Synthesis of trisaccharides containing 3-deoxy-D-manno-2-octulosonic acid residues related to the KDO-region of enterobacterial lipopolysaccharides

Glycosylation of methyl (allyl 7,8-O-carbonyl-3-deoxy-alpha-D-manno-2-octulo-pyranosid)o nate with an alpha-(2----4) linked per-O-acetylated KDO-disaccharide bromide derivative under Helferich conditions afforded a 2:1 mixture of the alpha- and beta-linked trisaccharide derivatives in 50% yield. Removal of the protecting groups gave sodium O-[sodium (3-deoxy-alpha-D-manno-2-octulopyranosyl)onate]-(2----4)-O-[ sodium (3-deoxy-alpha- and -beta-D-manno-2-octulopyranosyl)onate]-(2----4)-sodium (allyl 3-deoxy-alpha-D-manno-2-octulopyranosid)onate. Radical copolymerization of the allyl glycosides afforded artificial antigens, suitable for defining antibody specificities directed against the KDO-region of enterobacterial lipopolysaccharides.     

Characterization of monoclonal antibodies recognizing three distinct, phosphorylated carbohydrate epitopes in the lipopolysaccharide of the deep rough mutant I-69 Rd−/b+ of Haemophilus influenzae

Monoclonal antibodies against the lipopolysaccharide (LPS) of the deep rough mutant I-69 Rd⁻/b⁺ of Haemophilus influenzae were obtained after immunization of mice with sheep erythrocytes which had been coated with de- O -acylated LPS. Characterization of antibodies was performed by enzyme immuno assay (EIA) using LPS or neoglycoconjugates containing partial structures of LPS as solid-phase antigens and by haemagglutination with sheep erythrocytes coated with de- O -acylated LPS. Binding data were confirmed by EIA inhibition experiments using deacylated LPS or synthetic partial structures thereof. Three antibodies were specific for 3-deoxy- d - manno -octulopyranosonic acid- (Kdo) 5-phosphate, one for Kdo-4-phosphate, and one required, in addition to a Kdo-phosphate, parts of the phosphorylated glucosamine backbone of lipid A. All antibodies also bound in (i) Western blots to bacterial whole-cell lysates or isolated LPS separated by SDS–PAGE, (ii) bacterial colony blots, and (iii) immunofluorescence with live bacteria. The latter result indicated that Kdo-4- and Kdo-5-phosphate are synthesized by the bacteria and are not the result of phosphate migration.     

A nuclear magnetic resonance spectroscopic investigation of Kdo-containing oligosaccharides related to the genus-specific epitope of Chlamydia lipopolysaccharides.

The 1H- and 13C-NMR parameters, chemical shifts and coupling constants, for the pentasaccharide of the genus-specific epitope of Chlamydia lipopolysaccharide and related di-, tri-, and tetra-saccharides have been measured and assigned completely using 1D and 2D techniques, and their structures have been confirmed. NOE experiments indicated the preferred conformation of the pentasaccharide and the component oligosaccharides. The 3JH,H demonstrate a change in conformation by rotation of the C-6-C-7 bond of the side chain of the (2----8)-linked Kdo (unit b) in alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcN-(1--- -6)- GlcNol, alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcNAc-(1- ---O)- allyl, and alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----O)-allyl relative to that preferred in alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcNAc-(1----O)-allyl, alpha-Kdo-(2----8)-alpha-Kdo-(2----O)-allyl, alpha-Kdo-(2----4)-alpha-Kdo-(2----O)-allyl, and alpha-Kdo-(2----6)-beta-GlcNAc-(1----O)-allyl, irrespective of the size of the aglycon, e.g., allyl or beta-D-GlcN residues. The conformational results have been substantiated by computer calculations using the HSEA approach.     

“Hypermethylation” of anthranilic acid-labeled sugars confers the selectivity required for liquid chromatography-mass spectrometry

Analysis of the monosaccharides of complex carbohydrates is often performed by liquid chromatography with fluorescence detection. Unfortunately, methylated sugars, unusual amino- or deoxysugars and incomplete hydrolysis can lead to erroneous assignments of peaks. Here, we demonstrate that a volatile buffer system is suitable for the separation of anthranilic acid labeled sugars. It allows off-line examination of peaks by electrospray mass spectrometry. Approaches towards on-line mass spectrometric detection using reversed-phase or porous graphitic carbon columns fell short of achieving sufficient separation of the relevant isobaric sugars. Adequate chromatographic performance for isomeric sugars was achieved with reversed-phase chromatography of “hyper”-methylated anthranilic acid-labeled monosaccharides. Deuteromethyl iodide facilitates the discovery of naturally methylated sugars and identification of their parent monosaccharide as demonstrated with N-glycans of the snail Achatina fulica, where two thirds of the galactoses and a quarter of the mannoses were methylated.     

