Detection and characterization of endogenous protease associated with desquamation of stratum corneum.
In order to identify the endogenous protease associated with stratum corneum (SC) desquamation, we examined properties of proteases in the stratum corneum of normal human skin. SC were obtained by tape stripping, washed in toluene and then dried. The proteolytic activity in SC was measured using peptidyl 4-methyl-coumaryl-7-amides (MCAs). The SC was dispersed uniformly in the reaction mixture with dimethylformamide and Triton X-100 and incubated with the peptidyl MCAs. The protease in the SC hydrolysed both Boc-Phe-Ser-Arg-MCA and Boc-Gln-Ala-Arg-MCA (substrates for trypsin) very effectively. The hydrolytic activity was inhibited by the serine protease inhibitors diisopropyl fluorophosphate (DFP), aprotinin, antipain and leupeptin, but not by chymostatin, a chymotrypsin inhibitor. These results show that one or more trypsin-like serine protease is present in the SC of normal human skin. Casein-acrylamide electrophoresis showed that the molecular weight of this serine protease was about 30 kDa. We have previously shown that cells dissociate from human SC sheets in a detergent mixture (N,N-dimethyldodecylamine oxide and sodium lauryl sulphate). This cell dissociation was inhibited by aprotinin and leupeptin. In addition, the proteolytic activity in the outer SC was higher than that in the inner SC, and the activity in the SC of scaly skin induced by SLS treatment was higher than that of untreated skin. These results strongly suggest that the trypsin-like serine protease described here is involved in SC desquamation.
Suzuki
Y
Y
Shiseido Research Center, Yokohama, Japan.
Nomura
J
J
Hori
J
J
Koyama
J
J
Takahashi
M
M
Horii
I
I
D000328:Adult; D000595:Amino Acid Sequence; D002448:Cell Adhesion / Q000502:physiology; D003374:Coumarins / Q000737:chemistry; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D012867:Skin / Q000166:cytology / Q000201:enzymology*; D013379:Substrate Specificity
D003374:Coumarins; D009842:Oligopeptides; D011480:Protease Inhibitors; C048055:tertiary-butyloxycarbonyl-phenylalanyl-seryl-arginyl-4-methylcoumarin-7-amide; D010450:Endopeptidases