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 granular cells of epidermis where it is localized in keratohyalin. It consists of multiple copies of single filaggrin units plus N- and C-terminal sequences that differ from filaggrin. Profilaggrin is dephosphorylated and proteolytically processed during terminal differentiation to yield filaggrin, which associates with keratin intermediate filaments to form macrofibrils in the lower layers of the stratum corneum. The N-terminal 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 using 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 profilaggrin reacted with both B domain antibodies whereas it showed a weak and variable reaction with A domain antibodies. In addition to profilaggrin, a cationic 32-kDa protein was detected with all N-terminal antibodies. A similar-sized N-terminal peptide was also produced by in vitro proteolysis of human profilaggrin with endoproteinase 1 (PEP1), a protease 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) acidic and basic proteins with A and B domain antibodies, respectively. These results demonstrate that the N-terminal domain is an integral part of profilaggrin in keratohyalin but is proteolytically cleaved from profilaggrin during the terminal differentiation of keratinocytes to yield a 32-kDa peptide.