Publications

• Interruption of ganglioside synthesis produces central nervous system degeneration and altered axon-glial interactions.

  Yamashita T., Wu Y.P., Sandhoff R., Werth N., Mizukami H., Ellis J.M., Dupree J.L., Geyer R., Sandhoff K., Proia R.L.

  Gangliosides, which are sialylated glycosphingolipids, are the major class of glycoconjugates on neurons and carry the majority of the sialic acid within the central nervous system (CNS). To determine the role of ganglioside synthesis within the CNS, mice carrying null mutations in two critical ga ... >> More

  Gangliosides, which are sialylated glycosphingolipids, are the major class of glycoconjugates on neurons and carry the majority of the sialic acid within the central nervous system (CNS). To determine the role of ganglioside synthesis within the CNS, mice carrying null mutations in two critical ganglioside-specific glycosyltransferase genes, Siat9 (encoding GM3 synthase) and Galgt1 (encoding GM2 synthase), were generated. These double-null mice were unable to synthesize gangliosides of the ganglio-series of glycosphingolipids, which are the major ganglioside class in the CNS. Soon after weaning, viable mice developed a severe neurodegenerative disease that resulted in death. Histopathological examination revealed striking vacuolar pathology in the white matter regions of the CNS with axonal degeneration and perturbed axon-glia interactions. These results indicate that ganglioside synthesis is essential for the development of a stable CNS, possibly by means of the promotion of interactions between axon and glia. << Less

  Proc Natl Acad Sci U S A 102:2725-2730(2005) [PubMed] [EuropePMC]

• Both GA2, GM2, and GD2 synthases and GM1b, GD1a, and GT1b synthases are single enzymes in Golgi vesicles from rat liver.

  Pohlentz G., Klein D., Schwarzmann G., Schmitz D., Sandhoff K.

  Competition experiments using lactosylceramide, ganglioside GM3 and ganglioside GD3 as substrates, as well as mutual inhibitors for ganglioside N-acetylgalactosaminyltransferase, in Golgi vesicles derived from rat liver suggested that N-acetylgalactosamine transfer to these three respective compou ... >> More

  Competition experiments using lactosylceramide, ganglioside GM3 and ganglioside GD3 as substrates, as well as mutual inhibitors for ganglioside N-acetylgalactosaminyltransferase, in Golgi vesicles derived from rat liver suggested that N-acetylgalactosamine transfer to these three respective compounds, leading to gangliosides GA2, GM2, and GD2, respectively, is catalyzed by one enzyme. Analogous studies with gangliosides GA1, GM1, and GD1b as glycolipid acceptors in sialyltransferase assays indicated GM1b, GD1a, and GT1b synthases to be identical. These results are incorporated into a model for ganglioside biosynthesis and its regulation. << Less

  Proc. Natl. Acad. Sci. U.S.A. 85:7044-7048(1988) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Purification and characterization of UDP-N-acetylgalactosamine GM3/GD3 N-acetylgalactosaminyltransferase from mouse liver.

  Hashimoto Y., Sekine M., Iwasaki K., Suzuki A.

  A UDP-N-acetylgalactosamine: Sia alpha 2-3Gal beta 1-4Glc beta 1-/Sia alpha 2-8Sia alpha 2-3Gal beta 1-4Glc beta 1-1ceramide N-acetylgalactosaminyltransferase has been purified to apparent homogeneity from mouse liver. The purification procedure involved differential centrifugation for preparation ... >> More

  A UDP-N-acetylgalactosamine: Sia alpha 2-3Gal beta 1-4Glc beta 1-/Sia alpha 2-8Sia alpha 2-3Gal beta 1-4Glc beta 1-1ceramide N-acetylgalactosaminyltransferase has been purified to apparent homogeneity from mouse liver. The purification procedure involved differential centrifugation for preparation of Golgi membranes, extraction of the enzyme with Triton X-100, and sequential chromatography on phosphocellulose, UDP-aldehyde adipic acid hydrazone agarose, UDP-hexanolamine-Sepharose, CM-Sepharose, and DEAE-Sepharose. At the phosphocellulose column chromatography step, the recovery of the enzyme activity was less than 25%, but it was enhanced up to 70% when the enzyme assay was performed in the presence of the flow-through fraction from the phosphocellulose column. With this assay, the enzyme activity was found to be quantitatively recovered during all the column chromatographies, the enzyme finally being purified 171,000-fold with a specific activity of 3.6 mumol/min/mg protein. The apparent molecular mass of the purified enzyme is 65,000 daltons. The enzyme exhibits a pH optimum of 7.5-7.9 and requires 2.5-10 mM Mn2+ for the maximal activity. The Km value for UDP-N-acetylgalactosamine is 7 microM. Among the glycolipids tested as acceptor substrates, NeuGc alpha 2-3Gal beta 1-4Glc beta 1-1ceramide, NeuAc alpha 2-3 Gal beta 1-4Glc beta 1-1ceramide, NeuGc alpha 2-8NeuGc alpha 2-3Gal beta 1-4Glc beta 1-1ceramide, and NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1ceramide are good ones, the Km values for them being 160, 2,100, 27, and 350 microM, respectively, but sialyllactose, NeuAc alpha 2-3Gal beta 1-4Glc, is not. This suggests that the enzyme recognizes not only the oligosaccharide portion but also the ceramide of gangliosides. << Less

  J Biol Chem 268:25857-25864(1993) [PubMed] [EuropePMC]

• Role of a single amino acid in the evolution of glycans of invertebrates and vertebrates.

