concanavalin-a and lipoteichoic-acid

Graphene's suite of useful properties makes it of interest for use in biosensors. However, graphene interacts strongly with hydrophobic components of biomolecules, potentially altering their conformation and disrupting their biological activity. We have immobilized the protein Concanavalin A onto a self-assembled monolayer of multivalent tripodal molecules on single-layer graphene. We used a quartz crystal microbalance (QCM) to show that tripod-bound Concanavalin A retains its affinity for polysaccharides containing α-D-glucopyrannosyl groups as well as for the α-D-mannopyranosyl groups located on the cell wall of Bacillus subtilis. QCM measurements on unfunctionalized graphene indicate that adsorption of Concanavalin A onto graphene is accompanied by near-complete loss of these functions, suggesting that interactions with the graphene surface induce deleterious structural changes to the protein. Given that Concanavalin A's tertiary structure is thought to be relatively robust, these results suggest that other proteins might also be denatured upon adsorption onto graphene, such that the graphene-biomolecule interface must be considered carefully. Multivalent tripodal binding groups address this challenge by anchoring proteins without loss of function and without disrupting graphene's desirable electronic structure.

Topics: Adsorption; Bacillus subtilis; Canavalia; Cell Wall; Cells, Immobilized; Concanavalin A; Graphite; Lipopolysaccharides; Plant Proteins; Teichoic Acids

The lipoteichoic acid of Streptococcus sanguis DSM 20567 contains a poly(glycerophosphate) chain, with 49% of the glycerophosphate residues being substituted with D-alanine ester, 35% with alpha-D-glucopyranosyl and alpha-isomalto-oligosaccharide residues. Analysis of molecular species by affinity chromatography on concanavalin A showed all chains to be substituted and alanine ester and glycosyl residues to be present on the same rather than on separate chains. Molecular species varied in the length of the poly(glycerophosphate) chain, the extent of glycosylation, and had a constant alanine-ester content. An alkali-hydrolysis procedure revealed a distribution pattern between random and regular for the glycosyl substituents and suggested a similar distribution for the alanyl residues which occupy the free positions between the glycosyl substituents.

Topics: Chromatography, Affinity; Concanavalin A; Diglycerides; Glycerophosphates; Glycosides; Isomaltose; Lipopolysaccharides; Oligosaccharides; Streptococcus sanguis; Teichoic Acids

Hydrophobic interaction chromatography fractionated the lipoteichoic acid of Enterococcus faecalis into species of decreasing poly(glycerophosphate) chain length and decreasing extent of substitution with alpha-kojibiosyl residues (Glcp alpha 1----2Glcp alpha 1----). The chain length varied between 14 and 33 glycerophosphate residues per lipid anchor, the extent of glycosylation between 0.18 and 0.44 mol of alpha-kojibiosyl residues per mole of phosphorus, and, accordingly, the number of alpha-kojibiosyl substituents per chain between 3 and 15. Almost identical values were obtained when the same lipoteichoic acid was chromatographed on DEAE-Sephadex and concanavalin A, which separate molecular species according to increasing number of phosphate groups and alpha-kojibiosyl residues, respectively. Species from all three columns, which were identical in chain length and glycosylation, also had similar fatty acid patterns. These results prove the suitability of all three procedures for species analysis. One advantage of hydrophobic interaction chromatography over the other two procedures lies in its broader applicability since it is not dependent on negative charges or specifically binding oligosaccharide structures. Another advantage is the capacity of hydrophobic interaction chromatography to separate molecular species differing in the number of fatty acids [W. Fischer, H.U. Koch, and R. Haas (1983) Eur. J. Biochem. 133, 523-530] and render them accessible to molecular analyses.

