Preparation and characterisation of chitosans with oligosaccharide branches

The trimer 2-acetamido-2-deoxy-D-glucopyranosyl-beta-(1-->4)-2-acetamido-2-deoxy-D-glucopyranosyl-beta-(1-->4)-2,5-anhydro-D-mannofuranose (A-A-M) was reductively N-alkylated onto a fully de-N-acetylated chitosan (F(A)<0.001, DP(n)=25) to obtain branched chitosans with degree of substitution (DS) of 0.070, 0.23 and 0.40, as determined by 1H NMR spectroscopy. The apparent pK(a) values of the primary and secondary amines of the chitosans substituted with the trimer A-A-M were determined by monitoring the chemical shift of the H-2 of GlcN, and were determined as 6.5-6.9 for the primary (unsubstituted) amines and as 5.0-5.2 for the secondary (substituted) amines. The intrinsic pK(a) values (pK(int)) were found to be 7.3-7.4 for the substituted and 8.7 for the unsubstituted amines. The chitosan branched with A-A-M (DS 0.40) was found to be soluble in aqueous solution over the entire pH range. SEC-MALLS (size-exclusion chromatography with a multi-angle laser light scattering detector) further showed that addition of branches did not affect the molar hydrodynamic volume of the chitosan.     

Enzymatic synthesis of oligosaccharides

Abstract: The biological interest of oligosaccharides is growing very rapidly, and necessitates the development of efficient synthesis reactions. The stereo- and regio-selectivity of enzyme catalysis is a key advantage in this field, as a complementary tool to the chemical approach. Two types of enzymes can be applied to the obtention of oligosaccharides: Hydrolytic enzymes, which can catalyze either reverse hydrolysis (thermodynamic control) or transglycosylation (kinetic control) synthesis reactions; and transferase enzymes, which can use simple carbohydrates from agricultural origin as glycosyl donors.     

Screening for complex carbohydrates inhibiting hemagglutinations by CFA/I- and CFA/II-expressing enterotoxigenic Escherichia coli strains

Abstract Numerous structural families of naturally occurring glycopeptides and oligosaccharides have been evaluated as potential inhibitors of hemagglutinations mediated by CFA/I- and CFA/II-positive enterotoxigenic Escherichia coli strains. Among the preparations tested were glycopeptides with short O-linked (mucin-type) chains, various mixtures containing N-linked glycans (either oligomannoside-, hybrid- or complex-type), three fractions of human milk oligosaccharides, and glycopeptides derived from either pooled new-born meconiums or pooled human red blood cell membranes. In almost all cases, the same inhibitory preparations were active toward all E. coli strains. This emphasizes the close analogy between the carbohydrate specificities of the colonization factors concerned. Such inhibitors always contained lactosamine units in their oligosaccharide backbones, but this structural requirement alone was not sufficient for activity. The glycopeptide mixture derived from human erythrocyte membranes (known to contain blood group-related carbohydrate antigens carried by a lactosaminoglycan backbone) behaved as a potent hemagglutination inhibitor, especially towards CFA/II-expressing strains. This last result clearly indicates the structural family in which complex carbohydrates should be selected to establish precisely the specificity of these CFA/II adhesins.     

The effects of N-glycosylation sites and the N-terminal region on the biological function of beta1,3-N-acetylglucosaminyltransferase 2 and its secretion

Human beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) is thought to be an enzyme that extends the polylactosamine acceptor chains, but its function and structure analysis are unknown. To obtain insight into the structure of beta3GnT2, the effects of N-glycosylation on its biological function were evaluated using the addition of inhibitors, site-directed mutagenesis of potential N-glycosylation sites, and deletion of its N-terminal region using a fusion protein with GFP(uv) in a baculovirus expression system. Four of five potential N-glycosylation sites were found to be occupied, and their biological function and secretion were inhibited with the treatment of N-glycosylation inhibitor, tunicamycin. The N-glycosylation at Asn219 was necessary for the beta3GnT activity; moreover, N-glycosylation at Asn127 and Asn219 was critical for efficient protein secretion. When Ser221 was replaced with Thr, fusion protein was expressed as a single band, indicating that the double band of the expressed fusion protein was due to the heterogeneity of the glycosylation at Asn219. The truncated protein consisting of amino acids 82-397 (GFP(uv)-beta3GnT2Delta83), which lacked both one N-glycosylation site at Asn79 and the stem region of glycosyltransferase, was expressed as only a small form and showed no beta3GnT activity. These results suggest that the N-glycosylation site at Asn219, which is conserved throughout the beta1,3-glycosyltransferase family, is indispensable not only with regard to its biological function, but also to its secretion. The N-terminal region, which belongs to a stem region of glycosyltransferase, might also be important to the active protein structure.     

