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The human KLK14 gene is one of the newly identified serine protease genes belonging to the human kallikrein family, which contains 15 members. KLK14 , like all other members of the human kallikrein family, is predicted to encode for a secreted serine protease already found in various biological fluids. This new kallikrein is mainly expressed in prostate and endocrine tissues, but its function is still unknown. Recent studies have demonstrated that KLK14 gene expression is up-regulated in prostate and breast cancer tissues, and that higher expression levels correlate with more aggressive tumors. In this work, we used phage-display substrate technology to study the substrate specificity of hK14. A phage-displayed random pentapeptide library with exhaustive diversity was screened with purified recombinant hK14. Highly specific and sensitive substrates were selected from the library. We show that hK14 has dual activity, trypsin- and chymotrypsin-like, with a preference for cleavage after arginine residues. A SwissProt database search with selected sequences identified six potential human protein substrates for hK14. Two of them, laminin alpha-5 and collagen IV, which are major components of the extracellular matrix, have been demonstrated to be hydrolyzed efficiently by hK14.

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 10 7 M ؊1 . s ؊1 . Those derived from antithrombin III and ␣ 2 -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 P 2 and requires an arginyl residue at P 1 . 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 (1). These genes have been mapped to chromosome 19 (2) and are translated to give rise to proteinases hK1, hK2, and hK3 respectively (1). 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. 3 and 4). hK3 is produced mainly in the prostate and is known essentially as a marker of prostatic diseases (see Ref. 5 for a recent review). It participates in sperm liquefaction by cleaving fibronectin and semenogelins, the major components of the seminal vesicle coagulum after ejaculation (6, 7) and possibly in the processing of IGFBP-3 1 (8). 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 (9, 10), and it is present in seminal plasma as high M r inactive complexes with protein C inhibitor and as low M r inactive fragments (11). Its biological function remains unknown, but it has been suggested that it participates in the liquefaction of seminal coagulum (12). Interest has recently focused on this protein since it appears to be a novel marker of prostate cancer and/or benign prostate adenoma (...