Structural Analysis of the Interaction between Shiga Toxin B Subunits and Linear Polymers Bearing Clustered Globotriose Residues

Abstract

We previously developed linear polymers bearing clustered trisaccharides of globotriaosylceramide (Gb3) as orally applicable Shiga toxin (Stx) neutralizers. Here, using a Gb3 polymer with a short spacer tethering the trisaccharide to the core, we found that shortening the spacer length markedly reduced the binding affinity for Stx2 but not Stx1. Moreover, mutational analysis revealed that the essential binding sites of the terminal trisaccharides were completely different between Stx1 and Stx2. These results provide the molecular basis for the interaction between Stx B subunits and Gb3 polymers.

An Orally Applicable Shiga Toxin Neutralizer Functions in the Intestine To Inhibit the Intracellular Transport of the Toxin

“…Our findings also suggest that the tetravalent peptide accumulates in ileal epithelial cells only through the formation of a complex with Stx2. Thus, the mechanism of action of Ac-PPP-tet is likely to be different from that of other Stx neutralizers previously shown to function in the intestinal tract, all of which exert their inhibitory effects by binding to the toxin and inhibiting its incorporation into the epithelial cells (24,35,36). In Caco-2 cells, the formation of complexes of Ac-PPP-tet and Stx2 blocked the intracellular transport of Stx2 from the Golgi apparatus to the ER, probably resulting in the efficient degradation of this toxin.…”

The Crystal Structure of Shiga Toxin Type 2 with Bound Disaccharide Guides the Design of a Heterobifunctional Toxin Inhibitor

“…To be successful such approaches have to overcome the millimolar intrinsic affinity for Gb 3 analogues and, at least for oligosaccharide based antagonists, the short half-life of such polar molecules in circulation. Several interesting approaches and ligand designs have been reported (22)(23)(24)(25)(26)(27)(28)(29).…”

Polymer antidotes for toxin sequestration

“…The density of the Gb3os ligand and the length of the linker between the ligand and polymer backbone are important attributes in binding. Both Kobayashi et al and Nishikawa et al have synthesized linear acrylamide based polymers containing a variety of Gb3os substitution densities [40][41][42]. In each case, an increased neutralization of toxicity for both Stx1 and Stx2 isoforms was observed with increasing Gb3os ligand density in vitro.…”