Publications

• Purification and characterization of UDP-glucose:ceramide glucosyltransferase from rat liver Golgi membranes.

  Paul P., Kamisaka Y., Marks D.L., Pagano R.E.

  We present a method for solubilizing and purifying UDP-Glc:ceramide glucosyltransferase (EC 2.4.1.80; glucosylceramide synthase (GCS) from a rat liver and present data on its substrate specificity. A Golgi membrane fraction was isolated, washed with N-lauroylsarcosine, and subsequently treated wit ... >> More

  We present a method for solubilizing and purifying UDP-Glc:ceramide glucosyltransferase (EC 2.4.1.80; glucosylceramide synthase (GCS) from a rat liver and present data on its substrate specificity. A Golgi membrane fraction was isolated, washed with N-lauroylsarcosine, and subsequently treated with 3[3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate to solubilize the enzyme. GCS activity was monitored throughout purification using UDP-Glc and a fluorescent ceramide analog as substrates. Purification of GCS was achieved via a two-step dye-agarose chromatography procedure using UDP-Glc to elute the enzyme. This resulted in an enrichment > 10,000-fold relative to the starting homogenate. The enzyme was further characterized by sedimentation on a glycerol gradient, I labeling, and SDS-polyacrylamide gel electrophoresis. which demonstrated that two polypeptides (60-70 kDa) corresponded closely with GCS activity. Purified GCS was found to require exogenous phospholipids for activity, and optimal results were obtained using dioleoyl phosphatidylcholine. Studies of the substrate specificity of the purified enzyme demonstrated that it was stereospecific and dependent on the nature and chain length of the N-acyl-spingosine or -sphinganine substrate. UDP-Glc was the preferred hexose donor, but TDP-glucose and CDP-glucose were also efficiently used. This study provides a basis for molecular characterization of this key enzyme in glycosphingolipid biosynthesis. << Less

  J Biol Chem 271:2287-2293(1996) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• The absence of functional glucosylceramide synthase does not sensitize melanoma cells for anticancer drugs.

  Veldman R.J., Mita A., Cuvillier O., Garcia V., Klappe K., Medin J.A., Campbell J.D., Carpentier S., Kok J.W., Levade T.

  Conversion of ceramide, a putative mediator of anticancer drug-induced apoptosis, into glucosylceramide, by the action of glucosylceramide synthase (GCS), has been implicated in drug resistance. Herein, we compared GM95 mouse melanoma cells deficient in GCS activity, with cells stably transfected ... >> More

  Conversion of ceramide, a putative mediator of anticancer drug-induced apoptosis, into glucosylceramide, by the action of glucosylceramide synthase (GCS), has been implicated in drug resistance. Herein, we compared GM95 mouse melanoma cells deficient in GCS activity, with cells stably transfected with a vector encoding GCS (GM95/GCS). Enzymatic and metabolic analysis demonstrated that GM95/GCS cells expressed a fully functional enzyme, resulting in normal ceramide glycosylation. However, cytotoxicity assays, as well as caspase activation and cytochrome c release studies, did not reveal any difference between the two cell lines with respect to their sensitivity toward doxorubicin, vinblastine, paclitaxel, cytosine arabinoside, or short-chain ceramide analogs. Administration of doxorubicin resulted in ceramide accumulation in both cell lines, with similar kinetics and amplitude. Although glucosylceramide formation was detected in doxorubicin-treated GM95/GCS cells, metabolism of drug-induced ceramide did not appear to be instrumental in cell survival. Furthermore, N-(n-butyl)deoxynojirimycin, a potent and non-toxic GCS inhibitor, had no chemosensitizing effect on wild-type melanoma cells. Altogether, both genetic and pharmacological alterations of the cellular ceramide glycosylation capacity failed to sensitize melanoma cells to anticancer drugs, therefore moderating the importance of ceramide glucosylation in drug-resistance mechanisms. << Less

  FASEB J 17:1144-1146(2003) [PubMed] [EuropePMC]

• Histidine-193 of rat glucosylceramide synthase resides in a UDP-glucose- and inhibitor (D-threo-1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol)-binding region: a biochemical and mutational study.

  Wu K., Marks D.L., Watanabe R., Paul P., Rajan N., Pagano R.E.

