Abstract
The verotoxin (VT) (Shiga toxin) receptor globotriaosyl ceramide (Gb(3)), mediates VT1/VT2 retrograde transport to the endoplasmic reticulum (ER) for cytosolic A subunit access to inhibit protein synthesis. Adamantyl Gb(3) is an amphipathic competitive inhibitor of VT1/VT2 Gb(3) binding. However, Gb(3)-negative VT-resistant CHO/Jurkat cells incorporate adaGb(3) to become VT1/VT2-sensitive. CarboxyadaGb(3), urea-adaGb(3), and hydroxyethyl adaGb(3), preferentially bound by VT2, also mediate VT1/VT2 cytotoxicity. VT1/VT2 internalize to early endosomes but not to Golgi/ER. AdabisGb(3) (two deacyl Gb(3)s linked to adamantane) protects against VT1/VT2 more effectively than adaGb(3) without incorporating into Gb(3)-negative cells. AdaGb(3) (but not hydroxyethyl adaGb(3)) incorporation into Gb(3)-positive Vero cells rendered punctate cell surface VT1/VT2 binding uniform and subverted subsequent Gb(3)-dependent retrograde transport to Golgi/ER to render cytotoxicity (reduced for VT1 but not VT2) brefeldin A-resistant. VT2-induced vacuolation was maintained in adaGb(3)-treated Vero cells, but vacuolar membrane VT2 was lost. AdaGb(3) destabilized membrane cholesterol and reduced Gb(3) cholesterol stabilization in phospholipid liposomes. Cholera toxin GM1-mediated Golgi/ER targeting was unaffected by adaGb(3). We demonstrate the novel, lipid-dependent, pseudoreceptor function of Gb(3) mimics and their structure-dependent modulation of endogenous intracellular Gb(3) vesicular traffic.
Highlights
Verotoxin internalization and retrograde transport to the Golgi/endoplasmic reticulum (ER) is mediated by Gb3 glycolipid
The VT B subunit pentamer binding to Gb3 provides the basis for renal glomerular endothelial cell targeting following systemic verotoxemia and plays a central role in the pathology of hemolytic uremic syndrome (HUS) [17, 56], which remains a lifethreatening complication of gastrointestinal verotoxin-producing E. coli infection, an ever increasing threat in the devel
Verotoxin binding to cell surface Gb3 provides an index of the complex manner in which cell surface GSLs can be presented within a bilayer for ligand recognition and is a probe for GSL membrane organization [57]
Summary
Verotoxin internalization and retrograde transport to the Golgi/ER is mediated by Gb3 glycolipid. Substitution of the Gb3 fatty acid with an adamantane frame provided a watersoluble analog of Gb3 that retained high affinity VT1 binding in an aqueous environment [44, 45] This analog proved an effective competitor to prevent VT1 and VT2 cytotoxicity in vitro [12], in vivo, the analog was found to augment rather than reduce VT2 cytopathology [46]. We find that this adaGb3 pathway can hijack the endogenous cellular Gb3-mediated retrograde transport of VT in sensitive cells to reroute traffic and induce resistance This may relate to loss of interaction with cholesterol that we show for the adaGSL mimic. A dimeric Gb3 analog retains the VT1/VT2 inhibitory activity of adaGb3 in solution but is unable to insert into cell membranes to show VT1/VT2 pseudoreceptor function These studies demonstrate the importance of the lipid chemistry of Gb3 in membrane incorporation and intracellular trafficking and illustrate a new approach against VT-induced cytopathology. Exogenous GSL mimics can be functionally trafficked in cells and, according to their lipid structure, subvert endogenous intracellular membrane GSL trafficking pathways
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