Serpin-derived Peptide Substrates for Investigating the Substrate Specificity of Human Tissue Kallikreins hK1 and hK2

Abstract

The third human tissue kallikrein to be identified, hK2, could be an alternate or complementary marker to kallikrein hK3 (prostate-specific antigen) for prostate diseases. Most of the hK2 in seminal plasma forms an inactive complex with protein C inhibitor (PCI), a serpin secreted by seminal vesicles. As serpin inhibitors behave as suicide substrates that are cleaved early in the interaction with their target enzyme, and kallikreins have different sensitivities to serpin inhibitors, we prepared a series of substrates with intramolecularly quenched fluorescence based on the sequences of the serpin reactive loops. They were used to compare the substrate specificities of hK1 and hK2, which both have trypsin-like specificity, and thus differ from chymotrypsin-like hK3. The serpin-derived peptides behaved as kallikrein substrates whose sensitivities reflected the specificity of the parent inhibitory proteins. Substrates derived from PCI were the most sensitive for both hK1 and hK2 with specificity constants of about 107m−1. s−1. Those derived from antithrombin III and α2-antiplasmin were more specific for hK2 while a kallistatin-derived substrate was specifically cleaved by hK1. hK1 and hK2 substrates of greater specificity were obtained using chimeric peptides based on the sequence of serpin reactive loops.The main difference between specificities of hK1 and hK2 arise because hK2 can accommodate positively charged as well as small residues at P2 and requires an arginyl residue at P1. Thus, unlike hK1, hK2 does not cleave kininogen-derived substrates overlapping the region of N-terminal insertion of bradykinin in human kininogens. The third human tissue kallikrein to be identified, hK2, could be an alternate or complementary marker to kallikrein hK3 (prostate-specific antigen) for prostate diseases. Most of the hK2 in seminal plasma forms an inactive complex with protein C inhibitor (PCI), a serpin secreted by seminal vesicles. As serpin inhibitors behave as suicide substrates that are cleaved early in the interaction with their target enzyme, and kallikreins have different sensitivities to serpin inhibitors, we prepared a series of substrates with intramolecularly quenched fluorescence based on the sequences of the serpin reactive loops. They were used to compare the substrate specificities of hK1 and hK2, which both have trypsin-like specificity, and thus differ from chymotrypsin-like hK3. The serpin-derived peptides behaved as kallikrein substrates whose sensitivities reflected the specificity of the parent inhibitory proteins. Substrates derived from PCI were the most sensitive for both hK1 and hK2 with specificity constants of about 107m−1. s−1. Those derived from antithrombin III and α2-antiplasmin were more specific for hK2 while a kallistatin-derived substrate was specifically cleaved by hK1. hK1 and hK2 substrates of greater specificity were obtained using chimeric peptides based on the sequence of serpin reactive loops. The main difference between specificities of hK1 and hK2 arise because hK2 can accommodate positively charged as well as small residues at P2 and requires an arginyl residue at P1. Thus, unlike hK1, hK2 does not cleave kininogen-derived substrates overlapping the region of N-terminal insertion of bradykinin in human kininogens. The human kallikrein gene family contains three well-characterized members, hKLK1, hKLK2 and hKLK3 (1Berg T. Bradshaw R.A. Carretero O.A. Chao J. Chao L. Clements J.A. Fahnestock M. Fritz H. Gauthier F. MacDonald R.J. Margolius H.S. Morris B.J. Richards R.I. Scicli A.G. Agents Actions. 1992; 38: 19-25PubMed Google Scholar). These genes have been mapped to chromosome 19 (2Riegman P.H.J. Vlietstra R.J. Suurmeijer L. Cleutjens C.B.J.M. Trapman J. Genomics. 