The use of a genetic algorithm search for molecular mechanics (MM3)-based conformational analysis of oligosaccharides

We have implemented a system called glygal that can perform conformational searches on oligosaccharides using several different genetic algorithm (GA) search methods. The searches are performed in the torsion angle conformational space, considering both the primary glycosidic linkages as well as the pendant groups (C-5-C-6 and hydroxyl groups) where energy calculations are performed using the MM3(96) force field. The system includes a graphical user interface for setting calculation parameters and incorporates a 3D molecular viewer. The system was tested using dozens of structures and we present two case studies for two previously investigated O-specific oligosaccharides of the Shigella dysenteriae type 2 and 4. The results obtained using glygal show a significant reduction in the number of structures that need to be sampled in order to find the best conformation, as compared to filtered systematic search.     

Extremotrophs, extremophiles and broadband pigmentation strategies in a high arctic ice shelf ecosystem

Remnant ice shelves along the northern coast of Ellesmere Island, Nunavut, Canada ( approximately 83 degrees N) provide a habitat for cryo-tolerant microbial mat communities. Bioassays of bacterial and primary production were undertaken to quantify the short-term physiological response of the mats to changes in key variables that characterize this cryo-ecosystem (salinity, irradiance and temperature). The heterotrophic versus autotrophic community responses to these stressors differed markedly. The heterotrophic bacteria were extremophilic and specifically adapted to ambient conditions on the ice shelf, whereas the autotrophic community had broader tolerance ranges and optima outside the ambient range. This latter, extremotrophic response may be partly due to a diverse suite of pigments including oligosaccharide mycosporine-like amino acids, scytonemins, carotenoids, phycobiliproteins and chlorophylls that absorb from the near UV-B to red wavelengths. These pigments provide a comprehensive broadband strategy for coping with the multiple stressors of high irradiance, variable salinity and low temperatures in this extreme cryo-environment.     

Synthesis of oligosaccharides corresponding to Streptococcus pneumoniae type 9 capsular polysaccharide structures

Two trisaccharides, alpha-D-Galp-(1-->3)-beta-D-ManpNAc-(1-->4)-beta-D-Glcp and alpha-D-Glcp-(1-->3)-beta-D-ManpNAc-(1-->4)-beta-D-Glcp, corresponding to structures from Streptococcus pneumoniae capsular polysaccharides type 9A, L, V and type 9N, respectively, have been synthesised as 2-aminoethyl glycosides and as protected TMSE glycosides. Ethyl thioglycosides were used as glycosyl donors and NIS/TfOH (in CH(2)Cl(2) for beta-linkages) and DMTST (in Et(2)O for alpha-linkages) as promoters in the glycosylations. The beta-ManNAc motif was introduced at the disaccharide level by azide displacement of a 2-O-triflate with beta-D-gluco configuration. The protecting group patterns allow continued syntheses of larger structures.     

Synthesis of a tetrasaccharide related to the O-antigen from Azospirillum lipoferum SR65

Concise synthesis of a tetrasaccharide repeating unit of the LPS isolated from Azospirillum lipoferum SR65 has been accomplished through suitable protecting group manipulations and stereoselective glycosylation starting from commercially available l-rhamnose and d-glucose. The target oligosaccharide in the form of its p-methoxyphenyl glycoside is suitable for further glycoconjugate formation via selective cleavage of the OMP glycoside. Plant growth-promoting bacteria (PGPB) of genus Azospirillum plays important roles in the growth and development of plants. The interaction between the roots of the plants and the microbes is governed by the cell surface carbohydrate polymers (CPS, LPS, etc.). The present synthetic-based study elucidates aspects of plant-microbe interaction and future biofertiliser design.