Cystatin M/E expression is restricted to differentiated epidermal keratinocytes and sweat glands: a new skin-specific proteinase inhibitor that is a target for cross-linking by transglutaminase

Using serial analysis of gene expression on cultured human keratinocytes we found high expression levels of genes putatively involved in host protecti on and defense, such as proteinase inhibitors and antimicrobial proteins. O ne of these expressed genes was the recently discovered cysteine proteinase inhibitor cystatin M/E that has not been characterized so far at the prote in level with respect to tissue distribution and additional biologic proper ties. Here we report that cystatin M/E has a tissue-specific expression pat tern in which high expression levels are restricted to the stratum granulos um of normal human skin, the stratum granulosum/spinosum of psoriatic skin, and the secretory coils of eccrine sweat glands. Low expression levels wer e found in the nasal cavity, The presence of cystatin M/E in skin and the l ack of expression in a variety of other tissues was verified both at the pr otein level by immunohistochemistry or western blotting, and at the mRNA le vel by reverse transcriptase polymerase chain reaction or northern blotting . Using biotinylated hexapeptide probes we found that cystatin M/E is an ef ficient substrate for tissue type transglutaminase and for transglutaminase s extracted from stratum corneum, and that it acts as an acyl acceptor but not as an acyl donor, Western blot analysis showed that recombinant cystati n M/E could be cross-linked to a variety of proteins extracted from stratum corneum. In vitro, we found that cystatin M/E expression in cultured kerat inocytes is upregulated at the mRNA and protein level, upon induction of di fferentiation. We demonstrate that cystatin M/E, which has a putative signa l peptide, is indeed a secreted protein and is found in vitro in culture su pernatant and in vivo in human sweat by enzyme-linked immunosorbent assay o r western blotting, Cystatin M/E showed moderate inhibition of cathepsin B but was not active against cathepsin C, We speculate that cystatin M/E is u nlikely to be a physiologically relevant inhibitor of intracellular lysosom al cysteine proteinases but rather functions as an inhibitor of self and no nself cysteine proteinases that remain to be identified.