CA2331326A1 - Human glycoprotease-like proteins and dnas encoding these proteins - Google Patents

Abstract

A human glycoprotease-like protein comprising an amino acid sequence as shown in Sequence No. 1 and a DNA coding for said protein are provided. Said protein can be obtained by recombinant technology using cDNA having a nucleotide sequence as shown in Sequence No. 2 or 3 and is useful as a reagent for the study of sugar proteins.

Description

DESCRIPTION
HUrSAN GLYCOPROTEASIE-LTKE
PROTEINS AND DNAs ENCODING THESE PROTEINS
TECHNICAL FIELD
The present invention relates to glycoprotease-like proteins and DNAs coding for these proteins. The proteins of the present invention ca.n be employed as pharmaceuticals. The human cDNAs of the present invention can be utilized as probes for the gene diagnosis and gene sources for the gene therapy. Furthermore, the cDNAs can be utilized as gene sources for large-scale production of the proteins encoded by said cDNAs. The eucaryotic cells that introduce any of the expression vectors of said cDNAs can be used for secretory production of human glycoprotease-like proteins.
BACKGROUND ART
Glycoprotease is a kind of the neutral metalloprotease that is specific for glycoproteins. For instance, O-sialoglycoprotein endo~peptidase secreted by Pasteurella haemolytica that causes bovine pneumonia specifically cleaves O-sialoglycoproteins such as glycophorin A in the erythrocyte [Albdullah, K. M. et al . , J. Bacteriol. 173: 5597-5603 (1991,)]. This glycoprotease has two histidine residues to be putative as a zinc-binding site. There have been heretofore known several glycoproteases having sequences analogous to peripheral amino acid sequences of said glycoprotease. For example, there has been reported that glycoprotease-like proteins are encoded by genes of Escheric~hia coli, methanogen, thermophilic archaebacteria, mycoplasma, influenza virus, and so on. Also, there has been reported that budding yeast, an eucaryotic cell, has a gene coding for a protein highly analogous to these glycoprot:eases. Hereupon, there has not been any paper reporting that these glycoprotease analogues have been obtained from nnulticellular organisms including human beings.
Many glycoproteins exist on thE~ cell surface and play important roles in the information transmission between cells. Accordingly, glycoproteases that selectively cleave w such glycoproteins can be a powerful weapon on studies of the mechanism thereof. For instance, O-sialoglycoprotein endopeptidase obtained from the above-mentioned Pasteurella haemolytica has been utilized for the study of cell-surface membrane antigens of leukocytes [Sutherland, D. R. et al., J. Immunal. 148: 1458-:1464 (1992)].
DISCLOSURE OF INVENTION
The object of the present invention is to provide novel human glycoprotease-like proteins, DNAs coding for these proteins, and expression vectors of said DNAs as well as transformation eucaryotic cells expressing said DNAs.
As the result of intensive studies, the present inventors have been successful in cloning of human cDNAs coding for glycoprotease-like proteins, thereby completing the present invention. In other words, the present invention provides glycoprotease-like proteins, namely proteins containing the amino acid sequence represented by Sequence No. 1. Moreover, the present invention provides DNAs coding for the above-mentiane~d proteins and cDNAs containing the base sequence represented by Sequence No. 2 or 3 as well as expression vectora that are capable of expressing said DNAs by in vitro translation or in eucaryotic cells and transformation eucaryotic cells that are capable of expressing said DDfAs and producing the above-mentioned proteins. ' 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 prep;~ration of peptides by the chemical synthesis on the basis of the amino acid sequences of the present invention, or a method for production with the recombinant DNA. technology using the DNAs coding for the human glycoprot:ease-like proteins of the present invention, wherein the method for obtainment by the recombinant DNA technology i;s employed preferably.
For instance, in vitro expression 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 template. Also, recombination of the translation region into a suitable expression vector b;y the method known in the art leads to expression of a large amount of the encoded protein by using prokaryotic cells such as Escheriehia coli, Bacillus subtilis, etc., and eucaryotic cells such as yeasts, insect cells, mammalian cells, etc.
In the case in which a protein of the present invention is expressed by prokar~Yotic cells such as Escherio~hia coli, a recombinant expi:ession vector bearing the translation region in the c:DNA 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 prokaryotic cells, and, after transformation of the host cells with said expression vector, the thus-obtained transformant is incubated, whereby lthe protein encoded by said cDNA can be produced on a large scale in the prokaryotic cells. Alternatively, a fusion protein with another protein can be expressed. Only a protein portion encoded by said cDNA can be obtainErd by cleavage of said fusion protein with an appropriate protease. Said fusion protein, provided that it possesses the glycoprotease-like activity, shall come within the acope of the present invention.
In the case in which a protein of the present invention is subjected to secretary expression in eucaryotic cells, the proteins of the present invention can be produced by extracellular secretion, when the 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) addition site, etc., followed by introduction into the eucaryotic cells. The expression vector is exemplified by pKAl, pED6dpc2, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV
vector, pRS, pYES2, and so on. Examples of eucaryotic cells 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 so on, but any eucaryotic cells may be used, provided that they are capable of expressing the present proteins by secretion.
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 sequence represented by Sequence No. 1. These peptide fragments can be utilized as antigens for preparation of antibodies.
The DNAs of the present invention include all DNAs 5 coding 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 human cDNAs of the present invention can be cloned from cDNA libraries of the human cell origin. These cDNA libraries are constructed 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 cu_Lture cells. A poly(A)+
RNA isolated from the osteosarcoma cell line Saos-2 is l~ used in Examples. The cDNAs can b~e synthesized by using any method selected from the Okayam.a-Berg method [Okayama, H. and Berg, P., Mol. Cell. Biol. :?: 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}]
in order to obtain a full-length clone in an effective manner. The identification of the c~DNAs is carried out by the determination of the whole base sequence by the sequencing, the search of known proteins having sequences analogous to the amino acid sequence predicted from the base sequence, expression of proteins by in vitro translation, and expression by the culture cells.
