Dataset Information

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

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.

SUBMITTER: Cervin J

PROVIDER: S-EPMC7227030 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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

Cervin Jakob J Boucher Andrew A Youn Gyusaang G Björklund Per P Wallenius Ville V Mottram Lynda L Sampson Nicole S NS Yrlid Ulf U

ACS infectious diseases 20200319 5

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 gala ...[more]

PMID: 32134631

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Project description:Cholera toxin (CT) from Vibrio cholerae is responsible for the majority of the symptoms of the diarrheal disease cholera. CT is a heterohexameric protein complex with a 240-residue A subunit and a pentameric B subunit of identical 103-residue B polypeptides. The A subunit is proteolytically cleaved within a disulfide-linked loop to generate the A1 and A2 fragments. The B subunit of wild-type (wt) CT binds 5 cell surface ganglioside GM(1) (GM(1)) molecules, and the toxin-GM(1) complex traffics from the plasma membrane (PM) retrograde through endosomes and the Golgi apparatus to the endoplasmic reticulum (ER). From the ER, the enzymatic A1 fragment retrotranslocates to the cytosol to cause disease. Clustering of GM(1) by multivalent toxin binding can structurally remodel cell membranes in ways that may assist toxin uptake and retrograde trafficking. We have recently found, however, that CT may traffic from the PM to the ER by exploiting an endogenous glycosphingolipid pathway (A. A. Wolf et al., Infect. Immun. 76:1476-1484, 2008, and D. J. F. Chinnapen et al., Dev. Cell 23:573-586, 2012), suggesting that multivalent binding to GM(1) is dispensable. Here we formally tested this idea by creating homogenous chimeric holotoxins with defined numbers of native GM(1) binding sites from zero (nonbinding) to five (wild type). We found that a single GM(1) binding site is sufficient for activity of the holotoxin. Therefore, remodeling of cell membranes by mechanisms that involve multivalent binding of toxin to GM(1) receptors is not essential for toxicity of CT. Through multivalent binding to its lipid receptor, cholera toxin (CT) can remodel cell membranes in ways that may assist host cell invasion. We recently found that CT variants which bind no more than 2 receptor molecules do exhibit toxicity, suggesting that CT may be able to enter cells by coopting an endogenous lipid sorting pathway without clustering receptors. We tested this idea directly by using purified variants of CT with zero to five functional receptor-binding sites (BS). One BS enabled CT to intoxicate cells, supporting the conclusion that CT can enter cells by coopting an endogenous lipid-sorting pathway. Although multivalent receptor binding is not essential, it does increase CT toxicity. These findings suggest that achieving higher receptor binding avidity or affecting membrane dynamics by lipid clustering and membrane remodeling may be driving forces for evolution of AB(5) subunit toxins that can bind multivalently to cell membrane lipid receptors.

Dataset Information

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

Publications

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

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