EVIDENCE FOR SPECIFIC PROTEOLYTIC CLEAVAGE OF THE N-TERMINAL DOMAIN OF HUMAN PROFILAGGRIN DURING EPIDERMAL DIFFERENTIATION

Profilaggrin is a large phosphoprotein that is expressed in the granul ar cells of epidermis where it is localized in keratohyalin, It consis ts of multiple copies of single filaggrin units plus N- and C-terminal sequences that differ front filaggrin, Profilaggrin is dephosphorylat ed and proteolytically processed during terminal differentiation to yi eld filaggrin, which associates with keratin intermediate filaments to form macrofibrils in the lower layers of the stratum corneum, The N-t erminal sequence of human profilaggrin comprises two distinct domains; an acidic A domain of 81 amino acids that binds Ca2+, and a cationic B domain of 212 residues, In this report, we further characterize the N-terminal domain by immunohistochemistry and immunoblot analysis usin g anti-peptide antibodies raised to the A and B regions, All of these antibodies (n = 4) immunostained keratohyalin in the granular layer of human epidermis and also showed some reaction with the lower stratum corneum, In immunoblot studies, the high molecular weight human profil aggrin reacted with both B domain antibodies whereas it showed a weak and variable reaction with A domain antibodies, In addition to profila ggrin, a cationic 32-kDa protein was detected with all N-terminal anti bodies, A similar-sized N-terminal peptide was also produced by in vit ro proteolysis of human profilaggrin with endoproteinase I (PEP1), a p rotease involved in processing of mouse profilaggrin, and in cultured rat epidermal keratinocytes transfected with a human profilaggrin cDNA construct, Evidence for at least one additional cleavage within the N -terminal domain is shown by immunoreactivity of smaller (16-20 kDa) a cidic. and basic proteins with A and B domain antibodies, respectively , These results demonstrate that tile N-terminal domain is an integral part of profilaggrin in keratohyalin but is proteolytically cleaved f rom profilaggrin during the terminal differentiation of keratinocytes to yield a 32-kDa peptide.