The cDNAs of the present invention are characterized by containing the base sequence represented by Sequence No.
2 or 3. For example, that represented by Sequence No. 3 possesses a 1308-by base sequences with a 1008-by open reading frame. This open reading frame codes for a protein WO 99/67399 PCTIJP99l03358 consisting of 335 amino acid reaidues. This protein possesses an analogy of 31~ to O-sialoglycoprotein endopeptidase secreted by Pasteurella haemolytica and of 54~ to the glycoprotease-like protein. of the budding yeast, in the amino acid sequence level.
Hereupon, the same clones as thE: cDNAs of the present invention can be easily obtained by ;screening of the human cDNA libraries constructed from the human cells by the use of an oligonucleotide probe synthesized on the basis of IO the cDNA base sequence described in Sequence No. 3.
In general, the polymorphism dUe to the individual difference is frequently observed in human genes.
Accordingly, any cDNA that is subjeacted to insertion or deletion of one or plural nucleotides and/or substitution with other nucleotides in Sequence rfo. 2 or 3 shall come within the scope of the present invention.
In a similar manner, any protein that is formed by these modifications comprising inse:ction or deletion of one or plural amino acids and/or substitution with other amino acids shall come within the scope of the present invention, as far as the protein possesses the human glycoprotease-like activity.
The cDNAs of the present invention include cDNA
fragments (more than 10 bp) containing any partial base sequence in the base sequence represented by Sequence No.
3. Also, DNA fragments consisting of. a sense chain and an anti-sense chain shall came within this scope. 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 (including those associated with assays cited herein) identified below. Uses or activities described for proteins of the pre:~ent invention may be provided by administration or use of such proteins or by administration or use of polynuclE~otides encoding such proteins (such as, for example, :Ln 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 I5 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 gcsnetic disorders; 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 2~ discovering other novel polynucleoticles; for selecting and making oligomers for attachment to a "gene chip" or other support, including for examination oj= expression patterns;
to raise anti-protean antibodiesus.ing DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune resp~~nse. Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, WO 99/67399 PCT/Jf99/03358 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 polynucleotidea 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 (incluc~ng 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 product,.
Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without :Limitation "Molecular WO 99!67399 PCT/JP99/03358 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", Acadennic Press, Bergen, 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 rxse as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. zn 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 Prolife~ration/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 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 cytok:ine 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 far cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RBS, DA1, 123, T1165, HT2, CTLL2, TF-l, Mo7e and CM1:~.
5 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.
10 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; Bert:agnalli, 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 ar thymocytes include, without limitation, those described in: Polyclonal 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, Jahn Wiley and Sons, Toronto. 1994; and Measurement of mouse and human IntE:rferon y, Schreiber, R.D. In Current Protocols in Immunology. J.E.e.a. Coligan eds . Vol 1 pp . 6 . 8 .1-6 . 8 . 8 , John Wi:Ley and Sons, Toronto .
1994.
Assays for proliferation and differentiation of hematopoietic and lymphopoietic cE:lls include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleulkin 4, Bottomly, K., WO 99167399 PCT/,TP99/03358 Davis; L.S. and Lipsky, P.E. In Current Protocols in Immunology. J.E.a.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; Measuremesnt 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 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, 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 dirE~ct 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; Weinb~erger et al., Eur. J.
Immun. 11:405-411, 1981; Takai <at al., J. Immunol.
137:3494-3500, 1986; Takai et al., J.. Immunol. 140:508-512, X988.
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 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 e:~fecting 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, mycobacter.ia, 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 de:cirable; 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 rnay 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. Op<~rationally, 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 natura7L 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 <cn 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 1B 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 oaf a combination of B
lymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant rejection or C;VHD can be assessed using animal models than 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 5 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 10 useful for treating autoi.mmune diseases. Many autoimmune disorders are the result of inappropriate activation of T
cells that are reactive against self tissue and which promate the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the 15 activation of autoreactive T cells m.ay reduce or eliminate disease symptoms. Administration off: 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 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 erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis,, diabetes mellitus in NOD mice and BB rats, and murine e:Kperimental myasthenia gravis {see Paul ed., Fundamental Inu:nunology, 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 m.ay 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 in:Eection. In addition, systemic viral diseases such as infl.uen2a, the commoncoid, 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 expressa.ng 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 i~hat 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) transfecte~d 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 desiresd, 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 expre~~sion 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.
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 antigens) 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, yr 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 cc chain protein and (32 microglobulin protein or an MHC class IIa chain protein and an MHC class II(3 chain protein t:o 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

