(722c) Multivalent Binding of Lectins to Heterogeneous Gangliosides on Cell Mimicking Surfaces

Lectins, carbohydrate binding proteins, often consist of multiple binding subunits that specifically or semi-specifically target to various carbohydrates. However, their binding patterns to heterogeneous cell surfaces usually do not correlate to the amount of the preferred binding carbohydrates. It was hypothesized that the binding specificity and avidity can be modulated by altering the cooperative action between multiple bound receptors. To better understand the essential nature of binding cooperativity in a multivalent binding mechanism, quantitative analysis of multivalent membrane recruitment onto the cellular surface is critical. We have developed a nanocube sensor coupled with complex reaction analysis to quantitatively explore the multivalent binding mechanism. The nanocube sensor is surrounded by a lipid bilayer that possesses the same physical and chemical properties as cell membranes. This novel sensor is an ideal tool for studying binding cooperativities because receptors can freely diffuse and rotate on 2D fluidic cellular membranes allowing receptor self-organization to enable multivalent interactions. This label-free sensing platform can be conducted in standard 384-well microplate; therefore, its high-throughput utility enables the complex analysis of multivalent lectin binding. In addition, the simple protocol (â??mix-and-then-detectâ?) allows any end users to perform the analysis in their own laboratories.

Recently, we studied a pentameric lectin, cholera toxin subunit B (CTB), binding to mixed gangliosides present in lipid bilayers. Interestingly, very weak binding gangliosides (e.g. GM2, GM3, and asialo-GM1) could be activated by strong binding gangliosides (e.g. GM1, fucosyl-GM1, and GD1b) resulting in an up to ten fold higher CTB binding capacity. We also demonstrated that allosteric regulation is not the major cause of the observed binding enhancement. We hypothesize the increased effective local concentration of weak binding ligands on a fluidic bilayer improves the contact between the CTB binding subunit and the weak binding ligands. These unexpected discoveries indicate the multivalent lectin binding to a heterogeneous cell surface is not simply controlled by the specific ligands; the cooperative actions amongst gangliosides in a complex cell surface can influence the overall binding. Our novel nanocube-based cell membrane array and reaction analysis provides an excellent tool to dissect complex multivalent interactions. Its easy-to-use and high-throughput features will make this tool immediately available to many biology communities.