Hetero-multivalent binding of cholera toxin subunit B with glycolipid mixtures

Abstract

GM1 has generally been considered as the major receptor that binds to cholera toxin subunit B (CTB) due to its low dissociation constant. However, using a unique nanocube sensor technology, we have shown that CTB can also bind to other glycolipid receptors, fucosyl-GM1 and GD1b. Additionally, we have demonstrated that GM2 can contribute to CTB binding if present in a glycolipid mixture with a strongly binding receptor (GM1/fucosyl-GM1/GD1b). This hetero-multivalent binding result was unintuitive because the interaction between CTB and pure GM2 is negligible. We hypothesized that the reduced dimensionality of CTB-GM2 binding events is a major cause of the observed CTB binding enhancement. Once CTB has attached to a strong receptor, subsequent binding events are confined to a 2D membrane surface. Therefore, even a weak GM2 receptor could now participate in second or higher binding events because its surface reaction rate can be up to 104 times higher than the bulk reaction rate. To test this hypothesis, we altered the surface reaction rate by modulating the fluidity and heterogeneity of the model membrane. Decreasing membrane fluidity reduced the binding cooperativity between GM2 and a strong receptor. Our findings indicated a new protein-receptor binding assay, that can mimic complex cell membrane environment more accurately, is required to explore the inherent hetero-multivalency of the cell membrane. We have thus developed a new membrane perturbation protocol to efficiently screen receptor candidates involved in hetero-multivalent protein binding.

Crystal structures reveal that Lewis-x and fucose bind to secondary cholera toxin binding site – in contrast to fucosyl-GM1

“…The available data, however, suggest that compared to the villi, the deeper parts of the intestinal membranes are unaffected by the non-functional FUT2 of non-secretors, due to expression of an alternative 1,2-fucosyltransferase encoded by the H gene 65, 67 . Non-secretors cannot secrete soluble ABO(H) glycans, but might still present suitable docking sites (i.e., Le y ) on these deeper membrane regions, independent of their secretor status, and cooperative binding is expected to be enhanced for hetero-multivalent binding including GM1 55 . Indeed, Cervin et al observed tighter binding of CTB to the crypts than to the villi 32 .…”

“…A recently published hetero-multivalent binding model suggests that CTB first binds to a high-affinity ligand such as GM1. Subsequent binding occurs much more readily (up to 10,000-fold faster), also for low-affinity ligands like GM2, since binding is then confined to the 2D membrane surface 54 . In fact, cooperativity was found to be enhanced for a heterogeneous mixture of ligands 54 .…”

“…Subsequent binding occurs much more readily (up to 10,000-fold faster), also for low-affinity ligands like GM2, since binding is then confined to the 2D membrane surface 54 . In fact, cooperativity was found to be enhanced for a heterogeneous mixture of ligands 54 . In a biological context, it seems more likely that the toxins first bind to the broadly available fucosylated structures on the cell surface or mucus layer until they can bind to the few available GM1 receptors, preventing the flow in the small intestine to remove unbound toxins from the intoxication site 24 .…”

Carbohydrate supramolecular chemistry: beyond the multivalent effect

“…The contribution by Wu and coworkers has been fundamental in this line. In their seminal work, 111,112 Fig . 6 Schematic representation of the rebinding and sliding mechanisms in the interaction of heteromultivalent a-Man/b-Glc bCD-centered heteroglycoclusters with the a-Man-specific lectin ConA as discussed by García Fernández and coworkers.…”

“…9). 112 The experimental observations cannot be explained on the basis of allosteric regulation, but they are undoubtedly dynamic in nature and associated with the simultaneous presence of the high and low affinity ligands over a certain expression level in a relatively large peripheral area. The fact that going from the bulk to coating is a necessary requisite led the authors hypothesize that the heterocluster effect is mediated by a simple mechanism: reduction of dimensionality (RD).…”