title: Identification of the amino terminus of human filaggrin using differential LC/MS techniques: implications for profilaggrin processing.
abstract: Filaggrin, the intermediate filament aggregating protein of epidermis, is the product of proteolytic processing of the precursor profilaggrin, which consists of 10-20 tandem filaggrin domains. The proteolytic processing sites in mouse and rat profilaggrin have been previously reported. Mouse filaggrin is N-terminally blocked. Rat filaggrin is N-terminally ragged, making it heterogeneous. Human filaggrin, in addition to being N-terminally blocked and potentially ragged at the amino terminus, is heterogeneous due to sequence variation between one filaggrin domain and another along the profilaggrin gene. This complexity has made more difficult the analysis of processing sites in human profilaggrin. We have identified the amino terminus of human filaggrin by applying a general method we have developed for the recognition of amino-terminal peptides in digests of N-terminally blocked proteins. This method compares the peptides in an acetylated and an unacetylated tryptic digest of the protein during their separation by liquid chromatography on-line with electrospray mass spectrometry. In this comparison only the original blocked amino-terminal peptides appear unchanged between the two profiles. Human filaggrin was found to have a heterogeneous N-terminus, as a result both of sequence heterogeneity and of ragged processing; it is blocked by a pyrrolidonecarboxyl group derived from glutamine. By comparison to the termini of rat and mouse filaggrins, implications for the processing of human profilaggrin are discussed.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle 98195, USA.
authors.forename: C D
authors.initials: CD
authors.lastname: Thulin
authors.forename: K A
authors.initials: KA
authors.lastname: Walsh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004817:Epidermis / Q000737:chemistry; D000091344:Filaggrin Proteins; D006801:Humans; D007381:Intermediate Filament Proteins / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats
chemical_list: C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; D011498:Protein Precursors