Novel carbohydrate binding site recognizing blood group A and B determinants in a hybrid of cholera toxin and Escherichia coli heat-labile enterotoxin B-subunits

The B-subunits of cholera toxin (CTB) and Escherichia coli heat-labile ente rotoxin (LTB) are structurally and functionally related. However, the carbo hydrate binding specificities of the two proteins differ. While both CTB an d LTB bind to the GM1 ganglioside, LTB also binds to N-acetyllactosamine-te rminated glycoconjugates. The structural basis of the differences in carboh ydrate recognition has been investigated by a systematic exchange of amino acids between LTB and CTB. Thereby, a CTB/LTB hybrid with a gain-of-functio n mutation resulting in recognition of blood group A and B determinants was obtained. Glycosphingolipid binding assays showed a specific binding of th is hybrid B-subunit, but not CTB or LTB, to slowly migrating nonacid glycos phingolipids of human and animal small intestinal epithelium. A binding-act ive glycosphingolipid isolated from cat intestinal epithelium was character ized by mass spectrometry and proton NMR as GalNAc alpha 3(Fuc alpha 2)Gal beta 4(Fuc alpha 3)GlcNAc beta 3Gal beta 4Glc NAc beta 3Gal beta 4Glc beta 1Cer. Comparison with reference glycosphingolipids showed that the minimum binding epitope recognized by the CTB/LTB hybrid was Gal alpha 3(Fuc alpha 2)Gal beta 4(Fuc alpha 3)GlcNAc beta. The blood group A and B determinants bind to a novel carbohydrate binding site located at the top of the B-subun it interfaces, distinct from the GM1 binding site, as found by docking and molecular dynamics simulations.