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J. Biol. Chem., Vol. 281, Issue 23, 15893-15899, June 9, 2006

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Cystatin M/E Is a High Affinity Inhibitor of Cathepsin V and Cathepsin L by a Reactive Site That Is Distinct from the Legumain-binding Site

A NOVEL CLUE FOR THE ROLE OF CYSTATIN M/E IN EPIDERMAL CORNIFICATION^*

Tsing Cheng¹, Kiyotaka Hitomi¹, Ivonne M. J. J. van Vlijmen-Willems, Gys J. de Jongh, Kanae Yamamoto, Koji Nishi, Colin Watts^¶, Thomas Reinheckel^||, Joost Schalkwijk², and Patrick L. J. M. Zeeuwen

From the Department of Dermatology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands, the Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, 464-8601 Nagoya, Japan, the ^¶Division of Cell Biology and Immunology, School of Life Sciences, University of Dundee, DD1 5EH Scotland, United Kingdom, and the ^||Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University, D-79104 Freiburg, Germany

Cystatin M/E is a high affinity inhibitor of the asparaginyl endopeptidase legumain, and we have previously reported that both proteins are likely to be involved in the regulation of stratum corneum formation in skin. Although cystatin M/E contains a predicted binding site for papain-like cysteine proteases, no high affinity binding for any member of this family has been demonstrated so far. We report that human cathepsin V (CTSV) and human cathepsin L (CTSL) are strongly inhibited by human cystatin M/E. Kinetic studies show that K_i values of cystatin M/E for the interaction with CTSV and CTSL are 0.47 and 1.78 nM, respectively. On the basis of the analogous sites in cystatin C, we used site-directed mutagenesis to identify the binding sites of these proteases in cystatin M/E. We found that the W135A mutant was rendered inactive against CTSV and CTSL but retained legumain-inhibiting activity. Conversely, the N64A mutant lost legumain-inhibiting activity but remained active against the papain-like cysteine proteases. We conclude that legumain and papain-like cysteine proteases are inhibited by two distinct non-overlapping sites. Using immunohistochemistry on normal human skin, we found that cystatin M/E co-localizes with CTSV and CTSL. In addition, we show that CTSL is the elusive enzyme that processes and activates epidermal transglutaminase 3. The identification of CTSV and CTSL as novel targets for cystatin M/E, their (co)-expression in the stratum granulosum of human skin, and the activity of CTSL toward transglutaminase 3 strongly imply an important role for these enzymes in the differentiation process of human epidermis.

Received for publication, January 24, 2006 , and in revised form, March 13, 2006.

^* This work was supported by a grant from the Radboud University Nijmegen Medical Centre (to T. C.) and by VENI-Grant 916.56.117 from ZONMW, the Netherlands Organization for Health Research and Development (to P. L. J. M. Z.). 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 Both authors contributed equally to this study.

² To whom correspondence should be addressed. Tel.: 31-24-3614094; Fax: 31-24-3541184; E-mail: j.schalkwijk{at}derma.umcn.nl.

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