Mouse anti-ceramide antiserum: a specific tool for the detection of endogenous ceramide

Ceramide is a pivotal molecule in signal transduction and an essential stru ctural component of the epidermal permeability barrier. The epidermis is ma rked by a high concentration of ceramide and by a unique spectrum of cerami de species: Besides the two ceramide structures commonly found in mammalian tissue, N-acyl-sphingosine and N-2-hydroxyacyl-sphingosine, six additional ceramides differing in the grade of hydroxylation of either the sphingosin e base or the fatty acid have been identified in the epidermis, Here we rep ort on the characterization of an IgM-enriched polyclonal mouse serum again st ceramide, In dot blot assays with purified epidermal lipids the antiseru m bound to a similar extent to N-acyl-sphingosine (ceramide 2), N-acyl-4-hy droxy-sphinganine (ceramide 3), and N-(2-hydroxyacyl)-sphingosine (ceramide 5), whereas no specific reaction was detected with glycosylceramides, sphi ngomyelin, free sphingosine, phospholipids, or cholesterol, In contrast, a monoclonal IgM antibody, also claimed to be specific for ceramide, was show n to bind specifically to sphingomyelin and therefore was not further inves tigated, In thin-layer chromatography immunostaining with purified lipids a strong and highly reproducible reaction of the antiserum with ceramide 2 a nd ceramide 5 was observed, whereas the reaction with ceramide 1 and cerami de 3 was weaker and more variable. Ceramide 2 and ceramide 5 were detected in the nanomolar range at serum dilutions of up to 1:100 by dot blot and th in-layer immunostaining, In thin-layer chromatography immunostaining of cru de lipid extracts from human epidermis, the antiserum also reacted with N-( 2-hydroxyacyl)-4-hydroxy-sphinganine (ceramide 6) and N-(2-hydroxyacyl)-6-h ydroxy-sphingosine (ceramide 7), Furthermore, the suitability of the antise rum for the detection of endogenous ceramide by immunolight microscopy was demonstrated on cryoprocessed human skin tissue, Double immunofluorescence labeling experiments with the anti-ceramide antiserum and the recently desc ribed anti-glucosylceramide antiserum (Brade et al,, 2000, Glycobiology 10, 629) showed that both lipids are concentrated in separate epidermal sites, Whereas anti-ceramide stained the dermal and basal epidermal cells as well as the corneocytes, anti-glucosylceramide staining was concentrated in the stratum granulosum. In conclusion, the specificity and sensitivity of the reagent will enable studies on the subcellular distribution and biological functions of endogenous ceramide.