HUMAN TYPE-I MEMBRANE PROTEIN AND DNA ENCODING THIS PROTEIN

Abstract: Problems to be solved: to provide human type-I membrane proteins and human cDNAs encoding said proteins. Means to solve the problems: a protein containing the amino acid sequence represented by Sequence No. 1 and a DNA encoding said proteins exemplified by the cDNA containing the base sequence represented by Sequence No. 2. Said protein can be provided by expressing the human cDNA encoding the human type-I membrane protein and recombinants of this human cDNA.

Description: DESCRIPTION Human Type-I Membrane Protein and DNA Encoding this Protein TECHNICAL FIELD The present invention relates to a human type-I membrane protein and a cDNA encoding this protein. The protein of the present invention can be used as pharmaceuticals for the treatment and diagnosis or as an antigen for preparing an antibody against said protein. The cDNA of the present invention can be used as a probe for the gene diagnosis and a gene source for the gene therapy. Furthermore, the cDNA can be used as a gene source for large-scale production of the protein encoded by said cDNA. BACKGROUND ART Many of the type-I membrane proteins having a secretory signal sequence at the N-terminus and an inside transmembrane domain at one site function as signaling receptors. For example, all of interleukins receptors, colony-stimulating factors receptors, the erythropoietin receptor, and so on are type-I membrane proteins. These receptors play an important role in the cell proliferation, the cell differentiation, and so on. The ligand-binding sites existing on the cell surfaces of these type-I membrane proteins can be utilized as pharmaceuticals for trapping the ligands. Also, antibodies prepared against these antigens can be utilized as pharmaceuticals or diagnostic agents. Furthermore, the cells expressing large amounts of the membrane proteins by transfecting these human type-I membrane protein genes can be applied, for example, to the detection of the corresponding ligands, the screening of novel low-molecular medicines, and so on. Hereupon, the existence of many human type-I membrane proteins other than those mentioned above has been predicted, thereby isolation of novel genes encoding the human type-I membrane proteins has been desired for the above-mentioned purposes . DISCLOSURE OF INVENTION The object of the present invention is to provide a novel human type-I membrane protein and a DNA encoding said protein. As the result of intensive studies, the present inventors were successful in cloning of a human cDNA encoding a novel human type-I membrane protein, thereby completing the present invention. That is to say, the present invention provides a protein containing the amino acid sequence represented by Sequence No. 1 that is a human type-I membrane protein. The present invention, also, provides a DNA encoding said protein exemplified as a cDNA containing the base sequence represented by Sequence No. 2. The protein 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 the peptide by the chemical synthesis on the basis of the amino acid sequence of the present invention, or a method for production with the recombinant DNA technology using the DNA encoding the human type-I protein of the present invention, wherein the method for obtainment by the recombinant DNA technology is employed preferably. For example, an in vitro expression can be achieved by preparation of an RNA by the in vitro transcription from a vector having the cDNA of the present invention, followed by the in vitro translation using this RNA as a template. Also, the recombination of the translation domain to a suitable expression vector by the method known in the art leads to the expression of a large amount of the encoded protein by using Escherichia coli , Bacillus subtilis, yeasts, animal cells, and so on. In the case in which the protein of the present invention is expressed by a microorganism such as Escheri chia coli , the translation region of the cDNA of the present invention is constructed in an expression vector having an origin, a promoter, ribosome-binding site(s), cDNA-cloning site(s), a terminator, etc. that can be replicated in the microorganism and, after transformation of the host cells with said 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 that case, a partial protein appearing on the cell surface can be obtained by performing the expression with inserting an initiation codon and a termination codon before and after the translation region existing between the secretory signal sequence and the transmembrane domain. Alternatively, a fusion protein with another protein can be expressed. Only a protein portion encoding said cDNA can be obtained by cleavage of said fusion protein with an appropriate protease. In the case in which the protein of the present invention is secretory-expressed in animal cells, the protein of the present invention can be expressed in the form existing on the membrane, when the translation region of said cDNA is subjected to