Demonstration of the pH sensitive binding of multivalent carbohydrate ligands to immobilized Shiga-like toxin 1 B subunits.
The cytotoxic effects of Shiga-like toxins from enterohemorrhagic Escherichia coli O157:H7 depend on the recognition of carbohydrate determinants by B subunits. As a specific carbohydrate ligand, globotriaosylceramide has been characterized. We developed an alternative binding assay using multivalent carbohydrate ligands. We prepared globotriose-conjugated poly-lysine, and measured their binding to immobilized recombinant B subunits by an ELISA format. The signals representing ligand binding were dependent on the amount of immobilized B subunits as well as on the concentration of the ligands. The ligand binding activity was lost in an acidic environment, in which changes in the local conformation of the B subunits have been reported. Furthermore, pH dependent dissociation of the ligands from the B subunits was observed. We also demonstrate that antiserum from mice immunized with the B subunits specifically interferes with ligand binding. This suggests further potential for an assay to screen for blocking antibodies that could inhibit toxin internalization into host cells.
Imai
Y
Y
Department of Microbiology, University of Shizuoka School of Pharmaceutical Sciences, Shizuoka 422-8526, Japan. imai@u-shizuoka-ken.ac.jp
Matsuura
Y
Y
Ono
Y
Y
Ishikawa
T
T
Ito
Y
Y
D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005456:Fluorescent Dyes / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D017879:Isothiocyanates / Q000737:chemistry; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D011107:Polylysine / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000378:metabolism; D022622:Shiga Toxin 1 / Q000378:metabolism*; D014281:Trihexosylceramides / Q000737:chemistry / Q000378:metabolism*
D019138:Antibodies, Blocking; D005456:Fluorescent Dyes; D017879:Isothiocyanates; D008024:Ligands; D021122:Protein Subunits; D011994:Recombinant Proteins; D022622:Shiga Toxin 1; D014281:Trihexosylceramides; D011107:Polylysine; C037152:isothiocyanic acid; C018549:globotriaosylceramide