Role of endogenous cathepsin D-like and chymotrypsin-like proteolysis in human epidermal desquamation.
Even though the skin surface is acidic (about pH 5), most in vitro studies on desquamation have been performed at alkaline pH. We demonstrate that the standard in vitro model system, which achieves squame shedding upon incubation of plantar stratum corneum for 1 day in an alkaline buffer that must include a chelating agent, can be extended to a more realistic model in which the incubation is for 4 days, at varying pHs from 5 to 8, without exogenous chelators. Desmoglein I from stratum corneum was degraded by the squames shed at pH 5 as well as at pH 8. Squame shedding was inhibited to varying extents by the addition of proteinase inhibitors, whose specificity suggested that the crucial enzymatic activity at pH 8 was a chymotrypsin-like serine proteinase, while a similar activity at pH 5 was accompanied by an aspartic proteinase activity of comparable strength. Four degradation peaks were observed when the insulin B chain was reacted with shed squames at pH 5. Two of these peptides were suppressed by the addition of phenylmethylsulphonyl fluoride, the other two by pepstatin A; chymostatin inhibited all four, but E-64 and leupeptin showed no effect. The implied specificity was confirmed by reacting the insulin (without squames) with the standard enzymes human liver cathepsin D and pancreatic chymotrypsin, reproducing the expected degradation products. These results suggest that epidermal desquamation at acidic pH requires two proteolytic activities, one of which is an analogue of chymotrypsin and the other of cathepsin D. Endogenous proteinases corresponding to these activities have been previously identified, namely the stratum corneum chymotryptic enzyme and the mature active form of cathepsin D.
Horikoshi
T
T
Basic Research Laboratory, Kanebo Ltd, 5-3-28, Kotobuki-cho, Odawara, Kanagawa 250-0002, Japan. PXB12765@nifty.ne.jp
Igarashi
S
S
Uchiwa
H
H
Brysk
H
H
Brysk
M M
MM
D015153:Blotting, Western; D002402:Cathepsin D / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004817:Epidermis / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism; D012867:Skin / Q000166:cytology*
D007328:Insulin; D002918:Chymotrypsin; D002402:Cathepsin D