AU3537199A - Human proteins having transmembrane domains and dnas encoding these proteins - Google Patents

Description

WO 99/55862 PCT/JP99/02226 1 DESCRIPTION HUMAN PROTEINS HAVING TRANSMEMBRANE DOMAINS AND DNAs ENCODING THESE PROTEINS 5 TECHNICAL FIELD The present invention relates to human proteins having transmembrane domains, cDNAs coding for these proteins, and expression vectors of said cDNAs as well as eucaryotic cells 10 expressing said cDNAs. The proteins of the present invention can be employed as pharmaceuticals or as antigens for preparing antibodies against said proteins. The human cDNAs of the present invention can be utilized as probes for the gene diagnosis and gene sources for the gene therapy. Furthermore, 15 the cDNAs can be utilized as gene sources for large-scale production of the proteins encoded by said cDNAs. Cells, wherein these membrane protein genes are introduced and membrane proteins are expressed in large amounts, can be utilized for detection of the corresponding ligands, screening 20 of novel low-molecular pharmaceuticals, and so on. BACKGROUND ART Membrane proteins play important roles, as signal receptors, ion channels, transporters, etc. in the material 25 transportation and the information transmission which are mediated by the cell membrane. Examples thereof include receptors for a variety of cytokines, ion channels for the sodium ion, the potassium ion, the chloride ion, etc., transporters for saccharides and amino acids, and so on, where 30 the genes of many of them have been cloned already. It has been clarified that abnormalities of these membrane proteins are associated with a number of hitherto- WO 99/55862 PCT/JP99/02226 2 cryptogenic diseases. For instance, a gene of a membrane protein having twelve transmembrane domains was identified as the gene responsible for cystic fibrosis [Rommens, J. M. et al., Science 245: 1059-1065 (1989)]. In addition, it has been 5 clarified that several membrane proteins act as receptors when a virus infects the cells. For instance, HIV-1 is revealed to infect into the cells through mediation of a membrane protein fusin having a membrane protein on the T-cell membrane, a CD-4 antigen, and seven transmembrane domains [Feng, Y. et al., 10 Science 272: 872-877 (1996)]. Therefore, discovery of a new membrane protein is anticipated to lead to elucidation of the causes of many diseases, so that isolation of a new gene coding for the membrane protein has been desired. Heretofore, owing to difficulty in the purification, many 15 membrane proteins have been isolated by an approach from the gene side. A general method is the so-called expression cloning which comprises transfection of a cDNA library in eucaryotic cells to express cDNAs and then detection of the cells expressing the target membrane protein on the membrane by an 20 immunological technique using an antibody or a physiological technique on the change in the membrane permeability. However, this method is applicable only to cloning of a gene of a membrane protein with a known function. In general, membrane proteins possess hydrophobic 25 transmembrane domains inside the proteins, wherein, after synthesis thereof in the ribosome, these domains remain in the phospholipid membrane to be trapped in the membrane. Accordingly, the evidence of the cDNA for encoding the membrane protein is provided by determination of the whole base sequence 30 of a full-length cDNA followed by detection of highly hydrophobic transmembrane domains in the amino acid sequence of the protein encoded by said cDNA.

