetingand/orto facilitate uptake, e g capsid proteins orfragments thereof tropicfor a particular cell type, antibodies for proteins which undergo mternalization in cycling, proteins that target intracellular loca zation and enhance intracellular half-life The technique of receptor-mediated endocytosis is described, for example, by Wu etal , J Biol Chem 262, 4429-4432 (1987), and Wagner et al , Proc Natl Acad Sci USA 87, 3410-3414 (1990) For review of gene marking and gene therapy protocols see Anderson et al , Science 256, 808-613 (1992)
In a preferred embodiment, the Exo proteins, nucleic acids, modified proteins and cells containing the native or modified Exo proteins are used in screening assays Identification of this important exocytosis protein permits the design of drug screening assays for compounds that modulate Exo activity
Screens may be designed to first find candidate agents that can bind to Exo proteins, and then these agents may be used in assays that evaluate the ability of the candidate agent to modulate Exo activity Thus, as will be appreciatedby those in the art, there are a number of different assays which may be run, binding assays and activity assays
Thus, in a preferred embodiment, the methods comprise combining an Exo protein and a candidate bioactive agent, and determining the binding ofthe candidate agent to the Exo protein Preferred embodiments utilize the human Exo protein, although other mammalian proteins may also be used, including rodents (mice, rats, hamsters, guinea pigs, etc ), farm animals (cows, sheep, pigs, horses, etc ) and primates These latter embodiments may be preferredinthedevelopmentof animal modelsof human disease Insomeembodiments, as outlined herein, variant or derivative Exo proteins may be used, including deletion Exo proteins as outlined above
The term "candidatebioactiveagent" or "exogeneous compound" as used herein describes any molecule, e g , protein, oligopeptide, small organic molecule, polysacchaπde, polynucleotide, etc , with the capability of directly or indirectly altering the bioactivity of Exo Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations Typically, one of these concentrations serves as a negative control, i e , at zero concentration or below the level of detection
Candidateagents encompass numerouschemicalclasses, though typically they areorganic molecules, preferably small organic compounds having a molecular weight of more than
100 and less than about 2,500 daltons Candidate agents comprise functional groups necessaryforstructural teractionwith proteins, particularlyhydrogen bonding, and typically include at least an amme, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more ofthe above functionalgroups Candidateagentsare also found among biomolecules including peptides, saccha des, fatty acids, steroids, puπnes, pynmidines, derivatives, structural analogs or combinations thereof Particularly preferred are peptides
Candidate agents are obtained from a wide variety of sources including brariesof synthetic ornaturalcompounds Forexample.numerousmeansare available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extractsareavailableor readily produced Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, estenfication, amidification to produce structural analogs
In a preferredembodιment,thecandιdate bioactive agents are proteins By "protein" herein is meantatleasttwocovalentlyattachedaminoacids, which includesprotems, polypeptides, o gopeptides and peptides The protemmay be madeupofnaturallyoccurnngamino acids and peptide bonds, or synthetic peptidomimetic structures Thus "ammo acid", or "peptide residue", as used herein means both naturally occurring and synthetic ammo acids For example, homo-phenylalamne, citrullme and noreleucme are considered ammo acids for the purposes of the invention "Ammo acid" also includes imino acid residues such as prolme and hydroxypro ne The side chains may be in either the (R) or the (S) configuration lnthepreferredembodιment,theamιnoacιdsareιnthe(S)orL-configuratιon If non-naturally occurring side chains are used, non-ammo acid substituents may be used, for example to prevent or retard in vivo degradations
In a preferred embodiment, the candidate bioactive agents are naturally occurring proteins or fragments of naturally occunng proteins Thus, for example, cellular extracts containing proteins, or random or directed digests of protemaceous cellular extracts, may be used In this way libraries of procaryotic and eukaryotic proteins may be made for screening agamstExo Particularly preferred in this embodimentare libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred In a preferred embodiment, the candidate bioactive agents are peptides of from about δ to about 30 ammo acids, with from about δ to about 20 ammo acids being preferred, and from about 7 to about 16 being particularly preferred The peptides may be digests of naturally occunng proteins as is outlined above, random peptides, or "biased" random peptides By "randomized" or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotidesandaminoacids, respectively Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or ammo acid at any position The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive protemaceousagents
In one embodiment, the library is fully randomized, with no sequence preferences or constants at any position In a preferred embodiment, the library is biased That is, some positions within the sequenceareeitherheldconstant.orare selected from a limited number of possibilities For example, in a preferred embodiment, the nucleotides or am o acid residues are randomized within a defined class, for example, of hydrophobic am o acids, hydrophi c residues, stencally biased (either small or large) residues, towards the creation of cystemes, for cross-linking, prolines for SH-3 domains, sennes, threon es, tyrosmes or histidmes for phosphorylation sites, etc , or to purmes, etc
In a preferred embodiment, the candidate bioactive agents are nucleic acids By "nucleic acιd"or"olιgonucleotιde"or grammatical equivalents herein means at leasttwo nucleotides covalently linked together A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al , Tetrahedron 49(10) 1925 (1993) and references therein, Letsmger, J
Org Chem 35 3600 (1970), Spnnzl et al , Eur J Biochem δ1 579 (1977), Letsmger et al , Nucl Acids Res 14 3487 (1986), Sawai etal, Chem Lett 805 (1964), Letsmger etal , J Am Chem Soc 110 4470 (1988), and Pauwels etal , Chemica Scπpta 26 141 91986)), phosphorothioate (Mag et al , Nucleic Acids Res 19 1437 (1991), and U S Patent No 5,644,048), phosphorodithioate (Bπu et al , J Am Chem Soc 111 2321 (1989), O- methylphophoroamιdιtelιnkages(see Eckstein, Oligonucleotides and Analogues A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J Am Chem Soc 114 1895 (1992), Meier et al , Chem Int Ed Engl 31 1008 (1992), Nielsen, Nature, 365 566(1993), Carlssonetal , Nature380 207(1996), all of which are incorporated by reference) Other analog nucleic acids include those with positive backbones(Denpcyetal , Proc Natl Acad Sci USA 92 6097 (1995), non-ionicbackbones (U S Patent Nos 5,386,023, 5,637,634, 5,602,240, 5,216,141 and4,469,δ63, Kiedrowshi et al . Angew Chem Intl Ed English 30 423 (1991 ) Letsmger et al , J Am Chem Soc 110 4470 (1966), Letsmger et al , Nucleoside & Nucleotide 13 1597 (1994), Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research",
Ed Y S Sanghui and P Dan Cook, Mesmaeker etal , Bιoorganιc & Medicinal Chem Lett 4 395 (1994), Jeffs et al , J Biomolecular NMR 34 17 (1994), Tetrahedron Lett 37 743 (1996))and non-rιbosebackbones, ιncludιngthosedescrιbedιn U S PatentNos 5,235,033 and 5,034,606, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed Y S Sanghui and P Dan Cook Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al , Chem Soc Rev (1995) pp169-176) Several nucleic acid analogs are described in Rawls, C & E News June 2, 1997 page 35 All of these references are hereby expressly incorporated by reference These modifications of the ribose- phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments In addition, mixtures of naturally occurring nucleic acids and analogs can be made Alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occunngnucleicacidsand analogsmay be made The nucleicacids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleicacid contains any combination of deoxynbo-and nbo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosme, guanme, inosine, xathanme hypoxathanme, isocytosme, isoguanine, etc
As described above generally for proteins, nucleic acid candidate bioactive agents may be naturally occunng nucleicacids, randomnucleιcacιds,or"bιased"random nucleic acids Forexample.digestsofprocaryoticoreucaryoticgenomesmay beusedasisoutlinedabove for proteins
In a preferred embodiment, the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature
The assays provided utilize Exo proteins as defined herein In one embodiment, portions of Exo proteins are utilized, in a preferred embodiment, portions having Exo activity are used Exo activity is described further below and includes binding activity to GS27, rab7, rab9, snap-23, rab3a, rab11 , rab3d, rabδ, alpha-snap, und 8-1 , vamp3 or Exo protein modulatorsasfurtherdescribed below In addition, the assays described herein may utilize either isolated Exo proteins or cells comprising the Exo proteins
