Specificity Profiling of Seven Human Tissue Kallikreins Reveals Individual Subsite Preferences

Human tissue kallikreins (hKs) form a family of 15 closely related (chymo)trypsin-like serine proteinases. These tissue kallikreins are expressed in a wide range of tissues including the central nervous system, the salivary gland, and endocrine-regulated tissues, such as prostate, breast, or testis, and may have diverse physiological functions. For several tissue kallikreins, a clear correlation has been established between expression and different types of cancer. For example, the prostate-specific antigen (PSA or hK3) serves as tumor marker and is used to monitor therapy response. Using a novel strategy, we have cloned, expressed in Escherichia coli or in insect cells, refolded, activated, and purified the seven human tissue kallikreins hK3/PSA, hK4, hK5, hK6, hK7, hK10, and hK11. Moreover, we have determined their extended substrate specificity for the nonprime side using a positional scanning combinatorial library of tetrapeptide substrates. hK3/PSA and hK7 exhibited a chymotrypsin-like specificity preferring large hydrophobic or polar residues at the P1 position. In contrast, hK4, hK5, and less stringent hK6 displayed a trypsin-like specificity with strong preference for P1-Arg, whereas hK10 and hK11 showed an ambivalent specificity, accepting both basic and large aliphatic P1 residues. The extended substrate specificity profiles are in good agreement with known substrate cleavage sites but also in accord with experimentally solved (hK4, hK6, and hK7) or modeled structures. The specificity profiles may lead to a better understanding of human tissue kallikrein functions and assist in identifying their physiological protein substrates as well as in designing more selective inhibitors. Human tissue kallikreins (hKs) form a family of 15 closely related (chymo)trypsin-like serine proteinases. These tissue kallikreins are expressed in a wide range of tissues including the central nervous system, the salivary gland, and endocrine-regulated tissues, such as prostate, breast, or testis, and may have diverse physiological functions. For several tissue kallikreins, a clear correlation has been established between expression and different types of cancer. For example, the prostate-specific antigen (PSA or hK3) serves as tumor marker and is used to monitor therapy response. Using a novel strategy, we have cloned, expressed in Escherichia coli or in insect cells, refolded, activated, and purified the seven human tissue kallikreins hK3/PSA, hK4, hK5, hK6, hK7, hK10, and hK11. Moreover, we have determined their extended substrate specificity for the nonprime side using a positional scanning combinatorial library of tetrapeptide substrates. hK3/PSA and hK7 exhibited a chymotrypsin-like specificity preferring large hydrophobic or polar residues at the P1 position. In contrast, hK4, hK5, and less stringent hK6 displayed a trypsin-like specificity with strong preference for P1-Arg, whereas hK10 and hK11 showed an ambivalent specificity, accepting both basic and large aliphatic P1 residues. The extended substrate specificity profiles are in good agreement with known substrate cleavage sites but also in accord with experimentally solved (hK4, hK6, and hK7) or modeled structures. The specificity profiles may lead to a better understanding of human tissue kallikrein functions and assist in identifying their physiological protein substrates as well as in designing more selective inhibitors. The tissue kallikreins constitute a subgroup of the chymotrypsin-like serine proteinase family S1A of clan PA(S) (1Barrett A.J. Tolle D.P. Rawlings N.D. Biol. Chem. 2003; 384: 873-882Crossref PubMed Scopus (37) Google Scholar). They are highly homologous to the “true tissue kallikrein” K1, which is encoded in mammals, for example, by the human KLK1 or the mouse Klk1 genes (2Clements J.A. Hooper J.D. Odorico D.M. Dong Y. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. 