0023-6837/03/8303-397$03.00/0 LABORATORY INVESTIGATION Vol. 83, No. 3, p. 397, 2003 Copyright © 2003 by The United States and Canadian Academy of Pathology, Inc. Printed in U.S.A. Abnormal Expression of the Novel Epidermal Enzyme, Glucosylceramide Deacylase, and the Accumulation of its Enzymatic Reaction Product, Glucosylsphingosine, in the Skin of Patients with Atopic Dermatitis Mutsumi Ishibashi, Junko Arikawa, Reiko Okamoto, Makoto Kawashima, Yutaka Takagi, Kenji Ohguchi, and Genji Imokawa Department of Dermatology (MI, JA, MK, GI), Tokyo Women’s Medical University, Tokyo, and Kao Biological Science Laboratories (YT, KO, GI), Tochigi, Japan 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-14C] 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 14C-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). deficiency of ceramides in the stratum corneum is common substrate SM (Hara et al, 2000; Murata et al, A an causative factor of the barrier-disrupted and dry 1996). Recently, it was reported that omega-OH cer- skin of patients with atopic dermatitis (AD) (Imokawa et amide, an important acylceramide precursor, does not al, 1991). This dysfunction of the stratum corneum leads derive from SM in the mammalian stratum corneum, to a high vulnerability to irritants or allergens, which which suggests that it derives solely from glucosylcer- results in the induction, recurrence, or refractory nature amide (GC) precursors (Uchida et al, 2000). Thus, it is of the dermatitis. Thus, it is intriguing to determine the likely that acylceramides are biosynthesized not by the biochemical mechanism(s) underlying the ceramide de- hydrolysis of SM but through pathways involving the ficiency because it may be linked to the physiopatho- deglucosylation of acylglucosylceramides by genesis of AD. In the epidermis of patients with AD, we ␤-glucocerebrosidase (GlCdase). On the basis of these have previously demonstrated the accentuated expres- findings, the acylceramide deficiency observed in AD sion of a hitherto undiscovered epidermal enzyme, could not be explained in terms of the up-regulation of termed sphingomyelin (SM) deacylase, which was asso- SM deacylase. As a resolution for this discrepancy, we ciated with the reduced ceramide mass in the stratum hypothesized the existence of a novel epidermal en- corneum as a result of its competition with the ceramide- zyme, termed here GC deacylase, which hydrolyzes producing enzyme sphingomyelinase (SMase) for the (acyl)GC at the N-acyl site to yield its lysoform, glucosyl- sphingosine (GS), instead of the formation of (acyl)cer- amides by GlCdase. DOI: 10.1097/01.LAB.0000059931.66821.92 In this study, using [palmitic acid-14C] GC as a Received November 7, 2002. substrate, we examined the activity of GC deacylase This study was performed as part of a research project on the Control of in the stratum corneum and in the epidermis of pa- Atopic Dermatitis by Skin Care: Regulatory Factor of Cutaneous Perme- tients with AD and compared that with levels found in ability, and was supported by the special coordination funds from the healthy controls. As additional confirmation of the ministry of education, culture, sports, science, and technology of the Japanese functional relevance of GC deacylase to the acylcer- Government. Present address for Dr. Kenji Ohguchi, Gifu International amide deficiency, we quantified the amount (per mil- Institute of Biotechnology, Mitake, Kani-gun, Gifu 505-0116, Japan. Address reprint requests to: Dr. Genji Imokawa, Kao Biological Science ligram of stratum corneum) of the metabolic product Laboratories, 2606 Akabane, Ichikai-machi, Haga, Tochigi 321-34 Ja- GS produced by GC deacylase in the stratum cor- pan. E-mail: [email protected] neum at the nanomolar level. Here we report that Laboratory Investigation • March 2003 • Volume 83 • Number 3 397 Ishibashi et al extracts from the stratum corneum and from the epidermis of patients with AD have the potential to hydrolyze GC at the N-acyl site to release 14C-labeled free fatty acid. There are significantly higher levels of this enzymatic property in skin with AD than in healthy controls. Consistent with the up-regulation of GC deacylase in skin with AD, there was a significant increase in the content of GS, its enzymatic reaction product, in both the involved and the uninvolved stratum corneum from patients with AD compared with healthy controls. The up-regulation of GC deacy- lase and its