Characterization of UDP-glucose: ceramide glucosyltransferases from different organisms

Cerebrosides are typical membrane lipids of many organisms. They occur in p lants, fungi, animals, humans and some prokaryotes. Almost all of our knowl edge on the physiological functions of cerebrosides results from experiment al data obtained with mammalian cells. However, very little is known about the roles played by these lipids in plants and fungi. To initiate such inve stigations we have cloned and characterized a ceramide glucosyltransferase from the yeast Candida albicans. Functional expression of this gene in Sacc haromyces cevevisiae led to the accumulation of new glycolipids which were not present in wildtype baker's yeast. They were identified by MS and NMR s pectroscopy as beta -D-glucopyranosyl ceramides. The ceramide moieties of t hese cerebrosides comprised phytosphinganine and mainly long-chain (C-26) a lpha -hydroxy fatty acids in amide linkage. We also generated a ceramide gl ucosyltransferase-knock-out strain of C. albicans which was devoid of cereb rosides. The viability of this mutant showed that for this organism glucosy l ceramides are not essential for vegetative growth on complete or minimal media. In addition, we have cloned and functionally expressed one of the th ree putative glucosylceramide synthases from Caenorhabditis elegans, as wel l as a corresponding enzyme from Pichia pastoris.