2. Elsevier Academic Press, London2004: 1569-1577Google Scholar). The 15 human KLK genes code for the classical tissue kallikreins hK1, hK2, and the prostate-specific antigen (PSA, 5The abbreviations used are: PSA, prostate-specific antigen; hK, human tissue kallikrein; EK, enterokinase; AMC, 7-amino-4-methylcoumarine; uPA, urokinase-type plasminogen activator; PS-SCL, positional scanning with a synthetic combinatorial peptide library; BPTI, bovine pancreatic trypsin inhibitor. hK3) as well as for the 12 more recently discovered so-called new tissue kallikreins, hK4 to hK15, named in chronological order of their characterization (3Brattsand M. Egelrud T. J. Biol. Chem. 1999; 274: 30033-30040Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 4Yousef G.M. Luo L.Y. Diamandis E.P. Anticancer Res. 1999; 19: 2843-2852PubMed Google Scholar, 5Yousef G.M. Chang A. Scorilas A. Diamandis E.P. Biochem. Biophys. Res. Commun. 2000; 276: 125-133Crossref PubMed Scopus (189) Google Scholar, 6Harvey T.J. Hooper J.D. Myers S.A. Stephenson S.A. Ashworth L.K. Clements J.A. J. Biol. Chem. 2000; 275: 37397-37406Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 7Diamandis E.P. Yousef G.M. Clements J. Ashworth L.K. Yoshida S. Egelrud T. Nelson P.S. Shiosaka S. Little S. Lilja H. Stenman U.H. Rittenhouse H.G. Wain H. Clin. Chem. 2000; 46: 1855-1858Crossref PubMed Scopus (174) Google Scholar). These genes share common characteristics such as a similar exon/intron organization and encode single-chain pre-proproteinases, which consist of a signal peptide, a generally short, 4-9-residue-long pro-peptide (only hK5 harbors a rather long propeptide of 37 residues), and a chymotrypsin- or trypsin-like catalytic domain (Fig. 1) with amino acid sequence identity of about 80% for the classical and 40% among the new tissue kallikreins, respectively (8Clements J. Hooper J. Dong Y. Harvey T. Biol. Chem. 2001; 382: 5-14Crossref PubMed Scopus (127) Google Scholar, 9Yousef G.M. Diamandis E.P. Biol. Chem. 2002; 383: 1045-1057Crossref PubMed Scopus (90) Google Scholar). According to phylogenetic analyses, hK1, hK2, and hK3 form a subgroup of particularly closely related proteinases, whereas the tissue kallikreins hK4-hK15 diverge into several subgroups, namely hK4/hK5/hK7, hK6/hK13/hK14, hK8/hK10/hK12, and hK9/hK11/hK15 (2Clements J.A. Hooper J.D. Odorico D.M. Dong Y. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. 2. Elsevier Academic Press, London2004: 1569-1577Google Scholar, 10Yousef G.M. Diamandis E.P. Clin. Biochem. 2003; 36: 443-452Crossref PubMed Scopus (94) Google Scholar). In Table 1, the 15 human tissue kallikreins are listed together with their synonyms, their primary tissue localization, and their data base accession numbers (11Borgono C.A. Diamandis E.P. Nat. Rev. Cancer. 2004; 4: 876-890Crossref PubMed Scopus (565) Google Scholar).TABLE 1Human tissue kallikrein members, their main tissue expression, and GenBank™ accession numbers (8Clements J. Hooper J. Dong Y. Harvey T. Biol. Chem. 2001; 382: 5-14Crossref PubMed Scopus (127) Google Scholar, 24Yousef G.M. Diamandis E.P. Tumour Biol. 2002; 23: 185-192Crossref PubMed Scopus (84) Google Scholar, 78Borgono C.A. Michael I.P. Diamandis E.P. Mol. Cancer Res. 2004; 2: 257-280PubMed Google Scholar)Systematic names and synonymsTissue localizationGenBank™ accession numberReferenceshK1Pancreatic/renal kallikreinKidney, pancreas, salivary glandM2562979Fukushima D. Kitamura N. Nakanishi S. PubMed Scopus Google kallikrein PubMed Scopus Google A. Lilja H. PubMed Scopus Google serine 1, P.S. S. A. 1999; PubMed Scopus Google breast, M. Egelrud T. J. Biol. Chem. 1999; 274: 30033-30040Abstract Full Text Full Text PDF PubMed Scopus (194) Google pancreas, A. Stenman Mol. 2: PubMed Google M. A. A. Egelrud T. J. Biol. Chem. Full Text PDF PubMed Google serine S. M. A. Shiosaka S. PubMed Scopus Google G.M. Luo L.Y. Diamandis E.P. Anticancer Res. 1999; 19: 2843-2852PubMed Google breast, testis, Cancer Res. Google serine prostate, testis, S. T. A. H. N. Biochem. Biophys. Res. Commun. 2000; PubMed Scopus Google G.M. Luo L.Y. Diamandis E.P. Anticancer Res. 1999; 19: 2843-2852PubMed Google G.M. Chang A. Diamandis E.P. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google J.D. Harvey T.J. Myers S.A. Ashworth L.K. Clements J.A. 2001; PubMed Scopus Google gland, G.M. Scorilas A. Ashworth L.K. Diamandis E.P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google in a new In tissue kallikreins are expressed in tissues, such as pancreas, salivary gland, and central nervous G.M. Diamandis E.P. Clin. Biochem. 