WO 99/67399 PCT/,TP99103358 cell mediated immune response ag<~inst 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 t:o 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 th.e invention may, among other means, be measured by the following methods:
Suitable assays for thymocyte or splenacyte cytotoxicity include, without limit<~tion, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.ME. 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; Herrmarin 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, 1985; Bowmanet al., J. Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 19Fi8; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J.
Tmmunol. 153:3079-3092, 1994.
Assays for T-cell-dependent immunoglobulin responses WO 99!67399 PCTlJP99103358 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: Ma:Liszewski, J. Immunol.
144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production; Mond, ~T.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 (MLIt) 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 Experimenta~L 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 et al., 5cienc,e 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-WO 99/67399 PCT/JP99/0335$
640, 1990.
Assays for lymphocyte survival,/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate 5 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 10 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.
Assays for proteins that influE~nce early steps of T
cell commitment and development include, without 15 limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Gaiy et al., Blood 85:2770-2778, 1995;
Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
Hematopoiesis Regulating Activity 20 A proteW of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid ce:Ll deficiencies. Even marginal biological activity in support of colony forming cells or of 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 treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroi~d cells; in supporting the growth and proliferation of myeloid cells such as granuiocytes and imonocytes/macrophages (i.e., traditional CSF activity) useful, for example" in conjunction with chemotherapy to prevent or tre<~t 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 ~~apable 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 hennoglobinuria), as well as in repopulating the stem c<sll 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 c~_ted above.
Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesisj include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller e~t al., Molecular and Cellular Biology 13x473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.

WO 99/67399 PCTIJP991~3358 Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose co:Lony 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. J.-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. Val 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 WO 99/67399 PCT/,1P99/03358 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 reductian 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 ar induce differentiation of progenitors of bone-forming cells. A protein of t;he 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.j mediated by inflammai~ory 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 i.n 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 tendc~n/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 ito tendon or ligament tissue. De novo tendon/ligament-7Like 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 aurgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide an environment to attract tendon or ligament-farming e~ells, 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 tendi.nitis, 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 i;n accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and 5 cerebravascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable u;>ing a protein of the invention.
Proteins of the invention may also be useful to 10 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 15 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 20 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.
25 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 th.e invention may, among other means, be measured by the following methods:
Assays far tissue generation activity include, without limitation, those described ins International Patent Publication No. W095/160?~5 (bane, cartilage, tendon); International Patent Publ3.cation Na. W095/05846 (nerve, neuronal); International Patent Publication No.
W091/07491 (skin, endothelium ).
Assays far 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 (I978).
Activin/Inhibin Activitx A protein of the present invention may also exhibit activin- ar 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 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 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-(3 group, may be useful as a fertility inducing therapeutic, based upon tlhe ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for e~cample, 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 th.e invention may, among other means, be measured by the following methods:
Assays for activin/inhibin activity include, withaut limitation, those described in: Vale et al., Endocrinology 91x562-572, 1972; Ding et al., Nature 321:779-782, 1986; Vaie et al., Nature 321:776-779, 1986;
Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-31)95, 1986.
Chemotactic/Chemokinetic Activii~
A protein of the present invention may have chemotactic or chemokinetic activ~Lty (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 protE~ins 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 neutrophi.ls 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, t:he 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 protean 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. Marc~ulies, 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, /995;
Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber et al.
J. of Immunol. /52:5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768, 1994.
Hemostatic and Thrombolvtic Activity A protein of the inventior.~ 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 disarders, 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 prf:vention 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 t:hrombolytic activity include, without limitation, those diescribed 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/Li~and 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 receptors and lig~ands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor ~ahosphatases 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 inhibitars of receptor/ligand interactions.
The activity of a protein of thf~ invention may, among WO 99/b7399 PCT/JP99/03358 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. ICruisbeek, D.H.
5 Margulies, E.M. Shevach, W.Stro:ber, Pub. Greene Publishing Associates and Wiley-Intex:science (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.
10 168:1145-1156, 1988; Rosenstein ei~ al., J. Exp. Med.
169:149-160 1989; Stoltenbarg et al., J. Immunol.
Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
Anti-Inflammatory Activitv 15 Proteins of the present invenition 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 {:>uch as, for example, 20 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.
25 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-30 reperfusion injury, endotoxin ~_ethality, 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 ma~~ 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 ar 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 mEaabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, c<~rbohydrate, vitamins, minerals, cofactors or other nultritional 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 act:ivity (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 i.s cross-reactive with such protein.
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, WO 99167399 PCTlJP99J03358 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)x.
(1) cDNA Cloning Clone HP02053 was obtained as the result of a large scale sequencing of eDNA clones selected from the eDNA
library of the human osteosarcoma cell line Saos-2 (W097/33993). The present clone has a structure consisting of a 129-by 5'-nontranslation region, a 1008-by open reading frame, and a 171-by 3'-:nontranslation region (Sequence No. 3). The open reading frame coded for a protein consisting of 335 amino acid residues.
The search of the protein data base using the amino acid sequence of the present invention has revealed that the protein was analogous to O-sialoglycoprotein endopeptidase secreted by Pasteurella haemolytica [Abdullah, K. M. et al., J. BactESriol. 173: 5597-5603 (1991) and to a glycoprotease-like protein of the budding yeast {SWISS-PROT Accession No. P3611i3). Table 1 shows the comparison of the amino acid sequence between the human protein (HP) of the present invention and the O-sialoglycoprotein endopeptidase secreted by Pasteurella haemolytica (PA). Therein, the marks of - and * represent a gap and an amino acid residue identical with the protein of the present invention, respectively. The both proteins had a 31~ homology in the entire region.

