A membrane-bound
protease induced by
sulfur mustard in cultured normal human epidermal keratinocytes (NHEK) was purified and partially characterized. Maximum
enzyme stimulation occurred at 16 hr after normal human epidermal keratinocytes were exposed to 300 microM
sulfur mustard. Purification to homogeneity of the
protease was accomplished by
Triton X-100 solubilization, ultracentrifugation, and dialysis, followed by ion-exchange chromatography through
DEAE-cellulose and finally hydrophobic column chromatography through
phenyl Sepharose. Analysis of the purified
enzyme by SDS-PAGE revealed a single
polypeptide at the 80 kDa region. Further investigation of biochemical properties showed that a synthetic
serine-specific Chromozym TRY
peptide and the physiological
protein laminin were good substrates for this
enzyme. Moreover, this
enzyme was inhibited mostly by the
serine-protease inhibitors leupeptin and di-isopropyl
fluorophosphate and not by the
cysteine protease inhibitor E-64 or the
metalloprotease inhibitor
1,10-phenanthroline (Component H, CH), indicating the
serine protease nature of this
enzyme. This
enzyme had a pH optimum in the range of 7.0 to 8.0.
Amino acid sequencing of the purified
enzyme revealed that this
enzyme belongs to the
endopeptidase family (
serine protease), and is homologous with a mammalian-type bacterial
serine endopeptidase that can preferentially cleave K-X, including K-P. These results suggest that
serine-protease stimulation may be one of the mechanisms of mustard-induced skin
blister formation, and that some specific
serine-protease inhibitors may be useful for the treatment of this
sulfur mustard toxicity.