recombination to an expression vector for animal cells that has a promoter for the animal cells, a splicing domain, a poly(A) addition site, etc., followed by transfection into the animal cells. The thus-obtained recombinant cells can be used for the detection of a Iigand against the present protein and the screening of a low molecular compound substituting for the Iigand. The protein of the present invention includes peptide fragments (more than 5 amino acid residues) containing any partial amino acid sequence of the amino acid sequence represented by Sequence No. 1. These fragments can be used as antigens for preparation of the antibodies. Particularly, a sequence contained in a region between glutamic acid 28 and threonine 187 that appears putatively on the cell membrane surface is suitable for using as an antigen peptide. The DNA of the present invention includes all DNA encoding said protein. Said DNA can be obtained using the method by chemical synthesis, the method by cDNA cloning, and so on . The cDNA of the present invention can be cloned from, for example, a cDNA library of the human cell origin. The cDNA is synthesized using as a template a poly(A) RNA 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. A poly(A) RNA isolated from the human epidermal cancer cell line KB is used in Examples. The cDNA can be synthesized by using any method selected from the Okayama-Berg method [Okayama, H. and Berg, P., Mol . Cell. Biol. 2: 161-170 (1982)], the Gubler-Hoff an 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 illustrated in Examples in order to obtain a full-length clone in an effective manner. The cloning of the cDNA is performed by the sequencing of a partial base sequence of the cDNA clone selected at random from the cDNA library and the search of the protein data base by the amino acid sequence predicted from the base sequence. The identification of the cDNA is carried out by determination of the whole base sequence by the sequencing and the protein expression by the in vitro translation. The ascertainment of the cDNA of the present invention for encoding the protein having the secretory signal sequence is performed by using the signal sequence detection method [Yokoyama-Kobayashi, M. et al., Gene 163: 193-196 (1995)]. In other words, the ascertainment is provided by fusing a cDNA fragment encoding the N-terminus of the target protein with a cDNA encoding the protease domain of urokinase and then expressing the resulting cDNA in C0S7 cells to detect the urokinase activity in the cell culture medium. The cDNA of the present invention is characterized by containing the base sequence represented by Sequence No. 2, as exemplified by that represented by Sequence No. 3 possessing a 1408-bp base sequence with a 654-bp open reading frame. This open reading frame coded for a protein consisting of 217 amino acid residues and possessed a secretory signal sequence at the N-terminus and one transmembrane domain at the C-terminal site. Hereupon, the same clone as the cDNA of the present invention can be easily obtained by screening of the human cDNA library constructed from the cell line employed in the present invention, by the use of an oligonucleotide probe synthesized on the basis of the cDNA base sequence depicted in Sequence No . 2 or Sequence No . 3. In general, the polymorphism due to the individual difference is frequently observed in human genes. Therefore, any cDNA that is subjected to insertion or deletion of one or plural nucleotides and/or substitution with other nucleotides in Sequence No. 2 or Sequence No. 3 shall come within the scope of the present invention. In a similar manner, any protein that is produced by these modifications comprising insertion or deletion of one or plural nucleotides and/or substitution with other nucleotides shall come within the scope of the present invention, as far as said protein possesses the activity of the protein having the amino acid sequence represented by Sequence No . 1. The cDNA of the present invention includes cDNA fragments (more than 10 bp) containing any partial base sequence of the base sequence represented by Sequence No. 2 or No. 3. For example, as illustrated in Examples, the portion encoding the secretory signal sequence can be employed as means to secrete an optionally selected protein outside the cells by fusing with a cDNA encoding another protein. Also, DNA fragments consisting of a sense chain and an anti-sense chain shall come within this scope. These DNA fragments can be used 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 (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 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 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 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; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic ingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel 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. 