Identification of two GDP-6-deoxy-D-lyxo-4-hexulose reductases synthesizing GDP-D-rhamnose in Aneurinibacillus thermoaerophilus L420-91T

The glycan repeats of the surface layer glycoprotein of Aneurinibacillus thermoaerophilus L420-91T contain d-rhamnose and 3-acetamido-3,6-dideoxy-d-galactose, both of which are also constituents of lipopolysaccharides of Gram-negative plant and human pathogenic bacteria. The two genes required for biosynthesis of the nucleotide-activated precursor GDP-d-rhamnose, gmd and rmd, were cloned, sequenced, and overexpressed in Escherichia coli. The corresponding enzymes Gmd and Rmd were purified to homogeneity, and functional studies were performed. GDP-d-mannose dehydratase (Gmd) converted GDP-d-mannose to GDP-6-deoxy-d-lyxo-4-hexulose, with NADP+ as cofactor. The reductase Rmd catalyzed the second step in the pathway, namely the reduction of the keto-intermediate to the final product GDP-d-rhamnose using both NADH and NADPH as hydride donor. The elution behavior of the intermediate and end product was analyzed by high performance liquid chromatography. Nuclear magnetic resonance spectroscopy was used to identify the structure of the final product of the reaction sequence as GDP-alpha-d-rhamnose. This is the first characterization of a GDP-6-deoxy-d-lyxo-4-hexulose reductase. In addition, Gmd has been shown to be a bifunctional enzyme with both dehydratase and reductase activities. So far, no enzyme catalyzing these two types of reactions has been identified. Both Gmd and Rmd are members of the SDR (short chain dehydrogenase/reductase) protein family.     

p53-independent apoptosis in UV-irradiated mouse skin: possible inhibition by 50 Hz magnetic fields

Our recent results suggest that 50 Hz magnetic fields (MF) enhance ultraviolet (UV)-induced tumorigenesis in mouse skin. The aim of the present experiment was to study suppression of apoptosis as a possible mechanism for MF effects on skin tumorigenesis. Another aim was to test the importance of a UV and MF exposure schedule, particularly the role of MF exposure prior to UV irradiation. Female mice were exposed to a UV dose of 2 human MED and to 100 microT MF of 50 Hz, using the following exposure schedules: group 1 sham MF 24 h, UV 1 h, sham MF 24 h; group 2 sham MF 24 h, UV 1 h, MF 24 h; group 3 MF 24 h, UV 1 h, MF 24 h. Lamps emitting simulated solar radiation (SSR) were used for UV irradiation. Skin samples were analysed for apoptosis, expression of the p53 gene, activity of the enzyme ornithine decarboxylase (ODC) and polyamine concentrations. A significantly (p = 0.017) lower number of apoptotic cells was measured in group 2 compared to group 1. A similar but not statistically significant (p = 0.064) decrease was also detected in group 3. No p53 expression was detected in any sample. The levels of ODC and putrescine did not differ significantly between the UV-only and UV and MF-exposed groups. Spermidine and spermine levels were significantly (p = 0.014 and 0.014, respectively) lower in group 3 than in group 1, but no decrease was observed in group 2. Our findings suggest that SSR induces p53-independent apoptosis in mouse skin and that the apoptotic response may be inhibited by exposure to MF. The exposure schedule did not alter the MF effect. The results do not support a causal role for polyamines in MF effects on apoptosis.     

