Fucosylated Molecules Competitively Interfere with Cholera Toxin Binding to Host Cells

Abstract

Cholera toxin (CT) enters host intestinal epithelia cells, and its retrograde transport to the cytosol results in the massive loss of fluids and electrolytes associated with severe dehydration. To initiate this intoxication process, the B subunit of CT (CTB) first binds to a cell surface receptor displayed on the apical surface of the intestinal epithelia. While the monosialoganglioside GM1 is widely accepted to be the sole receptor for CT, intestinal epithelial cell lines also utilize fucosylated glycan epitopes on glycoproteins to facilitate cell surface binding and endocytic uptake of the toxin. Further, l-fucose can competively inhibit CTB binding to intestinal epithelia cells. Here, we use competition binding assays with l-fucose analogs to decipher the molecular determinants for l-fucose inhibition of cholera toxin subunit B (CTB) binding. Additionally, we find that mono- and difucosylated oligosaccharides are more potent inhibitors than l-fucose alone, with the LeY tetrasaccharide emerging as the most potent inhibitor of CTB binding to two colonic epithelial cell lines (T84 and Colo205). Finally, a non-natural fucose-containing polymer inhibits CTB binding two orders of magnitude more potently than the LeY glycan when tested against Colo205 cells. This same polymer also inhibits CTB binding to T84 cells and primary human jejunal epithelial cells in a dose-dependent manner. These findings suggest the possibility that polymeric display of fucose might be exploited as a prophylactic or therapeutic approach to block the action of CT toward the human intestinal epithelium.

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

“…Moreover, SPR experiments with toxin variants confirmed that disruption of the secondary binding site, but not the primary binding site, lowered the affinity to Le x (Figure 4, Table 2). We also found that Le x bound more weakly to CTB than Le y or A-Le y , which is in good agreement with inhibitor studies identifying Le y as the most potent small-molecule inhibitor of CTB 33 . The binding mode of Le x is highly similar to that of Le y tetraose and A-Le y pentaose 19 , which both feature an additional fucose residue (Figure 2 c).…”

“…Addition of a methyl group to OH1, locking the anomeric hydroxyl group in the P-configuration, resulted in less efficient CTB binding compared to L -fucose or α-locked fucose. Finally, the SAR study showed increased binding of α1,2 linked fucose compared to α1,3 linked fucose 33 .…”

“…Already twenty years ago, CTB was shown to bind fucose 43 . More recently, evidence was presented that BGAs may act as functional toxin receptors for the related LTB 44, 45 , and fucosylated glycan structures were shown to be functionally active and cause cellular uptake of CT 30,32,33 . For example, CTB binding to jejunal epithelial cells can be blocked by the Le x trisaccharide or a monoclonal antibody against Le x 32 Crosslinking and immunoprecipitation of CTB-bound cellular proteins revealed that CTB binds to glycoproteins modified with Le x 32 It was, however, unclear how Le x binds to CT. Also G33D, a CTB variant with greatly reduced affinity to GM1 15, 46 , showed weaker binding to Le x 32 Since Gly33 is located in the GM1 binding site, it was suggested that also Le x might bind to the primary binding site 32 .…”

“…L -fucose has been shown to block CTB binding in cell culture, serving as a promising starting point for the design of novel cholera inhibitors 33 . Recently, Wands et al described important molecular determinants of L -fucose by competition binding assays of CTB to various intestinal epithelial cells 33 . Their structure-activity relationship (SAR) studies showed that the stereochemical positioning of the terminal fucose is crucial for CTB binding.…”

“…Current research shows that fucosylated structures have an important role in cholera intoxication 30–33 . CT binds fucosylated human BGAs merely with millimolar affinity 19,23–25 , but nevertheless causes blood-group-dependent cellular intoxication 31 .…”