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 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 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 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 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 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 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, 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 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 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 cultured. Cytokine and Cell Proliferation/Differentiation Activitv 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 induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one 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 cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9 , B9/11, BaF3, MC9/G, M+ (preB M+ ) , 2E8, RB5, DAI, 123, T1165, HT2, CTLL2 , TF-1, Mo7e and CMK. The activity of a protein of the invention may, among 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 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; 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: Po lyclonal T cell stimulation, 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 I pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994. Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without 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, 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. 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 II - 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, 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 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. 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, 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. Immune Stimulating or Suppressing Activitv A protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are 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 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 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 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 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 asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may 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 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, 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 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 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 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 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 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 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 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 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 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, 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. Blocking antigen function may also be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells 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. Administration of reagents which block costimulation of T cells by disrupting receptor : Iigand 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 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 . Examples include murine experimental autoimmune encephalitis, systemic lupus eryth atosis 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 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 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 by the administration of stimulatory forms of B lymphocyte antigens systemically. Alternatively, anti-viral immune responses may be 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 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 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. 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 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 vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-l-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 . 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 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 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 chain protein and β 2 microglobulin protein or an MHC class Il chain protein and an MHC class Ilβ 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 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 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 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 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 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 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; 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 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. Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Thl 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 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. 149:3778-3783, 1992. Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that 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, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang etal., 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, 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 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 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 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 Activitv A protein of the present invention may be useful in 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 actor-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 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) 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 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 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 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: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above. Assays for embryonic stem cell differentiation (which 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 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 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 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, 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, 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 Activitv A protein of the present invention also may have utility 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 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 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 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 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 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 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 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 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 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 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 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 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 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, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, 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 . 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. 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 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. 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 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: Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium ). Assays for wound healing activity include, without 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 Activitv 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 follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin α family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease sper atogenesis in male mammals. Administration 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-β group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in 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 as cows, sheep and pigs . The activity of a protein of the invention may, among other means, be measured by the following methods: 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 318:659-663, 1985; Forage et al . , Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986. Chemotactic/Chemokinetic Activitv A protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine ) for 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 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 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 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 other means, be measured by the following methods: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis ) consist of assays 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, 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. 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 Activitv 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 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 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. 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. Receptor/Ligand Activity A protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/Iigand interactions. Examples of such 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, 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. 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: 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 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. Anti-Inflammatory Activitv Proteins of the present invention may also exhibit anti-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 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 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-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 antigenic substance or material. Tumor Inhibition Activitv 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 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 angiogenesis ) , by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, 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 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, 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; 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, 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 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 the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein. BRIEF DESCRIPTION OF DRAWINGS Figure 1: A figure depicting the structure of the plasmid pHP10035. Figure 2: A figure depicting the hydro- phobicity/hydrophilicity profile of the protein of the present invention. In the figure, the net-shaded bars indicate the secretory signal sequence as well as the transmembrane domain. BEST MODE FOR CARRING OUT INVENTION EXAMPLES The present invention is embodied in more detail by the 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]. 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 out according to the literature [Kato, S. et al., Gene 150: 243-250 (1994)]. (1) Preparation of Poly(A) + RNA The human epidermoid carcinoma cell line KB (ATCC CRL 17) was incubated at 37°C under the stream of 5% C0 2 in the MEM culture medium containing 10% fetal calf albumin to obtain 1 g of tissues. After they was dissolved in 16 ml of a 5.5 M guanidinium thiocyanate solution, mRNAs were prepared in accordance with the literature [Okayama, H. et al., "Methods in Enzymology" Vol. 164, Academic Press, 1987]. These RNAs were subjected to chromatography using an oligo(dT) -cellulose column washed with 20 mM Tris- hydrochloric acid buffer solution (pH 7.6), 0.5 M NaCl, and 1 mM EDTA to obtain 300 μg of a poly(A) RNA in accordance with the above-mentioned literature . (2) Construction of cDNA Library To a solution of 10 μg of the above-mentioned poly(A) RNA in 100 mM Tris-hydrochloric acid buffer solution (pH 8) was added one unit of an RNase-free, bacterium-origin alkaline phosphatase and the resulting solution was allowed to react at 37 °C for one hour. After the reaction solution underwent the phenol extraction followed by the ethanol precipitation, the obtained pellets were dissolved in a mixed solution of 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 pyrophosphatase (Epicenter Technologies) and the resulting solution at a total volume of 100 μl was allowed to react at 37 °C for one hour. After the reaction solution underwent the phenol extraction followed by the ethanol precipitation, the thus-obtained pellets were dissolved in water to obtain a decapped poly(A) RNA solution. To a solution of the decapped poly(A) RNA and 3 nmol of a DNA-RNA chimeric oligonucleotide ( 5 ' ~dG-dG-dG-dG-dA-dA-dT- dT-dC-dG-dA-G-G-A-3 ' ) in a mixed aqueous solution of 50 mM Tris-hydrochloric acid buffer solution (pH 7.5), 0.5 mM ATP, 5 mM MgCl 2 , 10 mM 2-mercaptoethanol, and 25% polyethylene glycol were added 50 units of T4 RNA ligase and the resulting solution at a total volume of 30 μl was allowed to react at 20°C for 12 hours. After the reaction solution underwent the phenol extraction followed by the ethanol precipitation, the thus-obtained pellets were dissolved in water to obtain a chimeric oligo-capped poly(A) RNA. After the vector pKAl developed by the present inventors (Japanese Patent Kokai Publication No. 1992-117292) was digested with Kpnl, an about 60-dT tail was inserted by a terminal transferase. This product was digested with EcoRV to remove the dT tail at one side and the resulting molecule was used as a vectorial primer. After 6 μg of the previously-prepared chimeric oligo- capped poly(A) RNA was annealed with 1.2 μg of the vectorial primer, the product was dissolved in a mixed solution of 50 mM Tris-hydrochloric acid buffer solution (pH 8.3), 75 mM KC1, 3 mM MgCl 2 , 10 mM dithiothreitol , and 1.25 mM dNTP (dATP + dCTP + dGTP + dTTP), mixed with 200 units of a reverse transferase (GIBCO-BRL), and the resulting solution at a total volume of 20 μl was allowed to react at 42 °C for one hour. After the reaction solution underwent the phenol extraction followed by the ethanol precipitation, the thus- obtained pellets were dissolved in a mixed solution of 50 mM Tris-hydrochloric acid buffer solution (pH 7.5), 100 mM NaCl, 10 mM MgCl 2 , and 1 mM dithiothreitol . Thereto were added 100 units of EcoRI and the resulting solution at a total volume of 20 μl was allowed to react at 37 °C for one hour. After the reaction solution underwent the phenol extraction followed by the ethanol precipitation, the obtained pellets were dissolved in a mixed solution of 20 mM Tris-hydrochloric acid buffer solution (pH 7.5), 100 mM KC1, 4 mM MgCl 2 , 10 mM (NH + ) 2 SO ή , and 50 μg/ml bovine serum albumin. Thereto were added 60 units of Escherichia coli DNA ligase and the resulting solution was allowed to react at 16 °C for 16 hours. To the reaction solution were added 2 μl of 2 mM dNTP, 4 units of Escherichia coli DNA polymerase I, and 0.1 unit of Escherichia coli DNase H and the resulting solution was allowed to react at 12°C for one hour and then at 22°C for one hour. Next, the cDNA-synthesis reaction solution was used to transform Escherichia coli