Carbohydrate Sequence

The structures of the lipopolysaccharide (LPS) core and O antigen of Bordetella bronchiseptica and Bordetella parapertussis are known, but how these two regions are linked to each other had not been determined. We have studied LPS from several strains of these ...

Previous studies have shown that the majority of the asparagine-linked high mannose oligosaccharide units present on the lysosomal enzymes of the slime mold Dictyostelium discoideum contain mannose 6-sulfate and mannose 6-phosphate residues, the latter being diesterified to methyl groups. Using a variety of techniques, including NMR spectroscopy and fast atom bombardment mass spectrometry, we have found that many of these oligosaccharides also contain an N-acetylglucosamine residue linked beta 1-4 to the mannose residue linked alpha 1-6 to the beta-linked core mannose. (Formula: see text) As far as we are aware, this is the first description of an intersecting N-acetylglucosamine residue in this position on a high mannose oligosaccharide.

UDP-GlcNAc:GlcNAc β1-2Manα1-6R (GlcNAc to Man) β1,6-N-acetylglucosaminyltransferase V (GlcNAc-T V) adds a GlcNAcβ1-6 branch to bi- and triantennaryN-glycans. An increase in this activity has been associated with cellular transformation, metastasis and differentiation. We have used synthetic substrate analogues to study the substrate specificity and inhibition of the partially purified enzyme from hamster kidney and of extracts from hen oviduct membranes and acute myeloid leukaemia leukocytes. All compounds with the minimum structure GlcNAcβ1-2Manα1-6Glc/Manβ-R were good substrates for GlcNAc-T V. The presence of structural elements other than the minimum trisaccharide structure affected GlcNAc-T V activity without being an absolute requirement for activity. Substrates with a biantennary structure were preferred over linear fragments of biantennary structures. Kinetic analysis showed that the 3-hydroxyl of the Manα1-3 residue and the 4-hydroxyl of the Manβ- residue of the Manα1-6(Manα1-3)Manβ-RN-glycan core are not essential for catalysis but influence substrate binding. GlcNAcβ1-2(4,6-di-O-methyl-)Manα1-6Glcβ-pnp was found to be an inhibitor of GlcNAc-T V from hamster kidney, hen oviduct microsomes and acute and chronic myeloid leukaemia leukocytes.

The mannose receptor (MR) is an endocytic protein on macrophages and dendritic cells, as well as on hepatic endothelial, kidney mesangial, tracheal smooth muscle, and retinal pigment epithelial cells. The extracellular portion contains two types of carbohydrate-recognition domain (CRD): eight membrane-proximal C-type CRDs and a membrane-distal cysteine-rich domain (Cys-MR). The former bind mannose-, N-acetylglucosamine-, and fucose-terminating oligosaccharides, and may be important in innate immunity towards microbial pathogens, and in antigen trapping for processing and presentation in adaptive immunity. Cys-MR binds to the sulfated carbohydrate chains of pituitary hormones and may have a role in hormonal clearance. A second feature of Cys-MR is binding to macrophages in marginal zones of the spleen, and to B cell areas in germinal centers which may help direct MR-bearing cells toward germinal centers during the immune response. Here we describe two novel classes of carbohydrate li...

Desalting of sugar samples is essential for the success of many techniques of carbohydrate analysis such as mass spectrometry, capillary electrophoresis, anion exchange chromatography, enzyme degradation and chemical derivatization. All desalting methods which are currently used have limitations: for example, mixed-bed ion-exchange columns risk the loss of charged sugars, precipitation of salt by a non-aqueous solvent can result in co-precipitation of oligosaccharides, and gel chromatography uses highly crosslinked packings in which separation of small oligosaccharides is difficult to achieve. We demonstrate that graphitized carbon as a solid phase extraction cartridge can be used for the purification of oligosaccharides (or their derivatives) from solutions containing one or more of the following contaminants: salts (including salts of hydroxide, acetate, phosphate), monosaccharides, detergents (sodium dodecyl sulfate and Triton X-100), protein (including enzymes) and reagents for ...

QS-21 is one of the most promising new adjuvants for immune response potentiation and dose-sparing in vaccine therapy given its exceedingly high level of potency and its favorable toxicity profile. Melanoma, breast cancer, small cell lung cancer, prostate cancer, HIV-1, and malaria are among the numerous maladies targeted in more than 80 recent and ongoing vaccine therapy clinical trials involving QS-21 as a critical adjuvant component for immune response augmentation. QS-21 is a natural product immunostimulatory adjuvant, eliciting both T-cell- and antibody-mediated immune responses with microgram doses. Herein is reported the synthesis of QS-21Aapi in a highly modular strategy, applying novel glycosylation methodologies to a convergent construction of the potent saponin immunostimulant. The chemical synthesis of QS-21 offers unique opportunities to probe its mode of biological action through the preparation of otherwise unattainable nonnatural saponin analogues.

Limnothrix redekei PUPCCC 116, a filamentous cyanobacterium, has been identified through 16S rRNA gene sequencing. Exopolysaccharides (EPS) of this organism have been isolated and characterized chemically, and its rheological properties were compared with commercial xanthan. The organism produced 304 µg EPS/ml culture in 21 days. The rate of EPS production was maximum (313 µg EPS/mg protein/day) during the initial days of growth, and it decreased to 140 µg EPS/mg protein/day during 18-21 days of growth. Chemical analysis of EPS revealed the presence of glucose/mannose, ribose, rhamnose, and uronic acid. Fourier transformed infrared spectrum of EPS further revealed the presence of methyl and carboxyl groups besides C–N groups indicating the presence of peptidyl moieties. Elemental analysis of EPS showed the presence of 4.97% N. The organism under continuous light produced 102% more EPS compared to when grown under a light/dark cycle of 14/10 h. The rheological properties of EPS were comparable with commercial xanthan gum.

This work presents the first part of a database of conformations for all the disaccharide fragments that are found inN-glycans. The conformational study of the five disaccharides found in the oligo-mannose type are presented here. For each disaccharide, several possible conformations are described. A method is presented to obtain realistic models of oligosaccharides using molecular mechanic methods. Analysis of some possible conformations of the oligo-mannose type glycan Man6-GlcNAc2 is given as an illustration of the possibilities.