NMR experiments reveal distinct antibody-bound conformations of a synthetic disaccharide representing a general structural element of bacterial lipopolysaccharide epitopes.
The recognition reactions between a synthetic disaccharide alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl and two monoclonal antibodies (mAbs) were studied by NMR, yielding two distinct bound conformations of the carbohydrate ligand. One mAb, S23-24, recognizes the disaccharides alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl and alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl with similar affinities, whereas mAb S25-2 binds to the disaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl with an approximately 10-fold higher affinity than to the disaccharide alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl. Compared to S25-2, S23-24 binds to alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl with an approximately 50-fold increased affinity. We used NMR experiments that are based on the transferred NOE effect, specifically, trNOESY, trROESY, QUIET-trNOESY, and MINSY experiments, to show that the (2-->8)-specific mAb, S25-2, stabilizes a conformation of the alpha-(2-->4)-linked disaccharide that is not highly populated in solution. S23-24 recognizes two conformations of alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl, one that is highly populated in aqueous solution and another conformation that is similar to the one bound by S25-2. This is the first example where it is experimentally shown that a carbohydrate ligand may adopt different bioactive conformations upon interaction with mAbs with different fine specificities. Our NMR studies indicate that a careful examination of spin diffusion is critical for the analysis of bioactive conformations of carbohydrate ligands.
Haselhorst
T
T
Institut für Chemie, Medizinische Universität Lübeck, Germany.
Espinosa
J F
JF
Jiménez-Barbero
J
J
Sokolowski
T
T
Kosma
P
P
Brade
H
H
Brade
L
L
Peters
T
T
D000907:Antibodies, Bacterial / Q000737:chemistry / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D001666:Binding Sites, Antibody; D002236:Carbohydrate Conformation; D002689:Chlamydia / Q000276:immunology; D004187:Disaccharides / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008070:Lipopolysaccharides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods
C114585:3-deoxymanno-oct-2-ulosonic acid-(2-8)-3-deoxymanno-oct-2-ulosonic acid-(2-O)-allyl; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D004187:Disaccharides; D000939:Epitopes; D008024:Ligands; D008070:Lipopolysaccharides