1992; 14: 6-11Crossref PubMed Scopus (136) Google Scholar) and are translated to give rise to proteinases hK1, hK2, and hK3 respectively (1Berg T. Bradshaw R.A. Carretero O.A. Chao J. Chao L. Clements J.A. Fahnestock M. Fritz H. Gauthier F. MacDonald R.J. Margolius H.S. Morris B.J. Richards R.I. Scicli A.G. Agents Actions. 1992; 38: 19-25PubMed Google Scholar). The only human tissue kallikrein with an undisputed function is hK1, which releases vasoactive kinins from kininogens, but these proteinases may have several other functions, including the processing of hormone and other peptide precursors (reviewed in Refs. 3MacDonald R.J. Margolius H.S. Erdös E.G. Biochem. J. 1988; 253: 313-321Crossref PubMed Scopus (130) Google Scholar and 4Bhoola K.D. Figueroa C.D. Worthy K. Pharmacol. Rev. 1992; 44: 1-80PubMed Google Scholar). hK3 is produced mainly in the prostate and is known essentially as a marker of prostatic diseases (see Ref. 5Catalona W.J. Prostate. 1996; 7: 64-69Crossref PubMed Scopus (73) Google Scholar for a recent review). It participates in sperm liquefaction by cleaving fibronectin and semenogelins, the major components of the seminal vesicle coagulum after ejaculation (6Lilja H. J. Clin. Invest. 1985; 76: 1899-1903Crossref PubMed Scopus (659) Google Scholar, 7Lilja H. Cockett A.T. Abrahamsson P.A. Cancer. 1992; 70: 230-234Crossref PubMed Scopus (100) Google Scholar) and possibly in the processing of IGFBP-3 1The abbreviations used are: IGFBP-3, insulin-like growth factor binding protein 3; ACT, α1-antichymotrypsin, α1-Pi, α1-proteinase inhibitor; α2-AP, α2-antiplasmin; ATIII, antithrombin III; PCI, protein C inhibitor; SBTI, soya bean trypsin inhibitor; TFA, trifluoroacetic acid; AMC, 7-amino-4-methylcoumarin; Abz, o-aminobenzoyl; EDDnp, N-(2,4-dinitrophenyl)ethylenediamine; pNA, paranitroanilide; PTH, phenylthiohydantoin; Bz, benzoyl; Fmoc,N-(9-fluorenyl)methoxycarbonyl; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight. (8Cohen P. Graves H.C. Peehl D.M. Kamarei M. Giudice L.C. Rosenfeld R.G. J. Clin. Endocrinol. Metab. 1992; 75: 1046-1053Crossref PubMed Scopus (371) Google Scholar). hK2 is also predominantly synthesized in the prostate epithelium. Its mRNA concentration in the prostate gland is 4–7 times lower than that of hK3 (9Chapdelaine P. Paradis G. Tremblay R. Dubé J.Y. FEBS Lett. 1988; 236: 205-208Crossref PubMed Scopus (131) Google Scholar, 10Henttu P. Lukkarinen O. Vihko P. Int. J. Cancer. 1990; 45: 654-660Crossref PubMed Scopus (89) Google Scholar), and it is present in seminal plasma as highM r inactive complexes with protein C inhibitor and as low M r inactive fragments (11Deperthes D. Chapdelaine P. Tremblay R.R. Brunet C. Berton J. Hébert J. Lazure C. Dubé J.Y. Biochim. Biophys. Acta. 1995; 1245: 311-316Crossref PubMed Scopus (85) Google Scholar). Its biological function remains unknown, but it has been suggested that it participates in the liquefaction of seminal coagulum (12Deperthes D. Frenette G. Brillard-Bourdet M. Bourgeois L. Gauthier F. Tremblay R.R. Dubé J.Y. J. Androl. 1996; 17: 659-665PubMed Google Scholar). Interest has recently focused on this protein since it appears to be a novel marker of prostate cancer and/or benign prostate adenoma (13Kumar A. Goel A.S. Hill T.M. Mikolajczyk S.D. Millar L.S. Kuus-Reichel K. Saedi M.S. Cancer Res. 1996; 56: 5397-5402PubMed Google Scholar, 14Young C.Y. Seay T. Hogen K. Charlesworth M.C. Roche P.C. Klee G.G. Tindall D.J. Prostate. 1996; 7: 17-24Crossref PubMed Scopus (35) Google Scholar, 15Tremblay R.R. Deperthes D. Têtu B. Dubé J.Y. Am. J. Pathol. 1997; 150: 455-459PubMed Google Scholar). hK2 has been obtained as a recombinant protease and used to prepare immunogens (11Deperthes D. Chapdelaine P. Tremblay R.R. Brunet C. Berton J. Hébert J. Lazure C. Dubé J.Y. Biochim. Biophys. Acta. 1995; 1245: 311-316Crossref PubMed Scopus (85) Google Scholar, 16Saedi M.S. Cass M.M. Goel A.S. Grauer L. Hogen K.L. Okaneya T. Griffin B.Y. Klee G.G. Young C.Y. Tindall D.J. Mol. Cell. Endocrinol. 