RAT EPIDERMAL CATHEPSIN-B - PURIFICATION AND CHARACTERIZATION OF PROTEOLYTIC PROPERTIES TOWARD FILAGGRIN AND SYNTHETIC SUBSTRATES

The aim of this study was to purify epidermal cathepsin B from rat skin and investigate its proteolytic activities on filaggrin and several synthetic substrates. The molecular weight of purified monomeric cathepsin B was estimated to be 30 kDa by SDS-polyacrylamide gel electrophoresis. The amino acid composition, similar to that of liver cathepsin B, indicated the enzyme to be an acidic protease. The enzyme had strong hydrolytic activity toward N-benzyloxy-carbonyl-L-arginyl-L-arginine-7-amido-4-methylcoumarin (Z-Arg-Arg-MCA) (152 mU/mg) and N-benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin (424 mU/mg), but had no proteolytic activity toward L-arginine-7-amido-4-methylcoumarin. The K-m value for Z-Arg-Arg MCA was 0.34 mM and pH optimun was 5.5. Cathepsin B degraded rat epidermal filaggrin into small fragments at pH 4.0 and 5.5, and was inhibited by a specific cysteine proteinase inhibitor, N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)L-leucyl]-agmatin. This study demonstrated that filaggrin was susceptible to degradation by cathepsin B. Such an action may have relevance to skin differentiation in which acid proteases are thought to participate.