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Background ABHD5 mutations cause Chanarin–Dorfman syndrome accompanied by ichthyosis. ω- O -Acylceramide (acylceramide) is essential for skin permeability barrier formation. Acylceramide production is impaired in Abhd5 knockout mice. The transacylase PNPLA1 catalyzes the final step of acylceramide production: transfer of linoleic acid in triglyceride to ω-hydroxyceramide. Objective We aimed to elucidate the role of ABHD5 in acylceramide production and the molecular mechanism of the ichthyosis symptoms of Chanarin–Dorfman syndrome. Methods We investigated how ABHD5 influences acylceramide production using an acylceramide-producing cell system. The effects of ABHD5 and PNPLA1 expression on the morphology of lipid droplets were examined by indirect immunofluorescent microscopy and immunoelectron microscopy. Results When ABHD5 was expressed in the acylceramide-producing cell system, acylceramide synthesis by PNPLA1 was enhanced. Dispersed localization of PNPLA1 was observed by immunofluorescent microscopy in HeLa cells under lipid droplet-forming conditions. Co-expression with ABHD5 caused PNPLA1 to localize on the lipid droplet membranes or their periphery. This staining pattern was observed in cells where PNPLA1 and ABHD5 were expressed at low levels. In contrast, lipid droplets disappeared in cells where PNPLA1 and ABHD5 were highly expressed. Immunoelectron microscopic analyses suggested that lipid droplets underwent morphological changes, transforming into vesicles or becoming incorporated into the endoplasmic reticulum. ABHD5 mutations found in Chanarin–Dorfman syndrome patients reduced ABHD5’s ability to promote PNPLA1-dependent acylceramide production. Conclusion ABHD5 enhances PNPLA1-catalyzed acylceramide production. We speculate that ABHD5 retains triglycerides in the endoplasmic reticulum, and presents them to PNPLA1 to promote substrate recognition.

Lipids are the primary components of the skin permeability barrier, which is the body's most powerful defensive mechanism against pathogens. Acylceramide (ω-O-acylceramide) is a specialized lipid essential for skin barrier formation. Here, we identify PNPLA1 as the long-sought gene involved in the final step of acylceramide synthesis, esterification of ω-hydroxyceramide with linoleic acid, by cell-based assays. We show that increasing triglyceride levels by overproduction of the diacylglycerol acyltransferase DGAT2 stimulates acylceramide production, suggesting that triglyceride may act as a linoleic acid donor. Indeed, the in vitro analyses confirm that PNPLA1 catalyses acylceramide synthesis using triglyceride as a substrate. Mutant forms of PNPLA1 found in patients with ichthyosis exhibit reduced or no enzyme activity in either cell-based or in vitro assays. Altogether, our results indicate that PNPLA1 is directly involved in acylceramide synthesis as a transacylase, and provide important insights into the molecular mechanisms of skin barrier formation and of ichthyosis pathogenesis., Loss-of-function mutations in an enzyme of unknown function, PNPLA1, cause dry and scaling skin in humans. Here Ohno et al. show that PNPLA1 is a transacylase that acts in the final step of acylceramide production- esterification between ω-hydroxyceramide and linoleic acid acylceramide, yielding a lipid essential for skin barrier function.

A skin permeability barrier is essential for terrestrial animals, and its impairment causes several cutaneous disorders such as ichthyosis and atopic dermatitis. Although acylceramide is an important lipid for the skin permeability barrier, details of its production have yet to be determined, leaving the molecular mechanism of skin permeability barrier formation unclear. Here we identified the cytochrome P450 gene CYP4F22 (cytochrome P450, family 4, subfamily F, polypeptide 22) as the long-sought fatty acid ω-hydroxylase gene required for acylceramide production. CYP4F22 has been identified as one of the autosomal recessive congenital ichthyosis-causative genes. Ichthyosis-mutant proteins exhibited reduced enzyme activity, indicating correlation between activity and pathology. Furthermore, lipid analysis of a patient with ichthyosis showed a drastic decrease in acylceramide production. We determined that CYP4F22 was a type I membrane protein that locates in the endoplasmic reticulum (ER), suggesting that the ω-hydroxylation occurs on the cytoplasmic side of the ER. The preferred substrate of the CYP4F22 was fatty acids with a carbon chain length of 28 or more (≥C28). In conclusion, our findings demonstrate that CYP4F22 is an ultra-long-chain fatty acid ω-hydroxylase responsible for acylceramide production and provide important insights into the molecular mechanisms of skin permeability barrier formation. Furthermore, based on the results obtained here, we proposed a detailed reaction series for acylceramide production.