Homeostasis of cell-surface glycosphingolipid content in B16 melanoma cells: Evidence revealed by an endoglycoceramidase

This paper describes the homeostasis of glycosphingolipid (GSL) on the cell surface as revealed for the first time by an application of endoglycoceramidase (EGCase) capable of hydrolyzing the linkage between the oligosaccharide and the ceramide of various GSLs. When cell-surface GSLs of B16 melanoma cells were hydrolyzed by the action of EGCase, the synthesis of GSLs was found to increase transiently, possibly due to the activation of UDP-glucose:ceramide glucosyltransferase. As a result, the cell-surface GSL content was restored quickly to exactly the same level found without the EGCase treatment, if EGCase was removed from the cell culture. Treatment of erythrocytes with EGCase was found to increase the ceramide content of the plasma membrane. Surprisingly, however, in B16 cells the increase of membrane ceramide by EGCase caused the suppression of de novo ceramide production, resulting in maintenance of the ceramide content of B16 cells at the same level even after EGCase treatment. The signal for homeostatic regulation could be the ceramide released by the action of EGCase, since C₂-ceramide was found to mimic in part the action of EGCase; it suppressed de novo production of ceramide and was directly converted to GSL, NeuAcα2,3Galβ,4Glcβ1,1 N-acetylsphingosine (C₂-ceramide GM₃). Our finding demonstrates a novel form of homeostatic regulation coupled to the GSL-synthesizing system in mammalian cells for maintaining the contents of both cell-surface GSLs and free ceramide. Since many opportunistic pathogens were found to produce EGCase extracellularly, this restoration mechanism could also be present as a defense mechanism against microbial EGCase.