WO 99/55862 PCT/JP99/02226 3 DISCLOSURE OF INVENTION The object of the present invention is to provide novel human proteins having transmembrane domains, DNAs coding for 5 said proteins, and expression vectors of said DNAs as well as transformation eucaryotic cells that are capable of expressing said DNAs. As the result of intensive studies, the present inventors have been successful in cloning of cDNAs coding for proteins 10 having transmembrane domains from the human full-length cDNA bank, thereby completing the present invention. In other words, the present invention provides human proteins having transmembrane domains, namely proteins containing any of the amino acid sequences represented by Sequence Nos. 1 to 9. 15 Moreover, the present invention provides DNAs coding for the above-mentioned proteins, exemplified by cDNAs containing any of the base sequences represented by Sequence Nos. 10 to 19, 21, 23, 25, 27, 29, 31, 33 and 35, as well as expression vectors that are capable of expressing any of said DNAs by in vitro 20 translation or in eucaryotic cells and transformation eucaryotic cells that are capable of expressing said DNAs and of producing the above-mentioned proteins. BRIEF DESCRIPTION OF DRAWINGS 25 Fig. 1: A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HPO2000. Fig. 2: A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by 30 clone HP02061. Fig. 3: A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by WO 99/55862 PCT/JP99/02226 4 clone HP02163. Fig. 4: A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP02219. 5 Fig. 5: A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP02256. Fig. 6: A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by 10 clone HP10390. Fig. 7: A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP10474. Fig. 8: A figure depicting the 15 hydrophobicity/hydrophilicity profile of the protein encoded by clone HP10527. Fig. 9: A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP10528. 20 BEST MODE FOR CARRYING OUT THE INVENTION The proteins of the present invention can be obtained, for example, by a method for isolation from human organs, cell lines, etc., a method for preparation of peptides by the 25 chemical synthesis, or a method for production with the recombinant DNA technology using the DNAs coding for the transmembrane domains of the present invention, wherein the method for obtainment by the recombinant DNA technology is employed preferably. For instance, in vitro expression of the 30 proteins can be achieved by preparation of an RNA by in vitro transcription from a vector having one of cDNAs of the present invention, followed by in vitro translation using this RNA as a WO 99/55862 PCT/JP99/02226 5 template. Also, recombination of the translation region into a suitable expression vector by the method known in the art leads to expression of a large amount of the encoded protein by using prokaryotic cells such as Escherichia coli, Bacillus subtilis, 5 etc., and eucaryotic cells such as yeasts, insect cells, mammalian cells, etc. In the case in which one of the proteins of the present invention is produced by expressing the DNA by in vitro translation, the protein of the present invention can be 10 produced in vitro, when the translation region of said cDNA is subjected to recombination to a vector having an RNA polymerase promoter, followed by addition to an in vitro translation system such as a rabbit riticulocyte lysate or a wheat germ extract, containing an RNA polymerase corresponding to the 15 promoter. RNA polymerase inhibitors are exemplified by T7, T3, SP6, and the like. The vectors containing these RNA polymerase inhibitors are exemplified by pKAl, pCDM8, pT3/7 18, pT7/3 19, pBluescript II, and so on. Furthermore, a membrane protein of the present invention can be expressed as the form incorporated 20 in the microsome membrane, when a dog pancreas microsome or the like is added into the reaction system. In the case in which a protein of the present invention is produced by expressing the DNA using a microorganism such as Escherichia coli etc., a recombinant expression vector bearing 25 the translation region in the cDNA of the present invention is constructed in an expression vector having an origin, a promoter, a ribosome-binding site, a cDNA-cloning site, a terminator etc., which can be replicated in the microorganism, and, after transformation of the host cells with said 30 expression vector, the thus-obtained transformant is incubated, whereby the protein encoded by said cDNA can be produced on a large scale in the microorganism. In this case, a protein WO 99/55862 PCT/JP99/02226 6 fragment containing an optional region can be obtained by carrying out the expression with inserting an initiation codon and a termination codon in front of and behind an optional translation region. Alternatively, a fusion protein with 5 another protein can be expressed. Only a protein portion coding for said cDNA can be obtained by cleavage of said fusion protein with a suitable protease. The expression vector for Escherichia coli is exemplified by the pUC system, pBluescript II, the pET expression system, the pGEX expression system, and 10 so on. In the case in which one of the proteins of the present invention is produced by expressing the DNA in eucaryotic cells, the protein of the present invention can be produced as a transmembrane protein on the cell-membrane surface, when the 15 translation region of said cDNA is subjected to recombination to an expression vector for eucaryotic cells that has a promoter, a splicing region, a poly(A) insertion site, etc., followed by introduction into the eucaryotic cells. The expression vector is exemplified by pKAl, pED6dpc2, pCDM8, 20 pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pYES2, and so on. Examples of eucaryotic cells to be used in general include mammalian culture cells such as simian kidney cells COS7, Chinese hamster ovary cells CHO, etc., budding yeasts, fission yeasts, silkworm cells, Xenopus laevis egg cells, and 25 so on, but any eucaryotic cells may be used, provided that they are capable of expressing the present proteins on the membrane surface. The expression vector can be introduced in the eucaryotic cells by methods known in the art such as the electroporation method, the potassium phosphate method, the 30 liposome method, the DEAE-dextran method, and so on. After one of the proteins of the present invention is expressed in prokaryotic cells or eucaryotic cells, the WO 99/55862 PCT/JP99/02226 7 objective protein can be isolated from the culture and purified by a combination of separation procedures known in the art. Such examples include treatment with a denaturing agent such as urea or a surface-active agent, sonication, enzymatic digestion, 5 salting-out or solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, ion-exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, and so on. 10 The proteins of the present invention include peptide fragments (more than 5 amino acid residues) containing any partial amino acid sequence in the amino acid sequences represented by Sequence Nos. 1 to 9. These peptide fragments can be utilized as antigens for preparation of antibodies. 15 Hereupon, among the proteins of the present invention, those having the signal sequence are secreted in the form of maturation proteins on the surface of the cells, after the signal sequences are removed. Therefore, these maturation proteins shall come within the scope of the present invention. 20 The N-terminal amino acid sequences of the maturation proteins can be easily identified by using the method for the cleavage site determination in a signal sequence [Japanese Patent Kokai Publication No. 1996-187100]. Furthermore, some membrane proteins undergo the processing on the cell surface to be 25 converted to the secretory forms. Such proteins or peptides in the secretory forms shall come within the scope of the present invention. When sugar chain-binding sites are present in the amino acid sequences, expression in appropriate eucaryotic cells affords proteins wherein sugar chains are added. 30 Accordingly, such proteins or peptides wherein sugar chains are added shall come within the scope of the present invention. The DNAs of the present invention include all DNAs coding WO 99/55862 PCT/JP99/02226 8 for the above-mentioned proteins. Said DNAs can be obtained by using a method by chemical synthesis, a method by cDNA cloning, and so on. The cDNAs of the present invention can be cloned, for 5 example, from cDNA libraries of the human cell origin. These cDNA are synthesized by using as templates poly(A)' RNAs extracted from human cells. The human cells may be cells delivered from the human body, for example, by the operation or may be the culture cells. The cDNAs can be synthesized by using 10 any method selected from the Okayama-Berg method [Okayama, H. and Berg, P., Mol. Cell. Biol. 2: 161-170 (1982)], the Gubler Hoffman method [Gubler, U. and Hoffman, J. Gene 25: 263-269 (1983) ], and so on, but it is preferred to use the capping method [Kato, S. et al., Gene 150: 243-250 (1994)], as 15 exemplified in Examples, in order to obtain a full-length clone in an effective manner. In addition, commercially available, human cDNA libraries can be utilized. Cloning of the cDNAs of the present invention from the cDNA libraries can be carried out by synthesis of an oligonucleotide on the basis of an 20 optional portion in the cDNA base sequences of the present invention, followed by screening using this oligonucleotide as the probe according to the colony or plaque hybridization by a method known in the art. In addition, the cDNA fragments of the present invention can be prepared by synthesis of an 25 oligonucleotide to be hybridized at both termini of the objective cDNA fragment, followed by the usage of this oligonucleotide as the primer for the RT-PCR method from an mRNA isolated from human cells. The cDNAs of the present invention are characterized by 30 containing either of the base sequences represented by Sequence Nos. 10 to 18 or the base sequences represented by Sequence Nos. 19, 21, 23, 25, 27, 29, 31, 33 and 35. Table 1 summarizes the WO 99/55862 PCT/JP99/02226 9 clone number (HP number), the cells affording the cDNA, the total base number of the cDNA, and the number of the amino acid residues of the encoded protein, for each of the cDNAs. 5 Table 1 Sequence HP No. Cell Number of Number of No. bases amino acids 1, 10, 19 HP02000 Liver 1705 268 2, 11, 20 HP02061 Saos-2 1759 236 3, 12, 21 HP02163 Saos-2 1069 261 4, 13, 22 HP02219 Stomach Cancer 1759 328 5, 14, 23 HP02256 Stomach Cancer 1697 300 6, 15, 24 HP10390 Stomach Cancer 814 182 7, 16, 25 HP10474 Saos-2 511 66 8, 17, 26 HP10527 Saos-2 1126 183 9, 18, 27 HP10528 Saos-2 2015 324 Hereupon, the same clones as the cDNAs of the present invention can be easily obtained by screening of the cDNA 10 libraries constructed from the human cell lines and human tissues utilized in the present invention by the use of an oligonucleotide probe synthesized on the basis of the cDNA base sequence described in any of Sequence Nos. 10 to 19, 21, 23, 25, 27, 29, 31, 33 and 35. 15 In general, the polymorphism due to the individual difference is frequently observed in human genes. Accordingly, any cDNA that is subjected to insertion or deletion of one or plural nucleotides and/or substitution with other nucleotides in Sequence Nos. 10 to 19, 21, 23, 25, 27, 29, 31, 33 and 35 20 shall come within the scope of the present invention. In a similar manner, any protein that is formed by these modifications comprising insertion or deletion of one or plural amino acids and/or substitution with other amino acids shall WO 99/55862 PCT/JP99/02226 10 come within the scope of the present invention, as far as the protein possesses the activity of any protein having the amino acid sequences represented by Sequence Nos. 1 to 9. The cDNAs of the present invention include cDNA fragments 5 (more than 10 bp) containing any partial base sequence in the base sequences represented by Sequence Nos. 10 to 18 or in the base sequences represented by Sequence Nos. 19, 21, 23, 25, 27, 29, 31, 33 and 35. Also, DNA fragments consisting of a sense chain and an anti-sense chain shall come within this scope. 10 These DNA fragments can be utilized as the probes for the gene diagnosis. In addition to the activities and uses described above, the polynucleotides and proteins of the present invention may exhibit one or more of the uses or biological activities 15 (including those associated with assays cited herein) identified below. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies 20 or vectors suitable for introduction of DNA). Research Uses and Utilities The polynucleotides provided by the present invention can be used by the research community for various purposes. The polynucleotides can be used to express recombinant protein for 25 analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome 30 markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; WO 99/55862 PCT/JP99/02226 11 as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel 5 polynucleotides; for selecting and making oligomers for attachment to a "gene chip" or other support, including for examination of expression patterns; to raise anti-protein antibodiesusing DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response. 10 Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify 15 polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction. The proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high-throughput 20 screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially 25 expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the 30 protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions WO 99/55862 PCT/JP99/02226 12 can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Any or all of these research utilities are capable of being developed into reagent grade or kit format for 5 commercialization as research products. Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation "Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, 10 Sambrook, J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987. Nutritional Uses Polynucleotides and proteins of the present invention can 15 also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the protein or polynucleotide of the invention can be added to the feed of 20 a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is 25 cultured. Cytokine and Cell Proliferation/Differentiation Activity A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may 30 induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one WO 99/55862 PCT/JP99/02226 13 or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent 5 cell proliferation assays for cell lines including, without limitation, 32D, DA2, DAlG, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAl, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK. The activity of a protein of the invention may, among 10 other means, be measured by the following methods: Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and 15 Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; 20 Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761, 1994. Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, 25 Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human Interferon y, Schreiber, R.D. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, 30 John Wiley and Sons, Toronto. 1994. Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without WO 99/55862 PCT/JP99/02226 14 limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, 5 Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6-Nordan, R. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 10 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11 - Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, 15 Toronto. 1991; Measurement of mouse and human Interleukin 9 Ciarletta, A., Giannotti, J., Clark,S.C. and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991. Assays for T-cell clone responses to antigens (which will 20 identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. 25 Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 30 1980; Weinberger et al., Eur. J. Immun. 11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.