1995; 109: 237-241Crossref PubMed Scopus (25) Google Scholar), but it has also been recently isolated as an active enzyme from seminal plasma (17Frenette G. Deperthes D. Tremblay R.R. Lazure C. Dubé J.Y. Biochim. Biophys. Acta. 1997; 1334: 109-115Crossref PubMed Scopus (57) Google Scholar). Preliminary investigations of its substrate specificity have shown that it has a trypsin-like specificity, cleaving chromogenic substrates with Arg and Lys, but not Phe, at P1(17). This agrees with the presence of an aspartyl residue at position 187 (chymotrypsin numbering) in the substrate binding pocket (18Vihinen M. Biochem. Biophys. Res. Commun. 1994; 204: 1251-1256Crossref PubMed Scopus (35) Google Scholar, 19Bridon D.P. Dowell B.L. Urology. 1995; 45: 801-806Abstract Full Text PDF PubMed Scopus (28) Google Scholar). hK2 seems to be more closely related to hK1 than to hK3 enzymatically, though it shares 78% sequence homology with the latter and only 57% with hK1 (16Saedi M.S. Cass M.M. Goel A.S. Grauer L. Hogen K.L. Okaneya T. Griffin B.Y. Klee G.G. Young C.Y. Tindall D.J. Mol. Cell. Endocrinol. 1995; 109: 237-241Crossref PubMed Scopus (25) Google Scholar). A recent in vitrostudy showed that both hK3 and hK2 cleaved proteins forming the seminal plasma coagulum, but at different sites (12Deperthes D. Frenette G. Brillard-Bourdet M. Bourgeois L. Gauthier F. Tremblay R.R. Dubé J.Y. J. Androl. 1996; 17: 659-665PubMed Google Scholar). hK2, therefore, differs in specificity from hK3, which means that the proteinases may have different physiological targets. Whether hK2 has some enzymatic properties in common with hK1, especially the ability to release vasoactive kinins, is not known. The restricted specificity of kallikreins, resulting from their extended site of interaction with the substrate, argues against this. This study was done to further investigate the substrate specificity of kallikrein hK2, to distinguish its activity from that of hK1 and therefore identify its physiological function in the prostate, in seminal fluid, and at metastatic sites. As kallikreins are inhibited differently by natural inhibitors of the serpin family (17Frenette G. Deperthes D. Tremblay R.R. Lazure C. Dubé J.Y. Biochim. Biophys. Acta. 1997; 1334: 109-115Crossref PubMed Scopus (57) Google Scholar), that probably all behave as suicide substrates (20Patston P.A. Gettins P. Beechem J. Schapira M. Biochemistry. 1991; 30: 8876-8882Crossref PubMed Scopus (173) Google Scholar, 21Engh R.A. Huber R. Bode W. Schulze A.J. Trends Biotechnol. 1995; 13: 503-510Abstract Full Text PDF PubMed Scopus (73) Google Scholar), we used the peptide sequences of several serpin reactive loops to prepare specific, sensitive substrates capable of discriminating between the activities of these proteinases. The following materials were obtained from commercial sources: DEAE-Sephadex A50 (Pharmacia Biotech Inc.); SBTI (Boehringer Mannheim); 4-methylumbelliferone, 7-amino-4-methylcoumarin, (D)PFR-AMC, IGEPAL CA-630 (Sigma); 4-methylumbelliferyl-4-guanidinobenzoate (Fluka Chemie AG); EDTA, N,N-dimethylformamide, acetonitrile (Merck); and TFA (Perkin-Elmer). All other reagents were of analytical grade. Kallikrein hK2 was purified from human seminal plasma after dissociation of the PCI-hK2 complex (17Frenette G. Deperthes D. Tremblay R.R. Lazure C. Dubé J.Y. Biochim. Biophys. Acta. 1997; 1334: 109-115Crossref PubMed Scopus (57) Google Scholar). Human kallikrein hK1 was purified to homogeneity from urine essentially as described (22Irie A. Takahashi S. Katayama Y. Ito K. Miyake Y. Biochem. Int. 1986; 13: 375-382PubMed Google Scholar). Briefly, 6.5 liters of human urine were concentrated to 200 ml by passage through an Amicon PM10 membrane, dialyzed against 20 mm phosphate buffer, pH 6.0, 150 mm NaCl, and applied to a DEAE A50 column (4 × 2.6 cm) equilibrated in the same buffer. The column was washed with 200 ml of equilibrium buffer and proteins were eluted with a 50–500 mm NaCl gradient. Fractions were assayed for their enzymatic activity toward (D)PFR-AMC in the presence of SBTI. Fractions resistant to SBTI inhibition were pooled and titrated with aprotinin (23Gutman N. Moreau T. Alhenc Gelas F. Baussant T. El Moujahed A. Akpona S. Gauthier F. Eur. J. Biochem. 