An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

Properties of extracellular polysaccharides of potato ring rot pathogen and corresponding recognition sites of potato cell walls

The properties of extracellular polysaccharides of the potato ring rot pathogen Clavibacter michiganensis subsp. sepedonicus (Cms) and the corresponding recognition sites isolated from cell walls of potato suspension cultures have been studied. Extracellular polysaccharides of Cms consist of 4-6 components, which differ greatly in molecular mass (from <1 kD to >700 kD), and are capable of formation of associates stabilized by electrostatic interactions in the presence of calcium. Using affinity column chromatography, sites possessing affinity for the total extracellular polysaccharide complex of Cms were isolated from cell walls of suspension cultures of three potato varieties with different resistance to the pathogen. The content of the receptor sites consisting of glycopeptides and sugars for the variety devoid of resistance was 10 times greater than that for the resistant variety. In the receptor fraction for the latter variety, only sugars were found. The molecular masses of the components of the receptor fraction of cell walls were from 39 to 86 kD. Polypeptides in the recognition sites for the resistant variety escaped detection in electrophoretic patterns. Study of the amino acid composition of the receptor sites of cell walls showed that the sites of the resistant variety contained trace amounts of only seven amino acids. In the sites of the receptive variety 14 amino acids were found, the content of polar amino acids being twice as large as that of nonpolar amino acids. Among polar amino acids, glutamine and glycine prevailed, whereas among nonpolar amino acids valine was dominant. We suggest that one of the reasons of variety-specific resistance of potato to Cms is the absence or a low content of the sites revealing the affinity for bacterial extracellular polysaccharides on the plant cell surface.     

Effect of self-association on the structural organization of partially folded proteins: inactivated actin

The propensity to associate or aggregate is one of the characteristic properties of many nonnative proteins. The aggregation of proteins is responsible for a number of human diseases and is a significant problem in biotechnology. Despite this, little is currently known about the effect of self-association on the structural properties and conformational stability of partially folded protein molecules. G-actin is shown to form equilibrium unfolding intermediate in the vicinity of 1.5 M guanidinium chloride (GdmCl). Refolding from the GdmCl unfolded state is terminated at the stage of formation of the same intermediate state. An analogous form, known as inactivated actin, can be obtained by heat treatment, or at moderate urea concentration, or by the release of Ca(2+). In all cases actin forms specific associates comprising partially folded protein molecules. The structural properties and conformational stability of inactivated actin were studied over a wide range of protein concentrations, and it was established that the process of self-association is rather specific. We have also shown that inactivated actin, being denatured, is characterized by a relatively rigid microenvironment of aromatic residues and exhibits a considerable limitation in the internal mobility of tryptophans. This means that specific self-association can play an important structure-forming role for the partially folded protein molecules.