title: Attenuated endocytosis and toxicity of a mutant cholera toxin with decreased ability to cluster ganglioside GM1 molecules.
abstract: Cholera toxin (CT) moves from the plasma membrane (PM) of host cells to the endoplasmic reticulum (ER) by binding to the lipid raft ganglioside GM(1). The homopentomeric B-subunit of the toxin can bind up to five GM(1) molecules at once. Here, we examined the role of polyvalent binding of GM(1) in CT action by producing chimeric CTs that had B-subunits with only one or two normal binding pockets for GM(1). The chimeric toxins had attenuated affinity for binding to host cell PM, as expected. Nevertheless, like wild-type (wt) CT, the CT chimeras induced toxicity, fractionated with detergent-resistant membranes extracted from toxin-treated cells, displayed restricted diffusion in the plane of the PM in intact cells, and remained bound to GM(1) when they were immunoprecipitated. Thus, binding normally to two or perhaps only one GM(1) molecule is sufficient for association with lipid rafts in the PM and toxin action. The chimeric toxins, however, were much less potent than wt toxin, and they entered the cell by endocytosis more slowly, suggesting that clustering of GM(1) molecules by the B-subunit enhances the efficiency of toxin uptake and perhaps also trafficking to the ER.
authors.affiliation: GI Cell Biology, Enders 720, Children's Hospital Boston, 300 Longwood Ave., Boston, MA 02115, USA.
authors.forename: Anne A
authors.initials: AA
authors.lastname: Wolf
authors.forename: Michael G
authors.initials: MG
authors.lastname: Jobling
authors.forename: David E
authors.initials: DE
authors.lastname: Saslowsky
authors.forename: Eli
authors.initials: E
authors.lastname: Kern
authors.forename: Kimberly R
authors.initials: KR
authors.lastname: Drake
authors.forename: Anne K
authors.initials: AK
authors.lastname: Kenworthy
authors.forename: Randall K
authors.initials: RK
authors.lastname: Holmes
authors.forename: Wayne I
authors.initials: WI
authors.lastname: Lencer
mesh_terms: D002460:Cell Line; D002772:Cholera Toxin / Q000235:genetics* / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D005677:G(M1) Ganglioside / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D021122:Protein Subunits; D021381:Protein Transport
chemical_list: D021122:Protein Subunits; D005677:G(M1) Ganglioside; D002772:Cholera Toxin