Generally, in a preferred embodiment of the methods herein, the Exo protein or the candidate agent is non-diffusibly bound to an insoluble support having isolated sample receiving areas (e g a microtiterplate, an array, etc ) The insolublesupports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening The surface of such supports may be solid or porous and of any convenient shape Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads These are typically made of glass, plastic (e g , polystyrene), polysacchandes, nylon or nitrocellulose, teflon™, etc Microtiterplatesandarraysareespeαallyconvenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples In some cases magnetic beads and the like are included The particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the compositionand is nondiffusable Preferred methods of binding mcludethe use of antibodies (which do not steπcally block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to "sticky" or ionic supports, chemical crosslιnkιng,thesynthesιsoftheproteιnoragentonthesurface,etc Insomeembodiments, GS27 can be used Other embodiments include using, rab7, rab3a, rab3d, snap23, rab9, rabδ, alpha snap, rab11 , und 8-1 or vamp3 Following binding of the protein or agent, excess unbound material is removed by washing The sample receiving areas may then be blocked through mcubationwith bovine serum albumin (BSA), casein or other innocuous protein or other moiety Also included in this invention are screening assays wherein solid supports are not used
In a preferred embodiment, the Exo protein is bound to the support, and a candidate bioactive agent is added to the assay Alternatively, the candidate agent is bound to the support and the Exo protein is added Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc Of particular interest are screening assays for agents that have a low toxicity for human cells A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc ) and the like The determination of the binding of the candidate bioactive agent to the Exo protein may be done in a number of ways In a preferred embodiment, the candidate bioactive agent is labelled, and binding determined directly For example, this may be done by attaching all or a portion ofthe Exo protein to a solid support, adding a labelled candidate agent (for example a fluorescent label) washing off excess reagent, and determining whether the label is present on the solid support Various blocking and washing steps may be utilized as is known in the art
By "labeled" herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e g radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc Specific binding molecules include pairs, such as biotm and streptavidm, digoxin and antidigoxm etc Forthe specific binding members, the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above The label can directly or indirectly provide a detectable signal
In some embodiments, only one of the components is labeled For example, the proteins (or proteinaceous candidate agents) may be labeled at tyrosine positions using 125l, or with fluorophores Alternatively, more than one component may be labeled with different labels, using 125l for the proteins, for example, and a fluorophor for the candidate agents
In a preferred embodiment, the binding of the candidate bioactive agent is determined through the use of competitive binding assays In this embodiment, the competitor is a binding moiety known to bind to the targetmolecule(ι e Exo), such as an antibody, peptide, binding partner, ligand, etc In a preferred embodiment, thecompetitoπs GS27, rab7, rab9, snap-23, rab3a, rab11 , rab3d, rabδ, alpha-snap, und δ-1, or vamp3 Under certain circumstances, there may be competitive binding as between the bioactive agent and the binding moiety, with the binding moiety displacing the bioactive agent This assay can be used to determinecandidateagents which interfere with binding between Exo proteins and GS27, rab7, rab9, snap-23, rab3a, rab11 , rab3d, rabδ, alpha-snap, undδ-1 , or vamp3
In one embodiment, the candidatebioactiveagent is labeled Eitherthe candidate bioactive agent, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present Incubations may be performed at any temperature which facilitates optimalactivity, typically between 4 and 40°C Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening Typically between 0 1 and 1 hour will be sufficient Excess reagent is generally removed or washed away The second component is then added, and the presence or absence ofthe labeled component is followed, to indicate binding
I n a preferred embodiment, the competitoπs added first, followed by the candidate bioactive agent Displacement of the competitor is an indication that the candidate bioactive agent is binding to the Exo protein and thus is capable of binding to, and potentially modulating, the activity ofthe Exo protein In this embodiment, either component can be labeled Thus, for example, if the competitor is labeled, the presence of label in the wash solution indicates displacement by the agent Alternatively, if the candidate bioactive agent is labeled, the presence of the label on the support indicates displacement
In an alternative embodiment, the candidate bioactive agent is added first, with incubation and washing, followed by the competitor The absence of binding by the competitor may indicate that the bioactive agent is bound to the Exo protein with a higher affinity Thus, if the candidate bioactive agent is labeled, the presenceof the label on the support, coupled with a lack of competitorbinding, may indicate that the candidate agent is capable of binding to the Exo protein
In a preferredembodιment,the methodscomprisedifferential screening to identity bioactive agents that are capable of modulating the activity ofthe Exo proteins In this embodiment, the methods comprise combining an Exo protein and a competitor in a first sample A second sample comprises a candidate bioactive agent, an Exo protein and a competitor
The binding ofthe competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the Exo protein and potentially modulating its activity That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the Exo protein
Alternatively, a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native Exo protein, but cannot bind to modified Exo proteins The structure of the Exo protein may be modeled, and used in rational drug design to synthesize agents that interact with that site Drug candidates that affect Exo bioactivity are also identified by screening drugs for the ability to either enhance or reduce the activity of the protein Positive controls and negative controls may be used in the assays Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results Incubation of all samples is for a time sufficient for the binding of the agent to the protein Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, generally labeled agentdetermmed For example, where a radiolabel is employed, the samples may be counted in a scintillation counterto determmethe amount of bound compound
A variety of other reagents may be included in the screening assays These include reagents like salts, neutral proteins, e g albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions Also reagents that otherwise improve the efficiency of the assay, such as proteasemhibitors.nuclease inhibitors, anti-microbialagents, etc , may be used The mixture of components may be added in any order that provides for the requisite binding
The components provided herein for the assays provided herein may also be combined to form kits The kits can be based on the use ofthe protein and/orthe nucleicacid encoding the Exo proteins Assays regarding the use of nucleic acids are further described below
Screening for agents that modulate the activity of Exo may also be done In a preferred embodiment, methods for screening for a bioactive agent capableofmodulatingthe activity of Exo comprise the steps of adding a candidate bioactive agent to a sample of Exo, as above, and determinmganalterationin the biologicalactivity of Exo "Modulating the activity of Exo" includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present Thus, in this embodiment, the candidate agent should both bind to Exo (although this may not be necessary), and alter its biological or biochemical activity as defined herein The methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution, activity or amount of Exo
Thus, in thisembodiment, the methods comprise combiningan Exo sampleand a candidate bioactive agent, and evaluating the effect on exocytosis By "Exo activity" or grammatical equivalents herein is meant one of Exo's biological activities, including, but not limited to, its ability to affect exocytosis, secretion and/or vesicular transport Included within exocytosis, secretion and/or vesicular transport activitiesmclude regulating or involvement in steps therein such as docking, fusion and targeting activities of proteins involved in the entire pathway of exocytosis, secretion and/or vesicular transport In one embodiment, vesicular refers to any vesicle including synaptic or secretory granules and vesicles those involved in exocytosis, endocytosis or the trans-golgi network In one embodiment, exo activity includes GTPase activity or regulation thereof One exo activity herein is binding to at least one protein selected from the group consisting of GS27, rab7, rab9, snap-23, rab3a, rabl 1 , rab3d, rabδ, alpha-snap, und 8-1 and vamp3 Other exo activities include the activities and regulation thereof of GS27, rab7, rab9, snap-23, rab3a, rab11 , rab3d, rabδ, alpha-snap, und 8-1 and vamp3
In a preferred embodiment, the activity ofthe Exo protein is increased, in another preferred embodiment, the activity ofthe Exo protein is decreased Thus, bioactive agents that are antagonists are preferred in some embodiments, and bioactive agents that are agonists may be preferred in other embodiments