WO 99/55862 PCT/JP99/02226 15 Immune Stimulating or Suppressing Activity A protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are 5 described herein. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other 10 cell populations. These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial orfungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of 15 the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, a protein of the present invention may also be useful where a boost to the immune system generally may 20 be desirable, i.e., in the treatment of cancer. Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary 25 inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft versus-host disease and autoimmune inflammatory eye disease. Such a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as 30 asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may WO 99/55862 PCT/JP99/02226 16 also be treatable using a protein of the present invention. Using the proteins of the invention it may also be possible to immune responses, in a number of ways. Down regulation may be in the form of inhibiting or blocking an 5 immune response already in progress or may involve preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. Immunosuppression of T cell responses is generally an active, 10 non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the 15 tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent. Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen 20 functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in 25 tissue transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a molecule which inhibits or blocks interaction of a B7 30 lymphocyte antigen with its natural ligand(s) on immune cells (such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a WO 99/55862 PCT/JP99/02226 17 peptide having an activity of another B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to -the natural ligand(s) on the immune cells without transmitting the 5 corresponding costimulatory signal. Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing 10 tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B 15 lymphocyte antigens. The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include 20 allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci USA, 25 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease. 30 Blocking antigen function may also be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells WO 99/55862 PCT/JP99/02226 18 that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms. 5 Administration of reagents which block costimulation of T cells by disrupting receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking 10 reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease. The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well characterized animal models of human autoimmune diseases. 15 Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New 20 York, 1989, pp. 840-856). Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing 25 immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the commoncold, and encephalitis might be alleviated 30 by the administration of stimulatory forms of B lymphocyte antigens systemically. Alternatively, anti-viral immune responses may be WO 99/55862 PCT/JP99/02226 19 enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble 5 peptide of the present invention and reintroducing the in vitro activated T cells into the patient. Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described 10 herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient. The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo. 15 In another application, up regulation or enhancement of antigen function (preferably B lymphocyte antigen function) may be useful in the induction of tumor immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least 20 one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides. For example, tumor cells obtained from a patient can be transfected ex vivo with an expression 25 vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-1 like activity and/or B7-3-like activity. The transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell. 30 Alternatively, gene therapy techniques can be used to target a tumor cell for transfection in vivo. The presence of the peptide of the present invention WO 99/55862 PCT/JP99/02226 20 having the activity of a B lymphocyte antigen(s) on the surface of the tumor cell provides the necessary costimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells which 5 lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I a chain protein and 12 microglobulin protein or 10 an MHC class IIa chain protein and an MHC class II chain protein to thereby express MHC class I or MHC class II proteins on the cell surface. Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T 15 cell mediated immune response against the transfected tumor cell. optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte 20 antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject. The activity of a protein of the invention may, among 25 other means, be measured by the following methods: Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing 30 Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA WO 99/55862 PCT/JP99/02226 21 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 5 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; 10 Brown et al., J. Immunol. 153:3079-3092, 1994. Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those 15 described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994. 20 Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W 25 Strober, Pub. Greene Publishing Associates and Wiley Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 30 149:3778-3783, 1992. Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that WO 99/55862 PCT/JP99/02226 22 activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 5 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062 4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 10 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990. Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte 15 homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et 20 al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992. Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et 25 al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991. Hematopoiesis Regulating Activity A protein of the present invention may be useful in 30 regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of WO 99/55862 PCT/JP99/02226 23 factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g. in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in 5 treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) 10 useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and 15 generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell 20 disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow 25 transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy. The activity of a protein of the invention may, among other means, be measured by the following methods: 30 Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above. Assays for embryonic stem cell differentiation (which WO 99/55862 PCT/JP99/02226 24 will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15:141 151, 1995; Keller et al., Molecular and Cellular Biology 5 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993. Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In 10 Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and Briddell, R.A. In Culture of 15 Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 1-21, 20 Wiley-Liss, Inc., New York, NY. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994; Long term culture initiating cell assay, 25 Sutherland, H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994. Tissue Growth Activity A protein of the present invention also may have utility 30 in compositions used for bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the WO 99/55862 PCT/JP99/02226 25 treatment of burns, incisions and ulcers. A protein of the present invention, which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone 5 fractures and cartilage damage or defects in humans and other animals. Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an 10 osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery. A protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair 15 processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells. A protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of 20 bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes. Another category of tissue regeneration activity that may be attributable to the protein of the present invention is 25 tendon/ligament formation. A protein of the present invention, which induces tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in 30 humans and other animals. Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament WO 99/55862 PCT/JP99/02226 26 tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue. De novo tendon/ligament-like tissue formation induced by a composition of the present invention 5 contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide an environment to attract tendon or 10 ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions of the invention may 15 also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art. The protein of the present invention may also be useful 20 for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More 25 specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, 30 amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, WO 99/55862 PCT/JP99/02226 27 such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention. 5 Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like. 10 It is expected that a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular 15 endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate. A protein of the invention may also exhibit angiogenic activity. 20 A protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage. A protein of the present invention may also be useful for 25 promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above. The activity of a protein of the invention may, among other means, be measured by the following methods: 30 Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. W095/16035 (bone, cartilage, tendon); WO 99/55862 PCT/JP99/02226 28 International Patent Publication No. W095/05846 (nerve, neuronal); International Patent Publication No. W091/07491 (skin, endothelium ). Assays for wound healing activity include, without 5 limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978). Activin/Inhibin Activity 10 A protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of 15 follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin a family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration 20 of sufficient amounts of other inhibins can induce infertility in these mammals. Alternatively, the protein of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin-0 group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in 25 stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798,885. A protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such 30 as cows, sheep and pigs. The activity of a protein of the invention may, among other means, be measured by the following methods: WO 99/55862 PCT/JP99/02226 29 Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659 5 663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091 3095, 1986. Chemotactic /Chemokinetic Activity A protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for 10 mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells. Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action. Chemotactic or 15 chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the 20 tumor or infecting agent. A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population. Preferably, the protein or peptide has the ability 25 to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis. The activity of a protein of the invention may, among 30 other means, be measured by the following methods: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis)consist of assays WO 99/55862 PCT/JP99/02226 30 that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, 5 without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 10 95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768, 1994. Hemostatic and Thrombolytic Activity 15 A protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result,such a protein is expected to be useful in treatment of various coagulation disorders (includinghereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating 20 wounds resulting from trauma, surgery or other causes. A protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system 25 vessels (e.g., stroke). The activity of a protein of the invention may, among other means, be measured by the following methods: Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. 30 Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.

WO 99/55862 PCT/JP99/02226 31 Receptor/Ligand Activity A protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions. Examples of such 5 receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, 10 integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses). Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. 15 A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions. The activity of a protein of the invention may, among other means, be measured by the following methods: 20 Suitable assays for receptor-ligand activity include without limitation those described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular 25 Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995. 30 Anti-Inflammatory Activity Proteins of the present invention may also exhibit anti- WO 99/55862 PCT/JP99/02226 32 inflammatory activity. The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by 5 inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response. Proteins exhibiting such activities can 10 be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia reperfusion injury, endotoxin lethality, arthritis, complement 15 mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of ytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an 20 antigenic substance or material. Tumor Inhibition Activity In addition to the activities described above for immunological treatment or prevention of tumors, a protein of the invention may exhibit other anti-tumor activities. A 25 protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting 30 angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, WO 99/55862 PCT/JP99/02226 33 eliminating or inhibiting factors, agents or cell types which promote tumor growth Other Activities A protein of the invention may also exhibit one or more 5 of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, 10 weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female subjects; 15 effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, 20 libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal 25 or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and 30 the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another WO 99/55862 PCT/JP99/02226 34 material or entity which is cross-reactive with such protein. Examples The present invention is embodied in more detail by the 5 following examples, but this embodiment is not intended to restrict the present invention. The basic operations and the enzyme reactions with regard to the DNA recombination are carried out according to the literature ["Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Laboratory, 1989]. 10 Unless otherwise stated, restrictive enzymes and a variety of modification enzymes to be used were those available from TAKARA SHUZO. The manufacturer's instructions were used for the buffer compositions as well as for the reaction conditions, in each of the enzyme reactions. The cDNA synthesis was carried 15 out according to the literature [Kato, S. et al., Gene 150: 243-250 (1994)]. (1) Preparation of Poly(A)+ RNA The osteosarcoma cell line Saos-2 (ATCC HTB 85), tissues of stomach cancer delivered by the operation, and the liver 20 were used for human cells to extract mRNAs. The cell line was incubated by a conventional procedure. After about 1 g of the human cells was homogenized in 20 ml of a 5.5 M guanidinium thiocyanate solution, a total mRNA was prepared according to the literature [Okayama, H. et al., 25 "Method in Enzymology", Vol. 164, Academic Press, 1987]. This was subjected to chromatography on oligo(dT)-cellulose column washed with a 20 mM Tris-hydrochloride buffer solution (pH 7.6), 0.5 M NaCl, and 1 mM EDTA to obtain a poly(A)* RNA according to the above-described literature. 30 (2) Construction of cDNA Library Ten micrograms of the above-mentioned poly(A)* RNA were WO 99/55862 PCT/JP99/02226 35 dissolved in a 100 mM Tris-hydrochloride buffer solution (pH 8), one unit of an RNase-free, bacterial alkaline phosphatase was added, and the reaction was run at 37 0 C for one hour. After the reaction solution was subjected to phenol extraction, followed 5 by ethanol precipitation, the resulting pellet was dissolved in a solution containing 50 mM sodium acetate (pH 6), 1 mM EDTA, 0.1% 2-mercaptoethanol, and 0.01% Triton X-100. Thereto was added one unit of a tobacco-origin acid pyrophosphatase (Epicentre Technologies) and a total 100 pl volume of the 10 resulting mixture was reacted at 37 0 C for one hour. After the reaction solution was subjected to phenol extraction, followed by ethanol precipitation, the resulting pellet was dissolved in water to obtain a solution of a decapped poly(A)* RNA. The decapped poly(A)* RNA and 3 nmol of a chimeric DNA-RNA 15 oligonucleotide (5' -dG-dG-dG-dG-dA-dA-dT-dT-dC-dG-dA-G-G-A-3') were dissolved in a solution containing 50 mM Tris hydrochloride buffer solution (pH 7.5), 0.5 mM ATP, 5 mM MgCl 2 , 10 mM 2-mercaptoethanol, and 25% polyethylene glycol, whereto was added 50 units of T4RNA ligase and a total 30 pl volume of 20 the resulting mixture was reacted at 20 0 C for 12 hours. After the reaction solution was subjected to phenol extraction, followed by ethanol precipitation, the resulting pellet was dissolved in water to obtain a chimeric-oligo-capped poly(A)* RNA. 25 After digestion of vector pKAl (Japanese Patent Kokai Publication No. 1992-117292) developed by the present inventors with KpnI, about 60 dT tails were added using a terminal transferase. A vector primer to be used below was prepared by digestion of this product with EcoRV to remove a dT tail at one 30 side. After 6 pg of the previously-prepared chimeric-oligo capped poly(A)* RNA was annealed with 1.2 pg of the vector WO 99/55862 PCT/JP99/02226 36 primer, the resulting product was dissolved in a solution containing 50 mM Tris-hydrochloride buffer solution (pH 8.3), 75 mM KCl, 3 mM MgCl 2 , 10 mM dithiothreitol, and 1.25 mM dNTP (dATP + dCTP + dGTP + dTTP), 200 units of a reverse 5 transcriptase (GIBCO-BRL) were added, and the reaction in a total 20 pl volume was run at 42 0 C for one hour. After the reaction solution was subjected to phenol extraction, followed by ethanol precipitation, the resulting pellet was dissolved in a solution containing 50 mM Tris-hydrochloride buffer solution 10 (pH 7.5), 100 mM NaCl, 10 mM MgCl 2 , and 1 mM dithiothreitol. Thereto were added 100 units of EcoRI and a total 20 pl volume of the resulting mixture was reacted at 37 0 C for one hour. After the reaction solution was subjected to phenol extraction, followed by ethanol precipitation, the resulting pellet was 15 dissolved in a solution containing 20 mM Tris-hydrochloride buffer solution (pH 7.5), 100 mM KCl, 4 mM MgCl 2 , 10 mM (NH 4

