Slavomir Bystricky

Physical, Chemical, Antigenic, and Immunologic Characterization of Polygalacturonan, Its Derivatives, and Vi Antigen from Salmonella typhi

Publisher Summary The immunogenicity, physical and chemical properties of three polygalacturonans:— pectin, O-acetylated pectin, and Vi— varied significantly depending on the presence of acetyl groups. The O-acetyl and N-acetyl groups that stabilize Vi to acid hydrolysis are also essential immunodeterminants. Cations at carboxyl groups have lesser effects on the immunologic properties of Vi. Based on the Courtald–Koltum space-filling model, it is observed that the bulky O- and N-acetyl groups dominate the molecular surface of the Vi or the O-acetylated pectin. As a result, these hydrophobic groups also shield the carboxyl groups from interaction with other molecules present as counterions in solvent-positive charged molecules or as receptors on cell surfaces. The binding of multivalent cations with polyanions consists of two major types: (1) the electrostatic binding of the cations with negative charges on the polyanions results in the same free energy of binding as the monovalent cation, and (2) intermolecular cooperative binding or the chelating effect. Potentiometric titration showed that when the Vi or O-acetylated pectin was highly O-acetylated, the binding of the carboxyl groups to Ca2+ and K+ was similar. The immunological properties of Vi are related to its content of O-acetylated groups. The binding activity of carboxyl groups may be affected sterically by the acetyl groups in close proximity.

Conformational analysis and molecular dynamics simulation of α-(1 → 2) and α-(1 → 3) linked rhamnose oligosaccharides: Reconciliation with optical rotation and NMR experiments

Molecular mechanics and dynamics calculations were carried out on the disaccharides alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->OMe) (1) and alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->OMe) (2), and the trisaccharide alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1--> 3)-alpha-L-Rhap-(1-->OMe) (3). The semiflexible conformational behavior of these molecules was characterized by the occupation of a combination of different glycosidic linkage and side-chain conformational positions whose relative occupations were sensitive to dielectric screening. Molecular dynamics simulations of the trisaccharide 3 showed little difference between the linkage conformations in the trisaccharide and the component disaccharides 1 and 2 Experimental optical rotation data of 1 and 2 were obtained as a function of temperature in varying solvents. The molecular models were combined with the semiempirical theory of Stevens and Sathyanarayana to yield calculated optical rotations. Interpretation of the data of both 1 and 2 implied that a combination of conformations, both in glycosidic and side-chain positions, could explain the experimental data. Solvents effects were important in influencing the conformational mix and averaged optical rotation. Three-bond heteronuclear coupling constants 3JC H were obtained for the glycosidic linkages of 1 and 2 in D2O and DMSO. Analysis of the coupling constants with a Karplus curve showed that small reductions in the glycosidic torsion angles of the conformations of the models used here of ca. 10 degrees-15 degrees in phi and 5 degrees-10 degrees in psi were required to give better agreement with experiment; a combination of conformations for both 1 and 2 was consistent with the data. There was a negligible influence on the coupling constants of 1 on changing the solvent from D2O to DMSO.

O-acetylation affects the binding properties of the carboxyl groups on the Vi bacterial polysaccharide

The capsular polysaccharide of Salmonella typhi and Citrobacter freundii (Vi) is a linear homopolymer of (1-->4)-linked 2-acetamido-2-deoxy-alpha-D-galactopyranosyluronic acid partially O-acetylated at the C-3 position. The physico-chemical properties of the carboxyl groups of the Vi polysaccharide, as a function of different degrees of O-acetylation, were studied by potentiometric titration, circular dichroism, and their reaction with the bulky nucleophile 2-nitro-phenylhydrazine (NPH). Potentiometric titrations with K+ and Ca2+ hydroxides showed that the difference in the free energy of binding between the two cations (delta GKCa) was inversely proportional to the degree of O-acetylation. Similar cationic effects were found when measuring circular dichroism. Moreover there was also an inverse relation between the degree of O-acetylation and the extent of binding of NPH to the carboxyl groups. These data all indicate that O-acetyl groups hinder the association of carboxyls with cations and nucleophilic reagents. This provides a possible explanation for the importance of the O-acetyl and the relative unimportance of the carboxyl groups in contributing to the immunologic properties of the Vi.

Characterization of colominic acid by circular dichroism and viscosity analysis

Conformations of oligo- and poly-(alpha (2-->8)-D-Neu pNAc) (colominic acid) and its derivatives were studied by circular dichroism (CD) spectroscopy and viscometry to understand the molecular basis of their unusual antigenic properties. No temperature-dependent conformational transition between 5 and 70 degrees C or divalent salt effect of Ca2+ or Mg2+ was observed in colominic acid or its N-deacetylated form by CD spectroscopy. However, CD spectroscopy indicated that the distribution of conformers in oligocolominic acid changes continuously from n = 2 to octamer, and there was no further change of the conformer distribution for n > 9. Colominic acid exhibited a much lower intrinsic viscosity compared with the values for other polyelectrolytes of similar linear charge density, such as polynucleic acids. The apparent absence of induced conformational transition by salt or temperature, and the high flexibility indicated that the binding of colominic acid to its antibodies may not contain a significant amount of specific conformationally controlled determinant. Instead, our data suggest that more than nine saccharide units are needed for a cooperative binding process.