We have previously demonstrated that there is abnormal expression of sphingomyelin (SM) deacylase-like enzyme in the epidermis of patients with atopic dermatitis (AD), which results in decreased levels of ceramides in their involved and uninvolved stratum corneum. For quantitation of the expression of SM deacylase in AD, we synthesized 16-(9-anthroyloxy) hexadecanoylsphingosylphosphorylcholine or [palmitic acid-14C] SM and used them as substrates to directly measure the activity of SM deacylase by detecting the release of labeled free fatty acid. Direct enzymatic measurements demonstrated that stratum corneum from lesional forearm skin (volar side) of AD patients has an extremely high SM deacylase activity that is at least five times higher than in the stratum corneum from healthy controls. In stratum corneum from nonlesional skin of AD patients, SM deacylase activity is still at least three times higher than in healthy controls. In contrast, stratum corneum from contact dermatitis patients shows levels of SM deacylase similar to healthy controls. In extracts of whole epidermis biopsies from AD patients, SM deacylase activities are significantly (3-fold) increased over healthy controls in the particulate fraction, whereas there is no significant difference in the activity of sphingomyelinase between AD and healthy control. In peripheral blood lymphocytes of AD patients, there is no increase in activity compared with healthy controls, indicating a possibility that the high expression of SM deacylase is highly associated with the skin of AD patients. These findings suggest that, in contrast to changes in sphingolipid metabolism due to aging, the hitherto undiscovered enzyme SM deacylase, is highly expressed in the epidermis of AD patients, and competes with sphingomyelinase or beta-glucocerebrosidase for the common substrate SM or glucosylceramide, which leads to the ceramide deficiency of the stratum corneum in AD.

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We have demonstrated previously that there is an abnormal expression of sphingomyelin (SM) deacylase in the epidermis of patients with atopic dermatitis (ADe). In the present study, we have prepared N-[palmitic acid-1-(14)C]SM and N-[palmitic acid-1-(14)C]glucosylceramide (GCer) to use as substrates and have quantified SM deacylase activity by detecting the release of [(14)C]palmitic acid in extracts of the stratum corneum or the epidermis of ADe patients. In studies using [palmitic acid-1-(14)C]SM as a substrate, a pH dependency of catalytic activity with a peak at pH 5.0 was found. Preparative SDS/PAGE using an extract of ADe epidermis revealed that the molecular mass of SM deacylase is 40000 Da, which is consistent with its apparent molecular mass of 42000 Da estimated by gel-filtration analysis of stratum corneum extracts. Analytical isoelectric focusing (IEF) chromatography demonstrated that the pI values of SM deacylase, beta-glucocerebrosidase (GlcCDase), sphingomyelinase (SMase) and acid ceramidase were 4.2, 7.4, 7.0 and 5.7, respectively. In enzymic analysis using pI-4.2 SM deacylase partially purified by IEF, which had no detectable contamination with acid ceramidase, GlcCDase or SMase, radio-TLC analysis revealed that radiolabelled sphingosylphosphocholine or [1-(14)C]palmitic acid was enzymically liberated from [choline-methyl-(14)C]SM or N-[palmitoyl-1-(14)C]GCer, respectively, used as substrates. Further the pI-4.2 protein purified from extracts of the stratum corneum of ADe patients was able to hydrolyse N-[palmitoyl-1-(14)C]SM and GCer, but not N-[palmitoyl-1-(14)C]ceramide. These results indicate that a hitherto undiscovered epidermal enzyme, termed here glucosylceramide sphingomyelin deacylase, is expressed in the skin of ADe patients, which plays an important role in ceramide deficiency (including acylceramides) in the stratum corneum.

