Characterization of protease processing sites during conversion of rat profilaggrin to filaggrin

Abstract

Profilaggrin is an intermediate filament-associated protein of cornified epithelia. It consists of multiple copies of similar filaggrin domains joined by peptide linker regions; during terminal differentiation of the epidermis, the linker regions are processed away in a regulated manner. In order to characterize the sites of proteolysis in rat profilaggrin, tryptic peptides of filaggrin and profilaggrin were fractionated by reverse-phase HPLC, and the HPLC fractions were analyzed by nebulization-assisted electrospray ionization mass spectrometry. Peptide sequences were confirmed or corrected by tandem mass spectrometry; in several cases, this was achieved by collisional activation of multiply charged precursor ions of peptides exceeding 3 kDa in mass. The tryptic peptides accounted for all of the sequence predicted by a partial cDNA sequence, with the exception of six arginines or dipeptides. Although the cDNA sequence predicted eight sites of heterogeneity among the filaggrin domains, only one of these was observed. An additional unpredicted site of heterogeneity was also seen. Comparison of the peptides from filaggrin with those of profilaggrin revealed several peptides unique to filaggrin, specifically at the new amino- and carboxyl-termini, that result from proteolytic processing of the linker region of profilaggrin. Both the amino- and carboxyl-termini were "ragged", suggesting that processing may involve exopeptidase action after an initial endopeptidase cleavage. The average mass of this mixture of filaggrins was determined by electrospray mass spectrometry to be 42 452 Da, in reasonable agreement with that predicted from the mass spectrometric analysis of the terminal sequences. The linker peptide of rat profilaggrin was found in two forms, which differed only in the phosphorylation state of serine 22.

Characterization of Profilaggrin Endoproteinase 1

“…Profilaggrin was purified as described previously (11,15). PEP1 activity was detected with an SDS-PAGE assay whereby profilaggrin was deposited as a thin film.…”

“…Mature profilaggrin is insoluble and stored in non-membrane-bound cytosolic keratohyalin granules. At terminal differentiation, the granule disperses; at the same time, profilaggrin undergoes dephosphorylation by phosphatase 2A (PP2A) and at least one other phosphatase (12) and proteolytic processing by two endoproteinases and at least two exopeptidases (13)(14)(15) to release the soluble filaggrin. The regulation of granule dispersal and the interaction of the processing products with keratins are not understood, although it seems likely that release is tightly regulated in order to prevent premature collapse of the keratin network.…”

“…Proteolytic processing occurs within a region referred to as the linker region and involves an initial cleavage at a hydrophobic site within this linker region, followed by carboxypeptidase and aminopeptidase cleavages at the new termini to produce the final amino and carboxyl termini of filaggrin (13,15). The actual amount of sequence removed during release of filaggrin varies in different situations, so that a specific linker sequence cannot be defined.…”

“…The first stage of processing involves cleavage of only the ␣ F linker regions. In rat profilaggrin, there are no differences in the primary sequence of the PIRs (15), requiring some other explanation to account for the two-stage processing observed in cultured rat keratinocytes (14).…”

Matrix Metalloproteinase 10 Degradomics in Keratinocytes and Epidermal Tissue Identifies Bioactive Substrates With Pleiotropic Functions*

“…Around three quarters (76%) of N termini could not be assigned to known mature protein N termini and thus were derived from proteolytic cleavage, whereby 26% resulted from aminopeptidase activity, a common phenomenon, e.g. for processing of linker regions during maturation of structural skin proteins such as filaggrin (22,48). From the remaining 50% we extracted 1173 N-terminally iTRAQ-labeled neo-N-terminal peptides representing true epidermal cleavages.…”