In a preferred embodiment, the invention providesmethodsforscreenmgforbioactiveagents capable of modulating the activity of an Exo protein The methods comprise adding a candidate bioactive agent, as defined above, to a cell comprising Exo proteins Preferred cell types include almost any cell The cells contama recombmantnucleicacid that encodes an Exo protein In a preferred embodiment, a library of candidate agents are tested on a plurality of cells
In some embodiments, the assays include exposing the cells to an exocytosis agent that will induce exocytosis in control cells, i e cells ofthe same type but that do not contain the exogeneous nucleic acid encoding an Exo Suitable exocytosis agents are known in the art and include but are not limited to lonomycin and CaN such as, but not limited to the Ca++ ιonophoreA23137 Alternatively, the cells may be exposedtoconditionsthatnormaliy result in exocytosis, and changes in the normal exocytosis progression are determined Alternatively, the cells into which the Exo nucleic acids are introduced normally under exocytosis, and thus changes (for example, inhibition of exocytosis) are determined
Optionally, the cells normally do not undergo exocytosis, and the introduction of a candidate agent causes exocytosis
Thus, the effect of the candidate agent on exocytosis is then evaluated
Detection of exocytosis may be done as will be appreciated by those in the art In one embodiment, indicators of exocytosis are used Suitable exocytosis labels include, but are not limited to, annexm Accordingly, these agents can be used as an affinity ligand, and attached to a solid support such as a bead, a surface, etc and used to pull out cells that are undergoing exocytosis Similarly, these agents can be coupled to a fluorescent dye such as PerCP, and then used as the basis of a fluorescent-activated cell sorting (FACS) separation Moreover, FACS or other optical methods can be used to detect exocytosis activity and the modulation thereof based on light scattering, light absorption, dye uptake and release, granule enzyme activity and quantification of granule specific proteins
In this way, bioactive agents are identified Compounds with pharmacological activity are able to enhance or interfere with the activity of the Exo protein The compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host, as previously described The agents may be administered in a variety of ways, orally, parenterally e g , subcutaneously, mtraperitoneally, mtravascularly, etc Depending upon the manner of introduction, the compounds may be formulatedin a variety of ways The concentration of therapeutically active compound in the formulation may vary from about 0 1-100 wt %
The pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds Diluents known to the art include aqueous media, vegetable and animal oils and fats Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents
Without being bound by theory, it appears that Exo is an important protein in exocytosis Accordingly, disorders based on mutant or variant Exo genes may be determined In one embodiment, the invention provides methods for identifying cells containing variant Exo genes comprising determining all or part ofthe sequence of at least one endogeneous Exo genes in a cell As will be appreciated by those in the art, this may be done using any number of sequencing techniques In a preferred embodiment, the invention provides methods of identifying the Exo genotype of an individual comprising determining all or part ofthe sequence of at least one Exo gene ofthe individual This is generally done in at least one tissue of the individual, and may include the evaluation of a number of tissues or different samples of the same tissue The method may include comparing the sequence of the sequenced Exo gene to a known Exo gene, i e a wild-type gene The sequence of all or part of the Exo gene can then be compared to the sequence of a known Exo gene to determine if any differences exist This can be done using any number of known sequence identity programs, such as Bestfit, etc In a preferred embodiment, the presenceof a difference in the sequence between the Exo geneof the patientand the known Exo gene is indicative of a disease state or a propensity for a disease state, as outlined herein
The present discovery relating to the role of Exo in exocytosis thus provides methods for inducing or preventing exocytosis in cells In a preferred embodiment, the Exo proteins, and particularly Exo fragments, are useful in the study or treatment of conditions which are mediated by exocytosis, i e to diagnose, treatorpreventexocytosis-mediateddisorders
Thus, "exocytosis mediated disorders" or "disease state" include conditions involving both insufficient or excessive exocytosis, vesicular transport, and/or secretion via the secretory pathway, including inflammatory mediator release from mast cells including asthma, allergies, and Chediak-Higashi Syndrome (CHS) Additionally, control of neurotransmitter release can be used to treat Alzheimer's disease, Parkinson's and Huntmgton's disease states as well as some skitzophrenia, thus these can also be included in exocytosis mediated disorders in some cases In other cases, fertilization and lactation disorders can be included as disease states which can be treated with the compositions provided and/or identified herein Additionally, some diabetes, digestion and wound healing disorders can be exocytosis mediated disorders
Thus, in one embodiment, methods of modulating exocytosis in cells or organisms are provided In one embodiment, the methods comprise administering to a cell an anti-Exo antibody that reduces or eliminates the biological activity ofthe endogeneous Exo protein Alternatively, the methods comprise administering to a cell or organism a recombinant nucleic acid encoding an Exo protein As will be appreciated by those in the art, this may be accomplished in any number of ways In a preferred embodiment, the activity of Exo is increased by increasing the amount of Exo in the cell, for example by overexpressmg the endogeneous Exo or by administering a gene encoding an Exo, using known gene- therapy techniques, for example In a preferred embodiment, the gene therapy techniques include the incorporation of the exogeneous gene using enhanced homologous recombination (EHR), for example as described in PCT/US93/03868, hereby incorporated by reference in its entireity
In one embodiment, the invention provides methods for diagnosing an exocytosis related condition in an individual The methods comprise measuring the activity of Exo in a tissue from the individual or patient, which may include a measurement ofthe amount or specific activity of Exo This activity is compared to the activity of Exo from either a unaffected second individual orfroman unaffectedtissuefromthefirstmdividual When these activities are different, the first individual may be at risk for an exocytosis mediated disorder
The proteins and nucleic acids provided herein can also be used for screening purposes wherein the protein-protein interactions of the Exo proteins can be identified Genetic systems have been described to detect protein-protein interactions The first work was done in yeast systems, namely the "yeast two-hybrid" system The basic system requires a protein-protein interaction in order to turn on transcnptionof a reportergene Subsequent work was done in mammalian cells See Fields et al , Nature 340 245 (1989), Vasavada et al , PNAS USA δδ 10666 (1991), Fearon et al , PNAS USA 69 7958 (1992), Dang et al . Mol Cell Biol 11 954 (1991), Chien etal , PNAS USA86 9676 (1991), and U S Patent Nos 5,283,173, 5,667,973, 5,468,614, 5,525,490, and 5,637,463 a preferred system is described in Serial No 09/050,863, filed March 30, 1998, entitled "Mammalian Protein InteractionCloning System" For use in conjunctionwith these systems, a particularlyuseful shuttle vector is described in Serial No 09/133,944, filed August 14, 1998, entitled "Shuttle Vectors"
In general, two nucleic acids are transformed into a cell, where one is a "bait" such as the geneencodιngGS27, rab7, rab9, snap-23, rab3a, rabl 1 , rab3d, rabδ, alpha-snap, und 8-1 , vamp3 or a portion thereof, and the other encodes a test candidate Only if the two expression products bind to one another will an indicator, such as a fluorescent protein, be expressed Expression of the indicator indicates when a test candidate binds to the GS27, rab7, rab9, snap-23, rab3a, rab11 , rab3d, rabδ, alpha-snap, und 8-1 orvamp3and can be identified as an Exo protein Using the same system and the identified Exo proteins the reverse can be performed Namely, the Exo proteins provided herein can be used to identify new baits, or agents which interact with Exo proteins Additionally, the two-hybrid system can be used wherein a test candidate is added in addition to the bait and the Exo protein encoding nucleic acids to determine agents which interfere with the bait, such as GS27, rab7, rab9, snap-23, rab3a, rabl 1 , rab3d, rabδ, alpha-snap, und 8-1 or vamp3, and the Exo protein
In one embodiment, a mammalian two-hybrid system is preferred Mammalian systems provide post-translational modifications of proteins which may contribute significantly to their ability to interact In addition, a mammalian two-hybrid system can be used in a wide variety of mammaliancell types to mimic the regulation, induction, processing, etc of specific proteins within a particularcell type Forexample, proteins involved in a disease state such as those descnbedabove could be tested in the relevant disease cells Similarly, for testing of random proteins, assaying them underthe relevant cellular conditionswill give the highest positive results Furthermore, the mammalian cells can be tested under a variety of experimental conditions that may affect intracellular protein-protein interactions, such as in the presence of hormones, drugs, growth factors and cytokines, cellular and chemical stimuli, etc , that may contribute to conditions which can effect protein-protein interactions, particularlythose involved in exocytosis, the secretory pathway, and/or vesiculartransport
