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.In the epidermis, the program of keratinocyte terminal differentiation is an oriented process during which cells of the basal layer undergo a series of metabolic and structural changes throughout their migration to the surface of the tissue. The stratum corneum, the outermost layer of the epidermis, is formed by the stacking of so-called corneocytes, the end products of the process. The stratum corneum functions as an effective barrier between the body and its outside environment, limiting skin dehydration and preventing the penetration of outside pathogens, UV radiation, and exogenous chemicals. It also contributes to mechanical protection of the body. So that this function can be achieved, peculiar structures are formed, such as the cornified cell envelope, a resistant and insoluble protein shell that replaces the plasma membrane, and the intracorneocyte fibrous matrix made by the aggregation of keratin intermediate filaments.

Peptidylarginine Deiminases and Protein Deimination in Skin Physiopathology

“…The effect of keratin deimination is not really known, but it is concomitant with and therefore could be involved in the modifications of the intracorneocyte fibrous matrix observed at the ultrastructural level. In addition, PAD1 and/or PAD3 could be involved in the deimination of filaggrin-2, a recently described protein of the S100-fused type protein family that may participate in the formation of the NMF (Hsu et al, 2011). The epidermal targets and the function of PAD2 in the epidermis have not been identified so far.…”

“…It has been found that filaggrin, filaggrin-2, keratin 1 (K1) and keratin 10 (K10) in the epidermis (Senshu et al, 1996;Kamata et al, 2009;Hsu et al, 2011), as well as trichohyalin and S100A3 in the hair follicles are the major deiminated proteins (Rogers et al, 1997;Tarcsa et al, 1996Tarcsa et al, , 1997. The presence of deiminated proteins mainly in the stratum corneum suggests a function for deimination during cornification, the last steps of terminal differentiation of keratinocytes.…”

“…However, filaggrin-2 degradation is strongly suspected to participate to the NMF formation (Hsu et al, 2011). Deimination of both filaggrin and filaggrin-2 increases the rate of their degradation by calpain 1 ad bleomycin hydrolases (Kamata et al, 20009;Hsu et al, 2011). Recently, non-sense mutations of the filaggrin gene have been defined as very strong predisposing factors for atopic dermatitis (OMIM 603165) and causative factors of ichtyosis www.intechopen.com vulgaris (OMIM 146700) Palmer et al, 2006;Smith et al, 2006;Nomura et al, 2007;Sandilands et al, 2007).…”

“…The amino acid content of NMF are nearly all produced from the degradation of filaggrin, their relative proportion being closely related to the filaggrin composition Simon et al, 2008;Kamata et al, 2009). However, filaggrin-2 degradation is strongly suspected to participate to the NMF formation (Hsu et al, 2011). Deimination of both filaggrin and filaggrin-2 increases the rate of their degradation by calpain 1 ad bleomycin hydrolases (Kamata et al, 20009;Hsu et al, 2011).…”

Eosinophilic Esophagitis: A Comprehensive Review

“…Later studies involving genomic wide associations studies (GWAS) have reported associations with variants at the c11orf30 locus (polysensitization of allegen) [41], STAT6 gene (allergic sensitization and serum IgE) [42], and CAPN14 (calcium regulated protease that is highly expressed in esophageal mucosa and is associated with filaggrin) [43]. Future studies will lead to more information of the role of genetics in this complicated interplay of genes and environment.…”