Identification of the sialosyl-sialosyl linkage in biosynthesized gangliosides

A technique was developed to examine the sialosyl-sialosyl linkage in the minute quantities of complex gangliosides biosynthesized in vitro. The disialoganglioside, G_D3¹ 1 The ganglioside nomenclature used here is that described by Svennerholm. The gangliosides are designated as follows: G_M3: II³NeuAc-LacCer; G_M1: II³NeuAc-GgOse₄Cer; G_D3: II³(NeuAc)₂LacCer; G_D1a: IV³NeuAc, II³NeuAc-GgOse₄Cer; and G_T1a: IV³(NeuAc)₂, II³NeuAc-GgOse₄Cer., and the trisialoganglioside, G_T1a, were biosynthesized as secondary products by the reaction of CMP-(4-¹⁴C)sialic acid with the glycolipid substrates, lactosyl ceramide and G_M1 ganglioside, respectively. Following periodate oxidation-borohydride reduction, the intact sialic acid residue, and the susceptible sialic acid residue converted into an N-acetylheptulosaminic acid residue, were released by hydrolysis, esterified, separated, and analyzed for radioactivity. Analysis of the biosynthesized G_D3 and G_T1a gangliosides indicated radioactivity in both the susceptible and the nonsusceptible sialic acid residues, demonstrating that a sialosyl-(2→8)-sialosyl linkage had been synthesized. When the primary products (G_M3 and G_D1a, respectively) of the synthesis just described were examined, radioactivity was detected, as expected, only in the susceptible sialic acid residue. Because the N-acetylneuraminic acid and N-acetylheptulosaminic acid derivatives formed in the reaction were identified by chromatography, the standards used for comparison were verified by g.l.c.-m.s.