Expression in various cell types, and assays for Exo activity are described above The activity assays, such as having an effect on exocytosis, secretion and/or vesiculartransport can be performed to confirm the activity of Exo proteins which have already been identified by their sequence identity/similarity or binding to GS27, rab7, rab9, snap-23, rab3a, rabl 1 , rab3d, rabδ, alpha-snap, und 8-1 or vamp3 as well as to further confirm the activity of lead compounds identified as modulators of exocytosis, secretion and/or vesicular transport
Assays involving binding such as the two-hybrid system may take into accountnon-specific binding proteins (NSB)
In one embodiment, the Exo proteins of the present invention may be used to generate polyclonal and monoclonalantibodiesto Exo proteins, which are useful as described herein Similarly, the Exo proteins can be coupled, using standard technology, to affinity chromatography columns These columns may then be used to purify Exo antibodies In a preferred embodiment, the antibodies are generated to epitopes unique to the Exo protein, that is, the antibodies show little or no cross-reactivity to other proteins These antibodies find use in a number of applications For example, the Exo antibodies may be coupled to standard affinity chromatography columns and used to purify Exo proteins as further described below The antibodies may also be used as blocking polypeptides, as outlined above, since they will specifically bind to the Exo protein
The anti-Exo protein antibodiesmay comprise polyclonal antibodies Methods of preparing polyclonal antibodies are known to the skilled artisan Polyclonal antibodies can be raised in a mammal, forexample, by one or more injections of an immunizing agent and, if desired, an adjuvant Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intrapentoneal injections The immunizing agent may include the Exo protein polypeptide or a fusion protein thereof It may be useful to conjugate the immunizing agent to a 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, bovinethyroglobu n, and soybean trypsin inhibitor Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant(monophosphorylLιpιd a, synthetic trehalosedicorynomycolate) The immunization protocol may be selected by one skilled in the art without undue experimentation
The anti-Exo protein antibodies may, alternatively, be monoclonal antibodies Monoclonal antibodies may be prepared using hybndoma methods, such as those described by Kohler and Milstem, Nature, 256 495 (1975) In a hybndoma 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 Alternatively, the lymphocytes may be immunized in vitro
The immunizing agent will typically include the Exo protein polypeptide or a fusion protein thereof Generally, either peripheral blood lymphocytes ("PBLs") are used if cellsof 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 hybndoma cell [Godmg, Monoclonal Antibodies Principles and Practice, Academic Press, (1986) pp 59-103] Immortalized cell lines are usually transformed mammaliancells, particularlymyeloma cells of rodent, bovmeand human origin Usually, rator mouse myeloma cell lines are employed The hybndoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival ofthe unfused.immortalizedcells For example, if the parental cells lack the enzyme hypoxanth e guanme phosphoribosyl transferase(HGPRTorHPRT), the culture medium forthe hybπdomas typically will include hypoxanthme, aminopteπn, and thymidme ("HAT medium"), which substances preventthe 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 immortalizedcell 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, Rockville, Maryland
Human myeloma and mouse-human heteromyeloma cell lines also have been described forthe production of human monoclonal antibodies [Kozbor, J Immunol , 133 3001 (1984), Brodeur et al , Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc , New York, (1987) pp 51-63] The culture medium in which the hybndoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against Exo protein Preferably, the binding specificity of monoclonal antibodies produced by the hybndoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent 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 (1960)
After the desired hybndoma cells are identified, the clones may be subcloned by limiting dilution proceduresand grown by standard methods[Godιng, supra] Suitableculturemedia forthis purpose include, forexample, Dulbecco'sModified Eagle's Medium and RPMI-1640 medium Alternatively, the hybndoma cells may be grown in vivo as ascites in a mammal
The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein a-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography
The monoclonalantibodiesmay also be made by recombinantDNA methods, such as those described in U S Patent No 4,616,567 DNA encoding the monoclonal antibodies ofthe 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 lightchamsof muπneantibodies) The hybndoma cells ofthe invention serve as a preferred source of such DNA Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cellsthatdo not otherwiseproduceimmunoglobulm protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells The DNAalsomay be modified, forexample, by substituting the coding sequencefor human heavy and light chain constant domains in placeofthehomologousmurιnesequences[U S Patent No 4,δ16,567, Morrison etal , supra] or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulm polypeptide Such a non-immunoglobu n polypeptide can be substituted for the constant domains of an antibody ofthe invention, or can be substituted forthe variable domains of one antigen- combining site of an antibody of the invention to create a chimeric bivalent antibody
The antibodies may be monovalent antibodies Methods for preparing monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevantcysteineresiduesare substituted with another amino acid residue or are deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in the art.
The anti-Exo protein antibodies ofthe invention may furthercomprise humanizedantibodies or human antibodies. Humanizedforms of non-human(e.g., murine)antibodiesare chimeric immunoglobulins, immunoglobulin chains orfragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins(recipientantibody)in which residues from a complementary determining region (CDR) ofthe recipientare replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanizedantibodiesmayalsocomprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the
CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321 :522-626 (1936); Riechmann et al., Nature. 332:323-329 (19δδ); and Presta, Curr. Op.
Struct. Biol.. 2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typicallytakenfroman "import" variabledomain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature. 321:522-626 (1966); 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. Accordingly, such "humanized" antibodies are chimeric antibodies (U S Patent No 4,816,567), wherein substantially less thanan mtacthumanvariabledomain has been substituted by the correspondmgsequence from a non-human species In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies
Human antibodiescan also be produced using vanoustechniquesknown in the art, including phage display libraries [Hoogenboom and Winter, J Mol Biol , 227 381 (1991), Marks et al , J Mol Biol , 222 581 (1991)1 The techniques of Cole etal and Boerner etal are also available for the preparation of human monoclonal antibodies (Cole et al , Monoclonal Antibodies and Cancer Therapy, Alan R Liss, p 77 (1985) and Boerner etal . J Immunol ,
147(1 ) δ6-95 (1991 )] Similarly, human antibodies can be made by introducing of 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 5,545,607, 5,545,806, 5,669,625, 5,625, 126, 5,633,425, 5,661 ,016, and in the following scientific publications Marks et al , Bio/Technology 10.779-783 (1992). Lonberg etal . Nature 368856-869 (1994), Morrison, Nature 368, 812-13 (1994), Fishwild et al , Nature Biotechnology 14, 845-51 (1996), Neuberger, Nature Biotechnology 14, 826 (1996), Lonberg and Huszar, Intern Rev
Immunol 13 65-93 (1996)
Bispecific antibodies are monoclonal, preferably human or humanized, antibodiesthat have bindmgspecificitiesforatleasttwodifferentantigens In the present case, one ofthe binding specificities is for the Exo protein, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit
Methodsformaking bispecificantibodiesare 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 [Milstem and Cuello, Nature, 305 537-539 (1983)] Because of the random assortment of immunoglobulin heavy and light chains, these hybπdomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure The purification of the correct molecule is usually accomplished by affinitychromatographysteps Similarproceduresare disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al , EMBO J . 10 3655-3659 (1991 ) Antibodyvariabledomainswiththedesiredbindingspecificities(antibody-antigencombining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are cotransfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121 :210 (1986).
Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared/π vitro using known methods in syntheticprotein chemistry, includingthoseinvolvingcrosslinkingagents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolateand methyl-4-mercaptobutyrimidateand those disclosed, forexample, in U.S. Patent No. 4,676,980.
