In vitro synthesis of disialoganglioside (G_d1α) from asialo‐G_m1 using sialyltransferases in rat liver Golgi vesicles

Abstract

Two gangliosides were efficiently synthesized from asialo-G,, (Gal~1-3GalNAc~l-4Gal~l-4GlcP1-1 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 a2-3 N-acetylneuraminic acid (NeuAca2-3) linkages, TLC immunostaining, and 'H-NMR spectroscopy. One of the gangliosides was identified as GDla [NeuAca2-3Gal~l-3(NeuAca2-6)GalNAc~l-4Gal~l-4Glc~1-1 Cer] . The other ganglioside was determined to be G,, , (NeuAca2-3Galp1-3GalNAcal-4Galpl-4Glcp1-1 Cer), which has been reported in a previous study [Pohlentz, G., Klein, D., Schmitz, D., Schwarzmann, G., Peter-Katalinic, J. & Sandhoff, K. (1988) Bid. Chem. Hoppe-Seyler 369, 55-63]. Finally, G,,, and GDla were obtained from asialo-G,, as a starting material in 8.1% and 1.2% overall yields, respectively. This study also suggests that the novel synthetic pathway aSialo-GMl--'G,, b+GDln may exist in rat liver.It is well known that gangliosides comprise one of the ubiquitous components of the eukaryotic plasma membrane. More than one hundred molecular species of gangliosides have been identified on the basis of the diversity of their carbohydrate structures [4]. Gangliosides have been reported to be tumor antigens, regional and temporal markers in early embryos, and perhaps play essential roles in cellular phenomena such as cellkell interactions, differentiation, and signal transduction via receptors [5 -81. Most gangliosides involved in these phenomena exist as minor components in the plasma membrane. These minor gangliosides have putative functional roles in the immune and nervous systems [9-131. For Abbreviations. The classification of gangliosides, including asialo-and a-series gangliosides, is based on Pohlentz et al. [l] and Taki et al. [2]. The nomenclature used for gangliosides is based on the system of Svennerholm [3]. Cer, ceramide; NeuAc, N-acetylneuraminic acid ; lactosylceramide, GalPl-4GlcPl-1 Cer ; asialo-G,,, GalPl3GalNAcPl-4GalPl-4Glcpl-l Cer ; G,,,, NeuAca2-3GalPl-3Gal-NAc/~l-4Gal~I-4Glc~l-l Cer ; IV'NeuAcGg,Cer, NeuAca2-6GalPl-3GalNAcpI -4GalPl-4Glcpl-1 Cer; G,,,', GalP1-3(NeuAcn2-6)GalNAcPl-4GalPl-4GlcPl-1 Cer; G,, c, NeuAca2-8NeuAca2-3Galfil-3GalNAcPl-4GalP1 -4GlcP1-1 Cer ; G,,,, NeuAcn2-3GalPl-3(NeuAca2-6)GalNAc/ll-4(NeuAca2-3)Gal~1-4Glc/ll-1 Cer ; G,,,, NeuAca2-3Gal~l-3(NeuAca2-6)GalNAc~l-4Gal~l-4Glc~1-l Cer ; G,,,, II'NeuAc-GgOse,Cer ; G,,, II'NeuAc-LacCer ; G,, a, IV'NeuAc, IPNeuAc-GgOse,Cer ; G,, b, II'(NeuAc),-GgOse,Cer; G,,, IIYNeuAc),-LacCer ; G,,, II'NeuAc-GgOse,Cer.Enzymes. Peroxidase (EC 1.11.1.7); sialidase (EC 3.2.1.18); sialyltransferase (EC 2.4.99.-).4Glcpl-lCer) is known as a specific and common marker in the mouse immune and nervous systems [9-121. G,,, (NeuAca2-3Galpl-3GalNAcpl-4Galpl-4Glcp1-1 C...

Combinatorial Ganglioside Biosynthesis

“…The complex gangliosides with sialic acid moieties ␣2,6-glycosidically linked to N-acetylgalactosamine residues have been added later to the biosynthetic scheme (44). There has been some confusion on the identity of the enzymes that catalyze the formation of gangliosides GD3 and GT3.…”

Association of Src Family Tyrosine Kinase Lyn with Ganglioside GD3 in Rat Brain

“…We have been investigating a biosynthesis of gangliosides during normal neuronal development and oncogenic transformation (12)(13)(14)(15)(16)(17)(18)(19)(20). Ganglioside biosynthesis takes place in the Golgi apparatus, where glucosylceramide is glycosylated by sequential addition of galactose, sialic acid, and N-acetylgalactosamine.…”

Expression cloning of a CMP-NeuAc:NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer alpha 2,8-sialyltransferase (GD3 synthase) from human melanoma cells.

“…However, the product was insensitive to S. typhimurium LT2 sialidase, whereas substrate GM3 was degraded by S. typhimurium LT2 sialidase (data not shown). S. typhimurium LT2 sialidase shows a high kinetic preference for sialyl a2-3 linkage compared with a2-6 and a2-8 linkage and does not cleave inner a2-3-sialyl linkages (35,43). Therefore, these data showed the [14C]NeuAclabeled product from GM3 was protected from digestion with S. typhimurium LT2 sialidase by additional sialyl linkage, suggesting that the sialyltransferase transferred sialic acid to the terminal sialic acid residue of GM3.…”

“…Both GM3 and the [14C]NeuAclabeled product from GM3 ganglioside were sensitive to A. ureafaciens sialidase (35). However, the product was insensitive to S. typhimurium LT2 sialidase, whereas substrate GM3 was degraded by S. typhimurium LT2 sialidase (data not shown).…”