In vitro binding characteristics and affinity for sulfatide of Escherichia coli STb enterotoxin.
It has previously been demonstrated that sulfatide (3'-sulfogalactosyl-ceramide), present at the surface of epithelial cells of the small intestine of pigs, interacts with the thermostable enterotoxin b (STb) produced by ETEC, and that this molecule is implicated in the mechanism of action of the toxin. However, few things are known about the affinity and physical characteristics of the interaction between these two macromolecules. In this study, using a microtiter plate binding assay (MPBA), we showed that STb toxin has a strong specificity for sulfatide and that this binding is dose-dependent and saturable. A very weak binding occurred with galactosyl-ceramide whereas attachment to 3'-sulfolactosyl-ceramide corresponded to 76% of the binding to sulfatide. STb toxin was shown to possess a lectin-like property; a significative binding was observed when a terminal beta-galactose was present in the glycosphingolipids tested and an increased binding was observed in presence of a sulfate group in position 3 on the galactose. These findings suggest that a sulfated galactosyl residue seems to represent the epitope recognized by the toxin. The reaction between sulfatide and STb toxin is also time and temperature dependent and is not affected by pH. The interaction was not inhibited by free sugars, sulfated polymers, glycolipids or free ions, but was partly inhibited by high concentrations of charged sugars. STb-sulfatide binding process is a low affinity interaction, as demonstrated by the determined Kd of 2-6 +/- 1.5 microM.
Beausoleil
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HE
Département de pathologie et microbiologie, faculté de médecine vétérinaire, Université de Montréal, 3200 Sicotte, P.O. Box 5000, Saint-Hyacinthe, Québec, Canada J2S 7C6.
Dubreuil
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JD
D001427:Bacterial Toxins / Q000378:metabolism*; D004768:Enterotoxins / Q000378:metabolism*; D004926:Escherichia coli / Q000472:pathogenicity*; D029968:Escherichia coli Proteins; D006162:Guanylate Cyclase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011485:Protein Binding / Q000187:drug effects; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000378:metabolism; D013433:Sulfoglycosphingolipids / Q000378:metabolism*; D013696:Temperature
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D018000:Receptors, Peptide; D013433:Sulfoglycosphingolipids; C034151:heat stable toxin (E coli); D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled