Mapping the binding of synthetic disaccharides representing epitopes of chlamydial lipopolysaccharide to antibodies with NMR.
A NMR study of the binding of the synthetic disaccharides alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl 1 (Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid) and alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl 2, representing partial structures of the lipopolysaccharide epitope of the intracellular bacteria Chlamydia, to corresponding monoclonal antibodies (mAbs) S23-24, S25-39, and S25-2 is presented. The conformations of 1 bound to mAbs S25-39 and of 2 bound to mAbs S23-24 and S25-39 were analyzed by employing transfer-NOESY (trNOESY) and QUIET-trNOESY experiments. A quantitative analysis of QUIET-trNOESY buildup curves clearly showed that S25-39 recognized a conformation of 1 that was similar to the global energy minimum of 1, and significantly deviated from the conformation of 1 bound to mAb S25-2. For disaccharide 2, only a qualitative analysis was possible because of severe spectral overlap. Nevertheless, the analysis showed that all mAbs most likely bound to only one conformational family of 2. Saturation transfer difference (STD) NMR experiments were then employed to analyze the binding epitopes of the disaccharide ligands 1 and 2 when binding to mAbs S23-24, S25-39, and S25-2. It was found that the nonreducing pyranose unit was the major binding epitope, irrespective of the mAb and the disaccharide that were employed. Individual differences were related to the engagement of other portions of the disaccharide ligands.
Maaheimo
H
H
Institut für Chemie, Medizinische Universität Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany.
Kosma
P
P
Brade
L
L
Brade
H
H
Peters
T
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D001666:Binding Sites, Antibody; D002236:Carbohydrate Conformation; D002692:Chlamydia trachomatis / Q000276:immunology*; D004187:Disaccharides / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D004187:Disaccharides; D000939:Epitopes; D008070:Lipopolysaccharides