Table 1 HP MPAVLGFEGSANKIGVGWRDGKVL AN--PRRTYVTPPGTGFLPGDTARHFiRAVILDLL 57 * ** * * ** * * ** * * * * * *
PA M-RILGIETSCDETGVAIYDEDKGLVANQLYSQIDMHADY'GGt7VPELASRDHIRKTLPLI 59 HP QEALTESGLTSQDIDCIAYTKGPG-MGAPLVSVAWARTU'AQLWNKPLVGVNHCIGHIEM 116 **** * * *** **** *** ** ** ** * ** * ** * **
PA QEALKEANLQPSDIDGIAYTAGPGLVGALLV-GSTIARSI~AYAWN~7PALGVHI~GHLLA 118 HP GRLITGA-TSPTV-LYVSGGNTQ-VIAYSEHRYRIFGETI:DIAVGNCLDRFARVLKISND 173 * * * * * *** ** * * ** ** * *
PA PMLEENAPEFPFVALLISGGHTQLVKVDGVGQYELLGESI:DDAAGEAFDKTGKLL--GLD 176 HP PSPGYNIEQMAKRG----KKLVELPYTVK(~IDVSFSGILS~F----IEDVAHRI~ATGECT 225 * * * * * * ****
PA YPAGVAMSKLAESGTPNRFKFPRPMTDRPGLDFSFSGLK'I'FAANTIKANLNENGELDEQT 236 HP PEDLCFSLQETVFAMLVEITERAMAHCGSQEALIVGGVGC:NVRLQENIMATMCQERGARLF 285 * * * ** * *** * * * *
PA KCDIAHAFQQAVVDTILIKCIaZALEQTGYKRLVMAGGVSF~NFCQLRADLAEN~tfQ~KGEVF 296 HP ATDEFLFCIDNGAMIA~rGWEMFRAGHRTPLSDSGVTQRYP;TDE~IEV'TWRD 335 ** ******* *
PA YPRPQFCTDNGAMIAYTG----------------FLRLR;T----MNKPT 325 Also, Table 2 shows the comparison of the amino acid sequence between the human protein. (HP) of the present invention and glycoprotease-like protein of the budding yeast (SC). The both proteins had a 54~ homology in the entire region.

WO 99/67399 PCT/,1P99/03358 Table 2 Hp M____________________________pAV-_______,_____________~~A 11 * ** ** ****
5 SC MMSSRVTIFTSCNDAYIYLLREGNSQAFPNAVGSIQ~1VNLNrIPPKNGRDYYIALGLEGSA 60 HP NKIGVGW-_______________g~~pRRTyVTPiPGTGFLPGDTARHF~AVILD 55 ** *** * * * * *****~** **** *******

10 * ** * * *** * ********** ** *** ** ***********
SC LIKQALAEADIKSPTLDIDVICFTKGPGMGAPLHSWIAAIEtTCSLLGVDVPLVGVNHCIGH 180 ***** ** * * **************** ******** *** ********* ***
SC IEMGREITKAQNPVVLYVSGGNTQVIAYSEKRYRIFGETLiDIAIGNCLDRFARTLKIPNE 240 15 HP PSPGYNIEQMAK-__RGKKLVELPYTVKGMDVSFSGILSF:IEDVA-___________~M 218 *****,r*** ** ************ * **** *
SC PSPGYNIEQLAIQ~APHKENLVELPYTVKGMDLSMSGILAS:IDLLAKDLFKGNKKNKILFD 300 HP LATGE--CTPEDLCFSLQETVFAI~VEITEHAMA~iCGSQE;ALIVGGVGCNVRLQEI~ll~IATM 276 *** * **** **** ************** * *****************
20 SC KTTGEQKVTVEDLCYSLQENLFAMLVEITERAMAfHVNSNQVLIVGGVGCNVRLQEMMAQM 360 HP CQER-GARLFATDERFCIDNGAMIAQAG-WENIE'RAGHRTPGSDSGVTQRYRTDEVEVTWR 334 * * *** ******* ****** * * * *** ***** **
SC CKDRANGQVHATDNRFCIDNGVMIAQAGLLEYRMGGIVKD:E'SETVVTQKFRTDEVYAAWR 420 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, WO 9916'7399 PCTLTP99103358 since- they are partial sequences, it can not be judged whether or not any of these sequenc:es codes for the same protein as the protein of the present invention.
(2) Protein Synthesis by In Vitro Translation Vector pHP02053 bearing the cDNA of the present invention was used for in vitro translation with a TNT
rabbit reticulocyte lysate kit (Promega). In this case, [3sS]methionine was added to label the expression product with a radioisotope. Each of the reactions was carried out according to the protocols attached to the kit. Two micrograms of plasmid pHP02053 was reacted at 30°C for 90 minutes in a total 100 ul volume of the reaction solution containing 50 ul of TNT rabbit reticulocyte lysate, 4 ul of a buffer solution (attached to kit), 2 ul of an 1~ amino acid mixture (methionine-free), 8 ~1 of [35S]methionine (Amersham) (0.37 MB~q/~l), 2 ul of T7RNA
polymerise, and 80 U of RNasin. To 3 ~l of the resulting reaction solutir~n was added 2 ul of the SDS sampling buffer (125 mM Tris-hydrochloric acid buffer, pH 6.8, 120 mM 2-mercaptoethanol, 2~ SDS solution, 0.025 bromophenol blue, and 20~ glycerol) and the resulting mixture was heated at 95°C for 3 minutes and ithen subjected to SDS-polyacrylamide gel electrophoresis. Determination of the molecular weight of the translation product by carrying out the autoradiography indicated that the eDNA of the present invention yielded the transJLation product with the molecular mass of about 36 kDa. This value is identical with the molecular weight of 36,403 predicted for the putative protein from the base sE~quence represented by Sequence No. 1, thereby indicalting that this cDNA
certainly codes for the protein represented by Sequence No.
1.