DH12S (GIBCO-BRL) . The transformation was carried out by the electroporation method. A portion of the transformant was inoculated on a 2xYT agar culture medium containing 100 μg/ml ampicillin, which was incubated at 37°C overnight. A colony grown on the culture medium was randomly picked up and inoculated on 2 ml of the 2xYT culture medium containing 100 μg/ml ampicillin, which was incubated at 37 °C overnight. The culture medium was centrifuged to separate the cells, from which a plasmid DNA was prepared by the alkaline lysis method. After the plasmid DNA was double-digested with EcoRI and NotI, the product was subjected to 0.8% agarose gel electrophoresis to determine the size of the cDNA insert. In addition, by the use of the obtained plasmid as a template, the sequence reaction using M13 universal primer labeled with a fluorescent dye and Taq polymerase (a kit of Applied Biosystems Inc. ) was carried out and the product was analyzed by a fluorescent DNA-sequencer (Applied Biosystems Inc.) to determine the base sequence of the cDNA 5 '-terminal of about 400 bp. The sequence data were filed as a homo-protein cDNA bank data base. (3) cDNAs Cloning The base sequence registered in the homo-protein cDNA bank was converted to three frames of amino acid sequences, which were then employed for the search of the protein data base. GENETYX-MAC (Software Development) was used for the software for the analysis. As the result, it has been revealed that a protein encoded by the pHP10035 contained by clone HP10035 is homologous with the T1/ST2 receptor-binding protein, one of type-I membrane proteins. Figure 1 depicts the structure of this plasmid. Determination of the whole base sequence for the cDNA insert revealed the structure consisting of a 5 ' -non-translation region of 128 bp, an open reading frame of 654 bp, and a 3' -non-translation region of 754 bp (Sequence No. 3). The open reading frame codes for a protein consisting of 217 amino acid residues and the search of the protein data base using this sequence revealed that the protein possessed a homology of 33.2% with the amino acid sequence of the human T1/ST2 receptor-binding protein among the entire regions . Table 1 indicates the comparison of the amino acid sequences between the type-I membrane protein of the present invention (HP) and the T1/ST2 receptor-binding protein (TI). - represents a gap, * represents an amino acid residue identical to that in the protein of the present invention, and . represents an amino acid residue analogous to that in the protein of the present invention. Also, Figure 2 depicts the hydrophobicity/hydrophilicity profile of the present protein obtained by the Kyte-Doolittle method. There are observed a hydrophobic region of a putative secretory signal protein at the N-terminal and a highly hydrophobic region of a putative transmembrane domain at the C-terminal. Application of the (-3,-1) rule, a method for predicting the signal sequence cleavage site [von Heijne, G., Nucl. Acid Res. 14: 4683-4690 (1986)], allows to expect that the maturation protein starts from glutamic acid at position 28. Table 1 HP MGSTVPRSASVL LLLLLRRABQPCGAELTFELPDNAKQCFHEEVBQGVKFS DY TI MMAAGAALALALM PPVBVGGAGPPPI QDGBFTFLLPAGRKQCPYQSAPANASLETEY HP αVITGGHYDVDCYVEDPQGNTI YRETK QYDSFTYRAEVKGVYQFCFSNBFSTFSHKTVY TI QVI GGAGLDVDFTLESPQGV LVSESRKADGVHTVEPTEAGOYKLCPDNSFSTI SEKLVF HP FD— F — QVGDEPPI LPDMGNRVTAL TQMESACVT I HEAL TV I DSQTHYRLREA *. * * . . * * *. . . *. *. * * ** TI FEIFDSL Q DDEEVEGWAEAVEPEEMLDVKMEDIKBSIBTMRTRLERSIQMLTLLRAFEA HP Q DRARAEDLNSRVSYWSVGETIALFVSFSQVLLLKSFFTBKRPISRAVHS TI RDRNL Q EGNLERVNFWSAVNVAVLLLVAVLQVCTLKRFFQDKRPVPT The search of GenBank using the base sequence of the present cDNA revealed that there existed some ESTs possessing the homology of 90% or more with the present cDNA and also containing the initiation codon (for example, Accession No. T79526), but many sequences were not distinct and the same ORF as that in the present cDNA was not identified. (4) Functional Verification of Secretory Signal After cleavage with NotI of the vector pHP10035 carrying the cDNA of the present invention, followed by deletion with the ExoIII nuclease at the 3 '-terminal of cDNA and digestion with Hindlll, a DNA fragment of about 1.1 kbp (containing about 500 bp from the 5 '-terminal of cDNA) was isolated by agarose gel electrophoresis . This fragment was inserted between Hindlll and EcoRV of pSSDl [Yokoyama-Kobayashi, M. et al., Gene 163: 193-196 (1995)], thereby constructing a vector expressing a fusion protein of the secretory signal portion of cDNA of the present invention and the urokinase protease domain . After each of 10 clones of Escherichia coli (host: JM109) bearing the fusion-protein expression vector was incubated at 37 °C for 2 hours in 2 ml of the 2xYT culture medium containing 100 μg/ml ampicillin, the helper phage M13K07 (50 μl ) was added and the incubation was continued at 37 °C overnight. A supernatant separated by centrifugation underwent precipitation with polyethylene glycol to obtain