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Novel Trypsin Family Serine Proteases Neue Serinproteasen Der Trypsin-Familie Nouvelles Sérines Protéases De La Famille De La Trypsine

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Novel Trypsin Family Serine Proteases Neue Serinproteasen Der Trypsin-Familie Nouvelles Sérines Protéases De La Famille De La Trypsine (19) & (11) EP 1 895 001 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 9/64 (2006.01) C12N 15/57 (2006.01) 03.08.2011 Bulletin 2011/31 C12N 5/10 (2006.01) C12P 21/02 (2006.01) G01N 33/573 (2006.01) C07K 16/40 (2006.01) (2006.01) (2006.01) (21) Application number: 07023492.7 C12P 21/08 C12Q 1/37 C12R 1/91 (2006.01) (22) Date of filing: 02.11.1999 (54) Novel trypsin family serine proteases Neue Serinproteasen der Trypsin-Familie Nouvelles sérines protéases de la famille de la trypsine (84) Designated Contracting States: (56) References cited: AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU WO-A-98/36054 MC NL PT SE • CHUNG,D.W., FUJIKAWA,K., MCMULLEN,B.A., (30) Priority: 04.11.1998 JP 31336698 DAVIE,E.W.,: "Human plasma prekallikrein, a zymogen to a serine protease that contains four (43) Date of publication of application: tandem repeats." BIOCHEMISTRY, vol. 25, no. 9, 05.03.2008 Bulletin 2008/10 1986, pages 2410-2407, XP001056849 • MCMULLEN,B.A., FUJIKAWA,K., DAVIE,E.W.: (62) Document number(s) of the earlier application(s) in "Locationof the disulfide bonds in human plasma accordance with Art. 76 EPC: prekallikrein: the presence of four novel apple 99951213.0 / 1 136 548 domains in the amino-terminal portion of the molecule." BIOCHEMISTRY, vol. 30, no. 8, 1991, (73) Proprietor: CHUGAI SEIYAKU KABUSHIKI pages 2050-2056, XP002190836 KAISHA • LUM LAWRENCE ET AL: "Evidence for distinct Tokyo, 115-8543 (JP) serine protease activities with a potential role in processing the sperm protein fertilin" (72) Inventors: DEVELOPMENTAL BIOLOGY, vol. 191, no. 1, 1 • Senoo, Chiaki November 1997 (1997-11-01), pages 131-145, Kanagawa 247-8530 (JP) XP009095029 ISSN: 0012-1606 • Numata, Mariko Kanagawa 247-8530 (JP) Remarks: Thefile contains technical information submitted after (74) Representative: Vossius & Partner the application was filed and not included in this Siebertstrasse 4 specification 81675 München (DE) Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 1 895 001 B1 Printed by Jouve, 75001 PARIS (FR) EP 1 895 001 B1 Description Technical Field 5 [0001] The present invention relates to novel trypsin-family serine proteases, the genes encoding them, and the production and uses thereof. Background Art 10 [0002] In the testis, the male reproductive organ, sperm, i.e. male gametes, are primarily formed through the following three-step process: (1)’ the self-reproduction of spermatogonium as the germ-line stem cell and the initiation of differ- entiation thereof to the sperm, (2) meiotic division of spermatocyte and the associated gene recombination, and (3) morphogenesis of the haploid spermatid to the sperm. The sperms formed in this manner are expelled into a female body by coitus, pass along the oviduct, and bind to an egg, the female gamete, to achieve fertilization (Yomogida, K. 15 and Nishimune, Y. (1998) Protein, Nucleic acid and Enzyme, 511-521) . To achieve fertilization, it is necessary for a sperm to move through the oviduct, adhere to and penetrate the zona pellucida on the egg surface, and then fuse with the egg. [0003] A variety of proteases participate in these steps of the fertilization process. For example, an analysis using knockout mice (Krege, J.H. et al. (1995) Nature 375: 146-148; Esther Jr, C.R. et al. (1996) Lab. Invest. 74: 953-965) 20 has revealed that sperm angiotensin-converting enzyme (testis ACE) plays an important role in the process of sperm transportation within the oviduct (Hagaman, J.R. et al. (1998) Proc. Natl. Acad. Sci. USA 95: 2552-2557). Fertilizing ability is markedly reduced in the male knockout mice that lack proprotein convertase 4 (PC4) (M. Mbikay et al. (1997) Proc.’ Natl. Acad. Sci. USA, 94: 6842-6846). [0004] Regarding serine proteases, a variety of trypsin inhibitors inhibit in vitro fertilization, suggesting that trypsin- 25 like serine proteases present in the sperm (the acrosome in particular) may digest the zona pellucida when the sperm penetrates the zona pellucida (Saling, P.M. (1981) Proc. Natl. Acad. Sci. USA, 78: 6231-6235; Benau, D.A. and Storey, B.T. (1987) Biol. Reprod., 36: 282-292; Liu D.Y. and Baker, H.W. (1993) Biol. Reprod., 48: 340-348). Previously, acrosin, a trypsin-family serine protease in the acrosome, was assumed to play this role (Brown, C.R. (1983) J. Reprod. Fertil., 69: 289-295; Kremling, H. et al. (1991) Genomics, 11: 828-834; Klemm, U. et al., (1990) Differentiation, 42: 160-166). 