Volume 272, Number 9, Issue of February 28, 1997 pp. 5533-5538
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Quantitative Analysis of Bacterial Toxin Affinity and Specificity for Glycolipid Receptors by Surface Plasmon Resonance

(Received for publication, August 21, 1996, and in revised form, November 19, 1996)

From the Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6

The primary virulence factors of many pathogenic bacteria are secreted protein toxins which bind to glycolipid receptors on host cell surfaces. The binding specificities of three such toxins for different glycolipids, mainly from the ganglioside series, were determined by surface plasmon resonance (SPR) using a liposome capture method. Unlike microtiter plate and thin layer chromatography overlay assays, the SPR/liposome methodology allows for real time analysis of toxin binding under conditions that mimic the natural cell surface venue of these interactions and without any requirement for labeling of toxin or receptor. Compared to conventional assays, the liposome technique showed more restricted oligosaccharide specificities for toxin binding. Cholera toxin demonstrated an absolute requirement for terminal galactose and internal sialic acid residues (as in G_M1) with tolerance for substitution with a second internal sialic acid (as in G_D1b). Escherichia coli heat-labile enterotoxin bound to G_M1 and tolerated removal or extension 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 residues 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 assays. 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 interactions.

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