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

Abstract

The influence of 9-O-acetylation of G_D1a, yielding G_D1a (^eNeu5,9Ac₂ 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 (^eNeu5,9Ac₂) 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 (^eNeu5,9Ac₂) 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.