)

2

SO

4 , and 50 pg/ml of the bovine serum albumin. Thereto were added 60 units of an Escherichia coli DNA ligase and the resulting mixture was reacted at 16 0 C for 16 hours. To the reaction 20 solution were added 2 pl of 2 mM dNTP, 4 units of Escherichia coli DNA polymerase I, and 0.1 unit of Escherichia coli RNase H and the resulting mixture was reacted at 12 0 C for one hour and then at 22 0 C for one hour. Next, the cDNA-synthesis reaction solution was used for 25 transformation of Escherichia coli DH12S (GIBCO-BRL). The transformation was carried out by the electroporation method. A portion of the transformant was sprayed on the 2xYT agar culture medium containing 100 pg/ml ampicillin and the mixture was incubated at 37 0 C overnight. A colony formed on the agar 30 medium was picked up at random and inoculated on 2 ml of the 2xYT culture medium containing 100 pg/ml ampicillin. After incubation at 37 0 C overnight, the culture mixture was WO 99/55862 PCT/JP99/02226 37 centrifuged to separate the mycelia, from which a plasmid DNA was prepared by the alkaline lysis method. The plasmid DNA was subjected to double digestion with EcoRI and NotI, followed by 0.8% agarose gel electrophoresis, to determine the size of the 5 cDNA insert. Furthermore, using the thus-obtained plasmid as a template, the sequence reaction was carried out by using an M13 universal primer labeled with a fluorescent dye and a Taq polymerase (a kit of Applied Biosystems) and then the product was examined with a fluorescent DNA sequencer (Applied 10 Biosystems) to determine an about 400-bp base sequence at the 5'-terminus of the cDNA. The sequence data were filed as the homo/protein cDNA bank database. (3) Selection of cDNAs Encoding Proteins Having Transmembrane Domains 15 A base sequence registered in the homo/protein cDNA bank was converted to three frames of amino acid sequences and the presence or absence of an open reading frame (ORF) beginning from the initiation codon was examined. Then, the selection was made for the presence of a signal sequence that is 20 characteristic to a secretory protein at the N-terminus of the portion encoded by the ORF. These clones were sequenced from the both 5' and 3' directions by the use of the deletion method using exonuclease III to determine the whole base sequence. The hydrophobicity/hydrophilicity profiles were obtained for 25 proteins encoded by the ORF by the Kyte-Doolittle method [Kyte, J. & Doolittle, R. F., J. Mol. Biol. 157: 105-132 (1982)] to examine the presence or absence of a hydrophobic region. In the case in which there is a hydrophobic region of a putative transmembrane domain in the amino acid sequence of an encoded 30 protein, this protein was judged as a membrane protein. (4) Functional Verification of Secretory Signal Sequence or Transmembrane Domains WO 99/55862 PCT/JP99/02226 38 It was verified by the method described in the literature [Yokoyama-Kobayashi, M. et al., Gene 163: 193-196 (1995) ] that the N-terminal hydrophobic region in the secretory protein clone candidate obtained in the above-mentioned steps functions 5 as a secretory signal sequence. First, the plasmid containing the target cDNA was cleaved at an appropriate restriction enzyme site existing at the downstream of the portion expected for encoding the secretory signal sequence. In the case in which this restriction site was a protruding terminus, the site 10 was blunt-ended by the Klenow treatment or treatment with the T4DNA polymerase. Digestion with HindIII was further carried out and a DNA fragment containing the SV40 promoter and a cDNA encoding the secretory signal sequence at the downstream of the promoter was separated by agarose gel electrophoresis. The 15 resulting fragment was inserted between HindIII in pSSD3 (DDBJ/EMBL/GenBank Registration No. AB007632) and a restriction enzyme site selected so as to match with the urokinase-coding frame, thereby constructing a vector expressing a fusion protein of the secretory signal sequence of the target cDNA and 20 the urokinase protease domain. After Escherichia coli (host: JM109) bearing the fusion protein expression vector was incubated at 37 0 C for 2 hours in 2 ml of the 2xYT culture medium containing 100 pg/ml of ampicillin, the helper phage M13KO7 (50 pl) was added and the 25 incubation was continued at 37 0 C overnight. A supernatant separated by centrifugation underwent precipitation with polyethylene glycol to obtain single-stranded phage particles. These particles were suspended in 100 pl of 1 mM Tris-0.1 mM EDTA, pH 8 (TE). Also, there were used as controls suspensions 30 of single-stranded phage particles prepared in the same manner from pSSD3 and from the vector pKAl-UPA containing a full length cDNA of urokinase [Yokoyama-Kobayashi, M. et al., Gene WO 99/55862 PCT/JP99/02226 39 163: 193-196 (1995)]. The culture cells originating from the simian kidney, COS7, were incubated at 37 0 C in the presence of 5% Co 2 in the Dulbecco's modified Eagle's culture medium (DMEM) containing 5 10% fetal calf albumin. Into a 6-well plate (Nunc Inc., 3 cm in the well diameter) were inoculated 1 X 10 5 COS7 cells and incubation was carried out at 37 0 C for 22 hours in the presence of 5% Co 2 . After the culture medium was removed, the cell surface was washed with a phosphate buffer solution and then 10 washed again with DMEM containing 50 mM Tris-hydrochloric acid (pH 7.5) (TDMEM). To the resulting cells was added a suspension of 1 pl of the single-stranded phage suspension, 0.6 ml of the DMEM culture medium, and 3 p1 of TRANSFECTAM (IBF Inc.) and the resulting mixture was incubated at 37 0 C for 3 hours in the 15 presence of 5% CO 2 . After the sample solution was removed, the cell surface was washed with TDMEM, 2 ml per well of DMEM containing 10% fetal calf albumin was added, and the incubation was carried out at 37 0 C for 2 days in the presence of 5% CO 2 To 10 ml of 50 mM phosphate buffer solution (pH 7.4) 20 containing 2% bovine fibrinogen (Miles Inc.), 0.5% agarose, and 1 mM calcium chloride were added 10 units of human thrombin (Mochida Pharmaceutical Co., Ltd.) and the resulting mixture was solidified in a plate of 9 cm in diameter to prepare a fibrin plate. Ten microliters of the culture supernatant of the 25 tansfected COS7 cells were spotted on the fibrin plate, which was incubated at 37 0 C for 15 hours. In the case in which a clear circle appears on the fibrin plate, it is judged that the cDNA fragment codes for the amino acid sequence functioning as a secretory signal sequence. On the other hand, in case in 30 which a clear circle is not formed, the cells were washed well, then the fibrin sheet was placed on the cells, and incubation was carried out at 37 0 C for 15 hours. In case in which a clear WO 99/55862 PCT/JP99/02226 40 portion is formed on the fibrin sheet, it indicates that the urokinase activity was expressed on the cell surface. In other words, the cDNA fragment is judged to code for the transmembrane domains. 5 (5) Protein Synthesis by In Vitro Translation The plasmid vector bearing the cDNA of the present invention was used for in vitro transcription/translation with a TNT rabbit reticulocyte lysate kit (Promega) . In this case,

