Binding of cholera toxin B-subunits to derivatives of the natural ganglioside, receptor, GM1

In a previous paper we showed that the B-pentamer of cholera toxin (CT-B) binds with reduced binding strength to different C(l) derivatives of iV-acetylneuraminic acid (NeuAc) of the natural receptor ganglioside, GM1. We have now extended these results to encompass two large amide derivatives, butylamide and cyclohexylmethylamide, using an assay in which the glycosphingolipids are adsorbed on hydrophobic PVDF membranes. The latter derivative showed an affinity approximately equal to that earlier found for benzylamide (~0.01 relative to native GM1) whereas the former revealed a approximately tenfold further reduction in affinity. Another derivative with a charged C(l)-amide group, amino-propylamide, was not bound by the toxin. Toxin binding to C(7) derivatives was reduced by about 50 % compared with the native ganglioside. Molecular modeling of C(l) and C(7) derivatives in complex with CT-B gave a structural rationale for the observed differences in the relative affinities of the various derivatives. Loss of or altered hydrogen bond interactions involving the water molecules bridging the sialic acid to the protein was found to be the major cause for the observed drop in CT-B affinity in the smaller derivatives, while in the bulkier derivatives, hydrophobic interactions with the protein were found to partly compensate for these losses. Key words: amide and amine derivatives, cholera toxin, GM1 receptor binding, molecular modeling, sialic acid chemical modification. The bacterium Vibrio cholerae produces a toxin (CT) that causes severe diarrhea in humans. This heterohexameric protein consists of a catalytic A-subunit anchored in the central pore of a B-pentamer responsible for binding to cell surface-located receptor molecules. Although the specific target cells in the intestinal epithelium to which the released toxin binds are not fully known, it is generally assumed that the receptor is the acidic glycosphingolipi