single-stranded phage particles. These particles were suspended in 100 μl of 1 mM Tris-0.1 mM EDTA, pH 8 (TE). Also, there was used as a control a suspension of single- stranded particles prepared in the same manner from the vector pKAl-UPA containing pSSDl and a full-length cDNA of urokinase [Yokoyama-Kobayashi, M. et al., Gene 163: 193-196 (1995)]. The simian-kidney-origin culture cells, C0S7, were incubated at 37°C in the presence of 5% C0 2 in the Dulbecco's modified Eagle's culture medium (DMEM) containing 10% fetal calf albumin. Into a 6-well plate (Nunc Inc., 3 cm in the well diameter) were inoculated 1 x 10 C0S7 cells and incubation was carried out at 37 °C for 22 hours in the presence of 5% C0 2 . After the culture medium was removed, the cell surface was washed with a phosphate buffer solution and then washed again with DMEM containing 50 mM Tris- hydrochloric acid (pH 7.5) (TDMEM) . To the cells were added 1 μl of the single-stranded phage suspension, 0.6 ml of the TM DMEM culture medium, and 3 μl of TRANSFECTAM ( IBF Inc. ) and the resulting mixture was incubated at 37°C for 3 hours in the presence of 5% C0 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°C for 2 days in the presence of 5% C0 2 . To 10 ml of 50 mM phosphate buffer solution (pH 7.4) containing 2% bovine fibrinogen (Miles Inc.), 0.5% agarose, and 1 mM potassium 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 transfected C0S7 cells were spotted on the fibrin plate, which was incubated at 37°C for 15 hours. The diameter of the thus-obtained clear circle was taken as an index for the urokinase activity. A clear circle is formed in three clones of the ten clones to identify that urokinase was secreted in the culture medium. These three clones possessed the cDNA open reading frame consistent with the cDNA frame of urokinase. The above result indicates that the cDNA of the present invention codes for the amino acid sequence that functions as the signal sequence. (5) Protein Synthesis by In Vitro Translation The vector pHP10035 carrying the cDNA of the present invention was utilized for the in vitro translation by the T N T rabbit reticulocyte lysate kit (Promega Biotec). In this 35 case, [ S]methionine was added and the expression product was labeled with the radioisotope. All reactions were carried out by following the protocols attached to the kit. Two micrograms of the plasmid pHP10035 was allowed to react at 30°C for 90 minutes in total 100 ml of a reaction solution containing 50 μl of the T N T rabbit reticulocyte lysate, 4 μl of the buffer solution (attached to the kit), 2 μl of an amino acid mixture (methionine-free) , 8 μl (0.37 MBq/μl) of [ 35S]methionine (Amersham Corporation), 2 μl of T7 RNA polymerase, and 80 U of RNasin. To 3 μl of the reaction solution was added 2 μl of an SDS sampling buffer (125 mM Tris-hydrochloric acid buffer solution, pH 6.8, 120 mM 2- mercaptoethanol , 2% SDS solution, 0.025% broraophenol blue, and 20% glycerol) and the resulting solution was heated at 95°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 to reveal that the cDNA of the present invention yielded a translation product having the molecular weight of about 27 kDa. This value was consistent, within the experimental error, with the expected molecular weight of 24,777 for the protein predicted from the base sequence represented by Sequence No. 1, thereby indicating that this cDNA definitely codes for the protein represented by Sequence No . 1. INDUSTRIAL APPLICATION The present invention provides a novel human type-I membrane protein, a DNA encoding said protein, and a human cDNA encoding said protein. The protein of the present invention can be used as pharmaceuticals or as an antigen for preparing an antibody against said protein. Furthermore, said DNA can be used for the expression of a large amount of said protein. SEQUENCE LISTING Sequence No. : 1 Sequence length: 217 Sequence type: Amino acid Topolog : Linear Sequence kind: Protein Hypothetical: No Original source: Organism species: Homo sapiens Cell kind: Epideπnoid carcinoma Cell line: KB Clone name: HP10035 Sequence description Met Gly Ser Thr Val Pro Arg Ser Ala Ser Val Leu Leu Leu Leu Leu 16 1 5 10 15 Leu Leu Arg Arg Ala Glu Gin Pro Cys Gly Ala Glu Leu Thr Phe Glu 32 20 25 30 Leu Pro Asp Asn Ala Lys Gin Cys Phe His Glu Glu Val Glu Gin Gly 48 35 40 45 Val Lys Phe Ser Leu Asp Tyr Gin Val He Thr Gly Gly His Tyr Asp 64 50 55 60 Val Asp Cys Tyr Val Glu Asp Pro Gin Gly Asn Thr He Tyr Arg Glu 80 65 70 75 80 Thr Lys Lys Gin Tyr Asp Ser Phe Thr Tyr Arg Ala Glu Val Lys Gly 96 85 90 95 Val Tyr Gin Phe Cys Phe Ser Asn Glu Phe Ser Thr Phe Ser His Lys 112 100 105 110 Thr Val Tyr Phe Asp Phe Gin Val Gly Asp Glu Pro Pro He Leu Pro 128 115 120 125 Asp Met Gly Asn Arg Val Thr Ala Leu Thr Gin Met Glu Ser Ala Cys 144 130 135 140 Val Thr He His Glu Ala Leu Lys Thr Val He Asp Ser Gin Thr His 160 145 150 155 160 Tyr Arg Leu Arg Glu Ala Gin Asp Arg Ala Arg Ala Glu Asp Leu Asn 176 165 170 175 Ser Arg Val Ser Tyr Trp Ser Val Gly Glu Thr He Ala Leu Phe Val 192 180 