Multiple copies of phosphorylated filaggrin in epidermal profilaggrin demonstrated by analysis of tryptic peptides

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Abstract
The precursor of mouse (c57/B16) epidermal filaggrin (profilaggrin) is a very large (.apprx. 500,000 daltons), highly phosphorylated protein containing multiple copies of filaggrin (26,000 daltons). The conversion of profilaggrin to filaggrin late in epidermal cell differentiation involves dephosphorylation and proteolysis to yield unphosphorylated filaggrin, which polymerizes with keratin filaments into macrofibrils. In order to gain insight into the nature of these processes, tryptic digests of profilaggrin were compared with those of filaggrin bt reverse-phase liquid chromatography. Approximately 80% of the profilaggrin mass consists of multiple copies of filaggrin. Twenty peptides purified in good yield from both profilaggrin and filaggrin accounted for most of the fialggrin seqence. A detailed analysis of the yield of several peptides provided an estimate of the size and frequency of the repeat unit within profilaggrin. These data indicate that the repeating substructure of profilaggrin contains about 265 amino acids and that about 50 residues are removed per filaggrin domain as the precursor is processed to filaggrin. Assuming a MW of 500,000 (as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis), this indicates there are 16 repeats. Analysis of phosphopeptides isolated from profilaggrin show that 66% of the phosphate was located on peptides that are unphosphorylated in filaggrin. Analysis of peptide recoveries confirmed the repeat size and showed that every copy of filaggrin was phopshorylated in profilaggrin.

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