A novel method for the determination of carbonyl groups in cellulosics by fluorescence labeling. 3. Monitoring oxidative processes

The fluorescence-based CCOA method for determination of carbonyl group profiles in cellulosic substrates was employed to study the mechanisms of various oxidative and degradation processes involving celluloses in greater detail. The approach comprises labeling with the marker carbazole-9-carboxylic acid [2-(2-aminooxyethoxy)ethoxy]amide (CCOA), followed by gel permeation chromatography in DMAc/LiCl with fluorescence, multiangle laser light scattering, and refractive index detection. At first, the CCOA method was applied to study solutions of pulp in N-methylmorpholine-N-oxide monohydrate (NMMO), as occurring in the production of Lyocell-type fibers. NMMO is a rather strong oxidant that on one hand converts reducing end groups to carboxyl structures, thus lowering the overall carbonyl content, but generates new keto structures along the chain by nonselective oxidation on the other hand. The CCOA method allowed for the first time to distinguish the carbonyl course in different molecular weight ranges. Second, alkalization and aging of pulp, which are used in the industrial preparation of cellulose derivatives, e.g., as an element of the preripening process in viscose rayon production, were investigated. The CCOA method shows a clear reduction of the molecular weight, accompanied by a fast loss of carbonyls in the first phase, which is due to removal of low-molecular weight material by dissolution, and a slow decrease in the second phase, which is caused by further oxidation of carbonyl groups. Also here, differences in the carbonyl course in different molecular weight regions were monitored. Third, electron beaming, proposed as a means of pulp activation, was shown to decrease and narrow the molecular weight distribution, under generation of comparatively low amounts of carbonyls, which, however, are also introduced into high molecular weight, crystalline domains, as shown by a comparison of homogeneous and heterogeneous CCOA labeling approach. Finally, as the fourth application, thermal treatment of cellulose at temperatures between 105 and 165 degrees C was shown to bring about a small reduction of the molecular weight, which only at higher drying temperatures is accompanied by an introduction of carbonyls over the whole molecular weight range.     

Structural Model of the Bilitranslocase Transmembrane Domain Supported by NMR and FRET Data

We present a 3D model of the four transmembrane (TM) helical regions of bilitranslocase (BTL), a structurally uncharacterized protein that transports organic anions across the cell membrane. The model was computed by considering helix-helix interactions as primary constraints, using Monte Carlo simulations. The interactions between the TM2 and TM3 segments have been confirmed by Förster resonance energy transfer (FRET) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, increasing our confidence in the model. Several insights into the BTL transport mechanism were obtained by analyzing the model. For example, the observed cis-trans Leu-Pro peptide bond isomerization in the TM3 fragment may indicate a key conformational change during anion transport by BTL. Our structural model of BTL may facilitate further studies, including drug discovery.     

The dTDP-4-dehydro-6-deoxyglucose reductase encoding fcd gene is part of the surface layer glycoprotein glycosylation gene cluster of Geobacillus tepidamans GS5-97T

The glycan chain of the S-layer protein of Geobacillus tepidamans GS5-97(T) consists of disaccharide repeating units composed of L-rhamnose and D-fucose, the latter being a rare constituent of prokaryotic glycoconjugates. Although biosynthesis of nucleotide-activated L-rhamnose is well established, D-fucose biosynthesis is less investigated. The conversion of alpha-D-glucose-1-phosphate into thymidine diphosphate (dTDP)-4-dehydro-6-deoxyglucose by the sequential action of RmlA (glucose-1-phosphate thymidylyltransferase) and RmlB (dTDP-glucose-4,6-dehydratase) is shared between the dTDP-D-fucose and the dTDP-L-rhamnose biosynthesis pathway. This key intermediate is processed by the dTDP-4-dehydro-6-deoxyglucose reductase Fcd to form dTDP-alpha-D-fucose. We identified the fcd gene in G. tepidamans GS5-97(T) by chromosome walking and performed functional characterization of the recombinant 308-amino acid enzyme. The in vitro activity of the enzymatic cascade (RmlB and Fcd) was monitored by high-performance liquid chromatography and the reaction product was confirmed by (1)H and (13)C nuclear magnetic resonance spectroscopy. This is the first characterization of the dTDP-alpha-D-fucopyranose biosynthesis pathway in a Gram-positive organism. fcd was identified as 1 of 20 open reading frames contained in a 17471-bp S-layer glycosylation (slg) gene cluster on the chromosome of G. tepidamans GS5-97(T). The sgtA structural gene is located immediately upstream of the slg gene cluster with an intergenic region of 247 nucleotides. By comparison of the SgtA amino acid sequence with the known glycosylation pattern of the S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a, two out of the proposed three glycosylation sites on SgtA could be identified by electrospray ionization quadrupole-time-of-flight mass spectrometry to be at positions Ser-792 and Thr-583.