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Human kallikrein 14 (hK14; encoded by the KLK14 gene) is a secreted serine protease, differentially expressed in breast, ovarian, prostate and testicular cancer. Several reports suggest that hK14 may have clinical utility as a cancer biomarker. However, its biological role is currently unknown. Thus, the aim of this study was to determine the substrate specificity and candidate physiological targets of hK14, as a first step towards delineating its function. Recombinant hK14 was expressed in P. pastoris as the mature enzyme and purified to homogeneity from the culture supernatant by cation exchange chromatography. The substrate specificity of hK14 was then defined by screening: 1) a phage-displayed random pentapeptide library with exhaustive diversity and 2) combinatorial fluorogenic synthetic tetrapeptide positional scanning libraries with amino acid diversity at P1-P4 positions. Six rounds of phage-displayed library screening yielded 32 candidate pentapeptide substrates for hK14. Twenty-two of the selected peptides had Arg or Lys residues at the P1 position while the remaining 10 had Tyr, suggesting that hK14 has both trypsin and chymotrypsin-like specificity. The SwissProt database was screened with the most efficiently cleaved pentapeptides and several putative protein substrates for hK14 were identified, including matrilin-4, laminin and collagen IV. Full-length laminin and collagen IV proteins were shown to be efficiently fragmented by hK14 in vitro. The N-terminal P1-P4 specificity of hK14 was further assessed with combinatorial fluorogenic tetrapeptide libraries. hK14 demonstrated trypsin-like specificity with a P1 preference for Arg over Lys, in accordance with the phage display data. However, chymotrypsin-like specificity was not observed. Furthermore, hK14 exhibited broad specificity at P2 and P3, with a preference for aromatic amino acids (Tyr, Trp and Phe) at P4. These results were confirmed using several synthetic fluorogenic tetrapeptides. Two substrate cleavage motifs were designed based on the P1-P4 residues most preferred by hK14 and used to screen the non-redundant protein databases of the National Center for Biotechnology Information for potential hK14 targets, using the “Pattinprot” algorithm (http://npsa-pbil.ibcp.fr). Many putative substrates for hK14 were retrieved including adhesion molecules (cadherins, integrins), structural components of the extracellular matrix (collagens, laminins) and proteases [matrix metalloproteases (MMPs), transmembrane serine proteases (TMPRSSs)]. Collectively, our findings suggest that hK14 may preferentially act upon extracellular matrix proteins and might therefore be implicated in tissue remodeling and/or tumour progression, acting alone or synergistically with other proteases. Further studies are required to verify these results and the role of hK14 in vivo.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]