Acylation of exogenous glycosylsphingosines by intact neuroblastoma (NCB-20) cells

Acylation of exogenously added galactosylsphingosine was demonstrated in intact NCB-20 neuroblastoma cells, a cell line that normally does not synthesize galactosylceramide. Labeling of cells with [3H]palmitic acid for 6 h in the presence of 100 microM exogenous galactosylsphingosine (GalSph) resulted in a more than 3-fold increase in the incorporation of label into the ceramide monohexoside fraction relative to controls. This increase, which was almost entirely due to the incorporation of labeled nonhydroxy fatty acid into galactosylceramide, was linear over a concentration range of 1-100 microM galactosylsphingosine and for the first 5 h after the addition of galactosylsphingosine. Similarly, the addition of 100 microM glucosylsphingosine resulted in a 3-fold increase of label incorporated into glucosylceramide. Incubation of cells with 100 microM GalSph and labeled fatty acids of various chain lengths revealed that the acylation of GalSph was specific for medium chain (C16-C18) nonhydroxy fatty acids, suggesting that this was an enzyme-mediated reaction. The enzymatic nature of GalSph acylation was further demonstrated when cells were incubated for 72 h with 15 microM [3H]galactosylsphingosine labeled in the galactose moiety. [3H]Galactosylceramide containing only medium chain non-hydroxy fatty acids accumulated linearly with time reaching a maximum at 48 h and was observed to be further metabolized to ceramide dihexoside. This acylation reaction may be potentially important for the removal of glycosylsphingosines in the cell.