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...

The zinc endopeptidase meprin (EC 3.4.24.18) is expressed in brush border membranes of intestine and kidney tubules, intestinal leukocytes, and certain cancer cells, suggesting a role in epithelial differentiation and cell migration. Here we show by RT-PCR and immunoblotting that meprin is also expressed in human skin. As visualized by immunohistochemistry, the two meprin subunits are localized in separate cell layers of the human epidermis. Meprin alpha is expressed in the stratum basale, whereas meprin beta is found in cells of the stratum granulosum just beneath the stratum corneum. In hyperproliferative epidermis such as in psoriasis vulgaris, meprin alpha showed a marked shift of expression from the basal to the uppermost layers of the epidermis. The expression patterns suggest distinct functions for the two subunits in skin. This assumption is supported by diverse effects of recombinant meprin alpha and beta on human adult low-calcium high-temperature keratinocytes. Here, beta induced a dramatic change in cell morphology and reduced the cell number, indicating a function in terminal differentiation, whereas meprin alpha did not affect cell viability, and may play a role in basal keratinocyte proliferation.