Evidence for Specific Proteolytic Cleavage of the N-Terminal Domain of Human Profilaggrin During Epidermal Differentiation

Abstract

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.

Bricks and mortar of the epidermal barrier

“…Interestingly, the amino terminus of profilaggrin contains two functional calcium binding EF hand motifs (Presland et al, 1992;Markova et al, 1993). Filaggrin is released by proteolysis from profilaggrin (Presland et al, 1997), and functions to bind keratin intermediate filaments into tight arrays typically seen in corneocytes (Dale et al, 1978;Mack et al, 1993). Some filaggrin becomes cross-linked to CE proteins (Richards et al ., 1988;Steinert and Marekov, 1995;Simon et al ., 1996), presumably together with and at the same time as the keratins.…”

“…Some filaggrin becomes cross-linked to CE proteins (Richards et al ., 1988;Steinert and Marekov, 1995;Simon et al ., 1996), presumably together with and at the same time as the keratins. It has also been proposed that the amino terminal parts of profilaggrin are also incorporated into CEs after proteolytic cleavage from the filaggrin units (Presland et al, 1997).…”

Structural organization of cornified cell envelopes and alterations in inherited skin disorders

“…Small amounts of filaggrin, however, are cross-linked to CEs (14, 122, 123). The amino-terminal sequence comprises two distinct domains; an acidic A domain which contains two calcium-binding motifs and a cationic B domain (124). The amino-terminal domain is proteolytically cleaved from profilaggrin during the terminal differentiation of keratinocytes.…”

“…This processing raises the intriguing possibility that the amino-terminal profilaggrin domain (as well as other S100 family members) may be involved in cell differentiation in the very late stages of keratinization. Immunoelectron microscopy analysis has further implicated a role for the amino-terminal profilaggrin domains in CE-assembly (124).…”

Clinical Impact of Current Genetics Findings

“…The EDC gene filaggrin, which displays a complex highly repetitive sequence, encodes a key protein in the formation of the outermost keratin layer of the skin [47]. The large precursor of filaggrin, profilaggrin, is expressed in the keratohyalin granules of the stratum granulosum, and during formation of the cornified cell envelope it is dephosphorylated and proteolytically cleaved into the N-terminal S-100 protein and 10-12 individual filaggrin peptides [48,49]. After cleavage, the amino-terminal domain of filaggrin enters the nucleus, where it has been suggested to have a role in regulating terminal differentiation, whereas the liberated filaggrin peptides participate in the aggregation of keratin filaments into bundles thereby promoting the flattened shape of corneocytes.…”