  Glucosylceramide synthase (GCS) catalyses the transfer of glucose from UDP-glucose (UDP-Glc) to ceramide to form glucosylceramide, the common precursor of most higher-order glycosphingolipids. Inhibition of GCS activity has been proposed as a possible target of chemotherapeutic agents for a number ... >> More

  Glucosylceramide synthase (GCS) catalyses the transfer of glucose from UDP-glucose (UDP-Glc) to ceramide to form glucosylceramide, the common precursor of most higher-order glycosphingolipids. Inhibition of GCS activity has been proposed as a possible target of chemotherapeutic agents for a number of diseases, including cancer. Design of new GCS inhibitors with desirable pharmaceutical properties is hampered by lack of knowledge of the secondary structure or catalytic mechanism of the GCS protein. Thus we cloned the rat homologue of GCS to begin studies to identify its catalytic regions. The histidine-modifying agent diethyl pyrocarbonate (DEPC) inhibited recombinant rat GCS expressed in bacteria; this inhibition was rapidly reversible by hydroxylamine and could be diminished by preincubation of GCS with UDP-Glc. These data suggest that DEPC acts on histidine residues within or near the UDP-Glc-binding site of GCS. Mutant proteins were expressed in which the eight histidine residues in GCS were individually replaced by other amino acids. H193A (His193-->Ala) and H193N (His193-->Asn) mutants were unaffected by 0.1 mM DEPC, a concentration that inhibited other histidine mutants and the wild-type enzyme by at least 60%. These results indicate that His193 is the primary target of DEPC and is at, or near, the UDP-Glc-binding site of GCS. His193 mutants were also insensitive to the GCS inhibitor d-threo-1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol, at concentrations which inhibited the wild-type enzyme by >80%. These results have significance for both an understanding of the GCS active site and also for the possible design of new and specific inhibitors of GCS. << Less

  Biochem. J. 341:395-400(1999) [PubMed] [EuropePMC]

• Glucosylceramide synthases, a gene family responsible for the biosynthesis of glucosphingolipids in animals, plants, and fungi.

  Leipelt M., Warnecke D., Zahringer U., Ott C., Muller F., Hube B., Heinz E.

  Glucosylceramides are membrane lipids in most eukaryotic organisms and in a few bacteria. The physiological functions of these glycolipids have only been documented in mammalian cells, whereas very little information is available of their roles in plants, fungi, and bacteria. In an attempt to esta ... >> More

  Glucosylceramides are membrane lipids in most eukaryotic organisms and in a few bacteria. The physiological functions of these glycolipids have only been documented in mammalian cells, whereas very little information is available of their roles in plants, fungi, and bacteria. In an attempt to establish appropriate experimental systems to study glucosylceramide functions in these organisms, we performed a systematic functional analysis of a glycosyltransferase gene family with members of animal, plant, fungal, and bacterial origin. Deletion of such putative glycosyltransferase genes in Candida albicans and Pichia pastoris resulted in the complete loss of glucosylceramides. When the corresponding knock-out strains were used as host cells for homologous or heterologous expression of candidate glycosyltransferase genes, five novel glucosylceramide synthase (UDP-glucose:ceramide glucosyltransferase) genes were identified from the plant Gossypium arboreum (cotton), the nematode Caenorhabditis elegans, and the fungi Magnaporthe grisea, Candida albicans, and P. pastoris. The glycosyltransferase gene expressions led to the biosynthesis of different molecular species of glucosylceramides that contained either C18 or very long chain fatty acids. The latter are usually channeled exclusively into inositol-containing sphingolipids known from Saccharomyces cerevisiae and other yeasts. Implications for the biosynthesis, transport, and function of sphingolipids will be discussed. << Less

  J. Biol. Chem. 276:33621-33629(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Cloning and functional characterization of two key enzymes of glycosphingolipid biosynthesis in the amphibian Xenopus laevis.

  Luque M.E., Crespo P.M., Monaco M.E., Aybar M.J., Daniotti J.L., Sanchez S.S.

  Gangliosides are a subfamily of complex glycosphingolipids (GSLs) with important roles in many biological processes. In this study, we report the cDNA cloning, functional characterization, and the spatial and temporal expression of Xlcgt and Xlgd3 synthase during Xenopus laevis development. Xlcgt ... >> More

  Gangliosides are a subfamily of complex glycosphingolipids (GSLs) with important roles in many biological processes. In this study, we report the cDNA cloning, functional characterization, and the spatial and temporal expression of Xlcgt and Xlgd3 synthase during Xenopus laevis development. Xlcgt was expressed both maternally and zigotically persisting at least until stage 35. Maternal Xlgd3 synthase mRNA could not be detected and showed a steady-state expression from gastrula to late tailbud stage. Xlcgt is mainly present in involuted paraxial mesoderm, neural folds, and their derivatives. Xlgd3 synthase transcripts were detected in the dorsal blastoporal lip, in the presumptive neuroectoderm, and later in the head region, branchial arches, otic and optic primordia. We determined the effect of glycosphingolipid depletion with 1-phenyl-2-palmitoyl-3-morpholino-1-propanol (PPMP) in mesodermal layer. PPMP-injected embryos showed altered expression domains in the mesodermal markers. Our results suggest that GSL are involved in convergent-extension movements during early development in Xenopus. << Less

  Dev. Dyn. 237:112-123(2008) [PubMed] [EuropePMC]

• Ceramide glucosyltransferase of the nematode Caenorhabditis elegans is involved in oocyte formation and in early embryonic cell division.