(3) Expression by COS7 pHP02053 of the present invention was introduced in the culture cells originating from the simian kidney, COS7, by the iipopolyamine method.. After incubation at 37°C for 2 days in the presence of 5~ CO2, the incubation was continued for one hour in the culture medium containing [ 355 ] cystine . Condensation of the cu_Lture medium, followed by subjecting to SDS-PAGE, allowed to observe the presence of a band expressed by the protein of the present invention, which did not exist in the C057 cells.
INDUSTRIAL APPLICABILITY
The present invention provides (human cDNAs coding for glycoprotease-like proteins and proteins encoded by these human cDNAs. The cDNAs of the present invention can be utilized as probes for the gene diagnosis and gene sources for the gene therapy and, further, as gene sources for large-scale production of the proteins encoded by said cDNAs. Eucaryotic cells, wherein the expression vectors of said cDNAs are introduced, can be ui~ilized for production of the human glycoprotease-like proteins. Said recombinant proteins can be utilized as pharmaceuticals/research reagents, particularly as research reagents useful for researches of glycoproteins.
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 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 to coding sequences, 5' and 3'- untranslated regions, alternatively spliced exons, introns, promoters, e;nhancers, and silencer or suppressor elements. The corresponding genes can be isolated in accordance with kno~m methods using the sequence 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 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 genes) corresponding to the polynucleotide sequences disclosed herein are provided.
The desired change in gene expre:~sion 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 I?harmacol. Sci. 15(7):
20. 250-254; Lavarosky et al., 1997, Biochem. Mol. Med. 62(lj:
II-22; and Hampel, 1998, Prog. Nucleic Acid Res. Mol. Biol.
58: 1-39; all of which are incorporated by reference herein). Transgenic animals that have multiple copies of the genes) corresponding to the polynucleotide sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells anoL their progeny, are provided. Transgenic animals that have modified genetic control regions that increase or reduce gene expression levels, or that change temporal o:r spatial patterns of gene expression, are also provided (see European Patent No.
0 649 464 Bl, incorporated by reference herein). In addition, organisms are provided in which the genes) corresponding to the polynucleotide sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding genes) or through deletion of all or part of the corresponding gene(s). Partial or complete gene inactivation can be accomplished through insertion, 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 homologous recombination, preferably detected by positivefnegative 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). 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 gen.e(s}, and for the development of assay systems for the identification of molecules that interact with the protein product{s) of the corresponding genes}.
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 ar 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 determination of such domains from sequence information.
Proteins and protein fragments of the present 5 invention include proteins with amino acid sequence lengths that are at 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 10 at least 90~ or 95~ identity) with 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 15 and protein fragments that contain a segment preferably comprising 8 or mare (more preferably 20 or more, must 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 20 any such segment of any of the disclosed proteins.
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 25 that of a given protein or polynucleotide, but with significant sequence similarity to the given protein or polynucleotide, as determined by those of skill in the art.
Species homologs may be isolated an<i identified by making suitable probes or primers from the sequences provided 30 herein and screening a suitable nucleic acid source from thedesired species.
The invention also encompasse:~ allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative foams of the isolated polynucleotide which also encode proteins which are identical, homologous, or related to that encoded by the polynucleotides.
The invention also includes polynucleotides with sequences complementary to those o~f the polynucleotides disclosed herein.
The present invention also includes polynucleotides capable of hybridizing under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent 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, f:or example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.