1988; 171: 577-582Crossref PubMed Scopus (32) Google Scholar). The active site of hK2 was titrated using 4-methylumbelliferyl-4-guanidinobenzoate (24Knight C.G. Methods Enzymol. 1995; 248: 85-101Crossref PubMed Scopus (75) Google Scholar). The intramolecularly quenched fluorogenic peptides were synthesized by classical solution methods; glutamine was the C-terminal residue in all peptides due to a requirement of synthesis strategy (25Hirata I.Y. Cezari M.H.S. Nakaie C.R. Boschcov P. Ito A.S. Juliano M.A. Lett. Pept. Sci. 1994; 1: 299-308Crossref Scopus (200) Google Scholar). All syntheses were performed with Fmoc methodology using a multiple automated peptide synthesizer (PSSM-8, Shimadzu Co.). Intramolecularly quenched fluorogenic substrates were prepared as 2 mm stock solutions inN,N-dimethylformamide and diluted with activation buffer. Substrate purity was checked by MALDI-TOF mass spectrometry (TofSpec-E, Micromass) and by reverse-phase chromatography on a C18 column eluted with a 10-min linear gradient of 0–60% acetonitrile in 0.075% TFA at 2.0 ml/min. Bz-VKKR-AMC and (D)PFF-AMC were synthesized as described previously (26Alves L.C. Almeida P.C. Franzoni L. Juliano J. Juliano M.A. Peptide Res. 1996; 9: 92-96PubMed Google Scholar). All assays were carried out at 37 °C in 50 mm Tris-HCl buffer, pH 8.3, containing 1 mm EDTA and 0.02% IGEPAL constants were for and substrates using a substrate concentration A. N. M. Gauthier F. FEBS Lett. 1990; PubMed Scopus (35) Google Scholar). were done using the was and was and were carried out using for and were and for with intramolecularly quenched fluorogenic The was using prepared by of and its concentration was from the at Substrate of were by the at for constants were using substrate the of hK1 and hK2 were to on the substrate peptides were with purified kallikreins at 37 °C in 50 buffer, pH 8.3, containing 1 mm EDTA, 0.02% were at different times to on the peptide and enzyme The was by of TFA, and the was on a C18 column × eluted with a linear gradient of acetonitrile in 0.075% TFA for at a of 2.0 with at and which the of peptides to N-terminal All sites of by were by N-terminal for all substrates used in this The of the were on an column × using a linear gradient of acetonitrile 0–60% in 0.075% TFA for at a of ml/min. sites were by N-terminal The sequences were using an liquid with the and by the were using an Preliminary to identify the biological of hK2 have shown that including and are cleaved at Arg sites by hK2 and at sites by (12Deperthes D. Frenette G. Brillard-Bourdet M. Bourgeois L. Gauthier F. Tremblay R.R. Dubé J.Y. J. Androl. 1996; 17: 659-665PubMed Google Scholar). It is not hK2 participates in sperm liquefaction the to which it proteins. hK2 is present in seminal plasma mainly as a complex with protein C inhibitor (11Deperthes D. Chapdelaine P. Tremblay R.R. Brunet C. Berton J. Hébert J. Lazure C. Dubé J.Y. Biochim. Biophys. Acta. 1995; 1245: 311-316Crossref PubMed Scopus (85) Google Scholar), whose concentration in seminal plasma is more than times that in the plasma F. J. A. A. J. A. Res. 1991; Full Text PDF PubMed Scopus (131) Google Scholar). The of inhibition of hK2 in seminal plasma that of after ejaculation (12Deperthes D. Frenette G. Brillard-Bourdet M. Bourgeois L. Gauthier F. Tremblay R.R. Dubé J.Y. J. Androl. 1996; 17: 659-665PubMed Google Scholar). PCI also human kallikreins hK3 and hK1, forming complexes A. H. Eur. J. Biochem. 1994; PubMed Scopus Google Scholar, F. J. J. A. K. Eur. J. Biochem. 1995; PubMed Scopus (35) Google Scholar). This inhibitor to the serpin family and with target proteinases to This the of the reactive and insertion of the cleaved a of the to the of the as a complex R.A. Huber R. Bode W. Schulze A.J. Trends Biotechnol. 