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 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.
van der Bijl
P
P
Department of Cell Biology, Faculty of Medicine, Utrecht University, The Netherlands.
Strous
G J
GJ
Lopes-Cardozo
M
M
Thomas-Oates
J
J
van Meer
G
G
D000818:Animals; D016466:CHO Cells; D002451:Cell Compartmentation; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D004075:Diglycerides / Q000096:biosynthesis* / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D038983:Galactolipids; D005699:Galactosylceramides / Q000096:biosynthesis* / Q000737:chemistry; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism*; D006017:Glycolipids / Q000096:biosynthesis* / Q000737:chemistry; D006031:Glycosylation; D051146:N-Acylsphingosine Galactosyltransferase; D011994:Recombinant Proteins / Q000378:metabolism; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013379:Substrate Specificity; D014162:Transfection
D018076:DNA, Complementary; D004075:Diglycerides; D005227:Fatty Acids; D038983:Galactolipids; D005699:Galactosylceramides; D006017:Glycolipids; D011994:Recombinant Proteins; C009909:monogalactosyldiacylglycerol; D005700:Galactosyltransferases; D051146:N-Acylsphingosine Galactosyltransferase