2-Hydroxy-ceramide synthesis by ceramide synthase family: enzymatic basis for the preference of FA chain length

Akio Kihara,Hiromasa Tojo,Hiroko Chiba,Yukiko Mizutani,Yasuyuki Igarashi

doi:10.1194/jlr.m800158-jlr200

Akio Kihara, Hiromasa Tojo + Show 3 more

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https://doi.org/10.1194/jlr.m800158-jlr200

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Abstract

Ceramide is unusually abundant in epidermal stratum corneum and is important for permeability barrier function. Ceramides in epidermis also comprise an unusual variety, including 2-hydroxy (alpha-hydroxy)-ceramide. Six mammalian ceramide synthase/longevity assurance homologue (CerS/LASS) family members have been identified as synthases responsible for ceramide (CER) production. We reveal here that of the six, CerS3/LASS3 mRNA is the most predominantly expressed in keratinocytes. Moreover, its expression is increased upon differentiation. CerS family members have known substrate specificities for fatty acyl-CoA chain length and saturation, yet their abilities to produce 2-hydroxy-CER have not been examined. In the present study, we demonstrate that all CerS members can produce 2-hydroxy-CER when overproduced in HEK 293T cells. Each produced a 2-hydroxy-CER with a chain length similar to that of the respective nonhydroxy-CER produced. In HeLa cells overproducing the FA 2-hydroxylase FA2H, knock-down of CerS2 resulted in a reduction in total long-chain 2-hydroxy-CERs, confirming enzyme substrate specificity for chain length. In vitro CerS assays confirmed the ability of CerS1 to utilize 2-hydroxy-stearoyl-CoA as a substrate. These results suggest that all CerS members can synthesize 2-hydroxy-CER with specificity for 2-hydroxy-fatty acyl-CoA chain length and that CerS3 may be important in CER and 2-hydroxy-CER synthesis in epidermis.

Highlights

Ceramide is unusually abundant in epidermal stratum corneum and is important for permeability barrier function
We previously demonstrated that human primary keratinocytes produce such unique CERs and, when differentiated in vitro to an advanced stage, express mRNA for the phytoSph, producing C4-hydroxylase DES2 [21]
To investigate whether ceramide synthase (CerS) family members might have a role in the production of these unique CERs, we examined the expression of CerS family members during keratinocyte differentiation using real-time quantitative PCR

Summary

Introduction

Ceramide is unusually abundant in epidermal stratum corneum and is important for permeability barrier function. CerS family members have known substrate specificities for fatty acylCoA chain length and saturation, yet their abilities to produce 2-hydroxy-CER have not been examined. In vitro CerS assays confirmed the ability of CerS1 to utilize 2-hydroxy-stearoyl-CoA as a substrate These results suggest that all CerS members can synthesize 2-hydroxy-CER with specificity for 2-hydroxy-fatty acylCoA chain length and that CerS3 may be important in CER and 2-hydroxy-CER synthesis in epidermis.—Mizutani, Y., A. FAs in CER are typically nonhydroxylated, in certain tissues, including skin and brain, a-hydroxy (2-hydroxy) FAs are abundant [5, 6] In these tissues, 2hydroxy-FA is converted from FFA by the hydroxylase FA2H [7, 8], which is known to be induced during keratinocyte differentiation [9]. This study was performed through special coordination funds for promoting science and technology from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government

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