SYNTHESIS OF NON-HYDROXY-GALACTOSYLCERAMIDES AND GALACTOSYLDIGLYCERIDES BY HYDROXY-CERAMIDE GALACTOSYLTRANSFERASE

Galactosylceramide (GalCer) is the major glycolipid in brain. In order to characterize the activity of brain UDPgalactose:ceramide galactosy ltransferase (CGalT), it has been stably expressed in CGalT-negative C hinese hamster ovary (CHO) cells. After fractionation of transfected c ells, CHO-CGT, on sucrose gradients, the activity resides at the densi ty of endoplasmic reticulum and not of Golgi. A lipid chromatogram fro m CHO-CGT cells revealed two new iodine-staining spots identified as G alCer, since they comigrate with GalCer standards, can be metabolicall y labelled with [H-3]galactose, are recognized by anti-GalCer antibodi es, and are resistant to alkaline hydrolysis. A third [H-3]galactose l ipid was identified as galactosyldiglyceride. In the homogenate CGalT displays a 25-fold preference for hydroxy fatty acid-containing cerami des. Remarkably, endogenous GalCer of transfected cells contains exclu sively non-hydroxy fatty acids: fast atom bombardment and collision-in duced dissociation mass spectrometric analysis revealed mainly C-16:0 in the lower GalCer band on TLC and mainly C-22:0 and C-24:0 in the up per band. Our results suggest that CGalT galactosylates both hydroxy- and non-hydroxy fatty acid-containing ceramides and diglycerides, depe nding on their local availability. Thus, CGalT alone may be responsibl e for the synthesis of hydroxy- and non-hydroxy-GalCer, and galactosyl diglyceride in myelin.