QUANTITATIVE-ANALYSIS OF BACTERIAL TOXIN AFFINITY AND SPECIFICITY FORGLYCOLIPID RECEPTORS BY SURFACE-PLASMON RESONANCE

The primary virulence factors of many pathogenic bacteria are secreted protein toxins which bind to glycolipid receptors on host cell surfac es, The binding specificities of three such toxins for different glyco lipids, mainly from the ganglioside series, were determined by surface plasmon resonance (SPR) using a liposome capture method, Unlike micro titer plate and thin layer chromatography overlay assays, the SPR/lipo some methodology allows for real time analysis of toxin binding under conditions that mimic the natural cell surface venue of these interact ions and without any requirement for labeling of toxin or receptor. Co mpared to conventional assays, the liposome technique showed more rest ricted oligosaccharide specificities for toxin binding, Cholera toxin demonstrated an absolute requirement for terminal galactose and intern al sialic acid residues (as in G(M1)) with tolerance for substitution with a second internal sialic acid (as in G(D1b)), Escherichia coli he at-labile enterotoxin bound to G(M1) and tolerated removal or extensio n of the internal sialic acid residue (as in asialo-G(M1) and G(D1b), respectively) but not substitution of the terminal galactose of G(M1). Tetanus toxin showed a requirement for two internal sialic acid resid ues as in G(D1b). Extension of terminal galactose with a single sialic acid was tolerated to some extent, The SPR analyses also yielded rate and affinity constants which are not attainable by conventional assay s, Complex binding profiles were observed in that the association and dissociation rate constants varied with toxin:receptor ratios, The sub -nanomolar affinities of cholera toxin and heat-labile enterotoxin for liposome-anchored gangliosides were attributable largely to very slow dissociation rate constants, The SPR/liposome technology should have general applicability in the study of glycolipid-protein interactions and in the evaluation of reagents designed to interfere with these int eractions.