Theanti-Exoprotein antibodies ofthe invention have various utilities. Forexample, anti-Exo protein antibodies may be used in diagnostic assays for an Exo protein, e.g. , detecting its expression in specif iccells, tissues, orserum. Various diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in eitherheterogeneousor homogeneous phases [Zola, Monoclonal Antibodies: a Manual of Technigues, CRC Press, Inc. (1987) pp. 147-158]. The antibodiesused in the diagnosticassays can be labeled with a detectable moiety. The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as
3H, 14C, 32P, 35S, or 125l, a fluorescent or chemiluminescent compound, such asfluorescein isothiocyanate.rhodamine, orluciferin, or an enzyme, such as alkaline phosphatase, beta- galactosidase or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter etal.. Nature. 144:946 (1962): David et al.. Biochemistry. 13:1014 (1974): Pain et al., J. Immunol. Meth., 40:219 (1981 ); and Nygren.J. Histochem. and Cvtochem., 30:407 (1982). Anti-Exo protein antibodies also are useful for the affinity purification of Exo protein from recombinant cell culture or natural sources In this process, the antibodies against Exo protein are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art The immobilized antibody then is contacted with a sample containmgthe Exo protemto be purified, and thereafterthesupportis washed with a suitable solvent that will remove substantially all the material in the sample except the Exo protein, which is bound to the immobilized antibody Finally, the support is washed with another suitable solvent that will release the Exo protein from the antibody
The anti-Exo protein antibodies may also be used in treatment In one embodiment, the genes encoding the antibodies are provided, such that the antibodies bind to and modulate the Exo protein within the cell
In one embodiment.a therapeuticallyeffectivedose of an Exo protein, agonist orantagonist is administered to a patient By "therapeutically effective dose" herein is meant a dose that produces theeffectsforwhich it is administered The exact dose will depend on the purpose ofthe treatment, and will be ascertainabie by one skilled in the art using known techniques
As is known in the art, adjustments for Exo degradation, systemicversus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainabie with routine experimentation by those skilled in the art
A "patient" for the purposes of the present invention includes both humans and other animals, particularly mammals, and organisms Thus the methods are applicable to both human therapy and veterinary applications In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is human
The administration of the Exo protein, agonist or antagonist of the present invention can be done in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, ιntranasally,transdermally,ιntrapeπtoneally,ιntramuscularly, intrapulmonary, vaginally, rectally, or intraocularly In some instances, for example, in the treatment of wounds and inflammation, the Exo may be directly applied as a solution or spray
The pharmaceuticalcompositionsof the present invention comprise an Exo protein, agonist orantagonist in a form suitable for administration to a patient In the preferred embodiment, the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. "Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, 5 sulfuric acid, nitric acid, phosphoricacid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. "Pharmaceuticallyacceptablebaseadditionsalts"includethosederivedfrom inorganic 0 bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularlypreferredaretheammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptableorganicnon-toxicbasesincludesaltsof primary, secondary, and tertiaryamines, substitutedaminesincludingnaturallyoccurringsubstitutedamines.cyclicaminesand basic δ ion exchange resins, such as isopropy lamine, trimethy lamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
The pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring 0 agents; and polyethylene glycol. Additives are well known in the art, and are used in a variety of formulations.
All references cited herein are incorporated by reference in their entireity. The following examples are merely for illustration.
EXAMPLES
δ The yeast two-hybrid cDNA cloning technology is a powerful in vivo protein-protein interaction assay first introduced in Fields S, Song O (1989) Nature 340:246-247 (Chevray PM, Nathans D (1992) Proc Natl Acad Sci USA 89:5789-6793; Chien CT, Bartel PL, Stemglanz R, Fields S (1991 ) Proc Natl Acad Sci USA 88:9578-9682; Durfee T, Becherer K, Chen PL, Yeh SH, Yang Y, Kilburn AE, Lee WH, Elledge S (1993) Genes Dev 7:566-669; 0 Fields S, Song O (1989) Nature 340:245-247; Mendelsohn AR, Brent R (1994)
Biotechnology 5:482-486; Zervos A, Gyuris J, Brent R (1993) Cell 72:223-232). It is based on co-expression of two proteins, X and Y, fused to GAL4 DNA binding domain (GAL4B) and GAL4 transcription activation domain (GAL4A) respectively (Figure 1 A). If the protein X interacts with the protein Y, the GAL4 transcription activation domain will be brought to the promoter containing the GAL4 DNA binding sites and will activate the transcription of reporter gene HIS3 or lacZ. The two-hybrid system can be used to clone cDNA encoding a novel protein that interacts with a known protein (bait) in yeast. It can also be used to study protein-proteininteractionsbetweentwo known proteins.Theyeasttwo-hybridsystem has several advantages over other conventional methods to study protein-protein interactions: Protein-protein interactions are studied in eukaryotic cells (yeast); cDNA clones are immediately available after screening.
Screening is very fast and convenient. No protein purification is necessary. Both growth selection and lacZcolorselection are very sensitive. No radioisotope is needed. However, this technology also presents a challenge to new users due to certain technical difficulties: Depending on the bait protein used in screening, false positive rates vary significantly. Yeast transformation efficiency is difficult to control.
High quality cDNA libraries can be very difficult to construct. The major difference between the yeast one-hybrid system and the yeast-two hybrid system is that the one-hybrid system is used to clone cDNA encoding DNA-bound proteins, rather than protein-bound proteins (Figure 1 B). DNA sequences of interest are inserted upstream of the minimal promoter controlling the expression of either HIS or lacZ gene. cDNA fragments are fused to the C- terminal of GAL4 transcription activation domain (GAL4A) to construct cDNA library. If the protein encoded by the cDNA can bind to the specific DNA sequences of interest, the transcription of HIS/lacZis activated. The yeast one-hybrid system is widely used in cloning new transcription factors (Lehming N, Thanos D, Brickman JM, Ma J, Maniatis T, Ptashne M (1994) Nature 371:175-179; Li JJ, Herskowitz I (1993) Science 262:1870-1873; Luo Y, Stile J, Zhu L (1996) BioTechniques 20:564-568; Shang J, Luo Y, Clayton D (1997) Developmental Dynamics 209:242-253; Strubin M., Newell JW, Matthias P (1995) Cell
80:497-506; Wilson TE, Fahmer TJ, Johnston M, Milbrandt J (1991) Science 252:1296- 1300; Wang MM, Reed RR (1993) Cell 74:205-214). Experimental protocols between these two methods are very similar except one notable exception. The one-hybrid system yeast reporterstrainsneedto be constructed by individual researcher. The expression of reporter genes (HIS/lacZ) should be underthe control of specific DNA sequences of interest, rather than the GAL4 DNA-binding sites in the yeast two-hybrid system. cDNA libraries used for two-hybrid screening can also be used for one-hybrid screening.
The experimental flow-chart of a yeast two-hybrid cDNA screening experiment is outlined in Figure 2. The experimental flow-chart of a yeast one-hybrid cDNA screening experiment is outlined in Figure 3.
MATERIALS
Medium and Yeast Strains All yeast culture mediums, including YPD, YPD Agar, DOB, DOBA, CSM-TRP,
CSM-LEU, CSM-HIS, CSM-URA, CSM-LYS, CSM-LEU-TRP, CSM-LEU-HIS, and CSM-LEU-TRP-HIS, are available from Bio101 , Inc. 3AT (3-amino-1 ,2,4-triazol) is available from Sigma (Cat no.: A-8066, St. Louis, MO, USA). Yeast two-hybrid system reporter strain Y190 (MATa, ura3-52, his3-200, Iys2-801 , ade2-101, trp1-901 , leu2-3, 112, gal4Δ, galδOΔ, cyhr2, LYS2::GAL1UAS-HIS3TATA-
HIS3, URA3::GAL1UAS-GAL1TATA-lacZ) and yeast one-hybrid system reporter strain YM4271 (MATa, ura3-δ2, his3-200, Iys2-801 , ade2-101 , trp1-903, leu2-3, 112, tyr1-601) are available from Clontech Laboratories, Inc. (Cat no.K1603-1, Clontech, Palo Alto, CA, USA). Plasmids and cDNA Libraries pAS2 and pACT2 series were originally constructed by Elledge lab (Durfee et al. 1993) and are available from Clontech laboratories (Cat no.K1604-A, K1604-B). Other GAL4 based two-hybrid vectors, such as pGBT9 and pGAD424 series, were originally published by Fields lab and are available from both Stratagene, Inc. (Cat no.235700,235722) and Clontech Laboratories, Inc. (Cat no.K1605-A, K1605-B).
