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

Biochemistry. 1993 Sep 28;32(38):10036-45. doi: 10.1021/bi00089a020.

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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Animals, Newborn
  • Chromatography, High Pressure Liquid
  • DNA, Complementary
  • Endopeptidases / metabolism*
  • Epidermis / metabolism*
  • Humans
  • Intermediate Filament Proteins / biosynthesis*
  • Intermediate Filament Proteins / chemistry
  • Intermediate Filament Proteins / genetics
  • Intermediate Filament Proteins / isolation & purification
  • Intermediate Filament Proteins / metabolism*
  • Mass Spectrometry
  • Mice
  • Molecular Sequence Data
  • Molecular Weight
  • Peptide Fragments / isolation & purification
  • Phosphoproteins / metabolism*
  • Protein Precursors / chemistry
  • Protein Precursors / isolation & purification
  • Protein Precursors / metabolism*
  • Protein Processing, Post-Translational*
  • Protein Structure, Secondary*
  • Rats
  • Sequence Homology, Amino Acid
  • Trypsin

Substances

  • DNA, Complementary
  • Intermediate Filament Proteins
  • Peptide Fragments
  • Phosphoproteins
  • Protein Precursors
  • filaggrin
  • Endopeptidases
  • Trypsin