title: Deimination of human filaggrin-2 promotes its proteolysis by calpain 1.
abstract: Filaggrin-2 (FLG2), a member of the S100-fused type protein family, shares numerous features with filaggrin (FLG), a key protein implicated in the epidermal barrier functions. Both display a related structural organization, an identical pattern of expression and localization in human epidermis, and proteolytic processing of a large precursor. Here, we tested whether FLG2 was a substrate of calpain 1, a calcium-dependent protease directly involved in FLG catabolism. In addition, deimination being critical for FLG degradation, we analyzed whether FLG2 deimination interfered with its proteolytic processing. With this aim, we first produced a recombinant form of FLG2 corresponding to subunits B7 to B10 fused to a COOH-terminal His tag. Incubation with calpain 1 in the presence of calcium induced a rapid degradation of the recombinant protein and the production of several peptides, as shown by Coomassie Blue-stained gels and Western blotting with anti-FLG2 or anti-His antibodies. MALDI-TOF mass spectrometry confirmed this result and further evidenced the production of non-immunoreactive smaller peptides. The degradation was not observed when a calpain 1-specific inhibitor was added. The calpain cleavage sites identified by Edman degradation were regularly present in the B-type repeats of FLG2. Moreover, immunohistochemical analysis of normal human skin revealed colocalization of FLG2 and calpain 1 in the upper epidermis. Finally, the FLG2 deiminated by human peptidylarginine deiminases was shown to be more susceptible to calpain 1 than the unmodified protein. Altogether, these data demonstrate that calpain 1 is essential for the proteolytic processing of FLG2 and that deimination accelerates this process.
authors.affiliation: UMR5165 CNRS, F-31059 Toulouse, France.
authors.forename: Chiung-Yueh
authors.initials: CY
authors.lastname: Hsu
authors.forename: Julie
authors.initials: J
authors.lastname: Henry
authors.forename: Anne-Aurélie
authors.initials: AA
authors.lastname: Raymond
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Méchin
authors.forename: Valérie
authors.initials: V
authors.lastname: Pendaries
authors.forename: Dany
authors.initials: D
authors.lastname: Nassar
authors.forename: Britta
authors.initials: B
authors.lastname: Hansmann
authors.forename: Stéfana
authors.initials: S
authors.lastname: Balica
authors.forename: Odile
authors.initials: O
authors.lastname: Burlet-Schiltz
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Schmitt
authors.forename: Hidenari
authors.initials: H
authors.lastname: Takahara
authors.forename: Carle
authors.initials: C
authors.lastname: Paul
authors.forename: Guy
authors.initials: G
authors.lastname: Serre
authors.forename: Michel
authors.initials: M
authors.lastname: Simon
mesh_terms: D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002154:Calpain / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004817:Epidermis / Q000737:chemistry / Q000378:metabolism*; D000091344:Filaggrin Proteins; D006801:Humans; D007381:Intermediate Filament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C000717249:FLG protein, human; C000717250:FLG2 protein, human; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; D011994:Recombinant Proteins; D002154:Calpain; D002118:Calcium