  Ramakrishnan B., Qasba P.K.

  Structures of glycoconjugate N-glycans and glycolipids of invertebrates show significant differences from those of vertebrates. These differences are due largely to the vertebrate beta1,4-galactosyltransferase-1 (beta4Gal-T1), which is found as a beta1,4-N-acetylgalactosaminyltransferase (beta4Gal ... >> More

  Structures of glycoconjugate N-glycans and glycolipids of invertebrates show significant differences from those of vertebrates. These differences are due largely to the vertebrate beta1,4-galactosyltransferase-1 (beta4Gal-T1), which is found as a beta1,4-N-acetylgalactosaminyltransferase (beta4GalNAc-T1) in invertebrates. Mutation of Tyr285 to Ile or Leu in human beta4Gal-T1 converts the enzyme into an equally efficient beta4GalNAc-T1. A comparison of all the human beta4Gal-T1 ortholog enzymes shows that this Tyr285 residue in human beta4Gal-T1 is conserved either as Tyr or Phe in all vertebrate enzymes, while in all invertebrate enzymes it is conserved as an Ile or Leu. We find that mutation of the corresponding Ile residue to Tyr in Drosophila beta4GalNAc-T1 converts the enzyme to a beta4Gal-T1 by reducing its N-acetylgalactosaminyltransferase activity by nearly 1000-fold, while enhancing its galactosyltransferase activity by 80-fold. Furthermore, we find that, similar to the vertebrate/mammalian beta4Gal-T1 enzymes, the wild-type Drosophila beta4GalNAc-T1 enzyme binds to a mammary gland-specific protein, alpha-lactalbumin (alpha-LA). Thus, it would seem that, during the evolution of vertebrates from invertebrates over 500 million years ago, beta4Gal-T1 appeared as a result of the single amino acid substitution of Tyr or Phe for Leu or Ile in the invertebrate beta4GalNAc-T1. Subsequently, the pre-existing alpha-LA-binding site was utilized during mammalian evolution to synthesize lactose in the mammary gland during lactation. << Less

  J Mol Biol 365:570-576(2007) [PubMed] [EuropePMC]

• Beta-1,4-N-acetylgalactosaminyltransferase can synthesize both asialoglycosphingolipid GM2 and glycosphingolipid GM2 in vitro and in vivo: isolation and characterization of a beta-1,4-N-acetylgalactosaminyltransferase cDNA clone from rat ascites hepatoma cell line AH7974F.

  Hidari J.K., Ichikawa S., Furukawa K., Yamasaki M., Hirabayashi Y.

  We have cloned a cDNA encoding beta 1-4N-acetylgalactosaminyltransferase (EC 2.4.1.92) (GalNAc-T) from rat ascites hepatoma of the free-cell type AH7974F. The cell line only expressed asialo-series glycosphingolipids (GSLs) including asialo-GM2 [Taki, T., Hirabayashi, Y., Ishiwata, Y., Matsumoto, ... >> More

  We have cloned a cDNA encoding beta 1-4N-acetylgalactosaminyltransferase (EC 2.4.1.92) (GalNAc-T) from rat ascites hepatoma of the free-cell type AH7974F. The cell line only expressed asialo-series glycosphingolipids (GSLs) including asialo-GM2 [Taki, T., Hirabayashi, Y., Ishiwata, Y., Matsumoto, M., and Kojima, K. (1979) Biochim. Biophys. Acta 572, 113-120]. The cDNA, pGNA56, was isolated by screening AH7974F cDNA library in lambda gt10 with a probe. The probe was obtained from AH7974F cDNA by PCR using primers with the nucleotide sequence of the human GalNAc-T cDNA. The amino acid sequence deduced from the nucleotide sequence of pGNA56 exhibited 88% similarity to the human GalNAc-T sequence. The enzyme was a typical type II membrane protein, which consisted of a short N-terminal residue, a transmembrane region, and a long C-terminal residue, including the catalytic domain. The substrate specificity of rat GalNAc-T was determined using homogenates from cells into which the cDNA clone was transfected. The enzyme catalysed not only the formation of GM2 and GD2 from GM3 and GD3 respectively, but also asialo-GM2 from CDH. It also acted on GSL substrates, including GM1b, sialylparagloboside and GD1 alpha. On the other hand, the enzyme did not transfer GalNAc to soluble substrates such as glycoproteins and oligosaccharide. The GSL compositional and immunocytochemical analyses of stable transfectants obtained by transfection of the cDNA showed simultaneous expression of asialo-GM2 and GM2 on the plasma membrane. Therefore, we concluded that the formation of asialo-GM2, GM2 and GD2 was catalysed by the single GalNAc-T. Northern-blot hybridization showed that the GalNAc-T mRNA was strongly expressed in rat brain, testis, and spleen. The gene was also expressed in rat normal liver to a lesser extent. We found the GSLs in asialo- and alpha-pathways such as asialo-GM1 and GD1 alpha in the rat tissues by using a sensitive t.l.c.-immunostaining method. These observations also supported our conclusion that the single GalNAc-T synthesizes asialo-GM2, GM2 and GD2 in vivo. << Less

