CHARACTERIZATION OF PROTEASE PROCESSING SITES DURING CONVERSION OF RAT PROFILAGGRIN TO FILAGGRIN

Profilaggrin is an intermediate filament-associated protein of cornifi ed epithelia. It consists of multiple copies of similar filaggrin doma ins 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 prof ilaggrin, tryptic peptides of filaggrin and profilaggrin were fraction ated by reverse-phase HPLC, and the HPLC fractions were analyzed by ne bulization-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 multipl y charged precursor ions of peptides exceeding 3 kDa in mass. The tryp tic peptides accounted for all of the sequence predicted by a partial cDNA sequence, with the exception of six arginines or dipeptides. Alth ough the cDNA sequence predicted eight sites of heterogeneity among th e filaggrin domains, only one of these was observed. An additional unp redicted site of heterogeneity was also seen. Comparison of the peptid es from filaggrin with those of profilaggrin revealed several peptides unique to filaggrin, specifically at the new amino- and carboxyl-term ini, that result from proteolytic processing of the linker region of p rofilaggrin. Both the amino- and carboxyl-termini were ''ragged'', sug gesting that processing may involve exopeptidase action after an initi al endopeptidase cleavage. The average mass of this mixture of filaggr ins was determined by electrospray mass spectrometry to be 42 452 Da, in reasonable agreement with that predicted from the mass spectrometri c analysis of the terminal sequences. The linker peptide of rat profil aggrin was found in two forms, which differed only in the phosphorylat ion state of serine 22.