  Nomura K.H., Murata D., Hayashi Y., Dejima K., Mizuguchi S., Kage-Nakadai E., Gengyo-Ando K., Mitani S., Hirabayashi Y., Ito M., Nomura K.

  Ceramide glucosyltransferase (Ugcg) [uridine diphosphate (UDP)-glucose:N-acylsphingosine D-glucosyltransferase or UDP-glucose ceramide glucosyltransferase (GlcT): EC 2.4.1.80] catalyzes formation of glucosylceramide (GlcCer) from ceramide and UDP-glucose. There is only one Ugcg gene in the mouse g ... >> More

  Ceramide glucosyltransferase (Ugcg) [uridine diphosphate (UDP)-glucose:N-acylsphingosine D-glucosyltransferase or UDP-glucose ceramide glucosyltransferase (GlcT): EC 2.4.1.80] catalyzes formation of glucosylceramide (GlcCer) from ceramide and UDP-glucose. There is only one Ugcg gene in the mouse genome, which is essential in embryogenesis and brain development. The nematode Caenorhabditis elegans has three Ugcg genes (cgt-1, cgt-2 and cgt-3), and double RNAi of the cgt-1 and cgt-3 genes results in lethality at the L1 larval stage. In this study, we isolated knockout worms for the three genes and characterized the gene functions. Each gene product showed active enzymatic activity when expressed in GM95 cells deficient in glycosphingolipids (GSLs). When each gene function was disrupted, the brood size of the animal markedly decreased, and abnormal oocytes and multinucleated embryos were formed. The CGT-3 protein had the highest Ugcg activity, and knockout of its gene resulted in the severest phenotype. When cgt-3 RNAi was performed on rrf-1 worms lacking somatic RNAi machinery but with intact germline RNAi machinery, a number of abnormal oocytes and multinucleated eggs were observed, although the somatic phenotype, i.e., L1 lethal effects of cgt-1/cgt-3 RNAi, was completely suppressed. Cell surface expression of GSLs and sphingomyelin, which are important components of membrane domains, was affected in the RNAi-treated embryos. In the embryos, an abnormality in cytokinesis was also observed. From these results, we concluded that the Ugcg gene is indispensable in the germline and that an ample supply of GlcCer is needed for oocytes and fertilized eggs to maintain normal membranes and to proceed through the normal cell cycle. << Less

  Glycobiology 21:834-848(2011) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis.

  Ichikawa S., Sakiyama H., Suzuki G., Hidari K.I.-P., Hirabayashi Y.

  We have isolated a cDNA encoding human ceramide glucosyltransferase (glucosylceramide synthase, UDP-glucose:N-acylsphingosine D-glucosyltransferase, EC 2.4.1.80) by expression cloning using as a recipient GM-95 cells lacking the enzyme. The enzyme catalyzes the first glycosylation step of glycosph ... >> More

  We have isolated a cDNA encoding human ceramide glucosyltransferase (glucosylceramide synthase, UDP-glucose:N-acylsphingosine D-glucosyltransferase, EC 2.4.1.80) by expression cloning using as a recipient GM-95 cells lacking the enzyme. The enzyme catalyzes the first glycosylation step of glycosphingolipid synthesis and the product, glucosylceramide, serves as the core of more than 300 glycosphingolipids. The cDNA has a G+C-rich 5' untranslated region of 290 nucleotides and the open reading frame encodes 394 amino acids (44.9 kDa). A hydrophobic segment was found near the N terminus that is the potential signal-anchor sequence. In addition, considerable hydrophobicity was detected in the regions close to the C terminus, which may interact with the membrane. A catalytically active enzyme was produced from Escherichia coli transfected with the cDNA. Northern blot analysis revealed a single transcript of 3.5 kb, and the mRNA was widely expressed in organs. The amino acid sequence of ceramide glucosyltransferase shows no significant homology to ceramide galactosyltransferase, which indicates different evolutionary origins of these enzymes. << Less

  Proc. Natl. Acad. Sci. U.S.A. 93:4638-4643(1996) [PubMed] [EuropePMC]