metabolic product GS in skin with AD strongly suggests the physiologic relevance of GC deacylase to the acylceramide deficiency in the stra- tum corneum of patients with AD. Results Distinction of GC Deacylase from GlCdase In assays to measure the release of [1-14C] palmitic acid as a criterion for GC deacylase activity, the released palmitic acid would not necessarily represent the activity of GC deacylase if both GlCdase and ceramidase (CDase) are present in extracts of the stratum corneum from patients with AD. This is be- Figure 1. cause the release of radiolabeled palmitic acid could Distinction of glucosylceramide (GC) deacylase from ␤-glucocerebrosidase also result from a two-step reaction sequence wherein (GlCdase). A, Chromatographic separation of GC deacylase and GlCdase using GC would be initially hydrolyzed by GlCdase to yield extracts of the stratum corneum from patients with atopic dermatitis (AD). An radiolabeled palmitoylsphingosine (ceramide), which extract of the stratum corneum was applied to CM-Sepharose equilibrated with in turn could be converted by acid CDase to radiola- elution buffer consisting of acetate buffer (pH 5.0 and 0.2 M NaCl) to obtain GC deacylase as the pass-through fraction; the column was then eluted with 0.5 beled palmitic acid. Thus, to determine whether the M NaCl to yield GlCdase. B, GC deacylase and GlCdase activities of fractions release of radiolabeled palmitic acid reflects the direct separated by CM Sepharose. Enzymatic activities were evaluated for each deacylation of GC or the two-step reaction sequence eluted fraction according to the methods described in “Materials and Meth- of deglucosylation and subsequent deacylation, we ods,” and results are expressed as picomoles of free fatty acid (FFA) or separated GC deacylase from GlCdase using cationic ceramide (CER) released in the final or first upper phase per fraction. exchange chromatography. This was based on their different pI values, presumably near 4.2 versus 8.0 GlCdase and GC Deacylase Activities in the (Higuchi et al, 2000), respectively. The release of Stratum Corneum radiolabeled free fatty acid and ceramide was then 14 characterized. Chromatographic analysis (Fig. 1A) re- Assays using [palmitic acid- C] GC as a substrate for vealed that whereas the unadsorbed fraction was able GlCdase (Fig. 2A) demonstrated that there were no to release radiolabeled free fatty acid but not cer- significant differences in GlCdase activity between the amide, the adsorbed fraction eluted by an increase in stratum corneum from uninvolved or involved skin of salt concentration was able to release radiolabeled patients with AD and from healthy controls. In con- 14 ceramide but not free fatty acid. Analysis of radiola- trast, assays using [palmitic acid- C] GC as a sub- beled ceramide or free fatty acid released by the crude strate for GC deacylase (Fig. 2B) revealed that the extract and by unadsorbed fractions from the stratum stratum corneum from involved forearm skin of pa- corneum of five patients with AD (Fig. 1B) demon- tients with AD has an extremely high level of GC strated that whereas the crude extracts were able to deacylase activity that is 5.4 times higher than that in release both radiolabeled ceramide and free fatty acid, healthy controls. In uninvolved skin with AD, GC the unadsorbed fraction was able to release radiola- deacylase activity is still 2.8 times higher than that beled free fatty acid to an extent similar to the crude found in healthy controls. In contrast, the stratum extract but was not able to release radiolabeled cer- corneum from patients with chronic eczema shows amide. This suggests that despite that the unadsorbed levels similar for both enzyme activities to healthy fractions have been depleted of GlCdase, they still controls (Fig. 3). contain GC deacylase. This strongly suggests that the GC deacylase activity seen in crude extracts from the GC Deacylase in the Epidermis of Patients with AD stratum corneum of patients with AD is mainly attrib- utable to the direct deacylation of GC but not the Assays using [palmitic acid-14C] GC as a substrate for two-step reaction sequence consisting of deglucosy- GlCdase (Fig. 4A) demonstrated that there was no lation and subsequent deacylation. significant difference in GlCdase activity between the 398 Laboratory Investigation • March 2003 • Volume 83 • Number 3 Glucosylceramide Deacylase and Glucosylsphingosine in Atopic Dermatitis Figure 2.