Structural basis for differential receptor binding of cholera and Escherichia coli heat-labile toxins: influence of heterologous amino acid substitutions in the cholera B-subunit.
The closely related B-subunits of cholera toxin (CTB) and Escherichia coli heat-labile enterotoxin (LTB) both bind strongly to GM1 ganglioside receptors but LTB can also bind to additional glycolipids and glycoproteins. A number of mutant CT B-subunits were generated by substituting CTB amino acids with those at the corresponding positions in LTB. These were used to investigate the influence of specific residues on receptor-binding specificity. A mutated CTB protein containing the first 25 residues of LTB in combination with LTB residues at positions 94 and 95, bound to the same extent as native LTB to both delipidized rabbit intestinal cell membranes, complex glycosphingolipids (polyglycosylceramides) and neolactotetraosylceramide, but not to non-GM1 intestinal glycosphingolipids. In contrast, when LTB amino acid substitutions in the 1-25 region were combined with those in the 75-83 region, a binding as strong as that of LTB to intestinal glycosphingolipids was observed. In addition, a mutant LTB with a single Gly-33-->Asp substitution that completely lacked affinity for both GM1 and non-GM1 glycosphingolipids could still bind to receptors in the intestinal cell membranes and to polyglycosylceramides. We conclude that the extra, non-GM1 receptors for LTB consist of both sialylated and non-sialylated glycoconjugates, and that the binding to either class of receptors is influenced by different amino acid residues within the protein.
Bäckström
M
M
Department of Medical Microbiology and Immunology, Göteborg University, Sweden.
Shahabi
V
V
Johansson
S
S
Teneberg
S
S
Kjellberg
A
A
Miller-Podraza
H
H
Holmgren
J
J
Lebens
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002772:Cholera Toxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004768:Enterotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005677:G(M1) Ganglioside / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007421:Intestine, Small / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D017354:Point Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014734:Vibrio cholerae / Q000235:genetics / Q000378:metabolism
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C034993:ganglioside receptor; D005677:G(M1) Ganglioside; D002772:Cholera Toxin; C027538:heat-labile enterotoxin, E coli