To clarify the functional relevance of sphingomyelin (SM) deacylase to the ceramide deficiency seen in atopic dermatitis (AD), we developed a new highly sensitive method and measured the metabolic intermediate sphingosylphosphorylcholine (SPC) that accumulates in the stratum corneum. SPC in intercellular lipids extracted from stratum corneum was reacted with [ 14 C]acetic anhydride to yield [ 14 C-C 2 ]SM, which was then analyzed by TLC. In both the lesional and non-lesional stratum corneum obtained from patients with AD, there was a significant increase in the content of SPC over that of healthy control subjects. There was a reciprocal relationship between increases in SPC and decreases in ceramide levels of stratum corneum obtained from healthy controls, and from lesional and non-lesional skin from patients with AD. Comparison with other sphingolipids present in the stratum corneum demonstrated that there is a significant positive correlation between SPC and glucosylsphingosine, another lysosphingolipid derived from glucosylceramide by another novel epidermal enzyme, termed glucosylceramide deacylase. In contrast, there was no correlation between SPC and sphingosine, a degradative product generated from ceramide by ceramidase. These findings strongly suggest the physiological relevance of SM deacylase function in vivo to the ceramide deficiency found in the skin of patients with AD. Intercellular lipids in the stratum corneum have been implicated as important determinants in the water-retaining properties (1, 2) and in the barrier function (3, 4) of the skin. Ceramides are major constituents of intercellular lipids, comprising more than 50% of them. Previously, we demonstrated that there is a marked reduction in the amount of ceramides in the stratum corneum in lesional and in non-lesional forearm skin of patients with atopic dermatitis (AD) (5), which suggested that the ceramide deficiency is an important etiologic factor for the barrierdisrupted and dry skin seen in AD. Subsequently, we found that the causative factor behind the ceramide deficiency in the stratum corneum of patients with AD is an abnormal expression of sphingomyelin (SM) deacylase in their epidermis (6). This enzyme hydrolyzes SM at the acyl site to yield free fatty acid and sphingosylphosphorylcholine (SPC) instead of the formation of ceramide and phosphorylcholine (PC) by sphingomyelinase (SMase). Direct enzymatic analysis of the stratum corneum or of the epidermis of patients with AD revealed that there are 9-fold or 3-fold increases, respectively, in the activity of SM deacylase in patients with AD compared with healthy normal controls (7). The sum of those findings demonstrates that the novel epidermal enzyme SM deacylase is expressed at high levels in the epidermis of patients with AD. The competition of this enzyme with SMase for the common substrate SM leads to the ceramide deficiency in the stratum corneum of patients with AD. Thus in this study, in order to clarify the physiologic and functional relevance of SM deacylase t...

SUMMARY:To clarify mechanisms underlying acylceramide deficiency as an causative factor of the permeability barrier disruption seen in the skin of patients with atopic dermatitis (AD), we hypothesized and then demonstrated the presence of a novel epidermal enzyme, termed glucosylceramide (GC) deacylase. This enzyme hydrolyzes (acyl)GC at the N-acyl site to yield its lysoform, glucosylsphingosine (GS), instead of the formation of (acyl)ceramides by ␤-glucocerebrosidase. Assays of enzymatic activity using [palmitic acid-14 C] GC as a substrate revealed that extracts from the stratum corneum and from the epidermis (but not from the dermis) of patients with AD have the significantly higher potential to hydrolyze GC at the N-acyl site to release 14 C-labeled free fatty acid than of healthy controls. To determine the in vivo physiologic function of this novel enzyme, we measured the metabolic product GS in the upper stratum corneum. In both the involved and the uninvolved stratum corneum from patients with AD, there were significant increases in the amounts of GS compared with healthy controls and there was a significant inverse correlation with the decreased content of ceramides or ceramide-1 (acylceramide). Thus, collectively these results strongly suggest the physiologic relevance of GC deacylase to the acylceramide deficiency seen in the stratum corneum of patients with AD. (Lab Invest 2003, 83:397-408).A deficiency of ceramides in the stratum corneum is an causative factor of the barrier-disrupted and dry skin of patients with atopic dermatitis (AD) (Imokawa et al, 1991). This dysfunction of the stratum corneum leads to a high vulnerability to irritants or allergens, which results in the induction, recurrence, or refractory nature of the dermatitis. Thus, it is intriguing to determine the biochemical mechanism(s) underlying the ceramide deficiency because it may be linked to the physiopathogenesis of AD. In the epidermis of patients with AD, we have previously demonstrated the accentuated expression of a hitherto undiscovered epidermal enzyme, termed sphingomyelin (SM) deacylase, which was associated with the reduced ceramide mass in the stratum corneum as a result of its competition with the ceramideproducing enzyme sphingomyelinase (SMase) for the common substrate SM Murata et al, 1996). Recently, it was reported that omega-OH ceramide, an important acylceramide precursor, does not derive from SM in the mammalian stratum corneum, which suggests that it derives solely from glucosylceramide (GC) precursors (Uchida et al, 2000). Thus, it is likely that acylceramides are biosynthesized not by the hydrolysis of SM but through pathways involving the deglucosylation of acylglucosylceramides by ␤-glucocerebrosidase (GlCdase). On the basis of these findings, the acylceramide deficiency observed in AD could not be explained in terms of the up-regulation of SM deacylase. As a resolution for this discrepancy, we hypothesized the existence of a novel epidermal enzyme, termed here GC deacylase, which hydrolyzes (acyl)GC at t...