title: A cholera toxin B-subunit variant that binds ganglioside G(M1) but fails to induce toxicity.
abstract: Entry of cholera toxin (CT) into target epithelial cells and the induction of toxicity depend on CT binding to the lipid-based receptor ganglioside G(M1) and association with detergent-insoluble membrane microdomains, a function of the toxin's B-subunit. The B-subunits of CT and related Escherichia coli toxins exhibit a highly conserved exposed peptide loop (Glu(51)-Ile(58)) that faces the cell membrane upon B-subunit binding to G(M1). Mutation of His(57) to Ala in this loop resulted in a toxin (CT-H57A) that bound G(M1) with high apparent affinity, but failed to induce toxicity. CT-H57A bound to only a fraction of the cell-surface receptors available to wild-type CT. The bulk of cell-surface receptors inaccessible to CT-H57A localized to detergent-insoluble apical membrane microdomains (lipid rafts). Compared with wild-type toxin, CT-H57A exhibited slightly lower apparent binding affinity for and less stable binding to G(M1) in vitro. Rather than being transported into the Golgi apparatus, a process required for toxicity, most of CT-H57A was rapidly released from intact cells at physiologic temperatures or degraded following its internalization. These data indicate that CT action depends on the stable formation of the CT B-subunit.G(M1) complex and provide evidence that G(M1) functions as a necessary sorting motif for the retrograde trafficking of toxin into the secretory pathway of target epithelial cells.
authors.affiliation: Gastrointestinal Cell Biology, Department of Pediatrics, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: C
authors.initials: C
authors.lastname: Rodighiero
authors.forename: Y
authors.initials: Y
authors.lastname: Fujinaga
authors.forename: T R
authors.initials: TR
authors.lastname: Hirst
authors.forename: W I
authors.initials: WI
authors.lastname: Lencer
mesh_terms: D002772:Cholera Toxin / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D004705:Endocytosis / Q000502:physiology; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D014407:Tumor Cells, Cultured
chemical_list: D005677:G(M1) Ganglioside; D002772:Cholera Toxin