In vitro synthesis of disialoganglioside (GD1 alpha) from asialo-GM1 using sialyltransferases in rat liver Golgi vesicles.
Two gangliosides were efficiently synthesized from asialo-GM1 (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1 Cer) and cytidine 5'-phosphate-N-acetylneuraminic acid (CMP-NeuAc) by using sialyltransferases in rat liver Golgi vesicles in vitro. These gangliosides were rapidly purified by a combination of anion exchange and reverse-phase column chromatographies. The ganglioside structures were determined by TLC analysis, treatment with a sialidase from Salmonella typhimurium LT2, which specifically hydrolyzes alpha 2-3 N-acetylneuraminic acid (NeuAc alpha 2-3) linkages, TLC immunostaining, and 1H-NMR spectroscopy. One of the gangliosides was identified as GD1 alpha [Neu-Ac alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAc beta 1-4Gal beta 1-4Glc beta 1-1 Cer]. The other ganglioside was determined to be GM1b (NeuAc alpha 2-3Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1 Cer), which has been reported in a previous study [Pohlentz, G., Klein, D., Schmitz, D., Schwarzmann, G., Peter-Katalinic, J. & Sandhoff, K. (1988) Biol. Chem. Hoppe-Seyler 369, 55-63]. Finally, GM1b and GD1 alpha were obtained from asialo-GM1 as a starting material in 8.1% and 1.2% overall yields, respectively. This study also suggests that the novel synthetic pathway asialo-GM1-->GM1b-->GD1 alpha may exist in rat liver.
Hidari
K I
KI
Department of Biochemical Cell Research, Tokyo Metropolitan Institute of Medical Science, Japan.
Kawashima
I
I
Tai
T
T
Inagaki
F
F
Nagai
Y
Y
Sanai
Y
Y
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer; D005260:Female; D005677:G(M1) Ganglioside / Q000737:chemistry / Q000378:metabolism*; D005732:Gangliosides / Q000096:biosynthesis* / Q000737:chemistry; D006056:Golgi Apparatus / Q000201:enzymology*; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000378:metabolism; D051381:Rats; D012486:Salmonella typhimurium / Q000201:enzymology; D012799:Sialyltransferases / Q000378:metabolism*; D013379:Substrate Specificity
D005732:Gangliosides; C021298:ganglioside, GD1a; D005677:G(M1) Ganglioside; C018835:asialo GM1 ganglioside; D012799:Sialyltransferases; D009439:Neuraminidase