LexA based two-hybrid vectors are available from Origene Technologies, Inc. (Cat no.DPL-100, DPL-102). cDNA libraries for two-hybrid and one-hybrid screening are available from Origene, Stratagene, Clontech, and Invitrogen. Rigel also makes its own two-hybrid cDNA libraries from various tissues. All of these two- hybrid vectors share basic structures as shown in Figure 4A.
The cDNA library is to be amplified before screening. It is recommended that at least 200 15cm-plates should be used to grow up 10 million independent cDNA clones. High quality plasmid can be obtained with Qiagene DNA preparation kits.
Single-strand carrier DNA for yeast transformation is available from Origene or Clontech. Carrier DNA can also be made according to protocol by Ito et al. (Ito H,
Fukada Y, Murata K, Kimura A (1993) Journal of Bacteriology 163:163-163).
Special Buffers Z buffer pH7.0 (per liter) Na2HP04.7H20 16.1 g
NaH2P04.H20 6.60 9
MgS04.7H20 0.246 g
Kcl 0.75 g
Z buffer + X-Gal
Z buffer 1 ml
20mg/ml X-Gal 40 μl β-mercaptoethanol(optional) 2 μl
PEG/LiAc (10 ml)
50% PEG(3350) 8 ml
10X TE (pH7.δ) 1 ml
I M LiAc 1 ml
Equipment and Others
30°C incubators and liquid nitrogen containers are required. Nylon membrane and Whatman filter for lacZ color assay are available from Fisher Scientific. X-
Gal is from either Promega (Cat no.V3941 , Madison, Wl, USA) or Denville Scientific (Cat no.CX-3000-3, Metuchen, NJ, USA). All plastic wares are from Fisher Scientific or VWR.
PROCEDURE: Yeast Two-Hybrid System Screening Grow up yeast reporter strains on YPD plates from frozen stock.
Since no antibiotics are added into the yeast medium, very stringent sterilization procedures are required during inoculation. It is also recommended that yeast reporter strain be streaked on SD-W, SD-L, SD-H, SD-U, and SD-K plates to test other markers of the yeast before cDNA library screening. Reporter strain such as Y190 should be able to grow up on SD-K, SD-U and SD-H plates, but not on SD-
W, and SD-L plates. Growth on SD-H plate is due to leaky expression of HIS reporter gene.
There are many reporter strains available from different resources. In General, Y190 consistently showed higher sensitivity than other yeast strains such as HF7c. Yeast reporter strains with both lacZ reporter gene and HIS3 reporter gene are strongly recommended. HIS selection will ensure that only interaction positive clones will grow, which makes colony picking much easier later. Determine optimal 3AT concentration. 3AT can be used to suppress background expression from HIS reporter gene of Y190. 3AT concentration varies among different reporter strains and ranges from 0 mM (HF7c) to 15 mM (Y190). To test the optimal concentration of 3AT, one yeast colony should be re-suspended in 10 ml of TE. 100 μl of the re-suspended yeast is spread on SD-H+0mM3AT, SD-H+δmM3AT, SD-H+10mM3AT, SD-H+1δmM3AT,
SD-H+25mM3AT, and SD-H+40mM3AT plates. Although 15 mM 3AT is sufficient to suppress background HIS expression of Y190, higher concentrations of 3AT (30-40 mM) are routinely used in our cDNA library screening. Construct bait plasmid. pAS2/pACT2 series plasmids showed higher level of sensitivity than pGAD424
/pGBT9 series plasmids (Estojak J, Brent R, Golemis EA (1995) Molecular and Cellular Biology 15:6820-6829; Legrain P, Dokhelar MC, Transy C (1994) Nucleic Acids Research 22:3241-3242). The disadvantage of using pAS2 is the large size of this plasmid (8 kb), which may present a challenge to cloning large cDNA fragments into the plasmid. cDNA fragments should fused to the C-terminal of Gal4 binding domain in frame (Figure 4A). The junction sequence between GAL4 and cDNA should have a GGG amino acid sequence to avoid any interruption of domain structure. Either full-length cDNA or partial fragments can used to generate bait plasmid. Transform bait into yeast: 1st round.
1 μg of bait plasmid is transformed into Y190 with small-scale yeast transformation protocol (see SUBPROTOCOL section). Transformants should be plated on SD- W, SD-WH, and SD-WH+3AT(5-40mM) plates. LacZ color assay can also be done after colonies grow to a diameter of 1 mm. If colonies grow up on SD- WH+40mM3AT plates after 3 days incubation and/or LacZ color assay of these colonies show positive result after only 30 minutes incubation with X-Gal, the bait gene should be determined not suitable for two-hybrid screening without further modification. The bait gene itself may be able to activate transcription of reporter genes HIS/lacZ. Although co-transformation of bait plasmid and cDNA library can be done in a single step, co-transformation efficiency is at least 10 fold lower than single plasmid transformation. Mating approach may also be used to introduce cDNA library into yeast cells containing the bait vector. Please refer to protocol published by Finley and Brent (Finley R, Brent R (1994) Proc Natl Acad Sci USA 91:12980- 12984).
Transform cDNA library: 2nd round. Y190 containing bait plasmid is grown up for second round of transformation by cDNA library plasmid (see SUBPROTOCOL section) Incubation time after transformation varies significantly from 4 days to 11 days Identify positive clones Identification of positive clones needs experience It should also be pointed out that background colonies at lightly populated area of the plate tend to grow bigger, occasionally reaching the size of a positive colony in a dense area on the same plate The size of the positive colony should at least 4 times bigger than the neighboring background colonies Positive colonies may also turn red faster Perform lacZ color assay
Positive colonies should be re-streaked to another SD-LWH+3AT plate to isolated single colonies for color assay and plasmid retrieval See SUBPROTOCOL section for the lacZ color assay protocol If a colony does not turn blue after a 4-hour incubation, strong protein-protein interaction is highly unlikely It is not recommended to pick positive clones after 12 hours incubation, except that you know the protein-protein interaction you are studying is very weak Retrieve plasmids
There are several methods to retrieve plasmids from yeast, ranging from lyticase lysis to glass beads The glass beads method is listed in SUBPROTOCOL section Electroporation method is by far the most efficient method to transform plasmids from yeast mmiprep into E coli Bait and cDNA plasmid may carry different antibiotic selection markers to facilitate separation in E coli For example, Rigel's bait plasmid carries Kanr gene and the cDNA plasmid carries Ampr gene Verify positive clones cDNA clones recovered from positive HIS/lacZ positive colonies should be re- transformed into yeast with other non-specific bait control to rule non-specific binding In vitro protein binding assays and function assays should also be done to rule out false positive clones
PROCEDURE: Yeast One-Hybrid System Screening Construct HIS and lacZ reporter plasmids
Selection of a very-well-defined DNA sequence is the most important step for one- hybrid screening Many DNA sequences lead to significant elevation of the basal expression levels of the reporter genes in yeast even in the absence of the cDNA library Multiple copies (~3) of the DNA sequences of interest should be inserted into the multiple cloning sites of both HIS reporter plasmid pHISι-1 and pLacZi
(Clontech Cat no K1603-1 , Figure 4B) Grow up yeast reporter strains on YPD plates from frozen stock.
