Comparison of the Interaction of Mono- and Oligovalent Ligands with Cholera Toxin. Demonstration of Aggregate Formation at Low Ligand Concentrations

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Abstract
Stimulation of adenylate cyclase [EC 4.6.1.1] by cholera toxin in Chinese hamster ovarian cells was inhibited by various ligands. Ligands contained structural oligosaccharide entities required for binding to cholera toxin, established as Gal.beta.1 .fwdarw. 3GalNAc.beta.1 .fwdarw. 4Gal3 .rarw. 2.alpha.NeuAc. Different inhibitory potency correlated with the size of aggregates formed with the toxin, which depended on the valency of the ligands. Interaction of cholera toxin and its B-promoter with ganglioside II3NeuAc-GgOse4-Cer, the newly synthesized bis-(monosialo-gangliotetraityl)amine and monosialogangliotetraose were compared. In a double diffusion test cholera toxin B-protomer precipitated with the ganglioside II3NeuAc-GgOse4-Cer and the divalent ligand bis(monosialo-gangliotetraityl)amine, suggesting the formation of high molecular weight aggregates. No precipitation was observed with the monovalent monosialo-gangliotetraose. By ultracentrifugation analysis, aggregate formation of the cholera toxin B-protomer was demonstrated with the ganglioside II3NeuAc-GgOse4-Cer and bis(monosialo-gangliotetraityl)amine at a concentration at which the ganglioside was monodisperse. Ganglioside/cholera toxin B-protomer complexes sedimented faster than toxin and bis(monosialo-gangliotetraityl)amine, suggesting higher aggregation of cholera toxin B-promoter with the former. No sedimentation with monosialo-gangliotetraose was observed. By equilibrium displacement dialysis, a comparable high binding affinity to cholera toxin B-protomer of both the mono and divalent oligosaccharides was demonstrated. Values for the maximal concentration of the bound ligand with cholera toxin B-protomer established molar ratios of ligand/protein of 4-1 and 2-1 for monosialo-gangliotetraose and bis(monosialo-gangliotetraityl)amine, respectively.

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