Neutral cysteine protease bleomycin hydrolase is essential for the breakdown of deiminated filaggrin into amino acids.
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 beta-naphthylamide derivatives and hydrolyzed citrulline-beta-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.
Kamata
Yayoi
Y
Laboratory of Biochemistry, Graduate School of Nutritional Sciences, Sagami Women's University, Sagamihara, Kanagawa, Japan.
Taniguchi
Aya
A
Yamamoto
Mami
M
Nomura
Junko
J
Ishihara
Kazuhiko
K
Takahara
Hidenari
H
Hibino
Toshihiko
T
Takeda
Atsushi
A
D000596:Amino Acids / Q000378:metabolism*; D000626:Aminopeptidases / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D002154:Calpain / Q000378:metabolism; D053456:Caspase 14 / Q000378:metabolism; D002956:Citrulline / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D000091344:Filaggrin Proteins; D006801:Humans; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism; D007097:Imines / Q000737:chemistry / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D000080063:Protein-Arginine Deiminase Type 3; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012867:Skin / Q000201:enzymology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000596:Amino Acids; C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D007097:Imines; D007381:Intermediate Filament Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D002956:Citrulline; D006867:Hydrolases; D000626:Aminopeptidases; D002154:Calpain; D053456:Caspase 14; D003546:Cysteine Endopeptidases; C031262:bleomycin hydrolase; D000080063:Protein-Arginine Deiminase Type 3