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 enterotoxin (LTB) are structurally and functionally related. However, the carbohydrate binding specificities of the two proteins differ. While both CTB and LTB bind to the GM1 ganglioside, LTB also binds to N-acetyllactosamine-terminated glycoconjugates. The structural basis of the differences in carbohydrate recognition has been investigated by a systematic exchange of amino acids between LTB and CTB. Thereby, a CTB/LTB hybrid with a gain-of-function mutation resulting in recognition of blood group A and B determinants was obtained. Glycosphingolipid binding assays showed a specific binding of this hybrid Bsubunit, but not CTB or LTB, to slowly migrating nonacid glycosphingolipids of human and animal small intestinal epithelium. A binding-active glycosphingolipid isolated from cat intestinal epithelium was characterized by mass spectrometry and proton NMR as GalNAc3(Fuc2)Gal4(Fuc3)GlcNAc3Gal4Glc NAc3Gal4Glc1Cer. Comparison with reference glycosphingolipids showed that the minimum binding epitope recognized by the CTB/LTB hybrid was Gal3(Fuc2)Gal4(Fuc3)GlcNAc. The blood group A and B determinants bind to a novel carbohydrate binding site located at the top of the B-subunit interfaces, distinct from the GM1 binding site, as found by docking and molecular dynamics simulations.