1995; 13: 503-510Abstract Full Text PDF PubMed Scopus (73) Google Scholar). the reactive is not for enzyme that its by or with peptides the inhibitor a substrate S. D. J. 1994; Full Text PDF PubMed Google Scholar). peptides derived from the of serpin inhibitors are substrates for proteinases that are inhibited by A is that the of the the protein is not for of the of the reactive in the it is that is for interaction and by the The in the sequences of the reactive loops in serpin family that it be to specific substrates for of the sequences of human is at in a The is at PCI is the only serpin inhibitor known to with but it is also an hK1 inhibitor F. J. J. A. K. Eur. J. Biochem. 1995; PubMed Scopus (35) Google Scholar). Peptide substrates of the sequence of human PCI from to with to the site in the reactive were synthesized and assayed as substrates of hK1 and constants were by the substrate concentration and by linear were from This the that with peptide and with the inhibition of PCI, peptides were by the human proteinases with specificity constants the and for hK1 were lower than for hK2, that substrate binding and by the kallikreins probably because of the extended binding site of interaction with the substrate M. Moreau T. Gauthier F. Biochim. Biophys. Acta. 1995; PubMed Scopus Google Scholar, Juliano M.A. Juliano L. Biochem. J. 1995; PubMed Scopus Google Scholar). both cleaved all peptides at a site at the as in for the by hK1 and hK2 of fluorogenic peptides derived from of human not are means Chao L. Chao J. J. 1992; Full Text PDF PubMed Google not in a are means S.D. and are other serpin inhibitors of hK1 R. Fritz H. Enzymol. PubMed Scopus Google Scholar, Chao L. Chao J. J. 1992; Full Text PDF PubMed Google Scholar). is the more specific and hK1 than does α1-Pi, but its is by Chao L. Chao J. Biochem. J. 1990; PubMed Scopus Google Scholar). The reactive of is cleaved after an Chao L. Chao J. J. 1992; Full Text PDF PubMed Google Scholar), an that has been recently to sensitive hK1 substrates Juliano M.A. Juliano L. Biochem. J. 1997; Google Scholar). Whether hK2 is inhibited by and after the is not known. The tissue kallikrein substrates (D)PFR-AMC and (D)PFF-AMC were assayed with hK1 and hK2, but only (D)PFR-AMC was cleaved by both This that hK2 does not cleave the reactive at at the and therefore that this serpin does not A kallistatin-derived peptide could therefore between hK1 and hK2 a substrate including the was synthesized and assayed with both It was cleaved by hK1 with a specificity of about but was resistant to by hK2 hK1 cleaved at the as shown by N-terminal of the obtained from not for the by hK1 and hK2 of fluorogenic peptides derived from the and C-terminal insertion sequences of human not are means Chao L. Chao J. J. 1992; Full Text PDF PubMed Google not in a are means S.D. hK1, hK2 is inhibited by several other serpin inhibitors, including and (17Frenette G. Deperthes D. Tremblay R.R. Lazure C. Dubé J.Y. Biochim. Biophys. Acta. 1997; 1334: 109-115Crossref PubMed Scopus (57) Google Scholar). used the sequences of the reactive loops of these serpin inhibitors to hK2 substrates that are resistant to hK1 They were assayed with of titrated were in with the inhibitory properties of the serpin Peptide substrates derived from and were cleaved by hK2 but or by hK1, possibly because of the presence of small or charged residues at P2 The peptide was cleaved at the by hK2 which to the sensitive in the the peptide was cleaved at the which the sensitive in the The of the specificity for this substrate was due to by hK2 at the which further the specificity of this This because of the that was to the is not present in or other serpin reactive but was by the of which as (25Hirata I.Y. Cezari M.H.S. Nakaie C.R. Boschcov P. Ito A.S. Juliano M.A. Lett. Pept. Sci. 1994; 1: 299-308Crossref Scopus (200) Google Scholar). The of an arginyl residue from sequences may hK2 does not cleave these substrates to hK2 in (17Frenette G. Deperthes