Solution conformation of sialosylcerebroside (G_M4) and its NeuAc(α2→3)Galβ sugar component

Abstract

The solution conformations of GM4 ganglioside [NeuAc(a2+3)Gal(Pl -+l)Cer] in (ZH3C)zS0 and its component disaccharide in 'HzO were investigated with the aid of 'H-nuclear magnetic resonance spectroscopy (nuclear Overhauser effect, analysis of coupling constants) and by energy-minimum calculations. The existence of three low-energy conformers obtained by theoretical calculations was supported by experimental findings in the case of GM4, whereas the disaccharide appears to exist as a mixture of two conformers.The sialic-acid-containing glycolipids of cell membranes play an important role as binding sites for various toxins, hormones and viruses, e. g. cholera toxin, thyrotropin, polyoma virus, etc. [ 11. Recent studies concerning the binding affinity of a murine monoclonal antibody to various gangliosides have shown that the antibody recognizes only the NeuAc(a2+3)Galp fragment of monosialogangliosides, regardless of the structure of the remaining part of the molecule [2]. The conformation of different oligosaccharides containing this fragment has been investigated by several research groups [3 -51. Since the results obtained differed considerably, it was of interest to elucidate the conformation of the simplest ganglioside containing this disaccharide unit alone, ( 3~4 . Incidentally, this ganglioside showed the highest binding affinity to the murine monoclonal antibody [2]. Unfortunately, intact gangliosides form micelles under physiological conditions and thus cannot be investigated by high-resolution NMR. It is possible however, that the conformation of the water-solvated sugar moiety of a ganglioside, dispersed in a cell membrane, does not differ significantly from the conformation of the same sugar devoid of ceramide and dissolved in water. There is no direct way of proving this, but it would become more probable if the sugar dissolved in water could be shown to have a conformation similar to that adopted by the sugar moiety within the ganglioside dissolved in a solvent suitable for NMR, e.g. (CH3)zS0. To approach this problem, we investigated GM4 and NeuAc(a2-+3)Galp, both with the aid of nuclear Overhauser enhancement (NOE) and by energyminimum calculations. MATERIALS AND METHODSThe preparation of GM4 and NeuAc(a2-3)Galp was described previously [6]. For IH-NMR measurements, deuterium-exchanged GM4 (2 mg) and NeuAc(a2-+3)Galp (2 mg) were dissolved in 0.4 ml (C2H3)zS0 containing 2% 'HzO, and 0.4 ml 'HZ0 containing 0.1% (CH&CO, respectively. The solutions were then degassed by several freezepump-thaw cycles, flushed with argon and sealed. ' H -N M R spectroscopySpectra were obtained at a frequency of 500 MHz using a Bruker AM-500 spectrometer equipped with an Aspect 3000 computer, a process controller and an array processor. Chemical shifts were referenced indirectly to (CH3)& by setting the 'H signal of residual (CH2Hz)zS0 at 2.49 ppm and the 'H signal of (CH3)'C0 at 2.225 ppm.One-dimensional hoinonuclear Hartmann-Hahn spectroscopy (1 D-HOHAHA) experiments were performed in the reverse mode, by using the pu...

The binding properties of the H5N1 influenza virus neuraminidase as inferred from molecular modeling

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Ramachandran‐type plots for glycosidic linkages: Examples from molecular dynamic simulations using the Glycam06 force field

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