Demonstration of the pH Sensitive Binding of Multivalent Carbohydrate Ligands to Immobilized Shiga-Like Toxin 1 B Subunits

• Published in: Journal of biochemistry (Tokyo) Vol. 130; no. 5; pp. 665 - 670
• Main Authors: Imai, Yasuyuki, Matsuura, Yasumi, Ono, Yousuke, Ishikawa, Tomoyuki, Ito, Yukishige
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.11.2001
• Subjects: Animals; Antibodies, Blocking - pharmacology; blocking antibodies; carbohydrate recognition; enzyme-linked immunosor-bent assay; Enzyme-Linked Immunosorbent Assay - methods; Escherichia coli; Fluorescent Dyes - analysis; Hydrogen-Ion Concentration; Isothiocyanates - chemistry; Ligands; Mice; Mice, Inbred BALB C; Polylysine - chemistry; Protein Binding; Protein Conformation; Protein Subunits; Recombinant Proteins - metabolism; Shiga toxin; Shiga Toxin 1 - metabolism; Trihexosylceramides - chemistry; Trihexosylceramides - metabolism
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a003032

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 recombi-nant 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.