2003; 36: 443-452Crossref PubMed Scopus (94) Google as well as in the endocrine-regulated tissues testis, prostate, and breast, whereas and hK3 are expressed in the For example, hK6 has been in of breast, prostate, and and in the central nervous as well as in and G.M. Diamandis E.P. Biol. Chem. 2002; 383: 1045-1057Crossref PubMed Scopus (90) Google Scholar). expression in a wide range of tissues in a of physiological and J.A. Myers S.A. Dong Y. Rev. Clin. 2004; PubMed Scopus Google Scholar). for a tissue kallikreins a has been For example, a in of the but is also in physiological Rev. Google Scholar). hK3/PSA has been to the and in of the H. J. Clin. PubMed Scopus Google whereas A. Cancer Res. Google Scholar, J. M. Lilja H. Biochem. Biophys. Res. Commun. PubMed Scopus Google and hK4 Nelson P.S. M. 2001; PubMed Scopus Google in a physiological hK4 and to for and their or serine proteinase and Y. 2000; PubMed Scopus Google Scholar, M. T. Y. T. J. M. J.D. J. Res. PubMed Scopus Google Scholar, S. M. A. Shiosaka S. PubMed Scopus Google and hK5 and hK7 as well as are in and A. M. Egelrud T. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar, T. M. M. J. PubMed Scopus Google Scholar). more is the of tissue kallikreins with tissue kallikreins are expressed in prostate, and and for (11Borgono C.A. Diamandis E.P. Nat. Rev. Cancer. 2004; 4: 876-890Crossref PubMed Scopus (565) Google Scholar). hK3/PSA is used as a tumor marker for and for therapy 2001; PubMed Scopus Google Scholar). of the more recently discovered tissue kallikreins are clear of or G.M. Diamandis E.P. Tumour Biol. 2002; 23: 185-192Crossref PubMed Scopus (84) Google for breast, prostate, and in the Diamandis E.P. Cancer PubMed Scopus Google Scholar). are of tissue kallikreins are also in human such as in and hK4 in P.S. D. S. J. 2004; PubMed Scopus Google hK6 and in central nervous M. M. J. 2004; PubMed Scopus (94) Google and hK7 in A. A. M. J. H. Egelrud T. 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Chem. 2002; Full Text Full Text PDF PubMed Scopus Google and hK7, N. T. and for a and H. J. Mol. Biol. PubMed Scopus Google Scholar, J. Mol. Biol. PubMed Scopus Google D. A. J. Mol. Biol. 2002; PubMed Scopus Google M. J. Mol. Biol. PubMed Scopus Google and mouse tissue kallikreins J. Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google The extended substrate specificity has been for hK3 M. J.A. Chem. Biol. Full Text PDF PubMed Scopus Google and hK5 I.P. A. M. Diamandis E.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and more and for hK4 M. D. N. Nelson P.S. PubMed Scopus Google and C.A. T. J. Diamandis E.P. D. Biol. Chem. 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Y. A. Biol. Chem. PubMed Scopus Google Scholar). hK5 purified by and the of hK5 established by with trypsin to about at of the tissue kallikrein by at and Biochem. PubMed Scopus Google Scholar, T. 4: PubMed Scopus Google Scholar). The of tissue kallikrein with the substrates for hK3 and hK7 and and for hK4, hK5, hK6, hK7, hK10, and hK11 in and and the substrate at by the of the at and of and with a For substrate and of and with a diverse with the of and in which the or is and by of the amino and including whereas the an of amino (Fig. Y. M. D. J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the a of different tetrapeptide substrates. 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These data hK4 the physiological or of substrate of hK4 are in good with the of hK4 the large with an at is particularly well to a side whereas the and polar also side the hydrophobic and polar side the wide between the side of and is to a of side the hydrophobic the side is by and and by hydrophobic side Human in the of hK5 is for side and are whereas the amino in with large side and are In is and whereas among residues and are amino in and but are in whereas the has an preference for by and the human trypsin-like (3Brattsand M. Egelrud T. J. Biol. Chem. 1999; 274: 30033-30040Abstract Full Text Full Text PDF PubMed Scopus (194) Google hK5 is known to a trypsin-like with a strong preference for but accepting substrates with a or at P1 I.P. A. M. Diamandis E.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of substrates a preference for and at and for and at The substrates the of and The specificity of hK5 for cleavage of is also by with the and with the and I.P. A. M. Diamandis E.