Abstract: Protein conjugates of oligosaccharides or peptides that mimic complex bacterial polysaccharide antigens represent alternatives to the classical polysaccharide-based conjugate vaccines developed so far. Hence, a better understanding of the molecular basis ensuring appropriate mimicry is required in order to design efficient carbohydrate mimic-based vaccines. This study focuses on the following two unrelated sets of mimics of the Shigella flexneri 5a O-specific polysaccharide (O-SP): (i) a synthetic branched pentasaccharide known to mimic the average solution conformation of S. flexneri 5a O-SP, and (ii) three nonapeptides selected upon screening of phage-displayed peptide libraries with two protective murine monoclonal antibodies (mAbs) of the A isotype specific for S. flexneri 5a O-SP. By inducing anti-O-SP antibodies upon immunization in mice when appropriately presented to the immune system, the pentasaccharide and peptides p100c and p115, but not peptide p22, were qualified as mimotopes of the native antigen. NMR studies based on transferred NOE (trNOE) experiments revealed that both kinds of mimotopes had an average conformation when bound to the mAbs that was close to that of their free form. Most interestingly, saturation transfer difference (STD) experiments showed that the characteristic turn conformations adopted by the major conformers of p100c and p115, as well as of p22, are clearly involved in mAb binding. These latter experiments also showed that the branched glucose residue of the pentasaccharide was a key part of the determinant recognized by the protective mAbs. Finally, by using NMR-derived pentasaccharide and peptide conformations coupled to STD information, models of antigen-antibody interaction were obtained. Most interestingly, only one model was found compatible with experimental data when large O-SP fragments were docked into one of the mIgA-binding sites. This newly made available system provides a new contribution to the understanding of the molecular mimicry of complex polysaccharides by peptides and short oligosaccharides.

Abstract: Microtubules are major cytoskeletal components involved in numerous cellular functions such as mitosis, cell motility, or intracellular traffic. These cylindrical polymers of alphabeta-tubulin assemble in a closely regulated dynamic manner. We have shown that the stathmin family proteins sequester tubulin in a nonpolymerizable ternary complex, through their stathmin-like domains (SLD) and thus contribute to the regulation of microtubule dynamics. We demonstrate here that short peptides derived from the N-terminal part of SLDs impede tubulin polymerization with various efficiencies and that phosphorylation of the most potent of these peptides reduces its efficiency as in full-length stathmin. To understand the mechanism of action of these peptides, we undertook a NMR-based structural analysis of the peptide-tubulin interaction with the most efficient peptide (I19L). Our results show that, while disordered when free in solution, I19L folds into a beta-hairpin upon binding to tubulin. We further identified, by means of saturation transfer difference NMR, hydrophobic residues located on the beta2-strand of I19L that are involved in its tubulin binding. These structural data were used together with tubulin atomic coordinates from the tubulin/RB3-SLD crystal structure to model the I19L/tubulin interaction. The model agrees with I19L acting through an autonomous tubulin capping capability to impede tubulin polymerization and provides information to help understand the variation of efficiency against tubulin polymerization among the peptides tested. Altogether these results enlighten the mechanism of tubulin sequestration by SLDs, while they pave the way for the development of protein-based compounds aimed at interfering with tubulin polymerization.

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.

Abstract: Convergent syntheses of the methyl glycosides of the branched pentasaccharide alpha-L-Rhap-(1–>2)-[alpha-D-Glcp(1–>3)]-alpha-L-Rhap-(1–>3)-alpha- L-Rhap-(1–>3)-beta-D-GlcNAcp [A(E)BCD], featuring the biological repeating unit of the O-specific polysaccharide of Shigella flexneri serotype 5a, and of a related linear tetrasaccharide (EBCD) are described. The strategy, based on the trichloroacetimidate methodology, relied on the use of a key EB disaccharide donor and appropriate CD acceptors. The use of an isopropylidene acetal to block OH-4 and OH-6 of residue D was found to be a suitable alternative to the employment of the more commonly used benzylidene acetal. Conformational analysis of EBCD-OMe and A(E)BCD-OMe was based on analysis of H-1 and C-13 chemical shifts and inter-proton distances data obtained by NMR spectroscopy. The data showed that residue A had no influence on the conformational behaviour of residue E, although these two residues were involved in a 2,3-cis vicinal glycosylation pattern in A(E)BCD-OMe. Comparison of H-1 and C-13 chemical shifts of the two oligosaccharides with those of their corresponding sequences in the O-specific polysaccharide of S. flexneri 5a showed that the two oligosaccharides presented a distribution of solution conformations similar to that in the O-specific polysaccharide. The conformation of A(E)BCD-OMe was investigated by two approaches: (i) energy minimisation based on ROE-derived distances with the DISCOVER program and (ii) a conformational searching method (the CICADA algorithm interfaced with MM3 force-field). The minimised conformation obtained by the former approach was in total agreement with the average of the two major families of conformations resulting from the CICADA calculations.

Abstract: A convergent chemical synthesis of the methyl glycoside of the linear epitope alpha-D-Glcp-(1–>3)-alpha-L-Rhap-(1–>3)-alpha-L-Rhap-(1–>3)-beta-D-Gl cNAcp-(1–>2)-alpha-L-Rhap (EBCDA) corresponding to the ramification of the O-antigen of Shigella flexneri serotype 5a is described. The strategy relies on the preparation of a key EB trichloroacetimidate donor and that of an appropriate CDA trisaccharide acceptor. Trichloroacetimidate chemistry was used for the construction of all glycosidic linkages except that of DA, where a bromide donor was preferred. In depth analysis of the pentasaccharide EBCDA H-1 and C-13 NMR spectra shows that its conformation approaches that of the corresponding fragment in the native polysaccharide. (C) 2002 Elsevier Science Ltd. All rights reserved.