[

35 S]methionine was added to label the expression product with a 10 radioisotope. Each of the reactions was carried out according to the protocols attached to the kit. Two micrograms of the plasmid was reacted at 30 0 C for 90 minutes in a total 25 pl volume of the reaction solution containing 12.5 pl of TNT rabbit reticulocyte lysate, 0.5 pl of a buffer solution 15 (attached to kit), 2 pl of an amino acid mixture (methionine free), 2 pl of [ 35 S]methionine (Amersham) (0.37 MBq/pl), 0.5 pl of T7RNA polymerase, and 20 U of RNasin. To 3 pl of the resulting reaction solution was added 2 pl of the SDS sampling buffer (125 mM Tris-hydrochloric acid buffer, pH 6.8, 120 mM 2 20 mercaptoethanol, 2% SDS solution, 0.025% bromophenol blue, and 20% glycerol) and the resulting mixture was heated at 95 0 C for 3 minutes and then subjected to SDS-polyacrylamide gel electrophoresis. The molecular weight of the translation product was determined by carrying out the autoradiography. 25 (6) Expression by COS7 Escherichia coli bearing the expression vector of the protein of the present invention was infected with helper phage M13KO7 and single-stranded phage particles were obtained by the above-mentioned procedure. The thus-obtained phage was used for 30 introducing each expression vector in the culture cells originating from the simian kidney, COS7. After incubation at 37 0 C for 2 days in the presence of 5% C0 2 , the incubation was WO 99/55862 PCT/JP99/02226 41 continued for one hour in the culture medium containing

[

3 5 S]cystine or [ 3 S]methionine. Collection and dissolution of the cells, followed by subjecting to SDS-PAGE, allowed to observe the presence of a band corresponding to the expression 5 product of each protein, which did not exist in the COS7 cells. (7) Northern Blot Hybridization Northern blot hybridization was carried out in order to examine the expression pattern in the human tissues. Filters where poly(A)* RNAs isolated from each of human tissues are 10 blotted were purchased from Clontech. After excision of a cDNA fragment from the objective clone, followed by agarose-gel electrophoresis to isolate the cDNA fragment, labeling with

[

3 2 P]dCTP (Amersham) was carried out by using a random primer labeling kit (TAKARA SHUZO). The hybridization was carried out 15 by using a solution attached to the blot paper according to the protocol. (8) Clone Examples <HPO2000> (Sequence Nos. 1, 10, and 19) Determination of the whole base sequence of the cDNA 20 insert of clone HPO2000 obtained from cDNA libraries of human stomach cancer revealed the structure consisting of a 186-bp 5'-nontranslation region, an 807-bp ORF, and a 712-bp 3' nontranslation region. The ORF codes for a protein consisting of 268 amino acid residues and there existed two putative 25 transmembrane domains. Figure 1 depicts the hydrophobicity/hydrophilicity profile, obtained by the Kyte Doolittle method, of the present protein. In vitro translation resulted in formation of a translation product of 31 kDa that was almost identical with the molecular weight of 30,481 30 predicted from the ORF. When expressed in COS 7 cells, an expression product of about 32 kDa was observed in the membrane fraction.

WO 99/55862 PCT/JP99/02226 42 The search of the protein data base by using the amino acid sequence of the present protein revealed that the protein was analogous to the rat organic cation transporter (EMBL Accession No. Y09945). Table 2 shows the comparison of the 5 amino acid sequence between the human protein of the present invention (HP) and the rat organic cation transporter (RN). Therein, the marks of -, *, and . represent a gap, an amino acid residue identical with the protein of the present invention, and an amino acid residue analogous to the protein 10 of the present invention, respectively. The both proteins possessed a homology of 67.5% in the N-terminal 169 amino acid residues. Table 2 15 HS MAFEELLSQVGGLGRFQMLHLVFILPSTMLIPHILLENFAAAIPGHRCWVHMLDNNTGS ***.*** .*****.*...** ... *******.*.**.*****. .***.* * RN MAFQDLLNQVGSLGRFQILQMTFILIFNIIISPHSLLENFTAVIPNHRCWVPILDNDTVS HS GNETGILSEDALLRISIPLDSNLRPEKCRRFVHPQWQLLHLNGTIHSTSEADTEPCVDGW 20 **..* **.****.******.*************.*******. *..*.********* RN GNDNGNLSQDDLLRVSIPLDSDLRPEKCRRFVQPQWDLLHLNGTFSSVTEPDTEPCVDGW HS VYDQSYFPSTIVTKWDLVCDYQSLKSVVQFLLLTGMLVGGIIGGHVSDRWLVESARWLII ***** * ***.*.*****. ***.*..**.***.***.*. *..** RN VYDQSTFLSTIITEWDLVCESQSLDSIAKFLFLTGILVGNILYGPLTDRFGRRLILICAS 25 HS TNKLDEGLUALRKVARTNGIKNAEETLNIEVVRSTMQEETDAAQTKTTVCDLFRNPSMRK RN LQMAVTETCAAFAPTFLIYCSLRFLAGISFSTVLTNSALLIIEWTRPKFQALATGLLLCA HS RICILVFLRKKISRKRHKNDCYTKVTKF 30 RN GAIGQTVLAGLAFTVRNWHHLHLAMSVPIFFLLVPTRWLSESARWLIMTNKLQKGLKELI WO 99/55862 PCT/JP99/02226 43 Furthermore, the search of the GenBank using the base sequences of the present cDNA has revealed the registration of sequences that possessed a homology of 90% or more (for example, Accession No. AA680184) in EST, but any of the sequences was 5 shorter than the present cDNAs and was not found to contain the initiation codon. An investigation of the expression pattern in the tissues by northern blot hybridization using the cDNA fragment of the present invention has revealed the expression only in the liver. 10 The rat organic cation transporter has been found as a membrane protein associated with a drug excretion in the kidney [Grundemann, D. et al., Nature 372: 549-552 (1994)]. Accordingly, the protein of the present invention that is its homologue is considered to possess a similar function and can 15 be utilized for the diagnosis and treatment of diseases that are associated with abnormalities of this enzyme. Furthermore, this is considered to be associated with a drug excretion, so that the cells expressing this protein can be used as a tool for designing this drug. In addition, since this protein is 20 expressed specifically in the liver, a substance prepared so as to possess an affinity with this protein can be applied to the drug delivery system to the liver. <HP02061> (Sequence Nos. 2, 11, and 21) Determination of the whole base sequence of the cDNA 25 insert of clone HP02061 obtained from cDNA libraries of human osteosarcoma cell line Saos-2 revealed the structure consisting of a 141-bp 5'-nontranslation region, a 711-bp ORF, and a 907 bp 3'-nontranslation region. The ORF codes for a protein consisting of 236 amino acid residues and there existed two 30 putative transmembrane domains. Figure 2 depicts the hydrophobicity/hydrophilicity profile, obtained by the Kyte Doolittle method, of the present protein. In vitro translation WO 99/55862 PCT/JP99/02226 44 resulted in formation of a translation product of 26 kDa that was almost identical with the molecular weight of 25,593 predicted from the ORF. The search of the protein data base by using the amino 5 acid sequence of the present protein revealed that the protein was analogous to the human neuroendocrine-specific protein C (PIR Accession No. 160904). Table 3 shows the comparison of the amino acid sequence between the human protein of the present invention (HP) and the human neuroendocrine-specific protein C 10 (PC) . Therein, the marks of -, *, and . represent a gap, an amino acid residue identical with the protein of the present invention, and an amino acid residue analogous to the protein of the present invention, respectively. The C-terminal 187 amino acid residues possessed a homology of 59.9% with the 15 human neuroendocrine-specific protein C. Table 3 HS MAEPSAATQSHSISSSSFGAEPSAPGGGGSPGACPALGTKSCSSSCAVHDLIFWRDVKKT 20 **.***.*.* PC MQATADSTKMDCVWSNWKSQAIDLLYWRDIKQT HS GFVFGTTLIMLLSLAAFSVISVVSYLILALLSVTISFRIYKSVIQAVQKSEEGHPFKAYL * ** *.***.***.***.*****.****.**.***** PC GIVFGSFT.T.T.FSLTQFSVVSVVAYLALAALSATISFRIYKSVLQAVQKTDEGHPFKAYL 25 HS DVDITLSSEAFHNYMNAAMVHINRALKLIIRLFLVEDLVDSLKLAVFMWLMTYVGAVFNG .. **** *...* . .*..** .******** .* .** .* PC ELEITLSQEQIQKYTDCLQFYVNSTLKELRRLFLVQDLVDSLKFAVLMWLLTYVGALFNG HS ITLLILAELLIFSVPIVYEKYKTQIDHYVGIARDQTKSIVEKIQAKLPGIAKKKAE .***..* . .**.** *...***.*.*..*......*.*****.** **..** 30 PC LTLLMAVVSMFTLPVVYVKHQAQIDQYLGLVRTHINAVVAKIQAKIPG-AKRHAE WO 99/55862 PCT/JP99/02226 45 Furthermore, the search of the GenBank using the base sequences of the present cDNA has revealed the presence of sequences that possessed a homology of 90% or more (for example, Accession No. AA362885) in EST, but, since they are partial 5 sequences, it can not be judged whether or not any of these sequences codes for the same protein as the protein of the present invention. <HP02163> (Sequence Nos. 3, 12, and 23) Determination of the whole base sequence of the cDNA 10 insert of clone HP02163 obtained from cDNA libraries of human osteosarcoma cell line Saos-2 revealed the structure consisting of a 179-bp 5'-nontranslation region, a 786-bp ORF, and a 104 bp 3'-nontranslation region. The ORF codes for a protein consisting of 261 amino acid residues and there existed one 15 putative transmembrane domain. Figure 3 depicts the hydrophobicity/hydrophilicity profile, obtained by the Kyte Doolittle method, of the present protein. In vitro translation resulted in formation of a translation product of 30 kDa that was almost identical with the molecular weight of 29,932 20 predicted from the ORF. When expressed in COS 7 cells, an expression product of about 28 kDa was observed in the membrane fraction. The search of the protein data base using the amino acid sequence of the present protein has revealed the presence of 25 sequences that were analogous to a yeast hypothetical protein of 29.4 kDa (SWISS-PROT Accession No. P36039). Table 4 shows the comparison of the amino acid sequence between the human protein of the present invention (HP) and the yeast hypothetical protein of 29.4 kDa (SC). Therein, the marks of -, 30 *, and . represent a gap, an amino acid residue identical with the protein of the present invention, and an amino acid residue analogous to the protein of the present invention, respectively.