Table StringencyPolynucleotideHybrid Hybridization TemperatureWash ConditionHybrid ~ and Buffers Temperature ~gth p and Buffer A DNA : DNA >_50 65C; lxSSC -or- 65C; 0.3xSSC

42C; IxSSC,50% formamide B DNA : DNA <50 TB*; IxSSC T$*; lxSSC

C DNA : RNA ?50 67C; lxSSC -or- 67C; 0.3xSSC

45C; lxSSC,50/~ formamide D DNA : RNA <50 TD*; lxSSC TD*; lxSSC

E RNA : RNA ?50 ?0C; lxSSC -or- 70C; 0.3xSSC

50C; IxSSC,tiO/~ formamide F RNA : RNA <50 TF*; lxSSC TF.*; IxSSC

G DNA : DNA >_50 65C; 4xSSC -or- 65C; IxSSC

42C; 4xSSC,;iO/~ formamide H DNA : DNA <50 T~*; 4xSSC TH*; 4xSSC

I DNA : RNA ?50 67C; 4xSSC -~or- 67C; IxSSC

45C; 4xSSC,liO/~ formamide J DNA : RNA <50 T,,*; 4xSSC TJ*; 4xSSC

K RNA : RNA >_50 70C; 4xSSC -~or- 67C; lxSSC

50C; 4xSSC,;iO/~ formamide L RNA : RNA <50 TL*; 2xSSC TL*; 2xSSC

M DNA : DNA >_50 50C; 4xSSC -or- 50C; 2xSSC

40C; 6xSSC,50% formamide N DNA : DNA <50 TN*; 6xSSC TN*; 6xSSC

O DNA : RNA X50 55C~. ~xSSC ..or- - 55C; 2xSSC
-- __ -42C; 6xSSC,50% formamide P DNA : RNA <50 TP*; 6xSSC TP*; 6xSSC

RNA : RNA >_50 60C; 4xSSC -or- 60C; 2xSSC

45C; 6xSSC,50% formamide R RNA : RNA <50 TR*; 4xSSC TR*; 4xSSC

$ : The hybrid length is that anticipated for the hybridized regions) of the hybridizing 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 o~f known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity.
j~ : SSPE (lxSSPE is 0.15M NaCI, lOmM NaiH2P04, and 1.25mM EDTA;
pH7.4) can be substituted for SSC (lxSSC is 0.15M NaCI and lSmM sodium citrate) in the 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 5-10°C less than the melting temperature (T~ of the hybrid, where Tm is determined according to the following equations.
For hybrids less than 18 base pairs in length, Tm(°C)=2(#of A + T
bases) + 4(# of G +
C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C)=81.5 +
16.6(log,o[Na+]} + 0.41 (%G+C) - (600I1~, where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for lxSSC=0.1651V1).
la Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambraok, J., E.F. Fritsch, and T: Maniatis, 1989" Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cald Spring Harbor, NY, chapters 9 and 11, and Current Protocols in Molecular 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 pi:eferably at least 50~, and 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 which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing polynucleatides when aligned so as to maximize overlap and identity while minimizing sequence gaps.

WO 99!67399 PCTIJP99I03358 Sequence Listing <110> Sagami Chemical Research Center et al.
<120> Human Glycoprotease-Like Proteins and DNfAs Encoding these Proteins <130> 661100 <141> 1999-06-24 <150> ~'P 10-178247 <151> 1998-06-25 <160> 4 <170> Windows 95 (Word 98) <210> 1 <211> 335 <212> PRT
<213> Homo sapiens <400> 1 Met Pro Ala Val Leu Gly Phe Glu Gly Ser Ala A.an Lys Ile Gly Val Gly Val Val Arg Asp Gly Lys Val Leu Ala Asn Pi:o Arg Arg Thr Tyr Val Thr Pro Pro Gly Thr Gly Phe Leu Pro Gly Aap Thr Ala Arg His 30 His Arg Ala Val Ile Leu Asp Leu Leu Gln Glu A:La Leu Thr Glu Ser 50 55 Ii0 Gly Leu Thr Ser Gln Asp Ile Asp Cys Ile Ala T;~r Thr Lys Gly Pro WO 99/ti~399 PCT/JP99/03358 Gly Met Gly Ala Pro Leu Val Ser Val Ala Val Val Ala Arg Thr Val Ala Gln Len Trp Asn Lys Pro Leu Val Gly Val Aan His Cys Ile G1y His Ile G1u Met Gly Arg Leu Ile Thr Gly Ala Thr Ser Pro Thr Val Leu Tyr Val Ser Gly Gly Asn Thr Gln Val Ile A7La Tyr Ser Glu His Arg Tyr Arg Ile Phe G1y Glu Thr Ile Asp Tle A7La Val Gly Asn Cys Leu Asp Arg Phe Ala Arg Val Leu Lys Ile Ser A:>n Asp Pro Ser Pro Gly Tyr Asn Ile Glu Gln Met Ala Lys Arg Gly Lys Lys Leu Val Glu Leu Pro Tyr Thr Val Lys Gly Met Asp Val Ser Phe Ser Gly Ile Leu Ser Phe Ile Glu Asp Val Ala His Arg Met Leu A7La Thr Gly Glu Cys 210 215 2:?0 Thr Pro Glu Asp Leu Cys Phe Ser Leu Gln Glu Thr Val Phe Ala Met Leu Val Glu Ile Thr Glu Arg Ala Met Ala His Cys Gly Ser Gln Glu Ala Leu Ile Val Gly Gly Val Gly Cys Asn Val Arg Leu Gln Glu Met Met Ala Thr Met Cys Gln Glu Arg G1y Ala Arg LE:u Phe Ala Thr Asp Glu Arg Phe Cys Ile Asp Asn Gly Ala Met Ile Ala Gln Ala Gly Trp Glu Met Phe Arg Ala Gly His Arg Thr Pro Leu Se:r Asp Ser Gly Val Thr Gln Arg Tyr Arg Thr Asp Glu Val Glu Val Ttlr Trp Arg Asp WO 99!67399 PCTIJP99/03358 <210> 2 <211> 1005 <212> DNA
<213> Homo sapiens <400> 2 atgccggcgg tgctgggttttgaaggcagcgccaataagattggcgtgggcgtggtgcgg60 gatggcaagg tgctggcgaacccgcggcggacttacgtcacgcctcctggcacaggattc120 cttccaggtg atacagccaggcatcaccgagctgttatcctagacctgctgcaggaggca180 ctaacagagt ctggattaacctcccaggatatcgactgcattgcatacaccaagggecct240 ggcatgggtg ccccactggtttctgtggctgttgtggcccgtactgtggcccagctgtgg300 aataagccat tggtgggtgtgaaccactgtataggccacattgagatgggccgcctcatc360 actggagcca ccagcccaaccgtgttgtatgtgagtggaggaaatacgcaggtgattgca420 tactcggaac atcgttaccgtatctttggggaaaccatcgatattgcagtgggtaattgt480 ctggatcgtt ttgctcgagtgctgaagatttctaacgacccaagtccaggatacaacatt540 gaacagatgg caaagcgaggcaagaagctggttgagctgccatacactgtaaaggggatg600 gacgtctcat tctcagggatcctgtctttcattgaggatgtagcccatcggatgctggcc660 acaggcgagt gtactcctgaggatctgtgtttctccctgcaggaaactgtgtttgcaatg720 ctggtagaga tcacagagcgagccatggcacattgtggctcccaggaggccctcattgtg780 ggaggagtgg ggtgtaatgtgaggctacaggagatgatggcaacaatgtgccaggaacgt840 ggagcccggc tttttgctacagatgagagattctgtattgacaatggagcgatgatagcc900 caggctggct gggagatgtttcgggctggacacaggaccccactcagtgattctggggtt960 acacagaggt atcggacagatgaagtagaggtgacctggagggac 1005 <210> 3 <211> 1308 <212> DNA