It is also recommended that the yeast reporter strain be streaked on SD-W, SD-L, SD-H, SD-U, and SD-K plates to test other markers of the yeast before cDNA library screening. Reporter strain such as YM4271 should be able to grow up on SD-K, SD-U SD-H, SD-W, and SD-L plates.
Integrate HIS reporter into yeast.
To facilitate integration of pHISi reporter into yeast chromosome, pHISi-1 should be linearized at Xho I site. Since pHISi-1 has no yeast replication origin and can not survive in yeast without integration, no gel purification of digested plasmid is required. Transform 1 μg of digested plasmid into YM4271 using the small-scale yeast transformation protocol (see SUBPROTOCOL section). Use more plasmids if integration efficiency is low. Transformants should be plated on SD-H, and SD-H plates with different concentration of 3AT(δ-40 mM) and incubated at 30°C for at least 4 days. Determine optimal 3AT concentration.
If more than 40 mM 3AT is needed to suppress transformants growth, the DNA sequences inserted into pHISi are not suitable for one-hybrid screening. Note: Integration efficiency of pHISi is very low. 20-100 colonies are expected on SD-H plate. Integrate LacZ reporter plasmid into yeast.
Pick a colony from a SD-H plate from step 3 and freeze as single HIS reporter strain YM4271/H. Linearize pLacZi at Nco I site. Transform 1 μg of linearized plasmid into yeast YM4271/H and plate transformants on SD-U plates. This step of integration is very efficient. Several hundred to thousand colonies are expected to grow on each SD-U plates. Pick colonies and freeze as YM4271/HB.
Screen cDNA library for DNA binding protein.
Transform 100-200 μg of cDNA library into YM4271/HB with large-scale yeast transformation protocol (see SUBPROTOCOL section). Transformants should be plated on SD-LH+3AT ( concentration determined at step 4 ). Identify positive clones.
Same as step 6 in two-hybrid screening procedure. Perform lacZ color assays.
Same as step 7 in two-hybrid screening procedure. Retrieve cDNA plasmid. Same as step 8 in two-hybrid screening procedure.
Verify positive clones. DNA gel retardation assay and other function assays are required to verify one- hybrid screening results.
SUBPROTOCOL
Small Scale Yeast Transformation (105 transformants/μg DNA) Inoculate one colony of yeast in 100 ml YPD (without plasmid) or corresponding selection medium (SD-W for Y190 with pAS2) at 240 rpm in a 30°C shaker overnight. Check OD600 the next day. If OD600 is between 0.6 and 1.0, the yeast can be used to prepare competent cells. Otherwise, dilute to OD600=0.4 and grow another 3 to 4 hours.
Spin down cells in two 50 ml plastic tubes at 3000 rpm at room temperature for 5 minutes. Remove medium. Add 30 ml TE pH7.δ and re-suspend the cell pellet on vortex at high speed. Combine cell pellet. Spin down cells again in at 3000 rpm at room temperature for 6 minutes.
Remove TE. Estimate the size of the cell pellet and add TE up to total volume of 0.9 ml. Re-suspend cell completely by pipetting up and down. Add 100 μl 1M LiAc and mix well by pipetting. Competent cells are ready. Note: Competent cells can be kept on at room temperature for several hours without significant reduction of transformation efficiency, or at 4°C overnight with a slight reduction of transformation efficiency. In a clean eppendorf, mix 1 μg of plasmid with 10 μl 10 mg/ml carrier DNA. Add 100 μl competent cells from step 8 to the eppendorf and mix well with the DNA. Add 600 μl PEG/LiAc and mix well.
Note: PEG/LiAc should be freshly made. Pre-mixed PEG/LiAc of up to 2 weeks old can also be used if transformation efficiency is not critical. Incubate at 30°C for 30 minutes with or without shaking. Add 70 μl DMSO and mix well. Incubate in 42°C water bath for 15 minutes.
Put on ice for 2 minutes.
Spin down cells in an eppendorf centrifuge at 8000 rpm for 1 minute. Remove supernatant. Add 160 μl of TE to re-suspend cell pellet. Plate on selection medium plate, (e.g. SD-W for Y190 transformed by pAS2).
Incubate in a 30°C incubator for 2 to 3 days. Large scale cDNA library transformation (1-10X106 transformants/100 μg cDNA) Inoculate one colony of yeast in 200 ml YPD (one-hybrid screening) or corresponding selection medium (SD-W for two-hybrid screening) at 240 rpm in a 30°C shaker overnight, δ 1. Check OD600 the next day. If OD600 is between 0.8 and 1.0, the yeast can be used to prepare competent cells. Otherwise, dilute to OD600=0.6 and grow another 3 to 4 hours.
2. Spin down cells in a 260 ml bottles at 3000 rpm at room temperature for δ minutes.
3. Remove medium. 0 4. Add 50 ml TE pH7.5 and re-suspend the cell pellet on vortex at high speed, δ. Spin down cells again in at 3000 rpm at room temperature for 5 minutes.
6. Remove TE.
7. Repeat steps 4 to 7 one more time.
Estimate the size of cell pellet and add TE up to total volume of 1.8 ml. Re- δ suspend cell pellet completely by vortex.
Add 200 μl 1 M LiAc and mix well by vortex.
In a clean eppendorf, mix 100-200μg of plasmid with 200 μl 10 mg/ml carrier DNA. Add DNA to competent cells drop by drop on a vortex at 6000 rpm. To ensure sufficient mixture, vortex at the highest speed for 30 seconds. 0 Add 12 ml PEG/LiAc and mix well.
Note: PEG/LiAc should be freshly made. Pre-mixed PEG/LiAc of up to 2 weeks old can also be used if transformation efficiency is not critical. Incubate at 30°C for 30 minutes with shaking. Either an orbital shaker or rotator can be used. 5 Add 140 μl DMSO and mix well.
Incubate in 42°C water bath for 15 minutes. Invert several times during incubation. Put on ice for δ minutes to chill.
Spin down cells at 3000 rpm in a bench-top centrifuge for 1 minute. Remove supernatant. 0 Add 20 ml TE to re-suspend cell pellet by vortex.
Plate 400 μl on each 15 cm selection medium plates (50 plates total). SD-LWH+40 mM3AT plates are used for Y190 strain two-hybrid screening; SD-LH+3AT plates are used for one-hybrid screening. Plate 1 μl on a 10 cm plate of SD-LW for transformation efficiency control. 5 Incubate at 30°C for up to 8 days until big colonies appear. LacZ color assay.
Grow up fresh yeast colonies to a 1 mm diameter. Fill a container (e.g. ice bucket) with liquid nitrogen.
Use a nylon membrane to lift colonies up from the plate. No special replica-plating device is needed. Simply press the nylon membrane to the plate.
Immerse the nylon membrane (Cat no. N04HY08250, N04HY13260, Fisher Scientific,
PA, USA) colony side face down into the liquid nitrogen. Wait for 20 seconds, remove the nylon membrane and allow to dry on a paper towel for 5 minutes. In a 10 ml tube, add 40 μl X-Gal to each ml of Z buffer.
Add 1.5 ml Z buffer/X-Gal solution to a clean 10 cm petri-dish. For 15 cm diameter petri-dish, add 4 ml Z buffer/X-Gal solution. Add a Whatman circle to the petri-dish, ensuring it is evenly soaked any air bubbles are squeezed out. Use forceps to transfer the dried nylon membrane with colony side facing up to lie over the soaked Whatman circle (Cat no.09-805C, Fisher Scientific, PA, USA). Make sure no air bubble in between the membrane and the circle. Incubate the petri-dish with lid on in 37°C until blue color is visible. Yeast plasmid mini-isolation. Inoculate 3 ml of selection medium ( e.g. SD-L for cDNA library plasmid pACT ) with a yeast colony. Incubate in a 30°C shaker or rotator overnight or until confluent. Spin down yeast in a bench-top centrifuge at 3000 rpm at room temperature. Remove medium and re-suspend pellet in 200 μl lysis buffer. Transfer to an eppendorf. Add 200 μl volume glass beads.