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). hK5 has been to at a N. M. M. D. Egelrud T. M. J. 2004; Full Text Full Text PDF PubMed Scopus Google to also to According to their (Fig. and hK5 and hK4 have similar structures. the of residues and both specificity are well to side The more preference of hK5 (Fig. to the of the hK4 by which in hK5 an hydrophobic the the of hK5, which of hK4, the more The preference for more hydrophobic residues by the at the and the side a to the in agreement with a of hK5 for more polar side in hK6 a strong preference for which is as well as and to the hK6 is selective among the seven tissue kallikreins preferring of amino acid residues. In strong contrast, hK6 is selective and residues. hK6 specificity for well with the of substrates in hK6 N. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. 2. Elsevier Academic Press, London2004: and with the cleavage residues of the the hK6 Y. M. M. 2002; PubMed Scopus Google Scholar). According to the of hK6 M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google the is similar to of hK4 and bovine trypsin J. Mol. Biol. PubMed Scopus Google with the side at the basic P1 the residues at and the the side side and of peptide substrates by The of hydrophobic P1 side may by the of the by the side the of for the side is by the side the of the to as for the side of in with J. Mol. Biol. PubMed Scopus Google Scholar). is for the strong preference of hK6 for and residues. 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According to the of the of with an and a at and and at similar to of is in with the of and residues but the strong of and (Fig. hK11 a of the in the known of hK6, the a which the experimentally preference of hK11 for residues. side the and side in a polar by seven different tissue kallikreins have been expressed as proteinases, and their extended substrate specificity has been determined using a positional scanning combinatorial tetrapeptide substrate of tissue kallikreins and hK7) a chymotrypsin-like specificity for to large hydrophobic or polar P1 residues an for and and for the amino which in is by a clear preference for in which is by as the in of the tissue kallikreins (hK4, hK5, and a trypsin-like specificity, with a strong preference for hK4 whereas and a specificity, accepting both basic as well as large aliphatic P1 residues the of the the (hK4, hK6, and hK7) or In contrast, of the for and residues to of such as the of the with the but also the of the of the in is preference of human tissue kallikreins determined with combinatorial substrates with P1 residues in physiological substrate cleavage sites C.A. Michael I.P. Diamandis E.P. Mol. Cancer Res. 2004; 2: 257-280PubMed Google kallikrein sites in physiological in a new positional scanning data are to data and may in the for substrates. the substrates of hK3/PSA are the and with cleavage of the J. J. Lilja H. 2000; PubMed Scopus Google Scholar, M. 36: PubMed Scopus Google Scholar, S. M. T. Biophys. 2002; PubMed Scopus Google Scholar). residues are the in of the to the specificity of hK3 (Fig. Moreover, have been as substrates for hK3 S. S. N. T. J. Biochem. 2001; PubMed Scopus (94) Google Scholar). hK4 may a of substrates the and the such as N. M. S. J. M. D. Biol. Chem. PubMed Scopus Google which is with specificity for the (Fig. hK5 at the cleavage as well as hK7 and in a of the serves for M. Y. Egelrud T. J. Full Text Full Text PDF PubMed Scopus Google Scholar). substrate for hK5, such as the propeptide of hK3 which closely scanning library (Fig. have been recently I.P. J. Diamandis E.P. J. Biol. Chem. 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Full Text PDF PubMed Scopus Google Scholar). in the amino acid at the diverse library between the side which in between and discovered by synthetic substrates with For example, a hK6 specificity using peptide M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google clear in the with whereas is a for (Fig. In the data by similar to substrates. the of large of the and of the tissue kallikreins, by for and for selective inhibitors. the and of the tissue kallikreins may for their physiological to a better understanding of and the The of the as for hK5 and hK7 with in the range T. M. M. J. PubMed Scopus Google Scholar, Y. Y. A. Biol. Chem. PubMed Scopus Google Scholar, M. M. A. Y. A. A. Nat. PubMed Scopus Google the physiological and of the the specificity of the in the of for for protein and and for