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 B-subunit, but not CTB or LTB, to slowly migrating non-acid 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 GalNAcalpha3(Fucalpha2)Galbeta4(Fucalpha3)Glc NAcbeta3Galbeta4Glc NAcbeta3Galbeta4Glcbeta1Cer. Comparison with reference glycosphingolipids showed that the minimum binding epitope recognized by the CTB/LTB hybrid was Galalpha3(Fucalpha2)Galbeta4(Fucalpha3)GlcNAc beta. 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.
Angström
J
J
Institute of Medical Biochemistry, Göteborg University, P. O. Box 440, SE 405 30 Göteborg, Sweden.
Bäckström
M
M
Berntsson
A
A
Karlsson
N
N
Holmgren
J
J
Karlsson
K A
KA
Lebens
M
M
Teneberg
S
S
D000017:ABO Blood-Group System / Q000378:metabolism*; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry / Q000235:genetics; D002415:Cats; D002772:Cholera Toxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004768:Enterotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000017:ABO Blood-Group System; D001427:Bacterial Toxins; D002241:Carbohydrates; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D002772:Cholera Toxin; C027538:heat-labile enterotoxin, E coli