WO 99/55862 PCT/JP99/02226 46 The both proteins possessed a homology of 33.2% in the entire region. Table 4 5 HS MAGPELLLDSNIRLWVVLPIVIITFFVGMIRHYVSI .*. .***.... **.*** *. .*...*. SC MTINQHLQQLLFNRIDKTTSSIQQARAPQMLLDDQLKYWVLLPISIVMVLTGVLKQYIMT HS LL---QSDKKLTQEQVSDSQVLIRSRVLRENGKYIPKQSFLTRK-YYFNN-PEDGFFKKT 10 * ... .. .... ** .... * .** ..... * .. * . .. .*. . *. SC LITGSSANEAQPRVKLTEWQYLQWAQLLIGNGGNLSSDAFAAKKEFLVKDLTEERHLAKA HS KRK-----VVPPSPMTDPTM---LTDMMKGNVTNVLPMILIGGWINMTFSGFVTTKVPFP *. *.*. .**.* . * ***.. .*.* *.* *.**. .. *** SC KQQDGSQAGEVPNPFNDPSMSNAMMNMAKGNMASFIPQTIIMWWVNHFFAGFILMQLPFP 15 HS LTLRFKPMLQQGIELLTLDASWVSSASWYFLNVFGLRSIYSLI-LGQDNAADQSRMMQEQ ** .** *** ** .**.. **** ****..*.**...**...* SC LTAKFKEMLQTGIICQDLDVRWVSSISWYFISVLGLNPVYNLIGLNDQDMGIQAGIGGPQ HS MTGAAMAMPADTNKAFKTEWEALELTDHQWALDDVEEELMAKDLHFEGMFKKELQTSIF 20 SC APKALHNHRLTKQCMRWLTI Furthermore, the search of the GenBank using the base sequences of the present cDNA has revealed the registration of 25 sequences that possessed a homology of 90% or more (for example, Accession No. Z43161) in EST, but, since they are partial sequences, it can not be judged whether or not any of these sequences codes for the same protein as the protein of the present invention. 30 <HP02219> (Sequence Nos. 4, 13, and 25) Determination of the whole base sequence of the cDNA insert of clone HP02219 obtained from cDNA libraries of human WO 99/55862 PCT/JP99/02226 47 stomach cancer revealed the structure consisting of a 58-bp 5' nontranslation region, a 987-bp ORF, and a 714-bp 3' nontranslation region. The ORF codes for a protein consisting of 328 amino acid residues and there existed one putative 5 transmembrane domain. Figure 4 depicts the hydrophobicity/hydrophilicity profile, obtained by the Kyte Doolittle method, of the present protein. In vitro translation resulted in formation of a translation product of 39 kDa that was almost identical with the molecular weight of 37,299 10 predicted from the ORF. When expressed in COS 7 cells, an expression product of about 39 kDa was observed in the membrane fraction. The search of the protein data base using the amino acid sequence of the present protein has revealed that the protein 15 was analogous to Alabidopsis thaliana dTDP-glucose 4-6 dehydratase homologue (PIR Accession No. 558282). Table 5 shows the comparison of the amino acid sequence between the human protein of the present invention (HP) and the Alabidopsis thaliana dTDP-glucose 4-6-dehydratase homologue (AT). Therein, 20 the marks of * and . represent an amino acid residue identical with the protein of the present invention and an amino acid residue analogous to the protein of the present invention, respectively. The both proteins possessed a homology of 57.2% in 145 amino acid residues at the C-terminal region.

WO 99/55862 PCT/JP99/02226 48 Table 5 HS MVSKALJRLVSAVNRRRMKLLLGIALIAYVASVWGNFVNMSFLLNRSIQENGELKIE 5 AT RVVVTGGAGFVGSHLVDRLMARGDTVIVVDNFFTGRKENVMHHFSNPNFEMIRHDVVEPI HS SKIEEMVEPLREKIRDLEKSFTQKYPPVKFLSEKDRKRILITGGAGFVGSHLTDKLMMDG AT LLEVDQIYHLACPASPVHYKFNPVKTIKTNVVGTLNMLGLAKRVGARFLLTSTSEVYGDP HS HEVTVVDNFFTGRKRNVEHWIGHENFELINHDVVEPLYIEGVEVRVARIFNTFGPRMHMN 10 * . AT LQHPQVETYWGNVNPIGVRSCYDEGKRTAETLTMDYHRGSNVEVRIARIFNTYGPRMCID HS DGRVVSNFILQALQGEPLTVYGSGSQTRAFQYVSDLVNGLVALMNSNVSSPVNLGNPEEH ********. ***. *******.*.***.**.*****.**. **.. * ***. AT DGRVVSNFVAQALRKEPLTVYGDGKQTRSFQFVSDLVEGLMRLMEGEHVGPFNLGNPGEF 15 HS TILEFAQLIKNLVGSGSEIQFLSEAQDDPQKRKPDIKKAKLMLGWEPVVPLEEGLNKAIH *.**.*..... ...... *.* .... ************ .***** *.* .** AT TMLELAKVVQETIDPNANIEFRPNTEDDPHKRKPDITKAKELLGWEPKVSLRQGLPLMVK HS YFRKELEYQANNQYIPKPKPARIKKGRTRHS 20 AT DFRQRVFGDQKEGSSAAATTTKTTSA Furthermore, the search of the GenBank using the base sequences of the present cDNA has revealed the registration of 25 sequences that possessed a homology of 90% or more (for example, Accession No. U46355) in EST, but, since they are partial sequences, it can not be judged whether or not any of these sequences codes for the same protein as the protein of the present invention.

WO 99/55862 PCT/JP99/02226 49 <HP02256> (Sequence Nos. 5, 14, and 27) Determination of the whole base sequence of the cDNA insert of clone HP02256 obtained from cDNA libraries of human stomach cancer revealed the structure consisting of a 131-bp 5 5'-nontranslation region, a 903-bp ORF, and a 663-bp 3' nontranslation region. The ORF codes for a protein consisting of 300 amino acid residues and there existed one transmembrane domain at the N-terminus. Figure 5 depicts the hydrophobicity/hydrophilicity profile, obtained by the Kyte 10 Doolittle method, of the present protein. In vitro translation resulted in formation of a translation product of 33 kDa that was almost identical with the molecular weight of 32,943 predicted from the ORF. When expressed in COS cells, an expression product of about 30 kDa was observed in the membrane 15 fraction. The search of the protein data base using the amino acid sequence of the present protein has revealed that the protein was analogous to the Caenorhabditis elegans hypothetical protein TllF9.11 (PID Accession No. 1403260). Table 6 shows the 20 comparison of the amino acid sequence between the human protein of the present invention (HP) and the Caenorhabditis elegans hypothetical protein T11F9.11 (CE). Therein, the marks of -, *, and . represent a gap, an amino acid residue identical with the protein of the present invention, and an amino acid residue 25 analogous to the protein of the present invention, respectively.

