Fucose-Galactose Polymers Inhibit Cholera Toxin Binding to Fucosylated Structures and Galactose-Dependent Intoxication of Human Enteroids.

Jakob Cervin,Andrew Boucher,Ville Wallenius,Nicole S. Sampson,Per Björklund,Lynda Mottram,Ulf Yrlid,Gyusaang Youn

doi:10.1021/acsinfecdis.0c00009

Jakob Cervin, Andrew Boucher + Show 6 more

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https://doi.org/10.1021/acsinfecdis.0c00009

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Abstract

A promising strategy to limit cholera severity involves blockers mimicking the canonical cholera toxin ligand (CT) ganglioside GM1. However, to date the efficacies of most of these blockers have been evaluated in noncellular systems that lack ligands other than GM1. Importantly, the CT B subunit (CTB) has a noncanonical site that binds fucosylated structures, which in contrast to GM1 are highly expressed in the human intestine. Here we evaluate the capacity of norbornene polymers displaying galactose and/or fucose to block CTB binding to immobilized protein-linked glycan structures and also to primary human and murine small intestine epithelial cells (SI ECs). We show that the binding of CTB to human SI ECs is largely dependent on the noncanonical binding site, and interference with the canonical site has a limited effect while the opposite is observed with murine SI ECs. The galactose–fucose polymer blocks binding to fucosylated glycans but not to GM1. However, the preincubation of CT with the galactose–fucose polymer only partially blocks toxic effects on cultured human enteroid cells, while preincubation with GM1 completely blocks CT-mediated secretion. Our results support a model whereby the binding of fucose to the noncanonical site places CT in close proximity to scarcely expressed galactose receptors such as GM1 to enable binding via the canonical site leading to CT internalization and intoxication. Our finding also highlights the importance of complementing CTB binding studies with functional intoxication studies when assessing the efficacy inhibitors of CT.

Highlights

A promising strategy to limit cholera severity involves blockers mimicking the canonical cholera toxin ligand (CT) ganglioside GM1
We have previously shown that occupying this noncanonical site with fucosylated histo-blood group antigens (HBGAs) such as Lex or Ley or with polymers carrying fucose (Fuc) blocks CT B subunit (CTB) binding to primary human small intestine (SI)-ECs.[29]
A lectin that binds terminal galactose on human SI-ECs inhibited the toxic effects of CT, the same lectin showed no detectable block on CTB binding to SI-ECs, results that may point to an essential role for high-affinity Gal-terminated receptors such as GM1

Summary

Introduction

A promising strategy to limit cholera severity involves blockers mimicking the canonical cholera toxin ligand (CT) ganglioside GM1. Each subunit of CTB has a binding site facing the CTA subunit distally This site was shown almost 50 years ago to bind the GM1 glycosphingolipid with very high affinity.[9−11] Incorporating GM1 into membranes greatly increases the sensitivity of the cell to CT-mediated intoxication.[12] GM1 has been described as the main ligand for CTB, and several inhibitors aimed at blocking this binding to the canonical site of CTB have been generated and reviewed extensively in Kumar and Turnbull.[13] Some of these carbohydrates that are mimics of GM1 could, in monovalent form, block CTB binding to GM1.14,15 the multivalent display of these mimics or GM1 oligosaccharide (GM1-os) exhibited a greatly enhanced inhibitive capacity to CTB.[16−20] this multimeric display of GM1 or GM1 mimics has been shown to result in inhibition of the toxic effect of CT in vivo in mice and in human colonic cell lines and enteroids.[19−21]. The differential capacity of CT to bind to a patient’s variable levels of HBGAs via the noncanonical site has been suggested to be a potential underlying cause of disease severity.[27]

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