title: Analysis of carbohydrate-protein interactions with synthetic N-linked neoglycoconjugate probes.
abstract: Recently we have describe a simple efficient chemical method of generating an asparagine side-chain linker with beta-stereochemistry at the anomeric position of neutral oligosaccharides. We now report the 1-N-glycyl beta-derivatization of sialylated saccharides. Several neoglycoconjugates formed using these N-linked inter-mediates were investigated for their usefulness in probing carbohydrate-protein interactions. First, biotinyl derivatives of two xylose/fucose class plant-type oligosaccharides purified from horseradish peroxidase were effective in demonstrating the carbohydrate specificity of polyclonal anti-(horseradish peroxidase) antibodies. Secondly, a fluorescein-labelled asialo- and digalactosylated biantennary complex sugar was synthesized and shown to bind to a Ricinus communis agglutinin column. This galactose-specific recognition was abolished by treating this fluorescein-labelled oligosaccharide with jack bean beta-galactosidase. Finally, two 1-N-glycyl beta-saccharide derivatives were modified with thiophosgene to form their corresponding isothiocyanate derivatives. Coupling of these isothiocyanate derivatives of sugars to BSA, amino-derivatized polystyrene plates and glass-fibre discs resulted in multiple sugar presentation. The binding of an anti-N-acetylglucosamine monoclonal antibody to N,N'-diacetylchitobiose residues presented on BSA and solid supports was shown by e.l.i.s.a. Similarly the binding of concanavalin A to asialo-, agalactosylated biantennary complex oligosaccharide residues attached to BSA was demonstrated by a competitive e.l.i.s.a. Our results demonstrate that N-linked neoglycoconjugates could be made readily available and they are valuable tools for the detailed analyses of carbohydrates and carbohydrate-binding proteins.
authors.affiliation: Department of Biochemistry, University of Oxford, U.K.
authors.forename: S Y
authors.initials: SY
authors.lastname: Wong
authors.forename: I D
authors.initials: ID
authors.lastname: Manger
authors.forename: G R
authors.initials: GR
authors.lastname: Guile
authors.forename: T W
authors.initials: TW
authors.lastname: Rademacher
authors.forename: R A
authors.initials: RA
authors.lastname: Dwek
mesh_terms: D000906:Antibodies / Q000276:immunology; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D016650:Fluorescein-5-isothiocyanate; D006001:Glycoconjugates / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000276:immunology; D037102:Lectins; D008969:Molecular Sequence Data; D009844:Oligosaccharides; D011506:Proteins / Q000378:metabolism*
chemical_list: D000906:Antibodies; D002241:Carbohydrates; D000939:Epitopes; D006001:Glycoconjugates; D037102:Lectins; D009844:Oligosaccharides; D011506:Proteins; D006735:Horseradish Peroxidase; D016650:Fluorescein-5-isothiocyanate