WO 99/55862 PCT/JP99/02226 50 The both proteins possessed a homology of 41.7% in the entire region. Table 6 5 HS MKFLLDIT..TLLIVCSLESFVKLFIPK---RRKSVTGEIVLITGAGHGIGRLTAYEFA **. . . .. * * *.*. **** * ***** * ***** * *** CE MDRALDFVKMVVGTLFFIVLNFFKNFLPNGVLPRKSVEGKKVLITGSGSGIGRLMALEFA HS KLKSKLVLWDINKHGLEETAAKCKGLGAKVHTFVVDCSNREDIYSSAKKVKAEIGDVSIL 10 ** ... *.**.**.* *** .. *.*. ***** *. .**.. **..* ... **..** CE KLGAEVVIWDVNKDGAEETKNQVVKAGGKASTFVVDLSQYKDIHKVAKETKEAVGDIDIL HS VNNAGVVYTSDLFATQDPQIEKTFEVNVLAHFWTTKAFLPAMTKNNHGHIVTVASAAGHV .****.**. .* .***..**. **.*.*.***..*.****. CE INNAGIVTGKKLFDCPDELMEKTMAVNTNALFYTAKNFLPSMLEKDNGHLVTIASMAGKT 15 HS SVPFLLAYCSSKFAAVGFHKTLTDELAALQITGVKTTCLCPNFVNTG-F--IKNPSTSLG . *..**.** .** *... *. *. **.** .** *.*** .. .* CE GCVGLVDYCASKHGAIGCHDSIAMEILAQKKYGVNTTLVCPFFIDTGMFHGVTTKCPALF HS PTLEPEEVVNRLMHGILTEQKMIFIPSSIAFLTTLERILPERFLAVLKRKISVKFDAVIG *.**. .* .. . * .. . *. . .* .** 20 CE PILEANYAVECIVEAILTNRPLLCMPKASYLILALIGLLPIESQVMMADFFGTNESMNDF HS YKMKAQ CE KGRQKND 25 Furthermore, the search of the GenBank using the base sequences of the present cDNA has revealed the registration of sequences that possessed a homology of 90% or more (for example, Accession No. H61494) in EST, but, since they are partial 30 sequences, it can not be judged whether or not any of these sequences codes for the same protein as the protein of the WO 99/55862 PCT/JP99/02226 51 present invention. <HP10390> (Sequence Nos. 6, 15, and 29) Determination of the whole base sequence of the cDNA insert of clone HP10390 obtained from cDNA libraries of human 5 stomach cancer revealed the structure consisting of a 144-bp 5'-nontranslation region, a 549-bp ORF, and a 121-bp 3' nontranslation region. The ORF codes for a protein consisting of 182 amino acid residues and possessed one transmembrane domain in the N-terminus. Figure 6 depicts the 10 hydrophobicity/hydrophilicity profile, obtained by the Kyte Doolittle method, of the present protein. Introduction of an expression vector, wherein the HindIII-BstXI (treated with T4RNA polymerase) fragment containing a cDNA portion coding for the N-terminal 50 amino acid residues of the present protein 15 was inserted into the HindIII-SmaI site of pSSD3, into the COS7 cells revealed the urokinase activity on the surface of the cells to indicate that the present protein is the type-II membrane protein. In vitro translation resulted in formation of a translation product of 20 kDa that was almost identical with 20 the molecular weight of 20,639 predicted from the ORF. When expressed in COS cells, an expression product of about 19 kDa was observed in the supernatant fraction and the membrane fraction. The search of the protein data base using the amino acid 25 sequence of the present protein has not identified any known protein having an analogy. Furthermore, the search of the GenBank using the base sequences of the present cDNA has revealed the presence of sequences that possessed a homology of 90% or more (for example, Accession No. AA315322) in EST, but, 30 since they are partial sequences, it can not be judged whether or not any of these sequences codes for the same protein as the protein of the present invention.

WO 99/55862 PCT/JP99/02226 52 <HP10474> (Sequence Nos. 7, 16, and 31) Determination of the whole base sequence of the cDNA insert of clone HP10474 obtained from cDNA libraries of human osteosarcoma cell line Saos-2 revealed the structure consisting 5 of a 22-bp 5'-nontranslation region, a 201-bp ORF, and a 288-bp 3'-nontranslation region. The ORF codes for a protein consisting of 66 amino acid residues and possessed one transmembrane domain at the C-terminus. Figure 7 depicts the hydrophobicity/hydrophilicity profile, obtained by the Kyte 10 Doolittle method, of the present protein. In vitro translation resulted in formation of a translation product of 10 kDa that was almost identical with the molecular weight of 7,599 predicted from the ORF. The search of the protein data base using the amino acid 15 sequence of the present protein has not revealed the presence of any known protein having an analogy. Also, the search of the GenBank using the base sequences of the present cDNA has revealed the presence of sequences that possessed a homology of 90% or more (for example, Accession No. H30340) in EST, but, 20 since they are partial sequences, it can not be judged whether or not any of these sequences codes for the same protein as the protein of the present invention. <HP10527> (Sequence Nos. 8, 17, and 33) Determination of the whole base sequence of the cDNA 25 insert of clone HP10527 obtained from cDNA libraries of the human osteosarcoma cell line Saos-2 revealed the structure consisting of a 113-bp 5'-nontranslation region, a 552-bp ORF, and a 461-bp 3'-nontranslation region. The ORF codes for a protein consisting of 183 amino acid residues and possessed 30 three putative transmembrane domains. Figure 8 depicts the hydrophobicity/hydrophilicity profile, obtained by the Kyte Doolittle method, of the present protein. As the result of in WO 99/55862 PCT/JP99/02226 53 vitro translation, there was produced a translation product of about 21 kDa, which is nearly equal to a molecular weight of 21,111 as expected from ORF. The search of the protein data base using the amino acid 5 sequence of the present protein has not revealed the presence of any known protein having an analogy. Also, the search of the GenBank using the base sequences of the present cDNA has revealed the presence of sequences that possessed a homology of 90% or more (for example, Accession No. AA310892) in EST, but, 10 since they are partial sequences, it can not be judged whether or not any of these sequences codes for the same protein as the protein of the present invention. <HP10528> (Sequence Nos. 9, 18, and 35) Determination of the whole base sequence of the cDNA 15 insert of clone HP10528 obtained from cDNA libraries of the human osteosarcoma cell line Saos-2 revealed the structure consisting of a 53-bp 5'-nontranslation region, a 975-bp ORF, and a 987-bp 3'-nontranslation region. The ORF codes for a protein consisting of 324 amino acid residues and possessed 20 seven putative transmembrane domains. Figure 9 depicts the hydrophobicity/hydrophilicity profile, obtained by the Kyte Doolittle method, of the present protein. As the result of in vitro translation, there was produced a translation product of about 32 kDa, which is nearly equal to a molecular weight of 25 34,227 as expected from ORF. The search of the protein data base using the amino acid sequence of the present protein has revealed it had an analogy to the epithelial cell growth arrest-inducible gene product (PID Accession No. 998569). Table 7 shows the comparison of the 30 amino acid sequence between the human protein of the present invention (HP) and the epithelial cell growth arrest-inducible gene product (GA). Therein, the marks of -, *, and . represent WO 99/55862 PCT/JP99/02226 54 a gap, an amino acid residue identical with the protein of the present invention, and an amino acid residue analogous to the protein of the present invention, respectively. The both proteins possessed a homology of 34.7% in the entire region. 5 Table 7 HS MGPWGEPELLVWRPEAVASEPPVPVGLEVKLGALVLLLVLTLLCSLVPICVLRRPGANHE *..*** * ** *** * * *** ... 10 GA MEQLLGIKLGCLFALLALTLGCGLTPICFKWFQIDAAR HS GSASRQKALSLVSCFAGGVFLATCLLDLLPDYLAAIDEALAALHV-------------- * .* .*.*..*.. ****. . .. . *..*.. .. . * GA GHHRR--VLRLLGCISAGVFLGAGFMHMTAEALEEIESQIQKFMVQNRSASERNSSGDAD HS -- TLQFPLQEFILAMGFFLVLVMEQITLAYKEQSGPSPLEETRALLGTVNGGPQHWHDGP 15 ... * *.*...***** . * .. *.. *.. ...... .. **.. . GA SAHMEYPYGELIISLGFFLVFFLESLALQC----CPGA-AGGSTVQDEEWGGAHIF---E HS GVPQASGAPATPSALRACVLVFSLALHSVFEGLAVGLQRDRARAMELCLALLHKGILAV ..... .... ..*** **. .**. .************.. * .***** ***. GA LHSHGHLPSPSKGPLRALVLLLSLSFHSVFEGLAVGLQPTVAATVQLCLAVLAHKGLVVF 20 HS SLSLRLLQSHLRAQVVAGCGILFSCMTPLGIGLGAALAES-AGPLHQLAQSVLEGMAAGT .. **.. . .. .*. *.***..* *. . .. *** **** **** GA GVGMRLVHLGTSSRWAVFSILLLALMSPLGLAVGLAVTGGDSEGGRGLAQAVLEGVAAGT HS FLYITFLEILPQELASSEQRILKVILLLAGFALLTGLLFIQI ** ******* **** * * 25 GA ELYVTFLEILPRELASPEAPLAKWSCVAAGEAFMAFIALWA The search of the protein data base using the amino acid sequence of the present protein has not revealed the presence 30 of any known protein having an analogy. Also, the search of the GenBank using the base sequences of the present cDNA has revealed the presence of sequences that possessed a homology of WO 99/55862 PCT/JP99/02226 55 90% or more (for example, Accession No. AA206511) in EST, but, since they are partial sequences, it can not be judged whether or not any of these sequences codes for the same protein as the protein of the present invention. 5 INDUSTRIAL APPLICABILITY The present invention provides human proteins having transmembrane domains, cDNAs coding for these proteins, and expression vectors of said cDNAs as well as eucaryotic cells 10 expressing said cDNAs. All of the proteins of the present invention exist in the cell membrane, so that they are considered to be proteins controlling the proliferation and the differentiation of the cells. Accordingly, the proteins of the present invention can be employed as pharmaceuticals such as 15 carcinostatic agents relating to the control of the proliferation and the differentiation of the cells or as antigens for preparing antibodies against said proteins. The cDNAs of the present invention can be utilized as probes for the gene diagnosis and gene sources for the gene therapy. 20 Furthermore, the cDNAs can be utilized for large-scale expression of said proteins. Cells, wherein these membrane protein genes are introduced to possess said proteins on the membrane surface, can be utilized for detection of the corresponding ligands, screening of novel low-molecular 25 pharmaceuticals, and so on. The present invention also provides genes corresponding to the polynucleotide sequences disclosed herein. "Corresponding genes" are the regions of the genome that are transcribed to produce the mRNAs from which cDNA polynucleotide 30 sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited WO 99/55862 PCT/JP99/02226 56 to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can be isolated in accordance with known methods using the sequence 5 information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials. An "isolated gene" is a gene that has been 10 separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated. Organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein are provided. The desired change in 15 gene expression can be achieved through the use of antisense polynucleotides or ribozymes that bind and/or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15(7): 250-254; Lavarosky et al., 1997, Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid 20 Res. Mol. Biol. 58: 1-39; all of which are incorporated by reference herein). Transgenic animals that have multiple copies of the gene(s) corresponding to the polynucleotide sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably 25 maintained within the transformed cells and their progeny, are provided. Transgenic animals that have modified genetic control regions that increase or reduce gene expression levels, or that change temporal or spatial patterns of gene expression, are also provided (see European Patent No. 0 649 464 B1, 30 incorporated by reference herein). In addition, organisms are provided in which the gene(s) corresponding to the polynucleotide sequences disclosed herein have been partially WO 99/55862 PCT/JP99/02226 57 or completely inactivated, through insertion of extraneous sequences into the corresponding gene(s) or through deletion of all or part of the corresponding gene(s). Partial or complete gene inactivation can be accomplished through insertion, 5 preferably followed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14(9): 629-633; Zwaal et al., 1993, Proc. Natl. Acad. Sci. USA 90(16): 7431-7435; Clark et al., 1994, Proc. Natl. Acad. Sci. USA 91(2): 719-722; all of which are incorporated by reference herein), or through 10 homologous recombination, preferably detected by positive/negative genetic selection strategies (Mansour et al., 1988, Nature 336: 348-352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059; 5,631,153; 5,614, 396; 5,616,491; and 5,679,523; all of which are incorporated by reference herein). 15 These organisms with altered gene expression are preferably eukaryotes and more preferably are mammals. Such organisms are useful for the development of non-human models for the study of disorders involving the corresponding gene(s), and for the development of assay systems for the identification of 20 molecules that interact with the protein product(s) of the corresponding gene(s). Where the protein of the present invention is membrane bound (e.g., is a receptor), the present invention also provides for soluble forms of such protein. In such forms part 25 or all of the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed. The intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for 30 determination of such domains from sequence information. Proteins and protein fragments of the present invention include proteins with amino acid sequence lengths that are at WO 99/55862 PCT/JP99/02226 58 least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of a disclosed protein and have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with 5 that disclosed protein, where sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps. Also included in the present invention are proteins and protein fragments that contain a segment 10 preferably comprising 8 or more (more preferably 20 or more, most preferably 30 or more) contiguous amino acids that shares at least 75% sequence identity (more preferably, at least 85% identity; most preferably at least 95% identity) with any such segment of any of the disclosed proteins. 15 Species homologs of the disclosed polynucleotides and proteins are also provided by the present invention. As used herein, a "species homologue" is a protein or polynucleotide with a different species of origin from that of a given protein or polynucleotide, but with significant sequence similarity to 20 the given protein or polynucleotide, as determined by those of skill in the art. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the 25 desired species. The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous, or 30 related to that encoded by the polynucleotides. The invention also includes polynucleotides with sequences complementary to those of the polynucleotides WO 99/55862 PCT/JP99/02226 59 disclosed herein. The present invention also includes polynucleotides capable of hybridizing under reduced stringency conditions, more preferably stringent conditions, and most preferably 5 highlystringent conditions, to polynucleotides described herein. Examples of stringency conditions are shown in the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and 10 reduced stringency conditions are at least as stringent as, for example, conditions M-R.

