IN-VITRO SYNTHESIS OF DISIALOGANGLIOSIDE (G(D1-ALPHA)) FROM ASIALO-G(M1) USING SIALYLTRANSFERASES IN RAT-LIVER GOLGI VESICLES

Two gangliosides were efficiently synthesized from asialo-G(M1) (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer) 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 H-1-NMR spectroscopy. One of the gangliosides was identified as G(D1 alpha) [NeuAc 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 G(M1b) (NeuAc alpha 2-3Gal beta 1-3GalAc 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. and Sandhoff, K. (1988) Biol. Chem. Hoppe-Seyler 369, 55-63]. Finally, G(M1b) and G(D1 alpha) were obtained from asialo-G(M1) as a starting material in 8.1% and 1.2% overall yields, respectively. This study also suggests that the novel synthetic pathway asialo-G(M1)-->G(M1b)-->G(D1 alpha) may exist in rat Liver.