Structural evaluation of GM1-related carbohydrate–cholera toxin interactions through surface plasmon resonance kinetic analysis
Surface plasmon resonance (SPR) can provide kinetic information about an interaction, and it can also be used to rapidly monitor dynamic processes, such as adsorption and degradation, without the need for sample labeling. Here, we employed SPR to analyze carbohydrate–protein interactions, particularly GM1-related carbohydrate–Vibrio cholera toxin interactions. The interaction between cholera toxin subunits A (ctxA) and B (ctxB) was similar to general ligand–receptor interactions. After the direct immobilization of thiol-containing GM1 pentasaccharide on a gold surface, the GM1–ctxB interaction kinetics were evaluated, and they showed a similar degree of kinetics as reported in previous reports. We found that ctxA had a high affinity for the GM1–ctxAB complex, although its equilibrium dissociation constant was 10 times lower than that of GM1–ctxB binding. Comparative analyses of GM1-related carbohydrate–ctxAB interactions were also conducted to determine the kinetic values of several GM1 analogues with different structures, although their kinetic values were one order of magnitude lower than those of the GM1–ctxAB interaction. The kinetic analysis results for the interactions of GM1 analogues and ctxAB indicated that the sialic acid thumb is important for recognition, and the terminal galactose and N-acetylgalactosamine fingers are required to stabilize the GM1–ctxAB interaction. Taken together, our results indicate that the direct immobilization of carbohydrate in an SPR-based analytical system can be used to evaluate the structural contribution of carbohydrate moieties in carbohydrate–protein interactions, as well as provide valuable information that can be used to understand the interactions.