Abstract
As part of a program for the development of synthetic vaccines against the pathogen Shigella flexneri, we used a combination of NMR and molecular modeling methods to study the conformations of the O-specific polysaccharide (O-SP) of S. flexneri 5a and of four related synthetic pentasaccharide fragments. The NMR study, based on the analysis of 1H and 13C chemical shifts, the evaluation of inter-residue distances, and the measurement of one- and three-bond heteronuclear coupling constants, showed that the conformation of one of the four pentasaccharides is similar to that of the native O-SP in solution. Interestingly, inhibition enzyme-linked immunosorbent assay demonstrated that a protective monoclonal antibody specific for S. flexneri 5a has a greater affinity for this pentasaccharide than for the others. We carried out a complete conformational search on the pentasaccharides using the CICADA algorithm interfaced with MM3 force field. We calculated Boltzmann-averaged inter-residue distances and 3JC,H coupling constants for the different conformational families and compared the results with NMR data for all pentasaccharides. Our experimental data are consistent with only one conformational family. We also used molecular modeling data to build models of the O-SP with the molecular builder program POLYS. The models that are in agreement with NMR data adopt right-handed 3-fold helical structures in which the branched glucosyl residue points outwards.
Highlights
Capsular polysaccharides (CPS)1 and lipopolysaccharides (LPS) of Gram-negative bacteria are important virulence fac tors and major targets of the host’s immune response upon
Several laboratories are studying glycoconjugate vaccines derived from detoxified LPSs as protein conjugates involving the polysaccharide moiety of LPSs have been shown to be safe and immunogenic in humans [5]
It has been demonstrated that the Ospecific polysaccharide moiety (O-SP) of the LPS is the major target of the protective immune response
Summary
The specificity of serotype 5a is associated with the presence of an ␣-D-glucopyranosyl (␣-D-Glcp) residue (E) at position 3 of residue B (Fig. 1) [11]. Frame-shifted di-, tri-, tetra- and pentasaccharide fragments of the O-SP 5a, all bearing the characteristic EB segment, have been synthesized recently as their methyl glycosides with the natural anomeric configuration at their reducing end [13,14,15,16,17]. To determine whether short fragments have some of the conformational features of the O-SP 5a, we compared the conformations of the four possible pentasaccharides 1, 2, 3, and 4 specific for the O-SP 5a (Fig. 1) with that of the native O-SP. The NMR conformational study was based on the analysis of 1H and 13C chemical shifts, the calculation of inter-residue distances, and the measurement of heteronuclear 3JC,H coupling constants across the glycosidic linkages that are related to ⌽ and ⌿ torsion angles in a Karplus-type relationship [18]. The 3JC,H coupling constants were determined by two different techniques, EXSIDE (excitation-sculptured indirect-detection experiments) [19] and gradient-selected J-HMBC experiments
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