Abstract
A ceramide deficiency in the stratum corneum (SC) is an essential etiologic factor for the dry and barrier-disrupted skin of patients with atopic dermatitis (AD). Previously, we reported that sphingomyelin (SM) deacylase, which hydrolyzes SM and glucosylceramide at the acyl site to yield their lysoforms sphingosylphosphorylcholine (SPC) and glucosylsphingosine, respectively, instead of ceramide and/or acylceramide, is over-expressed in AD skin and results in a ceramide deficiency. Although the enzymatic properties of SM deacylase have been clarified, the enzyme itself remains unidentified. In this study, we purified and characterized SM deacylase from rat skin. The activities of SM deacylase and acid ceramidase (aCDase) were measured using SM and ceramide as substrates by tandem mass spectrometry by monitoring the production of SPC and sphingosine, respectively. Levels of SM deacylase activity from various rat organs were higher in the order of skin > lung > heart. By successive chromatography using Phenyl-5PW, Rotofor, SP-Sepharose, Superdex 200 and Shodex RP18-415, SM deacylase was purified to homogeneity with a single band of an apparent molecular mass of 43 kDa with an enrichment of > 14,000-fold. Analysis by MALDI-TOF MS/MS using a protein spot with SM deacylase activity separated by 2D-SDS-PAGE allowed its amino acid sequence to be determined and identified as the β-subunit of aCDase, which consists of α- and β-subunits linked by amino bonds and a single S-S bond. Western blotting of samples treated with 2-mercaptoethanol revealed that, whereas recombinant human aCDase was recognized by antibodies to the α-subunit at ~56 kDa and ~13 kDa and the β-subunit at ~43 kDa, the purified SM deacylase was detectable only by the antibody to the β-subunit at ~43 kDa. Breaking the S-S bond of recombinant human aCDase with dithiothreitol elicited the activity of SM deacylase with ~40 kDa upon gel chromatography. These results provide new insights into the essential role of SM deacylase expressed as an aCDase-degrading β-subunit that evokes the ceramide deficiency in AD skin.
Highlights
Atopic dermatitis (AD) is characterized pathophysiologically by accentuated cutaneous permeability [1] and deficient water reservoir functions even in the stratum corneum (SC) of non-lesional skin [2]
We succeeded in purifying SM deacylase from rat skin to homogeneity and found that it is identical to the β-subunit of acid ceramidase (aCDase), which consists of α- and β-subunits linked by a single S–S bond, disruption of which by dithiothreitol results in eliciting the activity of SM deacylase
Analysis by MALDI-TOF MS/MS using a protein spot with SM deacylase activity separated by 2D-SDS-PAGE allowed its amino acid sequence to be determined and identified as the β-subunit of aCDase, which consists of α- and β-subunits linked by one disulfide bond (C31/C340)
Summary
Atopic dermatitis (AD) is characterized pathophysiologically by accentuated cutaneous permeability [1] and deficient water reservoir functions even in the stratum corneum (SC) of non-lesional skin [2]. The essential role of the ceramide deficiency in the pathogenesis of AD is corroborated by our recent study demonstrating that repetitive topical applications of a synthetic pseudo-ceramide (pCer) to AD skin significantly improved inflammation and atopic dry skin as well as the SC barrier/water reservoir function by switching the ceramide profile to a healthy skin phenotype [18]. These clinical and functional improvements in the SC can be achieved without any recovery of the decreased levels of total endogenous ceramides but with applied and compensated pCer remaining at a similar level to existing endogenous ceramides. It is intriguing to know what biological factors would trigger the epidermis to down-regulate the synthesis of the SC ceramides in the AD skin
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