Synthesis of non-hydroxy-galactosylceramides and galactosyldiglycerides by hydroxy-ceramide galactosyltransferase

• Published in: Biochemical journal Vol. 317 ( Pt 2); no. 2; pp. 589 - 597
• Main Authors: van der Bijl, P, Strous, G J, Lopes-Cardozo, M, Thomas-Oates, J, van Meer, G
• Format: Journal Article
• Language: English
• Published: England 15.07.1996
• Subjects: Animals; Cell Compartmentation; CHO Cells; Cricetinae; Diglycerides - biosynthesis; Diglycerides - chemistry; DNA, Complementary - genetics; Endoplasmic Reticulum - enzymology; Fatty Acids - chemistry; Fatty Acids - metabolism; Galactolipids; Galactosylceramides - biosynthesis; Galactosylceramides - chemistry; Galactosyltransferases - genetics; Galactosyltransferases - metabolism; Glycolipids - biosynthesis; Glycolipids - chemistry; Glycosylation; N-Acylsphingosine Galactosyltransferase; Recombinant Proteins - metabolism; Spectrometry, Mass, Fast Atom Bombardment; Substrate Specificity; Transfection
• DOI: 10.1042/bj3170589
• ISSN: 0264-6021

Galactosylceramide (GalCer) is the major glycolipid in brain. In order to characterize the activity of brain UDPgalactose: ceramide galactosyltransferase (CGalT), it has been stably expressed in CGalT-negative Chinese hamster ovary (CHO) cells. After fractionation of transfected cells, CHO-CGT, on sucrose gradients, the activity resides at the density of endoplasmic reticulum and not of Golgi. A lipid chromatogram from CHO-CGT cells revealed two new iodine-staining spots identified as GalCer, since they comigrate with GalCer standards, can be metabolically labelled with [3H]galactose, are recognized by anti-GalCer antibodies, and are resistant to alkaline hydrolysis. A third [3H]galactose lipid was identified as galactosyldiglyceride. In the homogenate CGalT displays a 25-fold preference for hydroxy fatty acid-containing ceramides. Remarkably, endogenous GalCer of transfected cells contains exclusively non-hydroxy fatty acids: fast atom bombardment and collision-induced dissociation mass spectrometric analysis revealed mainly C16:0 in the lower GalCer band on TLC and mainly C22:0 and C24:0 in the upper band. Our results suggest that CGalT galactosylates both hydroxy- and non-hydroxy fatty acid-containing ceramides and diglycerides, depending on their local availability. Thus, CGalT alone may be responsible for the synthesis of hydroxy- and non-hydroxy-GalCer, and galactosyldiglyceride in myelin.