D. Tremblay R.R. Lazure C. Dubé J.Y. Biochim. Biophys. Acta. 1997; 1334: 109-115Crossref PubMed Scopus (57) Google Scholar). It could be that hK2 the reactive at a site from which is not present in the at several sites the reactive has been T. H. P.A. Sci. 1996; PubMed Scopus Google Scholar). specific for hK1, the kallistatin-derived substrate is well by this than derived from the PCI reactive to its by the to Arg as the presence of Arg at or by hK1 I.Y. Almeida P.C. Juliano M.A. Juliano L. Biochem. J. 1995; PubMed Scopus Google Scholar). at the same site as but the specificity was this chimeric substrate was also cleaved to some by hK2 was at the as for the peptide As the at in the kallistatin-derived peptide is mainly for the specificity of hK1, we the Arg with in the sensitive substrate The resulting substrate, was resistant to hK2 but an hK1 substrate, though of lower It thus between the kallikreins of hK1, which is by its is that it a residue at P2 for substrate and F. Eur. J. Biochem. PubMed Scopus Google Scholar). This for serpin inhibitors of hK1 that all have a residue at hK1, hK2 and peptides at sites a small residue at P2 carried out on several of the tissue kallikrein family showed that some of which also residues at cleaved substrates with positively charged residues at that position T. Brillard-Bourdet M. J. Gauthier F. J. 1992; Full Text PDF PubMed Google Scholar, D. Chao J. Biochem. Pharmacol. 1992; 45: Scopus Google Scholar). the of a substrate, by hK1 and The substrate was resistant to hK1, but was cleaved by hK2 The peptide sequence was therefore used as the of an intramolecularly quenched fluorogenic substrate the sequence of the substrates to the and specificity to The resulting chimeric was by hK2 at the site with a about 50 times greater than that for the hK1 also cleaved this substrate, mainly at the in with its P2 specificity and at the the at P2 in this substrate to by hK1. was more by hK1 but by hK2, that it is the most sensitive substrate for hK2 for the of fluorogenic peptides by hK1 and are means in a are means S.D. All of serpin-derived substrates by hK2 were at a site hK1 and kallistatin-derived substrates at a site and also at a site to release The restricted specificity of hK2 that this protease does not release a vasoactive from its at a different This could because the P2 specificity of hK2 is than that of hK1. this by the activities of hK1 and hK2 using fluorogenic substrates that the sequences at both of the bradykinin in human and on a peptide the bradykinin sequence with and C-terminal Juliano M.A. Juliano L. Biochem. J. 1995; PubMed Scopus Google Scholar). the latter peptide was for of the it was only with Abz, and were by reverse-phase and protein proteinases cleaved at the C-terminal site of bradykinin the This peptide was further by hK1 not by to after at the The intramolecularly quenched fluorogenic substrate was specifically cleaved by hK1 at the same These that hK2 has properties of biological The used that the specificity of hK2 differs from that of kallikrein hK1, and therefore that it has different biological targets. these to be The hK2 concentration in seminal plasma is at Deperthes and that of Chao L. Chao J. J. 1994; Full Text PDF PubMed Google Scholar), but at times lower than that of M.C. T.M. Invest. 17: Google Scholar). hK2 is by PCI in seminal that active enzyme is present in this The at which hK2 is inhibited by PCI is the same as that of the of the hK3 remains after protein (12Deperthes D. Frenette G. Brillard-Bourdet M. Bourgeois L. Gauthier F. Tremblay R.R. Dubé J.Y. J. Androl. 1996; 17: 659-665PubMed Google Scholar). hK2 and fibronectin in D. Frenette G. Brillard-Bourdet M. Bourgeois L. Gauthier F. Tremblay R.R. Dubé J.Y. J. Androl. 1996; 17: 659-665PubMed Google Scholar), it is not known the also in the concentration of hK3 is hK2 participates in sperm liquefaction or other active peptide from it be a of the of activity by the of of substrate and The was by O.