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.

Keywords: cholera toxin; fucose; galactose; inhibition; multivalent glycopolymer; norbornene.

Scheme 1. Norborneyl Glycopolymers Prepared

Catalyst 2 is dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene](benzylidene)bis(3-bromopyridine)ruthenium(II). Abbreviations used in the text: poly (1a′)₁₀₀, pGlc₁₀₀; poly (1b′)₁₀₀, pGal₁₀₀; poly (1c′)₁₀₀, pFuc₁₀₀; poly (1b′)_n-ran-poly(1c′)_n, pGal_nFuc_n; poly (1a)₁₀₀, Glc₁₀₀; poly (1b)₁₀₀ Gal₁₀₀; poly (1c)₁₀₀, Fuc₁₀₀; and poly (1b)_n-ran-poly(1c)_n, Gal_nFuc_n, where n = DP.

Figure 1

Polymer block in ELISA of CTB binding to triLe^x and GM1. (A and C) Plates were coated with triLe^x-HSA and probed with CTB preincubated with the different polymers. (B and D) Plates were coated with GM1-HSA and probed with CTB preincubated with the different polymers. In (A) and (B), all polymers used displayed 100 sugars. In (C) and (D), the length of the copolymer varies with the number of displayed sugars. Curve fits were made using a three-parameter fit to eq 1. Dotted lines indicate unblocked CTB binding. Graphs show a representative experiment out of three independent experiments, and error bars are the SD of intra-assay duplicates or quadruplicates.

Figure 2

Evaluation of glycopolymers’ capacity to block CTB binding to murine SI enterocytes and intoxication. (A–C) Cells were isolated from murine SI, stained for common cell markers and with CTB to analyze the polymer block by flow cytometry. Full gating can be seen in Figure S4. Panel (A) shows representative histograms of CTB binding to WT EpCAM⁺ cells with or without the polymer block. Panels (B) and (C) show graphs of gMFI for CTB binding after the polymer block. The values are normalized to the % of unblocked CTB gMFI. Data is pooled from three independent experiments with two to three mice in each experiment, and error bars represent the SD. (D) Representative pictures of ligated loops after 4 h of CT (10 μg/mL) treatment with or without Gal₅₀Fuc₅₀ polymer (40 μM) or just PBS in vivo. (E) Bar graph showing fluid accumulation as the length/weight ratio of the ligated loops. In each animal, two loops were created. Statistics were calculated using one-way ANOVA with Tukey correction. Two stars represent p < 0.01, and four stars represent p < 0.001. Error bars represent the SD, and each dot represents a loop.

Figure 3

Flow cytometry evaluation of glycopolymers’ capacity to block CTB and LTB binding in human SI enterocytes. Cells were isolated from human SI and stained for common cell markers and with CTB to analyze the polymer block by flow cytometry. Full gating can be seen in Figure S6. Panel (A) shows representative histograms of CTB binding to EpCAM+ cells with or without a polymer block. Panels (B) and (C) show graphs of gMFI for CTB binding after long polymer (B) and short polymer (C) blocks (n = 5–10 donors). (D) Representative (out of five donors) histogram of LTB binding to EpCAM+ cells with or without a polymer block. (E) Representative (out of five donors) histogram of CTB and LTB binding to EpCAM+ cells. The values are normalized to the % of unblocked CTB gMFI. Error bars represent the SD.

Figure 4

Enteroid characterization and functional evaluation of the polymer block. (A) Enteroid cells were evaluated using flow cytometry for the presence of Lrg5⁺ stem cells. (B–D) Enteroid cells were cultured on a transwell insert and differentiated into a non-stem-cell state for 5 days. For all panels, the DAPI stain is blue and the enterocyte marker phalloidin stain is red (for cell visualization). Markers were used to identify different cell types (green) such as goblet cells (B), mature enterocytes (C), and Paneth cells (D). (E) One representative histogram out of two independent experiments of polymer and the GM1-os block of CTB binding to enteroid cells (flow cytometry). Full gating can be seen in Figure S7. One representative analysis out of four donors. (F) Bar graph showing the % of CTB gMFI on cells from four different donors after preincubating CTB with GM1-os or polymers. Error bars are SD.

Figure 5

CT challenge of enteroid cells. Differentiated enteroid cells were used to evaluate the polymer (5 μM) block of CT (0.1 μg/mL) intoxication. The graph shows I_sc values pooled from four independent experiments using enteroids from one donor. Values are normalized to PBS-treated control cells at each time point. Statistics calculated using two-way ANOVA with Tukey correction in Prism software. *Represents a significant difference between the closest data point and CT only. One star represents p < 0.05, three stars represent p < 0.001, and four stars represent p < 0.0001. Error bars are the SEM.