The polysialic acid units of the neural cell adhesion molecule N-CAM form filament bundle networks.

Polysialic acid is a developmentally regulated component in the neural cell adhesion molecule N-CAM which also occurs as the capsular polysaccharide of bacteria causing meningitis. Polysialic acid has been considered as a repulsive element that regulates intermolecular and intercellular adhesion. Using atomic force microscopy we unexpectedly find that oligomers of polysialic acid assemble with each other into filament bundle networks. Filaments were formed from oligomers containing 12 or more N-acetylneuraminic acid residues, and they were sensitive to sialidase digestion. The networks were also formed by the polysialic acid-containing carbohydrate units of N-CAM. The formation of filament bundles is a novel and unexpected property of polysialic acid and of short carbohydrate oligomers in general and represents a previously unrecognized molecular interaction mechanism which impacts both eukaryotic and prokaryotic cell-cell adhesions.

Immunoblot analysis of bacterial polysaccharides: application to the type-specific polysaccharides of Streptococcus suis and Streptococcus agalactiae.

A method for the immunoblot analysis of the type-specific capsular polysaccharides of streptococci was developed. The capsular polysaccharides were extracted by sonication and subjected to polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulphate (SDS). After electrophoresis the polysaccharides were transferred to charged PVDF-N membranes and probed with the type specific antibodies. A characteristic ladder-like pattern of polysaccharide bands specific for the serotype (1, 2, 4, 7) was observed for capsular extracts of Streptococcus suis. Human immune sera against type-specific group B streptococcal polysaccharides reacted most strongly with the immunizing polysaccharide type (Ia, II, III). The previously observed crossreactions between the group B streptococcal type-specific capsular preparations were shown to be due to binding to the isomeric polysaccharide molecules. Thus, gel electrophoresis combined with immunoblot analysis seems to provide a novel method for the molecular and immunochemical characterization of bacterial polysaccharides and for the study of the specificity and properties of antibodies to capsular polysaccharides.

Antibodies to polysialic acid and its N-propyl derivative: binding properties and interaction with human embryonal brain glycopeptides.

There is no efficient vaccine against group B meningococcal meningitis because of tolerance induced by host tissue polysialic acid cross-reacting with the capsular polysaccharide. The specificities of polysialic acid-antibody interactions were studied using a ligand binding assay. Antibodies 735, 20-1, 2-1B, 2-2B, 5E1, and t5E1 and antibodies against N-propionylated group B meningococcal polysaccharide-tetanus toxoid conjugate (NP-4, 106-6) bound polysialylated human embryonal brain glycopeptides but not control glycopeptides or disialosyllactose, whereas antibodies 109-3 and I-627 were more specific for the N-propionylated polysaccharide. Antiganglioside antibodies (KM538, KM641) did not cross-react with polysialic acid. Human class-switched antibodies 5E1 (IgM) and t5E1 (IgG) reacted identically with all compounds tested and no temperature-dependent differences were observed. All anti-polysialosyl antibodies required a polysaccharide chain of 8-10 residues for binding independent of the immunizing antigen, animal species, or immunoglobulin class. The results suggest careful evaluation of polysialic acid cross-reactivity in vaccine development.

Interaction of meningococcal group B monoclonal antibody and its Fab fragment with alpha 2-8-linked sialic acid polymers: requirement of a long oligosaccharide segment for binding.

Mouse monoclonal IgG2a antibody (735D4) and other antibodies to the capsular polysaccharide of group B meningococci have been shown to require an unusually long segment of the alpha 2-8-linked N-acetylneuraminic acid polymer for binding. This property may be due to a conformational nature of the polysaccharide epitope recognized, or alternatively due to the requirement of bivalent binding of the antibody to the polysaccharide. In order to study the binding requirements, Fab fragments were prepared from the monoclonal antibody and their binding to alpha 2-8-linked sialic acid polymers of different lengths was studied. Both the intact antibody and its Fab fragment bound to sialic acid poly- and oligomers to similar extents, the critical chain length being about 10 sialyl units for both molecules. This excluded bivalency as the explanation for the requirement of a long oligosaccharide segment for binding. Although the binding was enhanced with increasing chain length, the first 10 monosaccharides were calculated to contribute to more than 90% of the total binding energy. This is in agreement with an oligosaccharide segment with defined conformational epitope binding to the antibody combining site. The antibody preparations also bound polysialic acid containing glycopeptides isolated from developing human and rat brain, suggesting, in quantitative binding assay, an average chain length of 10 or more sialic acid residues. The interaction of the antibody with both the bacterial and the tissue derived polysialic acids suggests that the conformational epitope critical for the interaction is formed by both classes of compounds.

Structural similarity of the type-specific group B streptococcal polysaccharides and the carbohydrate units of tissue glycoproteins: evaluation of possible cross-reactivity.

Type-specific capsular polysaccharides of group B streptococci show striking structural similarity with the terminal sugar sequences of tissue glycoconjugates. The polysaccharides have been put forward as vaccines against neonatal meningitis. A potential source of hazard in immunization of pregnant mothers may be the presence of the cross-reactive components in adult or fetal tissues. A radioactive ligand binding assay was used to test human immune sera to type Ia, II and III group B streptococcal polysaccharides for binding to tissue-derived glycopeptides showing structural similarities with the streptococcal polysaccharides. Of the 13 glycopeptides of human and rat tissues studied, representing a wide selection of structures known to occur in glycoproteins, only two showed some reactivity with the antisera. The reactivity with human small intestinal glycopeptides could be explained by the presence of natural blood group A antibodies, and was not related to the streptococcal group B antibodies. The basis of the reactivity of a high-molecular-weight glycopeptide from rat kidney with some of the sera was unknown, but was unrelated to the vaccination and clearly could not be inhibited with the streptococcal polysaccharides. Thus, no immunological cross-reactions of the tissue glycopeptides studied could be demonstrated with the group B streptococcal antisera.

Polyacrylamide gel electrophoresis of the capsular polysaccharides of Escherichia coli K1 and other bacteria.

Methods were developed for the polyacrylamide gel electrophoretic analysis of capsular polysaccharides of bacteria with Escherichia coli K1 as a model. Conditions were determined for the rapid and gentle extraction of the K1 polysaccharide by incubation of the bacteria in a volatile buffer and for the subsequent removal of the putative phospholipid moiety attached to the reducing end of the polysaccharide. Detection of the polysaccharides after gel electrophoresis was carried out by fluorography of samples labeled by sodium borotritiide reduction or by combined alcian blue and silver staining. The smallest components could be detected only by fluorography, owing to diffusion during staining. Components of the E. coli K1 polysialic acid capsule ranging from monomers to 80 sialic-acid-unit-containing polymers could be separated as distinct bands in a ladderlike pattern. A maximum chain length of 160 to 230 sialyl residues was estimated for the bulk of the K1 polysaccharide from the nearly linear reciprocal relationship between the logarithm of the molecular size and the distance of migration. Gel electrophoresis of capsular polysaccharides of other bacterial species revealed different electrophoretic mobilities for each polysaccharide, with a ladderlike pattern displayed by the fastest-moving components. There are many potential applications of this facile method for the determination of the sizes of molecules present in a polydisperse polysaccharide sample. When combined with the simple method for the isolation of the capsule, as in the case of the K1 capsule, it provides an efficient tool for the characterization and comparison of the capsular polysaccharides of bacteria.