title: Molecular mimicry in the recognition of glycosphingolipids by Gal alpha 3 Gal beta 4 GlcNAc beta-binding Clostridium difficile toxin A, human natural anti alpha-galactosyl IgG and the monoclonal antibody Gal-13: characterization of a binding-active human glycosphingolipid, non-identical with the animal receptor.
abstract: Glycoconjugates with terminal Gal alpha 3Gal beta 4GlcNAc beta sequences have been shown to be recognized by three carbohydrate-binding proteins; toxin A of Clostridium difficile, human natural anti alpha-galactosyl IgG and the monoclonal antibody Gal-13. However, the biological significance of this binding specificity in humans is unclear, since unsubstituted Gal alpha 3Gal beta 4GlcNAc beta sequences are not found in human tissues, due to suppression of the gene coding for the enzyme Gal beta 3-transferase. To explore this inconsistency, the binding of toxin A, human natural anti alpha-galactosyl IgG, and the Gal-13 monoclonal antibody to various glycosphingolipids was examined using the thin-layer chromatogram binding assay. The binding to Gal alpha 3Gal beta 4GlcNAc beta-terminated glycosphingolipids of rabbit erythrocytes was confirmed. A minor binding-active compound was also detected in the non-acid glycosphingolipid fraction of human erythrocytes. This glycosphingolipid was isolated and characterized by EI mass spectrometry, gas chromatography-EI mass spectrometry after degradation, and proton NMR spectroscopy, as GalNAc beta 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, corresponding to the x2 glycosphingolipid isolated before form this source. Two additional binding-active glycosphingolipids were found. One was GalNAc alpha 3Gal beta 4GlcNAc beta 3Gal beta 4 Glc beta 1 Cer, produced from blood group A-active GalNAc alpha 3 (Fuc alpha 2) Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1 Cer by acid-induced defucosylation. The other was GlcNAc beta 3 Gal beta 4 GlcNAc beta 3 Gal beta 4 Glc beta 1 Cer, generated from NeuGc alpha 3Gal beta 4GlcNAc beta 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer by enzymatic hydrolysis. A number of other glycosphingolipid sequences, including the human Le(x), Le(y), and I blood group determinants, suggested to act as receptors for toxin A, were not recognized by the three ligands. Despite the different terminal substituents and anomerity of the binding-active glycosphingolipids, calculated minimum energy conformations demonstrated topographical similarities in the spatial orientation of the terminal trisaccharides, possibly accounting for the cross-reactivity.
authors.affiliation: Department of Medical Biochemistry, Göteborg University, Sweden.
authors.forename: S
authors.initials: S
authors.lastname: Teneberg
authors.forename: I
authors.initials: I
authors.lastname: Lönnroth
authors.forename: J F
authors.initials: JF
authors.lastname: Torres López
authors.forename: U
authors.initials: U
authors.lastname: Galili
authors.forename: M O
authors.initials: MO
authors.lastname: Halvarsson
authors.forename: J
authors.initials: J
authors.lastname: Angström
authors.forename: K A
authors.initials: KA
authors.lastname: Karlsson
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001427:Bacterial Toxins; D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer; D004768:Enterotoxins / Q000378:metabolism*; D006001:Glycoconjugates / Q000737:chemistry / Q000378:metabolism*; D006028:Glycosphingolipids / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology*; D013058:Mass Spectrometry; D008958:Models, Molecular; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D011817:Rabbits
chemical_list: D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D004768:Enterotoxins; D006001:Glycoconjugates; D006028:Glycosphingolipids; D007074:Immunoglobulin G; C041348:tcdA protein, Clostridium difficile