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J. Biol. Chem., Vol. 281, Issue 35, 25678-25688, September 1, 2006

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Specificity Profiling of Seven Human Tissue Kallikreins Reveals Individual Subsite Preferences^*

Mekdes Debela, Viktor Magdolen, Norman Schechter^¶, Martina Valachova¹, Friedrich Lottspeich^||, Charles S. Craik^**2, Youngchool Choe^**2, Wolfram Bode³, and Peter Goettig⁴

From the Max-Planck-Institut für Biochemie, Proteinase Research Group, Am Klopferspitz 18, D-82152 Martinsried, Germany, Klinische Forschergruppe der Frauenklinik, Klinikum rechts der Isar der Technische Universität München, Ismaninger Strasse 22, D-81675 München, Germany, ^¶Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, ^||Max-Planck-Institut für Biochemie, Protein Analytics, Am Klopferspitz 18, D-82152 Martinsried, Germany, and ^**Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143

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.

Received for publication, March 14, 2006 , and in revised form, May 24, 2006.

^* This work was supported by the European Commission through SPINE Contract QLG2-CT-2002-00988 under the integrated program "Quality of Life and Management of Living Resources," through project CAMP LSHG-2006-018830 by the Fonds der Chemischen Industrie (to W. B.), and by the Graduiertenkolleg 333 of the Ludwig-Maximilians-Universitaet Muenchen (to M. D. and V. M). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a Madam Curie Training Site Program of the European Union within the Graduiertenkolleg 333 of the Ludwig-Maximilians-Universitaet Muenchen. Current address: Slovak University of Technology, Faculty of Chemical and Food Technology, Vazovova 5, 812 43 Bratislava 1, Slowakia.

² Supported by National Institutes of Health Grant CA 72006.

³ To whom correspondence may be addressed. Tel.: 49-89-8578-2676; Fax: 49-89-8578-3516; E-mail: bode{at}biochem.mpg.de. ⁴ To whom correspondence may be addressed. Tel.: 49-89-8578-2097; Fax: 49-89-8578-3516; E-mail: goettig{at}biochem.mpg.de.

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