<213> Homo Sapiens <400> 3 agtgctatct gcaggctggc cagcttcctc tgcgctccgg aa,agctgcgg cccagcgcgg 60 actagtgagg acctccacag ctcctgacat tgccaggagt cctgtcggcg ttttctccca 120 gcctccgcc atg ccg gcg gtg ctg ggt ttt gaa ggc agc gcc aat aag att 171 Met Pro Ala Val Leu Gly Phe Glu Gly Ser Ala Asn Lys Ile ggc gtg ggc gtg gtg cgg gat ggc aag gtg ctg gcg aac ccg cgg cgg 219 Gly Val Gly Val Val Arg Asp Gly Lys Val Leu Al.a Asn Pro Arg Arg act tac gtc acg cct cct ggc aca gga ttc ctt cc:a ggt.gat aca gcc 267 10 Thr Tyr Val Thr Pro Pro G1y Thr Gly Phe Leu Pro Gly Asp Thr Ala agg cat cac cga get gtt ate cta gac ctg ctg ce~g gag gca cta aca 315 Arg His His Arg Ala Val Ile Leu Asp Leu Leu Gln Glu Ala Leu Thr 15 gag tct gga tta acc tcc cag gat atc gac tgc ata gca tac acc aag 363 Glu Ser Gly Leu Thr Ser Gln Asp Ile Asp Cys ILe Ala Tyr Thr Lys ggc cet ggc atg ggt gcc cca ctg gtt tct gtg get gtt gtg gcc cgt 411 Gly Pro Gly Met G1y Ala Pro Leu Val Ser Val A:La Val Val Ala Arg s o e5 !a o act gtg gcc cag ctg tgg aat aag cca ttg gtg gdt gtg aac cac tgt 459 Thr Val Ala Gln Leu Trp Asn Lys Pro Leu Val G:Ly Val Asn His Cys ata ggc cac att gag atg ggc cgc ctc atc act gga gcc acc agc cca 507 Ile Gly His Ile Glu Met Gly Arg Leu Ile Thr G:Ly Ala Thr Ser Pro acc gtg ttg tat gtg agt gga gga aat acg cag gtg att gca tac tcg 555 Thr Val Leu Tyr Val Ser Gly Gly Asn Thr Gln Vsl I1e Ala Tyr Ser gaa cat cgt tac cgt atc ttt ggg gaa acc atc g~at att gca gtg ggt 603 Glu His Arg Tyr Arg Iie Phe Gly Glu Thr Ile Asp Ile Ala Val Gly aat tgt ctg gat cgt ttt get cga gtg ctg aag at:t tet aac gac cca 651 Asn Cys Leu Asp Arg Phe Ala Arg Val Leu Lys IJ.e Ser Asn Asp Pro agt cca gga tac aac att gaa cag atg gca aag cc~a ggc aag aag ctg 699 Ser Pro Gly Tyr Asn Ile Glu Gln Met Ala Lys Ai:g Gly Lys Lys Leu gtt gag ctg cca tac act gta aag ggg atg gac gi:.c tca ttc tca ggg 747 Val Glu Leu Pro Tyr Thr Val Lys Gly Met Asp Val Ser Phe Ser Gly IO atc ctg tct ttc att gag gat gta gcc cat cgg ai~g ctg gcc aca ggc 795 Ile Leu Ser Phe Ile Glu Asp Val Ala His Arg M<st Leu Ala Thr Gly gag tgt act cct gag gat ctg tgt ttc tcc ctg crag gaa act gtg ttt 843 Glu Cys Thr Pro Glu Asp Leu Cys Phe Ser Leu G:Ln Glu Thr Val Phe gca atg ctg gta gag atc aca gag cga gcc atg gca cat tgt ggc tcc 891 Ala Met Leu Val Glu Ile Thr Glu Arg Ala Met Ala His Cys Gly Ser 240 245 2:50 cag gag gcc ctc att gtg gga gga gtg ggg tgt a~at gtg agg eta cag 939 Gln Glu Ala Leu Ile Val Gly Gly Val Gly Cys Asn Val Arg Leu Gin gag atg atg gca aca atg tgc cag gaa cgt gga gec cgg ett ttt get 987 Glu Met Met Ala Thr Met Cys Gln Glu Arg Gly Ala Arg Leu Phe