Neutral Cysteine Protease Bleomycin Hydrolase Is Essential for the Breakdown of Deiminated Filaggrin into Amino Acids

Abstract

Filaggrin is a component of the cornified cell envelope and the precursor of free amino acids acting as a natural moisturizing factor in the stratum corneum. Deimination is critical for the degradation of filaggrin into free amino acids. In this study, we tried to identify the enzyme(s) responsible for the cleavage of deiminated filaggrin in vitro. First, we investigated citrulline aminopeptidase activity in the extract of newborn rat epidermis by double layer fluorescent zymography and detected strong activity at neutral pH. Monitoring the citrulline-releasing activity, we purified an enzyme of 280 kDa, comprised of six identical subunits of 48 kDa. The NH 2 terminus of representative tryptic peptides perfectly matched the sequence of rat bleomycin hydrolase (BH). The enzyme released various amino acids except Pro from ␤-naphthylamide derivatives and hydrolyzed citrulline-␤-naphthylamide most effectively. Thus, to break down deiminated filaggrin, another protease would be required. Among proteases tested, calpain I degraded the deiminated filaggrin effectively into many peptides of different mass on the matrix-assisted laser desorption/ionization-time of flight mass spectrum. We confirmed that various amino acids including citrulline were released by BH from those peptides. On the other hand, caspase 14 degraded deiminated filaggrin into a few peptides of limited mass. Immunohistochemical analysis of normal human skin revealed co-localization of BH and filaggrin in the granular layer. Collectively, our results suggest that BH is essential for the synthesis of natural moisturizing factors and that calpain I would play a role as an upstream protease in the degradation of filaggrin.The mammalian epidermal keratinocytes arise from proliferating basal cells and move outward through a series of distinct differentiation events to form the stratum corneum (1, 2). During this progressive epidermal differentiation, keratinocytes express different proteins such as keratins, profilaggrin/filaggrin, involucrin, small proline-rich proteins, loricrin, cystatin A, and elafin, which form the cornified envelope of mature corneocytes (3-7). Profilaggrin is synthesized as a large, extremely insoluble phosphoprotein that consists of a unique NH 2 -terminal Ca 2ϩ -binding protein of the S-100 family, linked to 10 -20 tandem filaggrin monomer repeats (8 -10). Each individual filaggrin repeat is completely removed by proteolysis to generate the mature filaggrin monomer (a molecular mass of 37 kDa in human). Then, filaggrin is completely degraded in the uppermost layer of the stratum corneum to produce a mixture of free and modified hygroscopic amino acids that are important for maintaining epidermal hydration (2, 11-13). In addition, a number of proteins are subjected to various post-translational modifications such as disulfide bonding, N-(␥-glutamyl)-lysine isopeptide cross-linking, and deimination during the terminal differentiation of epidermal keratinocytes (4, 6, 14, 15). Deimination is catalyzed by peptidylarginine deiminase (P...

Kallikrein-related Peptidase-7 Regulates Caspase-14 Maturation during Keratinocyte Terminal Differentiation by Generating an Intermediate Form

“…Many of the KLKs are trypsinlike serine proteases, although some are chymotrypsin-like (KLK3, KLK7, and KLK9) or mixed type (KLK11 and KLK14) (18). Calpain I was chosen as a representative of cysteine proteases and an interactant with caspase-14 in the natural moisturizing factor (NMF) generation pathway (19). Aspartic proteases and metalloproteases were not included, because processing at acidic pH is unlikely and the latter would not be expressed in SG.…”

Bleomycin Hydrolase Is Regulated Biphasically in a Differentiation- and Cytokine-dependent Manner

“…Cytokine-mediated Regulation of BH Gene Expression-Because BH is an NMF-generating enzyme (12), it may be involved in the pathophysiology of AD. Thus, we examined the effects of Th1, Th2, and Th17 cytokines on BH gene expression.…”

“…NMF consist primarily of amino acids, including citrulline, and their derivatives, such as pyrrolidone carboxylic acid and urocanic acids, together with lactic acid, urea, citrate, and sugars (9 -11). Previously, we identified the neutral cysteine protease bleomycin hydrolase (BH) as an NMF-generating enzyme and also showed that calpain I is an upstream protease in the filaggrin degradation pathway (12). BH inactivates the anti-tumor antibiotic bleomycin by hydrolyzing the amide bond in the ␤-aminoalanine moiety.…”

“…Although BH is widely expressed in mammalian tissues (13-16), skin seems to contain the highest amount, and relatively high levels of BH were detected in the kidney and liver of 6-week-old male rats (16). BH exhibits mainly broad specificity aminopeptidase activity, with citrulline being the most effective substrate (12,14). It is involved in homocysteinethiolactone metabolism (17), antigen presentation (18,19), and amyloid-␤-peptide clearance (20,21).…”

La désimination ou citrullination

“…La dégradation des monomères de filaggrine est alors assurée par une cascade de protéases faisant intervenir successivement la caspase 14, la calpaïne I et la bléomycine hydrolase. Ce processus aboutit à la production d'un pool d'acides aminés, constituants majeurs du facteur naturel d'hydratation (FNH) [5] (Figure 2C-D). Récemment, il a été montré que des mutations non-sens du gène FLG, codant la profilaggrine, sont responsables de l'ichtyose vulgaire 1 et constituent un facteur de forte prédisposition à la dermatite atopique (appelée communément eczéma atopique).…”