In vitro synthesis of disialoganglioside (Gd1α) from asialo‐Gm1 using sialyltransferases in rat liver Golgi vesicles

Abstract

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.