Surface plasmon resonance analysis of botulinum neurotoxin binding for lipid receptors

Abstract

Botulinum neurotoxins (BoNTs) are the most potent protein toxins. There are seven serotypes of BoNTs, termed A to G (BoNT/A to BoNT/G). The carboxyl-terminal half of the heavy chain (Hc) of the neurotoxin recognizes its specific receptor on the plasma membrane. We have previously demonstrated that BoNT/C binds to gangliosides GD1b and GT1b under physiological conditions, while BoNT/D interacts with phosphatidylethanolamine (PE). In this study, the binding specificities of BoNT-Hc for GT1b or PE were determined by surface plasmon resonance (SPR) using a receptor-including liposome. 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 of these interactions and without any requirement for labeling of toxin or receptor. BoNT/C-Hc showed the binding specificity to GT1b liposome but not PE liposome. On the other hand, BoNT/D-Hc bound to only PE liposome. The SPR analysis 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. The affinities of BoNTs for liposome-anchored gangliosides or PE were attributable largely to very fast dissociation rate constants. The SPR/liposome analysis should have general applicability in the study of lipid–protein interactions.