GM1 ganglioside-independent intoxication by Cholera toxin

Abstract

Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (LeX) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the LeX glycan in vitro when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to LeX. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants.

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

“…Likewise, CT variant H18A showed increased GM1os affinity compared to wild-type CT, even though the mutation is in the secondary binding site. Cross-talk between the two sites could also explain the partial competition of GM1 and fucosylated receptors and the reduced binding of G33D to fucosylated structures observed by Cervin et al 32 .…”

“…Recent studies suggested that Le x and similar structures might also bind to the GM1 binding site 32 . However, to date all relevant crystal structures of CTB contain fucosylated sugars in the secondary binding site 19, 20 .…”

“…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 . To explore this possibility, Cervin et al performed competition experiments with plate-bound GM1 or Le x and CTB pre-incubated with the free sugars 32 .…”

“…A large body of research points to GM1 and fucosylated structures facilitating toxin binding and uptake 10,14–18,30,32,33 , however, little is known about the exact localization and expression of specific fucosylated structures on healthy intestinal cells and tissues 32,50–54 . It is not clear which fucosylated structures are the major cellular attachment sites besides GM1.…”

“…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 . Due to the lack of soluble BGAs, non-secretors would moreover lack competition by soluble and mucin-bound CT receptors, leading to more efficient CT uptake and more severe cholera symptoms.…”