Determining the structure of protein-bound ceramides, essential lipids for skin barrier function

Abstract

Protein-bound ceramides, specialized ceramides covalently bound to corneocyte surface proteins, are essential for skin permeability barrier function. However, their exact structure and target amino acid residues are unknown. Here, we found that epoxy-enone (EE) ceramides, precursors of protein-bound ceramides, as well as their synthetic analog, formed stable conjugates only with Cys among nucleophilic amino acids. NMR spectroscopy revealed that the {beta}-carbon of the enone was attached by the thiol group of Cys via a Michael addition reaction. We confirmed the presence of Cys-bound EE ceramides in mouse epidermis by mass spectrometry analysis of protease-digested epidermis samples. EE-ceramides were reversibly released from protein-bound ceramides via sulfoxide elimination. We found that protein-bound ceramides with reversible release properties accounted for approximately 60% of total protein-bound ceramides, indicating that Cys-bound EE ceramides are the predominant protein-bound ceramides. Our findings provide clues to the molecular mechanism of skin barrier formation by protein-bound ceramides. HighlightsO_LIEpoxy-enone (EE) ceramides form stable conjugates with Cys. C_LIO_LICys binds to the {beta}-carbon of the enone via a Michael addition reaction. C_LIO_LICys-bound EE ceramides exist in the mouse epidermis. C_LIO_LICys-bound EE ceramides are the predominant protein-bound ceramides. C_LI In BriefProtein-bound ceramides are specialized ceramides essential for skin permeability barrier function. In the present study, we determined the exact structure of protein-bound ceramides as Cys-bound epoxy-enone ceramides where the {beta}-carbon of the epoxy-enone ceramides is attached by Cys residues of corneocyte surface proteins via a Michael addition reaction.