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 Cheng,Kiyotaka Hitomi,Ivonne M J.J Van Vlijmen-Willems,Gys J De Jongh,Kanae Yamamoto,Koji Nishi,Colin Watts,Thomas Reinheckel,Joost Schalkwijk,Patrick L J.M Zeeuwen

doi:10.1074/jbc.m600694200

Abstract

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 Ki 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.

Highlights

Cysteine proteases and have shown important regulatory and protective functions in cells and tissues against proteolysis by cysteine proteases of host, bacterial, and viral origin [1,2,3]
We show that the protease inhibitor cystatin M/E inhibits legumain and papain-like cysteine proteases due to two distinct nonoverlapping sites
The papain-like cysteine proteases cathepsin V (CTSV) and cathepsin L (CTSL) are strongly inhibited by cystatin M/E, suggesting that these proteases are new targets for cystatin M/E

Summary

EXPERIMENTAL PROCEDURES

Three-dimensional Modeling of Cystatin M/E—A three-dimensional model of human cystatin M/E was generated using MODELLER [23]. Each incubation was performed in the appropriate buffers as follows: for CTSL, CTSV, CTSB, and CTSS, 100 mM MES (pH 6.0), 16 mM dithiothreitol, 1.6 mM EDTA; and for CTSD, 100 mM sodium acetate (pH 4.0), 100 mM KCl. For controls, the same reaction mixtures were prepared but with the addition of pepstatin (Nacarai, Kyoto, Japan), which is an inhibitor of the aspartic protease CSTD, or E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, Peptide Institute Inc., Osaka, Japan), a synthetic inhibitor of the cysteine proteases CTSL, CTSV, CTSB, and CTSS. The same reaction mixtures were prepared but with the addition of pepstatin (Nacarai, Kyoto, Japan), which is an inhibitor of the aspartic protease CSTD, or E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, Peptide Institute Inc., Osaka, Japan), a synthetic inhibitor of the cysteine proteases CTSL, CTSV, CTSB, and CTSS Both controls were used at a final concentration of 10 ␮M. Ki values of wild type cystatin M/E and mutated variants for legumain, cathepsin V, and cathepsin L

Ki cathepsin L nM

RESULTS

DISCUSSION