  Biochem. J. 303:957-965(1994) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Beta1,4-N-acetylgalactosaminyltransferase--GM2/GD2 synthase: a key enzyme to control the synthesis of brain-enriched complex gangliosides.

  Furukawa K., Takamiya K., Furukawa K.

  Beta1,4-N-acetylgalactosaminyltransferase (GM2/GD2 synthase) is a key enzyme which catalyzes the conversion of GM3, GD3 and lactosylceramide (LacCer) to GM2, GD2 and asialo-GM2 (GA2), respectively. This step is critical for the synthesis of all complex gangliosides enriched in the nervous system o ... >> More

  Beta1,4-N-acetylgalactosaminyltransferase (GM2/GD2 synthase) is a key enzyme which catalyzes the conversion of GM3, GD3 and lactosylceramide (LacCer) to GM2, GD2 and asialo-GM2 (GA2), respectively. This step is critical for the synthesis of all complex gangliosides enriched in the nervous system of vertebrates. Following the cloning of cDNAs encoding GM2/GD2 synthase by an expression cloning approach, substantial evidence for the roles of complex gangliosides have been obtained. Above all, knock-out mice lacking all complex gangliosides revealed important roles of complex gangliosides in vivo, i.e., in the maintenance and repair of nervous tissues, in the intact differentiation of spermatocytes via the transport of testosterone, and in the regulation of interleukin-2 receptor complex. Molecular mechanisms for these functions of complex gangliosides in vivo remain to be clarified. << Less

  Biochim Biophys Acta 1573:356-362(2002) [PubMed] [EuropePMC]

• The enzymic synthesis of ganglioside. IV. UDP-N-acetylgalactosamine: (N-acetylneuraminyl)-galactosylglucosyl ceramide N-acetylgalactosaminyltransferase in rat brain.

  Dicesare J.L., Dain J.A.

  Biochim Biophys Acta 231:385-393(1971) [PubMed] [EuropePMC]

• Biosynthesis of monosulfogangliotriaosylceramide and GM2 by N-acetylgalactosaminyltransferase from rat brain.

  Nagai K., Ishizuka I.

  Some properties of the enzyme activity that catalyzes the transfer of N-acetylgalactosamine from UDP-N-acetylgalactosamine to exogenous lactosylceramide-II3-sulfate (SM3) and N-acetylneuraminosyllactosylceramide (GM3) were studied using the enzyme preparation solubilized from the 100,000 X g pelle ... >> More

  Some properties of the enzyme activity that catalyzes the transfer of N-acetylgalactosamine from UDP-N-acetylgalactosamine to exogenous lactosylceramide-II3-sulfate (SM3) and N-acetylneuraminosyllactosylceramide (GM3) were studied using the enzyme preparation solubilized from the 100,000 X g pellet of 6-day-old rat brain. The products from SM3 and GM3 were identified as gangliotriaosylceramide-II3-sulfate (SM2) and N-acetylneuraminosylgangliotriaosylceramide (GM2), respectively, by TLC-autoradiography. Optimal conditions for both activities were similar: pH (Hepes-NaOH), 7.0-7.5; detergent (heptylthioglucoside), 0.64% and Mn2+, 5-10 mM. The concentrations of the detergent optimal for both enzyme activities were also examined at various concentrations of the acceptors. The lower the amounts of acceptors, the less the amounts of detergent that were required, and vice versa, for the maximum activities. The acceptor-saturation curve for SM2 synthesis was triphasic, exhibiting a sigmoidal region at lower concentrations, a hyperbolic region and finally a descending region. For GM2 synthesis, the curve was biphasic without the descending region. The donor-saturation curves were classical hyperbolic ones for both syntheses. The Km values calculated for SM3 and GM3 were 0.37 and 0.19 mM, respectively, when the data corresponding to the hyperbolic regions were used for the double-reciprocal plots. The Km values for UDP-N-acetylgalactosamine in the SM2- and GM2-synthesis were 82 and 26 microM, respectively. SM3 and GM3 were the best acceptors for this enzyme preparation. From the results of the acceptor competition study, it was suggested that the two synthetic reactions are catalyzed by a single enzyme. << Less

  J Biochem 101:1115-1127(1987) [PubMed] [EuropePMC]