CHARACTERIZATION OF PROFILAGGRIN ENDOPROTEINASE-1 - A REGULATED CYTOPLASMIC ENDOPROTEINASE OF EPIDERMIS

Profilaggrin, an insoluble precursor of the intermediate filament-asso ciated protein filaggrin, contains multiple internal repeats (PIRs), A t terminal differentiation of epidermis, proteolytic processing within a ''linker'' region of each PIR releases soluble filaggrin in a two-s tage process, The first stage endoproteinase (PEP1, profilaggrin endop roteinase 1) cleaves mouse profilaggrin at a subset of the linkers, yi elding processing intermediates consisting of several filaggrin repeat s, An epidermal endoproteinase that cleaves the requisite linker subse t has been purified 4,966-fold from mouse epidermal extracts, SDS-poly acrylamide gel electrophoresis demonstrated a band of molecular mass o f 29.5 kDa that correlated with the activity, Labeling with [H-3]diiso propylfluorophosphate identified PEP1 as a serine protease; inhibitor studies suggest that it is similar to chymotrypsin, as expected from p revious in vivo studies, The purified PEP1 cleaved a peptide derived f rom profilaggrin (P1) at three residues within and adjacent to a multi ple tyrosine sequence, consistent with the in vivo processing sites, N o exopeptidase activity was detected, PEP1 is only active toward insol uble profilaggrin, resulting in partial solubilization, consistent wit h a role in dispersal of profilaggrin during terminal differentiation, In contrast to the specific cleavage of mouse profilaggrin, PEP1 clea ved all linker regions of rat profilaggrin, Studies with phosphorylate d P1 suggest that PEP1 specificity may be partly regulated by profilag grin phosphorylation.