Molecular dynamics-derived conformation and intramolecular interaction analysis of the N-acetyl-9-O-acetylneuraminic acid-containing ganglioside G(D1a) and NMR-based analysis of its binding to a human polyclonal inununoglobulin G fraction with selectivity for O-acetylated sialic acids

Siebert, Hans-Christian; von der Lieth, Claus-Wilhelm; Dong, Xin; Reuter, Gerd; Schauer, Roland; Gabius, Hans-Joachim; Vliegenthart, Johannes F.G.

The influence of 9-O-acetylation of G(D1a), yielding G(D1a) ((e)Neu5,9Ac(2) with a 9-O-acetylated sialic acid moiety linked to the outer galactose residue, on the spatial extension and mobility of the carbohydrate chain and on recognition by a natural human antibody is analysed. To study a potential impact of the O-acetyl group on the overall conformation of the carbohydrate chain, molecular dynamics (MD) simulations of oligosaccharide chain fragments of increasing length starting from the non-reducing end have been carried out for the first time in this study. They revealed a considerable loss in chain flexibility after addition of the internal N-acetylneuraminic acid onto the chain. Besides MD calculations with different dielectric constants, the conformational behaviour of the complete oligosaccharide chain of the 9-O-acetylated G(D1a) ganglioside was simulated in the solvents water and dimethyl sulfoxide. These solvents were also used in NMR measurements. The results of this study indicate that 9-O-acetylation at the terminal sialic acid does not influence the overall conformation of the ganglioside. An extended interaction analysis of energetically minimized conformations of G(D1a) ((e)Neu5,9Ac(2)) and G(D1a), obtained during molecular dynamics simulations, allowed assessment of the influence of the different parts of the saccharide chains on spatial flexibility. Noteworthy energetic interactions, most interestingly between the 9-O-acetyl group and the pyranose ring of N-acetylgalactosamine, were ascertained by the calculations. However, the strength of this interaction does not force the ganglioside into a conformation, where the 9-O-acetyl group is no longer accessible. Binding of G(D1a) ((e)Neu5,9Ac(2)) to proteins, which are specific for 9-O-acetylated sialic acids, should thus at least partially be mediated by the presence of this group. To experimentally prove this assumption, a NMR study of 9-O-acetylated G(D1a) in the presence of an affinitypurified polyclonal IgG fraction from human serum with preferential binding to 9-O-acetylated sialic acid was performed. The almost complete disappearance of the intensity of the 9-O-acetyl methyl signal of the G(D1a) (clearly indicates that the assumed interaction of the 9-O-acetyl group with the human protein takes place.