Note: The lid of eppendorf can be used as scoop to collect 200 μl glass beads. Add 200 μl phenol/chloroform/isoamyl alcohol (25:24:1). Vortex at the highest speed for 3 minutes. Spin in micro-centrifuge at 14000 rpm for 10 minutes. Transfer top water layer to another eppendorf, add 20 μl 3M NaAc and 500 μl ethanol.
Precipitate should be visible immediately. Put the eppendorf into a dry ice bath for 15 minutes or until frozen. Spin in a micro-centrifuge at 14000 rpm for 10 minutes. Remove supernatant and dry pellet. Wash pellet by 100 μl of 80% ethanol, and dry the pellet in air.
Re-suspend pellet in 30 μl H20 and use 1 ml for electroporation to transform E. coli. TWO-HYBRID SCREENING RESULTS
Bait peptide was cloned into pAS2-1 to screen for proteins that can bind thereto. Results are listed below. cDNA Library Human Lymphocyte
Bait Vector pAS2-1
Bait Protein Protein involved in exocytosis
Yeast Strain Y190
Number of Transformants 15 million
HIS7lacZ+ clones 2
Clone Identity PCNA (2)
PCNA is a published binding protein of the bait peptide. Yeast one-hybrid screening results were previously published ( Luo Y, Stile J, Zhu L (1996) BioTechniques 20:564- 568).

Claims

CLAIMS We claim
1 A recombinant nucleic acid encoding an Exo protein comprising a nucleic acid that hybridizes under high stringency conditions to a sequence set forth in SEQ ID NO 86, SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 91 , SEQ
ID NO 92, SEQ ID NO 93, SEQ ID NO 94, SEQ ID NO 96, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101 , SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 111, SEQ ID NO 112, SEQ ID NO 113, SEQ ID NO 114, SEQ ID NO 115, SEQ ID NO 116, SEQ ID
NO 117, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 120, SEQ ID NO 121, SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO 125, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 130, SEQ ID NO 131, SEQ ID NO 132, SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137, SEQ ID NO 138, SEQ ID NO 139, SEQ ID NO 140, SEQ ID NO 141 , SEQ ID
NO 142, SEQ ID NO 143 and each complement thereof, respectively, wherein said Exo protein binds to SNAP-23
2 A recombinant nucleic acid comprising a nucleic acid that is at least about 90% identical to a nucleic acid sequence selected forth in SEQ ID NO 86, SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 91, SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 94, SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101, SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 111, SEQ ID NO 112, SEQ ID NO 113, SEQ ID NO 114, SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO 117, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 120, SEQ ID NO 121, SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO 126, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131 , SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:13δ, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID N0:141 , SEQ ID NO:142, SEQ ID NO: 143 and complements, respectively, wherein said Exo protein binds to SNAP-23.
3. An expression vector comprising the recombinant nucleic acid according to claims 1 or 2 operably linked to regulatory sequences recognized by a host cell transformed with the nucleic acid.
4. A host cell comprising the recombinant nucleic acid according to claim 3.
5. A process for producing an Exo protein comprising culturing the host cell of claim 4 under conditions suitable for expression of an Exo protein.
6. A process according to claim 5 further comprising recovering said Exo protein.
7. A recombinant Exo protein encoded by the nucleic acid of claim 1 or 2.
8. A recombinant polypeptide comprising an amino acid sequence encoded by the first 100 nucleic acid residues of a sequence selected from the group consisting of the sequences set forth in SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89,
SEQ ID NO:90, SEQ ID NO:91 , SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID
NO:11δ, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121 , SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131 , SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141 , SEQ ID NO:142, SEQ ID N0.143 and each complement thereof, wherein said polypeptide binds to SNAP-23.
9. A recombinant polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence that will hybridize under high stringency to a nucleic acid selected from the group consisting of the sequences set forth SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID
NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101 , SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111 , SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121 , SEQ ID
NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143 and each complement thereof.
10. An isolated polypeptide which specifically binds to an Exo protein according to claim 9.
11. A polypeptide according to claim 10 that is an antibody.
12. A polypeptide according to claim 11 wherein said antibody is a monoclonal antibody.
13. The monoclonal antibody of claim 12 wherein said antibody reduces or eliminates the biological function of said Exo protein.
14. A method for screening for a bioactive agent capable of binding to an Exo protein, said method comprising combining an Exo protein and a candidate bioactive agent, and determining the binding of said candidate agent to said Exo protein.
15. A method for screening for agents capable of interfering with the binding of Exo and SNAP-23 comprising: a) combining an Exo protein, a candidate bioactive agent and an SNAP-23 protein; and b) determining the binding of said Exo protein and said SNAP-23 protein.
16. A method according to claim 15 wherein said Exo protein and said SNAP-23 protein are combined first.
17. A method for screening for an bioactive agent capable of modulating the activity of an Exo protein, said method comprising the steps of: a) adding a candidate bioactive agent to a cell comprising a recombinant nucleic acid encoding an Exo protein; b) determining the effect of the candidate bioactive agent on said cell.
18. A method according to claim 17 wherein a library of candidate bioactive agents are added to a plurality of cells comprising a recombinant nucleic acid encoding an Exo protein.
19. A method according to claim 17 or 18 further comprising adding a labeling agent that will label exocytosing cells.
20. A method according to claim 19 further comprising separating the exocytosing cells from the non-exocytosing cells.
21. A method according to claim 20 wherein said separation is done by FACS.
22. A method of treating an exocytosis related disorder comprising administering an agent that interferes with specific binding of a protein selected from a protein encoded by the group consisting of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID N0.71 , SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:7δ, SEQ ID NO:76, SEQ ID NO:77, SEQ ID
NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 , SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:9δ, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101 , SEQ ID NO:102, SEQ ID
NO:103, SEQ ID NO:104, SEQ ID NO:10δ, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111 , SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121 , SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:12δ, SEQ ID NO:126, SEQ ID NO:127, SEQ ID
NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131 , SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO: 143, with SNAP23 expressed in a tissue such that said disorder is ameolerated.
23. A method of treating an exocytosis related disorder comprising administering to a patient an agent that binds to a protein encoded by a sequence selected from the group consisting of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 , SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID
NO:79, SEQ ID NO:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 , SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101 , SEQ ID NO:102, SEQ ID NO:103, SEQ ID
NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111 , SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID
NO:129, SEQ ID NO:130, SEQ ID NO:131 , SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141 , SEQ ID NO:142, and SEQ ID NO:143 such that exocytosis is altered.
24. A method of reducing or inhibiting exocytosis in a cell comprising administering an agent that interferes with specific binding of a protein encoded by a sequence selected from the group consisting of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 , SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 , SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101 , SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID N0:111 , SEQ ID N0:112, SEQ ID
N0:113, SEQ ID N0:114, SEQ ID N0:115, SEQ ID N0:116, SEQ ID N0:117, SEQ ID N0:118, SEQ ID N0:119, SEQ ID NO:120, SEQ ID N0:121, SEQ ID N0:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID N0:125, SEQ ID N0:126, SEQ ID N0:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID N0:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID
NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID N0:141 , SEQ ID NO:142, and SEQ ID NO:143, with SNAP23 expressed in said cell such that exocytosis is inhibited.
25. A method of neutralizing the effect of a protein encoded by a sequence selected from the group consisting of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID
NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 , SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID
NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101 , SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID
NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131 , SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID N0.139, SEQ ID NO:140, SEQ ID NO:141 , SEQ ID NO:142, and SEQ ID NO:143, comprising contacting an agent specific for said protein with said protein in an amount sufficient to effect neutralization.
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