Ala aca gat gag aga ttc tgt att gac aat gga gcg atg ata gcc cag get 1035 Thr Asp Glu Arg Phe Cys Ile Asp Asn Gly Ala Met Ile Ala Gln Ala ggc tgg gag atg ttt egg get gga cac agg acc cca ctc agt gat tet 1083 Gly Trp Glu Met Phe Arg Ala Gly His Arg Thr Pro Leu Ser Asp Ser ggg gtt aca cag agg tat cgg aca gat gaa gta gag gtg acc tgg agg 1131 Gly Val Thr Gln Arg Tyr Arg Thr Asp Glu Val Glu Val Thr Trp Arg gac taataagatc aacagaatca gagtagatag ttccttaat:c ggaacccaaa ggaccc 1190 Asp cgtgcctcaa tctctatcct gatgtcatgg gagtcctagc a<~agctatag actccaagca 1250 aggcttgggg tcctttatgg aaccccagga tgactcagca ai:aaaatatt tttggttt 1308 <210> 4 <211> 335 <212> PRT
<2I3> Homo sapiens <400> 4 Met Pro Ala Val Leu Gly Phe Glu G1y S~er Ala Asn Lys Ile Gly Val Gly Val Val Arg Asp Gly Lys Va1 Leu Ala Asn Pro Arg Arg Thr Tyr Val Thr Pra Pro Gly Thr Gly Phe Leu Pro Gly Asp Thr Ala Arg His His Arg Ala Val Ile Leu Asp Leu Leu Gln Glu Ala Leu Thr Glu Ser Gly Leu Thr Ser Gln Asp Ile Asp Cys Ile Ala Tyr Thr Lys Gly Pro Gly Met Gly Ala Pro Leu Val Ser Val 1~3a Val Val Ala Arg Thr Val Ala Gln Leu Trp Asn Lys Pro Leu Val Gly Val Asn His Cys Ile Gly His Ile Glu Met Gly Arg Leu Tle Thr f:ly Ala Thr Ser Pro Thr Val Leu Tyr Val Ser Gly Gly Asn Thr Gln Val I1e Ala Tyr Ser Glu His Arg Tyr Arg Ile Phe Gly Glu Thr Ile Asp Ile Ala Val Gly 7l7 Asn Cys Leu Asp Arg Phe Ala Arg Val Leu Lys I:le Ser Asn Asp Pro 160 165 1.70 Ser Pro Giy Tyr Asn Ile Glu Gln Met Ala Lys p~rg Gly Lys Lys Leu Val Glu Leu Pro Tyr Thr Val Lys Gly Met Asp Val Ser Phe Ser Gly Ile Leu Ser Phe Ile Glu Asp Val Ala His Arg Met Leu Ala Thr Gly Glu Cys Thr Pro Glu Asp Leu Cys Phe Ser Leu (~ln Glu Thr Val Phe Ala Met Leu Val Glu Ile Thr Glu Arg Ala Met A.la His Cys Gly 5er Gln Glu Ala Leu Ile VaI Gly Gly Val Gly Cys ~~.sn Val Arg Leu Gln l~ 255 260 265 270 Glu Met Met Ala Thr Met Cys Gln Glu Arg Gly Al.a Arg Leu Phe Ala Thr Asp Glu Arg Phe Cys Ile Asp Asn G1y Ala trlet Ile Ala Gln Ala Gly Trp Glu Met Phe Arg Ala Gly His Arg Thr 3?ro Leu Ser Asp Ser Gly Val Thr Gln Arg Tyr Arg Thr Asp GIu Val s3lu Val Thr Trp Arg 320 325 :330 Asp

Claims (6)

1. A protein comprising the amino acid sequence represented by Sequence No. 1.

2. The DNA coding for the protein according to Claim 1.

3. A cDNA comprising the base: sequence represented by Sequence No. 2.

4. The cDNA according to Claim 3 comprising the base sequence represented by Sequence No. 3.

5. An Expression vector capable of expressing the DNA according to any of Claims 2 to 4 by in vitro translation or in eucaryotic cells.

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