WO 99/55862 PCT/JP99/02226 60 Table Stringency Polynucleotide Hybrid Hybridization Temperature Wash Condition Hybrid Length and Buffert Temperature (bp)- and Buffert A DNA: DNA 50 65 0 C; 1xSSC -or- 65 0 C; 0.3xSSC 429C; 1xSSC,50% formamide B DNA: DNA <50 TB *; 1XSSC TB*; 1XSSC C DNA: RNA 50 679C; 1xSSC -or- 679C; 0.3xSSC 459C; 1xSSC,50% formamide D DNA: RNA <50 TD *; 1XSSC TD*; 1XSSC E RNA: RNA 50 70 0 C; 1xSSC -or- 70 0 C; 0.3xSSC 50'C; 1xSSC,50% formamide F RNA: RNA <50 TF*; 1xSSC TF*; 1XSSC G DNA: DNA 50 659C; 4xSSC -or- 65'C; 1xSSC 429C; 4xSSC,50% formamide H DNA: DNA <50 TH*; 4xSSC TH*; 4XSSC I DNA: RNA 50 679C; 4xSSC -or- 67 0 C; 1xSSC 459C; 4xSSC,50% formamide J DNA: RNA <50 Tj*; 4xSSC Tj*; 4xSSC K RNA: RNA 50 709C; 4xSSC -or- 67 0 C; 1xSSC 509C; 4xSSC,50% formamide L RNA: RNA <50 TL*; 2xSSC TL*; 2xSSC M DNA: DNA 50 509C; 4xSSC -or- 50 0 C; 2xSSC 40 0 C; 6xSSC,50% formamide N DNA: DNA <50 TN*; 6xSSC TN*; 6xSSC 0 DNA: RNA 50 559C; 4xSSC -or- 559C; 2xSSC 429C; 6xSSC,50% formamide P DNA: RNA <50 Tp*; 6xSSC Tp*; 6xSSC Q RNA: RNA 50 609C; 4xSSC -or- 60 0 C; 2xSSC I I 45 0 C; 6xSSC,50% formamide R RNA: RNA <50 TR*; 4xSSC TR*; 4xSSC The hybrid length is that anticipated for the hybridized region(s) of the hybridizing 5 polynucleotides. When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridizing polynucleotide. When polynucleotides of known sequence are hybridized, the hybrid WO 99/55862 PCT/JP99/02226 61 length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity. t: SSPE (1xSSPE is 0.15M NaCl, 10mM NaH 2

PO

4 , and 1.25mM EDTA, pH7.4) can be substituted for SSC (1xSSC is 0.15M NaCl and 15mM sodium citrate) in the 5 hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete. *TB - TR: The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-109C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 10 base pairs in length, Tm( 0 C)=2(#of A + T bases) + 4(# of G + C bases). For hybrids between 18 and 49 base pairs in length, Tm( 0 C)=81.5 + 16.6(logio[Na*]) + 0.41 (%G+C) (600/N), where N is the number of bases in the hybrid, and [Na*] is the concentration of sodium ions in the hybridization buffer ([Na*] for 1xSSC=0.165M). 15 Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Current Protocols in Molecular 20 Biology, 1995, F.M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference. Preferably, each such hybridizing polynucleotide has a length that is at least 25%(more preferably at least 50%, and 25 most preferably at least 75%) of the length of the polynucleotide of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to 30 which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.

Claims (6)

1. A protein comprising any of the amino acid sequences represented by Sequence Nos. 1 to 9. 5

2. A DNA coding for any of the proteins as claimed in Claim 1.

3. A cDNA comprising any of the base sequences represented by Sequence Nos. 10 to 18.

4. The cDNA as claimed in Claim 3 comprising any of the 10 base sequences represented by Sequence Nos. 19, 21, 23, 25, 27, 29, 31, 33 and 35.

5. An expression vector capable of expressing the DNA as claimed in any of Claim 2 to Claim 4 by in vitro translation or in eucaryotic cells. 15

6. A transformation eucaryotic cell capable of expressing the DNA as claimed in any of Claim 2 to Claim 4 and producing the protein as claimed in Claim 1.