185 190 Val Ser Phe Ser Gin Val Leu Leu Leu Lys Ser Phe Phe Thr Glu Lys 208 195 200 205 Arg Pro He Ser Arg Ala Val His Ser 217 210 215 Sequence No. : 2 Sequence length: 651 Sequence type: Nucleic acid Strandedness: Double Topology: Linear Sequence kind: cDNA to mRNA Original source: Organism species: Homo sapiens Cell kind: Epidermoid carcinoma Cell line: KB Clone name: HP10035 Sequence description ATGGGCAGCA CTGTCCCGCG CTCCGCCTCC GTGCTGCTTC TGCTGCTGCT CCTGCGCCGG 60 GCCGAGCAGC CCTGCGGGGC CGAGCTCACC TTCGAGCTGC CGGACAACGC CAAGCAGTGC 120 TTCCACGAGG AGGTGGAGCA GGGCGTGAAG TTCTCCCTGG ATTACCAGGT CATCACTGGA 180 GGCCACTACG ATGTTGACTG CTATGTAGAG GACCCCCAGG GGAACACCAT CTACAGAGAA 240 ACGAAGAAGC AGTACGACAG CTTCACGTAC CGGGCTGAAG TCAAGGGCGT TTATCAGTTT 300 TGCTTCAGTA ATGAGTTTTC CACCTTCTCT CACAAGACCG TCTACTTTGA CTTTCAAGTG 360 GGCGATGAGC CTCCCATTCT CCCAGACATG GGGAACAGGG TCACAGCTCT CACCCAGATG 420 GAGTCCGCCT GCGTGACCAT CCATGAGGCT CTGAAAACGG TGATTGACTC CCAGACGCAT 480 TACCGGCTGC GGGAGGCCCA GGACCGGGCC CGAGCGGAAG ACCTTAATAG CCGAGTCTCT 540 TACTGGTCTG TTGGCGAGAC GATTGCCCTG TTCGTGGTCA GCTTCAGTCA GGTGCTACTG 600 TTGAAAAGCT TCTTCACAGA AAAACGACCC ATCAGCAGGG CAGTCCACTC C 651 Sequence No. : 3 Sequence length: 1408 Sequence type: Nucleic acid Strandedness: Double Topology: Linear Sequence kind: cDNA to mRNA Original source : Organism species: Homo sapiens Cell kind: Epidermoid carcinoma Cell line: KB Clone name: HP10035 Sequence characteristics Code representing characteristics: CDS Existence site: 129.. 782 Characterization method: E Sequence description AAGCAGAGCT CCGCTGGTGC CACGTTTATC CCCTTACATC CTCCTAGGAC CCGGTCGGTA 60 GTCGTCGCCC CAGCCCGCCG GGGGCGCAGC GCCCGAGCCG CGGCCCTCGA GACGGGACCG 120 AGAGCATC ATG GGC AGC ACT GTC CCG CGC TCC GCC TCC GTG CTG CTT CTG 170 Met Gly Ser Thr Val Pro Arg Ser Ala Ser Val Leu Leu Leu 1 5 10 CTG CTG CTC CTG CGC CGG GCC GAG CAG CCC TGC GGG GCC GAG CTC ACC 218 Leu Leu Leu Leu Arg Arg Ala Glu Gin Pro Cys Gly Ala Glu Leu Thr 15 20 25 30 TTC GAG CTG CCG GAC AAC GCC AAG CAG TGC TTC CAC GAG GAG GTG GAG 266 Phe Glu Leu Pro Asp Asn Ala Lys Gin Cys Phe His Glu Glu Val Glu 35 40 45 CAG GGC GTG AAG TTC TCC CTG GAT TAC CAG GTC ATC ACT GGA GGC CAC 314 Gin Gly Val Lys Phe Ser Leu Asp Tyr Gin Val He Thr Gly Gly His 50 55 60 TAC GAT GTT GAC TGC TAT GTA GAG GAC CCC CAG GGG AAC ACC ATC TAC 362 Tyr Asp Val Asp Cys Tyr Val Glu Asp Pro Gin Gly Asn Thr He Tyr 65 70 75 AGA GAA ACG AAG AAG CAG TAC GAC AGC TTC ACG TAC CGG GCT GAA GTC 410 Arg Glu Thr Lys Lys Gin Tyr Asp Ser Phe Thr Tyr Arg Ala Glu Val 80 85 90 AAG GGC GTT TAT CAG TTT TGC TTC AGT AAT GAG TTT TCC ACC TTC TCT 458 Lys Gly Val Tyr Gin Phe Cys Phe Ser Asn Glu Phe Ser Thr Phe Ser 95 100 105 110 CAC AAG ACC GTC TAC TTT GAC TTT CAA GTG GGC GAT GAG CCT CCC ATT 506 His Lys Thr Val Tyr Phe Asp Phe Gin Val Gly Asp Glu Pro Pro He 115 120 125 CTC CCA GAC ATG GGG AAC AGG GTC ACA GCT CTC ACC CAG ATG GAG TCC 554 Leu Pro Asp Met Gly Asn Arg Val Thr Ala Leu Thr Gin Met Glu Ser 130 135 140 GCC TGC GTG ACC ATC CAT GAG GCT CTG AAA ACG GTG ATT GAC TCC CAG 602 Ala Cys Val Thr He His Glu Ala Leu Lys Thr Val He Asp Ser Gin 145 150 155 ACG CAT TAC CGG CTG CGG GAG GCC CAG GAC CGG GCC CGA GCG GAA GAC 650 Thr His Tyr Arg Leu Arg Glu Ala Gin Asp Arg Ala Arg Ala Glu Asp 160 165 170 CTT AAT AGC CGA GTC TCT TAC TGG TCT GTT GGC GAG ACG ATT GCC CTG 698 Leu Asn Ser Arg Val Ser Tyr Trp Ser Val Gly Glu Thr He Ala Leu 175 180 185 190 TTC GTG GTC AGC TTC AGT CAG GTG CTA CTG TTG AAA AGC TTC TTC ACA 746 Phe Val Val Ser Phe Ser Gin Val Leu Leu Leu Lys Ser Phe Phe Thr 195 200 205 GAA AAA CGA CCC ATC AGC AGG GCA GTC CAC TCC TAGCCCCGGC A 790 Glu Lys Arg Pro He Ser Arg Ala Val His Ser 210 215 TCCTGCTCTA GGGCCCCTCA TGCCCCAGGC TGGAGCAGCT CTCCTAGGTC ACAGCCTGCT 850 GGGCTGGGTC GCGTAGCCCA GGGTGGAGGC AGAACGATGC TGCTGTGGTA GCCCTTTGCC 910 TTTCATGCCC ATGCTTGATT CTTGCACCTC AGCAGCTGAA GGTCTCAGAG ACCAGTAATC 970 AGAAGGCATC CGACTGCATT AAGTGTGCAG CGCTGAAAAG ACATTTACAA CTAGGCCAGG 1030 GATTAGCCAC TGTGGGAGGG TGGACAGGCA ATGGTTCAGT GGCCTGGCTG TTGGCAGGAA 1090 CTCCAAGTGC CCAGGCCTCT TGGGCAGCTT AGGGCCCTGC CTCTGTTTCA TGATGCATGG 1150 GTCATTTGTC TTGGGTGTCC TATCCCATAT GGAGAAGAAA GGGGCTCTAA GTTCTGGCTC 1210 TTCTTTCTTT GGGGTTCTCT GTACCTGAGG AAACCAGGCC CTGGGTGACT TTGCAGATCT 1270 GCTCACCCTC GGTGAGCAAC AGTGTCAGCC ATGCAAGCAG GACAGAATGG TGACTGGGTG 1330 CCCTTGGTGA GCTGTGTATT TCCTAGGAGG TAGAAAACTG TGGGAAACTG TGGCTAATAA 1390 AAACTAAGTG TGAGCGTC 1408

Claims: CLAIMS 1. A protein containing the amino acid sequence represented by Sequence No. 1. 2. A DNA encoding the protein as described in Claim 1. 3. A cDNA containing the base sequence represented by Sequence No . 2. 4. A cDNA described in Claim 3 which comprises the base sequence represented by Sequence No. 3.

Inventors: KATO SEISHI, KOBAYASHI MIDORI, SEKINE SHINGO, KATO, SEISHI, KOBAYASHI, MIDORI, SEKINE, SHINGO
Applicants: KATO SEISHI, KOBAYASHI MIDORI, PROTEGENE INC, SAGAMI CHEM RES, SEKINE SHINGO, KATO, SEISHI, KOBAYASHI, MIDORI, PROTEGENE INC., SAGAMI CHEMICAL RESEARCH CENTER, SEKINE, SHINGO
Application year: 1997
Main classification: C12N15
IPC codes: C07K14, C12N15