Discovery of Dihydroxy-Ketone-Type Protein-Bound Ceramides as the Dominant Type in Human Stratum Corneum

Protein-bound ceramides are a specialized subclass of ceramides that are essential for skin barrier function, and their defective formation leads to severe skin disorder ichthyosis. Despite their biological importance, the precise molecular structures of protein-bound ceramides have remained incompletely defined, largely due to the technical challenges arising from their unique covalent linkage between lipid and protein components with highly distinct physicochemical properties. We investigated whether epoxy-ketone (EpK)-type protein-bound ceramides present in mouse epidermis are conserved in human skin. Mass spectrometric analyses of epoxy-enone (EpE) acylceramides reversibly released from EpK-type protein-bound ceramides revealed only low levels in human stratum corneum. Instead, much higher amounts of dihydroxy-enone (DiHE) acylceramides were detected, indicating that dihydroxy-ketone (DiHK)-type protein-bound ceramides are predominant in human skin. In contrast, DiHK-type protein-bound ceramides were present in mouse epidermis at much lower levels. DiHE acylceramides appeared as two chromatographically distinct peaks, which likely correspond to putative stereoisomers with (9R,10S) and (9R,10R) configurations. Age-dependent increases in the (9R,10S) form in mouse epidermis closely paralleled changes in the expression levels of the epoxide hydrolase Ephx3, suggesting a role for EPHX3 in the conversion of epoxy-type ceramides to dihydroxy-type ceramides through epoxide ring opening. Together, these findings reveal molecular diversity in protein-bound ceramides and a fundamental difference between human and mouse epidermal lipid architectural organization.