Multiple copies of phosphorylated filaggrin in epidermal profilaggrin demonstrated by analysis of tryptic peptides.
The precursor of mouse (c57/B16) epidermal filaggrin (profilaggrin) is a very large (ca. 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 the unphosphorylated filaggrin, which polymerizes with keratin filaments into macrofibrils. In order to gain insight in the nature of these processes, we compared tryptic digests of profilaggrin with those of filaggrin by 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 filaggrin sequence. 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 molecular weight of 500 000 (as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis), this indicates there are 16 repeats. Analysis of phosphopeptides isolated from profilaggrin showed 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 phosphorylated in profilaggrin.
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D004817:Epidermis / Q000032:analysis*; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins / Q000302:isolation & purification*; D046911:Macromolecular Substances; D051379:Mice; D008810:Mice, Inbred C57BL; D010446:Peptide Fragments / Q000032:analysis; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011487:Protein Conformation; D011498:Protein Precursors / Q000302:isolation & purification; D012867:Skin / Q000032:analysis*; D014357:Trypsin
D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010748:Phosphopeptides; D011498:Protein Precursors; D014357:Trypsin