The recognition reactions between a synthetic disaccharide α-Kdo-(2→4)-α-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 α-Kdo-(2→4)-α-Kdo-(2→O)-allyl and α-Kdo-(2→8)-α-Kdo-(2→O)-allyl with similar affinities, whereas mAb S25-2 binds to the disaccharide α-Kdo-(2→8)-α-Kdo-(2→O)-allyl with an approximately 10-fold higher affinity than to the disaccharide α-Kdo-(2→4)-α-Kdo-(2→O)-allyl. Compared to S25-2, S23-24 binds to α-Kdo-(2→4)-α-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 α-(2→4)-linked disaccharide that is not highly populated in solution. S23-24 recognizes two conformations of α-Kdo-(2→4)-α-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.

Lipopolysaccharide, NMR, conformation, Bacterial, structural, epitope, epitopes, disaccharide, Synthetic

13C NMR data:
missing...
1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8
1,4	aXKdop	-	-	1.87-2.24	4.18	4.14	3.71	4.08	3.84-4.07
1	aXKdop	-	-	2.02-2.10	4.24	4.18	3.65	4.04	3.71-4.02
	Allyl

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

C=CCO[C@]1(C(=O)O)C[C@@H](O[C@]2(C(=O)O)C[C@@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O2)[C@@H](O)[C@@H]([C@H](O)CO)O1

498.434 g/mol (C₁₉H₃₀O₁₅)

The recognition reactions between a synthetic disaccharide α-Kdo-(2→4)-α-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 α-Kdo-(2→4)-α-Kdo-(2→O)-allyl and α-Kdo-(2→8)-α-Kdo-(2→O)-allyl with similar affinities, whereas mAb S25-2 binds to the disaccharide α-Kdo-(2→8)-α-Kdo-(2→O)-allyl with an approximately 10-fold higher affinity than to the disaccharide α-Kdo-(2→4)-α-Kdo-(2→O)-allyl. Compared to S25-2, S23-24 binds to α-Kdo-(2→4)-α-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 α-(2→4)-linked disaccharide that is not highly populated in solution. S23-24 recognizes two conformations of α-Kdo-(2→4)-α-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.

Lipopolysaccharide, NMR, conformation, Bacterial, structural, epitope, epitopes, disaccharide, Synthetic

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

C=CCO[C@]1(C(=O)O)C[C@@H](O)[C@@H](O)[C@@H]([C@H](O)CO[C@]2(C(=O)O)C[C@@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O2)O1

498.434 g/mol (C₁₉H₃₀O₁₅)

Transferred nuclear Overhauser enhancement (TRNOE) experiments have been performed to investigate the bound conformation of the trisaccharide repeating unit of the Streptococcus Group A cell-wall polysaccharide. Thus, the conformations of propyl 3-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-2-O-(α-L-rhamnopyran osyl)-α-L-rhamnopyranoside [C(A')B] (1) as a free ligand and when complexed to the monoclonal antibody Strep 9 were examined. Improved insights about the conformational preferences of the glycosidic linkages of the trisaccharide ligand showed that the free ligand populates various conformations in aqueous solution, thus displaying relatively flexible behavior. The NOE HNAc-H2A', which was not detected in previous work, accounts for a conformation at the β-(1→3) linkage with a phi angle of approximately 180 degrees. Observed TRNOEs for the complex are weak, and their analysis was further complicated by spin diffusion. With the use of transferred rotating-frame Overhauser enhancement (TRROE) experiments, the amount of spin diffusion was assessed experimentally, proving that all of the observed long-range TRNOEs arose through spin diffusion. Four interglycosidic distances, derived from the remaining TRNOEs and TRROEs, together with repulsive constraints, derived from the absence of TRROE effects, were used as input parameters in simulated annealing and molecular mechanics calculations to determine the bound conformation of the trisaccharide. Complexation by the antibody results in the selection of one defined conformation of the carbohydrate hapten. This bound conformation, which is a local energy minimum on the energy maps calculated for the trisaccharide ligand, shows only a change from a +gauche to a -gauche orientation at the psi angle of the α-(1→2) linkage when compared to the global minimum conformation. The results infer that the bound conformation of the Streptococcus Group A cell-wall polysaccharide is different from its previously proposed solution structure (Kreis et al., 1995)

NMR, conformation, Streptococcus, antibodies, monoclonal antibodies, NOE, antigen-antibody interaction

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CCC(=O)O[C@@H]1O[C@@H](C)[C@H](O)[C@@H](O[C@@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2NC(C)=O)[C@H]1O[C@@H]1O[C@@H](C)[C@H](O)[C@@H](O)[C@H]1O

569.557 g/mol (C₂₃H₃₉NO₁₅)