Specificity Profiling of Seven Human Tissue Kallikreins Reveals Individual Subsite Preferences

Abstract

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) (1). 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 (2). The 15 human KLK genes code for the classical tissue kallikreins hK1, hK2, and the prostate-specific antigen (PSA, 5 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 (3-7). 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 (8, 9). 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 (2, 10). In Table 1, the 15 human tissue kallikreins are listed together with their s...

Engineering Kunitz Domain 1 (KD1) of Human Tissue Factor Pathway Inhibitor-2 to Selectively Inhibit Fibrinolysis

“…2 and 3 are consistent with the concept that coagulation proteases prefer a nonpolar residue at P2Ј position in their substrates/inhibitors (6,17,23,42). In this context, KD1-L17R has no inhibitory activity toward pKLK, FXIa, and FVIIa/sTF.…”

“…Serine proteases are ubiquitous in living organisms and are involved in many physiologic processes, including digestion and respiration (1)(2)(3), blood coagulation and fibrinolysis (4,5), kinin formation and tumorigenesis (6), complement activation and phagocytosis (7), osteoarthritis and bone remodeling (8,9), as well as in ovogenesis and fertilization (10). Biologic activity of serine proteases is tightly regulated by their cognate inhibitors.…”

Activation Profiles and Regulatory Cascades of the Human Kallikrein-related Peptidases

“…non-P1 site) hydrolyses after Ala (pro-KLK9), Ser (pro-KLK13), and Leu (pro-KLK15) residues. These results show that KLK11 is capable of dual substrate specificity (both trypsinlike and chymotrypsin-like) and is in good agreement with the P1 specificity of KLK11 as determined using a positional scanning combinatorial library of tetrapeptides (52). The dual substrate specificity of KLK11 is similar to KLK1, but KLK11 exhibits a much broader specificity in contrast to KLK1.…”

Activation profiles of human kallikrein‐related peptidases by proteases of the thrombostasis axis

“…Characterization of the potential autolytic and cross-activation potential among the members of the KLK family, and the elucidation of activation cascades regulating function, has been a subject of intense investigation (Lovgren et al 1997;Denmeade et al 2001;Takayama et al 2001a;Brattsand et al 2005;Debela et al 2006;Michael et al 2006;Memari et al 2007;Yoon et al 2007;Emami and Diamandis 2008). These studies have demonstrated the potential for complex regulatory activation cascades between members of the KLK family; however, almost nothing is known regarding the intersection of the KLK axis with other protease families.…”