Topics: Chromatography, Affinity; Chromatography, Ion Exchange; Concanavalin A; Enterococcus faecalis; Lipopolysaccharides; Sepharose; Teichoic Acids; Water

This study shows for the first time microheterogeneity of 1,3-linked poly(glycerophosphate) lipoteichoic acids. The lipoteichoic acids investigated were those of Enterococcus faecalis Kiel 27738 (I), Enterococcus hirae (Streptococcus faecium) ATCC 9790 (II), and Leuconostoc mesenteroides DMS 20343 (III). Lipoteichoic acids II and III are partially substituted by mono-, di-, tri-, and tetra-alpha-D-glucopyranosyl residues with (1----2) interglycosidic linkages. Lipoteichoic acid I is substituted with alpha-kojibiosyl residues only. Lipoteichoic acids I and III additionally carry D-alanine ester. Lipoteichoic acids were separated on columns of concanavalin-A-Sepharose according to their increasing number of glycosyl substituents per chain. It was evident that all molecular species are usually glycosylated and that alanine ester and glycosyl residues occur on the same chains. The chain lengths of lipoteichoic acid I and II vary between 9-40 glycerophosphate residues, whereas those of lipoteichoic acid III appear to be uniform (33 +/- 2 residues). Molecular species differ in the extent of glycosylation but their content of alanyl residues is fairly constant. All lipoteichoic acids contain a small fraction (5-15%) different in composition from the bulk and most likely reflecting an early stage of biosynthesis. Two procedures for chain length determination of poly(glycerophosphate) lipoteichoic acids are described.

Topics: Chromatography, Affinity; Concanavalin A; Enterococcus faecalis; Glycerophosphates; Glycosylation; Leuconostoc; Lipopolysaccharides; Teichoic Acids

The location of lipoteichoic acid (LTA) on the surface of group A streptococci was studied by immunoelectron microscopic and ultrastructural cytochemical methods, i.e. by means of LTA antibodies labelled with ferritin, or concanavalin A labelled with ferritin or colloidal gold. All these methods proved the LTA to be located on the outer cell surface of most group A streptococcus strains. The differences in the intensity of labelling paralleled the hydrophobicity of the strains, being substantially higher in the strains exhibiting a high degree of hydrophobicity. Treatment of streptococci with pronase or trypsin led to a complete loss of surface-located LTA. On the other hand, pepsin treatment of streptococci under mild conditions resulted in an increased amount of surface-located LTA in some strains. On the isolated cell walls, LTA could be demonstrated only on the outer surface of the walls. These findings correlated well with the presumed role of group A streptococcus LTA in the adherence of streptococci to the epithelial cells which is accomplished with the aid of surface-located LTA molecules.

Topics: Bacterial Adhesion; Cell Wall; Concanavalin A; Ferritins; Gold; Immunohistochemistry; Lipopolysaccharides; Microscopy, Electron; Protoplasts; Streptococcus pyogenes; Teichoic Acids

We describe the use of lectins as specific stabilizing agents for the polysaccharide capsular components of two Gram-positive bacteria, Streptococcus agalactiae and Streptococcus bovis. Treatment of bacterial suspensions with wheatgerm agglutinin and concanavalin A allowed better morphological preservation as well as immunoelectron microscopic localization of a capsular component (lipoteichoic acid) by employing specific antibodies and the protein A-gold technique. Data obtained indicate that lectins are useful agents in preserving highly water-soluble capsular components during the electron microscopy procedures for both unembedded and embedded samples.

Topics: Animals; Concanavalin A; Immunohistochemistry; Lipopolysaccharides; Microscopy, Electron; Preservation, Biological; Streptococcus agalactiae; Teichoic Acids; Wheat Germ Agglutinins

We have used 31P-nuclear magnetic resonance spectroscopy to identify phosphorus-containing compounds in whole cells of two serotype c strains of the oral pathogen Streptococcus mutans. The major resonance, centered at 0 ppm in whole cells, was attributed to lipoteichoic acid on the basis of its chemical shift, insensitivity to pH changes, cellular localization and a comparison with spectra obtained with purified lipoteichoic acid from S. mutans. The linewidths of resonances observed for intact cells and purified lipoteichoic acid were moderately narrowed by increasing the ionic strength, and substantially broadened in the presence of the lectin concanavalin A. Experiments with purified lipoteichoic acid suggest that this compound in whole cells is complexed with divalent cations such as Mg2+. Intracellular pools of other phosphorus-containing metabolites were found to be low when compared to the lipoteichoic acid concentration in both starved and glycolyzing cells.

Topics: Cell Membrane; Cell Wall; Concanavalin A; Hydrogen-Ion Concentration; Lipopolysaccharides; Magnesium; Magnetic Resonance Spectroscopy; Osmolar Concentration; Phosphatidic Acids; Phosphorus; Streptococcus mutans; Teichoic Acids