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

Abstract

SummaryThe 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.

37-kDa Laminin Receptor Precursor Modulates Cytotoxic Necrotizing Factor 1–mediated RhoA Activation and Bacterial Uptake

“…However, this is the first demonstration that LRP acts as a cellular receptor for bacterial toxin in activation of RhoA and enhancement of bacterial uptake. Identification of receptors for bacterial toxins has been a challenge, and only a limited number of these receptors have been identified to date (31,32). The identification of a toxin receptor is essential for elucidating structure-functional analysis of a toxin as well as understanding the pathogenesis of toxin-induced diseases.…”

The mucosal adjuvanticity of two nontoxic mutants of Escherichia coli heat-labile enterotoxin varies with immunization routes

“…Although the mutant LTs resembled the holotoxin in terms of assembly and cell binding activity, the mutations in the A subunit may influence receptorbinding properties and hence exert different effects on lymphocytes of the mucosal immune system. In contrast to CT, which binds to only G M1 , LT exhibits a broad range of binding affinities including G M1 , G M2 , and asialo-G M1 (Tsuji et al, 1985;Spiegel, 1992;Bäckström et al, 1997). This suggests that different mutant forms of LT may require different immunization routes for adequate adjuvanticity, and that specific immunization route may elicit specific mechanisms of up-regulation of immune responses, independent of cAMP levels.…”

“…Both toxins are composed of two functionally distinct domains, the enzymatically active A subunit with ADP-ribosylating activity (Noda et al, 1989;Lycke et al, 1992;Dickinson et al, 1995), and the pentameric B subunit that contains a G M1 (monosialoganglioside) receptor-binding site (Tsuji et al, 1985;Spiegel, 1990;Bäckström et al, 1997). The A subunit intoxicates eucaryotic cells by activating Gs, a GTPbinding protein that regulates the levels of second messenger cAMP (Guerrant et al, 1974).…”

Development of two novel nontoxic mutants of Escherichia coli heat-labile enterotoxin

“…LT is a multimeric protein composed of two functionally distinct domains; the enzymatically active A subunit (LTA; Mr, ~30,000 daltons) with ADP-ribosylating a c t i v i t y, and the pentameric B subunits (LTB; Mr, ~11 , 6 0 0 daltons) that contain G M1 (monosialoganglioside) receptorbinding site (Bäckström et al, 1997). Upon thiol reduction, the A subunit dissociates into two polypeptide chains, A1 ( M r, 23,000 daltons) and A2 (Mr, 6,000 daltons) (Tsuji e t a l., 1985;Grant et al, 1994).…”