A cholera toxin B-subunit variant that binds ganglioside G(M1) but fails to induce toxicity.
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.
Rodighiero
C
C
Gastrointestinal Cell Biology, Department of Pediatrics, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Fujinaga
Y
Y
Hirst
T R
TR
Lencer
W I
WI
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
D005677:G(M1) Ganglioside; D002772:Cholera Toxin