Sphingomyelin Deacylase, the Enzyme Involved in the Pathogenesis of Atopic Dermatitis, Is Identical to the β-Subunit of Acid Ceramidase

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.

Cutting Edge of the Pathogenesis of Atopic Dermatitis: Sphingomyelin Deacylase, the Enzyme Involved in Its Ceramide Deficiency, Plays a Pivotal Role

“…These results confirmed that the single protein spot with SM deacylase activity separated by 2D-SDS-PAGE is identical to the β-subunit of aCDase. This identification was also corroborated by a gel chromatographic analysis demonstrating that breaking the disulfide bond (C31/C340) of recombinant human aCDase with the reducing agent dithiothreitol (DTT) provokes the activity of SM deacylase with ≈40 kDa upon gel chromatography ( Figure 31) [71]. ------------------------------------------------------------------------------------ In Western blotting analysis using our novel β-subunit specific antibodies under reduced or nonreduced conditions, the SM deacylase purified from rat skin had a distinct band of ≈40 kDa (Lane 1) which was consistent with the band detected for recombinant human aCDase (Lane 2) under reduced conditions (ME+) ( Figure 30).…”

“…Lane 1, purified rat SM deacylase (ME+); Lane 2, recombinant human aCDase (ME+); Lane 3, recombinant human aCDase (ME-). Lane 4, mock transfected (ME-); Lane 5, separated recombinant β-subunit of human aCDase (ME-) [71]. Figure 31.…”

“…To precisely measure SM deacylase activity, we developed a highly sensitive LC-MS/MS method which has the capacity to accurately measure the reaction product, SPC, over five orders of magnitude [ 71 ]. Since there was a distinct activity of SM deacylase in extracts after the centrifugation of homogenates of normal rat skin, we purified SM deacylase to homogeneity (a single spot by 2D-SDS-PAGE) ( Figure 28 ) after a five-step purification procedure consisting of hydrophobic chromatography, IEF chromatography, ion-exchange chromatography, gel filtration chromatography, and immune-affinity chromatography.…”

“…Since there was a distinct activity of SM deacylase in extracts after the centrifugation of homogenates of normal rat skin, we purified SM deacylase to homogeneity (a single spot by 2D-SDS-PAGE) ( Figure 28 ) after a five-step purification procedure consisting of hydrophobic chromatography, IEF chromatography, ion-exchange chromatography, gel filtration chromatography, and immune-affinity chromatography. The purified SM deacylase had an apparent molecular mass of 43 kDa, an enrichment of >14,000-fold, and maximal pH and pI values of 5.0 and around 7.0, respectively [ 71 ].…”

“…The purified SM deacylase followed normal Michaelis–Menten kinetics with V max and K m of 14.1 nmol/mg/h and 110.5 µM, respectively [ 71 ]. These enzymatic properties of pH dependency and molecular weight are consistent with those properties (pH = 4.7, MW = 40 kDa) obtained in our previous study of AD skin [ 55 ].…”