30 However, acrosin knockout mice have been shown to have almost normal fertilizing ability, suggesting that other serine proteases which are present in the sperm, apart from acrosin, digest zona pellucida (Baba, T. et al. (1994) J. Biol. Chem., 269: 31845-31849; Adham, I.M. et al. (1997) Mol. Reprod. Dev., 46: 370-376). In ascidians, a trypsin-family serine protease, called spermosin, is expressed in the sperm (Sawada, H. et al. (1984) J. Biol. Chem., 259: 2900-2904) . An antibody specific to this protease has been shown to inhibit fertilization in ascidians in a concentration-dependent manner 35 (Sawada, H. et al., (1996) Biochem. Biophys. Res. Commun., 222: 499-504). Recently, cDNAs of the trypsin-family serine proteases, TESP1 and TESP2, which are expressed specifically in mouse acrosome, were cloned (Kohno, N. et al., (1998) Biochem. Biophys. Res. Commun., 245: 658-665). However, the roles these genes play in the fertilization process remains to be clarified. Moreover, serine proteases existing in the sperm and capable of digesting the zona pellucida have not yet been reported. 40 Disclosure of the Invention [0005] An objective of the present invention is to provide novel trypsin-family serine proteases associated with sper- matogenesis and sperm functions, the genes encoding these proteases and a production method and use thereof. 45 [0006] The present inventors attempted to amplify a gene designated as 76A5sc2 by polymerase chain reaction, and eventually found a gene fragment having a nucleotide sequence different from that of 76A5sc2 gene. Using this gene fragment, the present inventors have cloned the cDNAs containing entire open reading frames (ORF) of two novel trypsin-family serine proteases ("Tespec PRO-1" and "Tespec PRO-2") expressed specifically in adult mouse testis. They have also analyzed the tissue-specific expression of these genes. 50 [0007] "Tespec PRO-1" (Testis specific expressed serine proteinase-1) is predicted to encode 321 amino acids. The deduced amino acid sequence contains trypsin-family serine protease motifs, "Trypsin-His" and "Trypsin-Ser" active sites, and exhibits significantly high homology to other trypsin- family serine proteases, such as acrosin, prostasin, trypsin and so on, in the regions of the two motifs and their neighboring regions. In the other regions, however, there are no known genes found to exhibit significant homology to this protein at the nucleotide or amino acid level. The foregoing 55 demonstrates that this protein is a novel trypsin-family seine protease. [0008] On the other hand, "Tespec PRO-2" is predicted to encode 319 amino acids. The protein has a "Trypsin-His" active site. With regard to the "Trypsin-Ser" active site, which consists of 12 amino acids, it is differs from that of the canonical motif by two amino acid residues. Such a difference is found in some other known trypsin-family serine 2 EP 1 895 001 B1 proteases, and, thus, "Tespec PRO- 2" is predicted to function as a protease. There are no known genes found to exhibit significant homology to "Tespec PRO- 2" at the nucleotide and amino acid levels. Thus this protein is also a novel trypsin- family serine protease. [0009] Interestingly, for "Tespec PRO-2", a splicing isoform was found that comprises the first half region of "Tespec 5 PRO-2" connected to the latter half region of "Tespec PRO- 1". This suggests that these two proteases are located very close to each other on the chromosome. Though a variety of splicing isoforms are found for "Tespec PRO-2", these "Tespec PRO-2" isoforms do not retain a long stretch of ORF, and thus do not encode any proteases at all. The homology between "Tespec PRO-1" and "Tespec PRO-2" is 52.2% at the nucleotide level and 33.1% at the amino acid level. [0010] The present inventors have also successfully cloned a cDNA for human "Tespec PRO-2" by RT-PCR and 10 RACE, based on the nucleotide sequence of mouse "Tespec PRO-2". Human "Tespec PRO-2" has been revealed to have 74.2% and 69.8% homology with mouse "Tespec PRO-2" at the nucleotide and amino acid levels, respectively. Further it has been clarified that human "Tespec PRO-2" is encoded on chromosome 8. [0011] The present inventors have further succeeded in cloning a cDNA encoding human "Tespec PRO-3" by RT- PCR and RACE, based on the nucleotide sequence of mouse "Tespec PRO-1". In addition, they also succeeded in 15 cloning a cDNA that encodes mouse "Tespec PRO-3", a mouse counterpart to human "Tespec PRO-3". [0012] Northern blot analysis using the coding region for "Tespec PRO-1" as a probe revealed that this gene is expressed merely in adult mouse testis, but it failed to identify the expression in other tissues or in the fetal stage.
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