Novel Trypsin Family Serine Proteases
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Europäisches Patentamt *EP001136548A1* (19) European Patent Office Office européen des brevets (11) EP 1 136 548 A1 (12) EUROPEAN PATENT APPLICATION published in accordance with Art. 158(3) EPC (43) Date of publication: (51) Int Cl.7: C12N 9/64, C12N 15/57, 26.09.2001 Bulletin 2001/39 C12N 5/10, C12P 21/02, G01N 33/573, C07K 16/40, (21) Application number: 99951213.0 C12P 21/08, C12Q 1/37 (22) Date of filing: 02.11.1999 // C12P21:02, C12R1:91 (86) International application number: PCT/JP99/06111 (87) International publication number: WO 00/26352 (11.05.2000 Gazette 2000/19) (84) Designated Contracting States: (72) Inventors: AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU • Senoo, Chiaki, Chugai Res. Inst. f. Mol. Med. Inc MC NL PT SE Nihari-gun, Ibaraki 300-4101 (JP) Designated Extension States: • Numata, Mariko, Chugai Rs. Inst. f. Mol. Med. Inc AL LT LV MK RO SI Niihari-gun, Ibaraki 300-4101 (JP) (30) Priority: 04.11.1998 JP 31336698 (74) Representative: VOSSIUS & PARTNER Siebertstrasse 4 (71) Applicant: Chugai Research Institute for 81675 München (DE) Molecular Medicine Inc. Niihari-gun, Ibaraki 300-4101 (JP) (54) NOVEL TRYPSIN FAMILY SERINE PROTEASES (57) Two novel trypsin-family serine proteases spe- "Tespec PRO-3") have been isolated. It has been sug- cifically expressed in adult mouse testis ("Tespec PRO- gested that these proteins are involved in sperm differ- 1" and "Tespec PRO-2"), and a novel trypsin-family ser- entiation and maturation, and sperm functions (e.g., fer- ine protease derived from mouse ("Tespec PRO-3") tilization). Therefore, these proteins are useful for de- have been isolated. Also, two novel trypsin-family serine velopment of novel therapeutics and diagnostics for in- proteases derived from human ("Tespec PRO-2" and fertility, as well as for development of novel contracep- tives. EP 1 136 548 A1 Printed by Jouve, 75001 PARIS (FR) EP 1 136 548 A1 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 35 manner (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 fertili- zation 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 55 foregoing demonstrates that this protein is a novel trypsin-family serine 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 1 136 548 A1 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 "TespecPRO-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. Likewise, RT-PCR analysis also showed that expression of "Tespec PRO-1" is distinctly high in the adult testis. In addition, "Tespec PRO-1" was verified to have increased expression in the testis of 18 day-old mice or older, but it was 20 not expressed in the testis of 12 day-old mice or younger or in the spermatogenesis-defect mutant mice. Similar analysis was carried out for "Tespec PRO-2" and revealed that expression pattern of this gene is identical to that of "Tespec PRO-1".