Expression and binding properties of a soluble chimeric protein containing the N-terminal domain of the Duffy antigen.

The blood group Duffy antigen of human erythrocytes, which exists in two allelic forms, Fy(a) and Fy(b), is a promiscuous chemokine receptor. In this report we describe the expression and purification of a chimeric protein composed of the amino-terminal extracellular domain of the Duffy antigen (aa 3-60), C-terminal intracellular fragment of glycophorin A (GPA, aa 104-131), and the hexahistydyl tag. We obtained two forms of the recombinant protein containing the Fy(a) or Fy(b) epitope, denoted Fy(a)/GPA and Fy(b)/GPA, respectively. These constructs were expressed in Escherichia coli as periplasmic proteins and were purified by affinity chromatography on the Ni-NTA-agarose. Both proteins bound the monoclonal antibodies recognizing the common Fy6 epitope of the Duffy antigen and an epitope of the C-terminal fragment of GPA, and only the Fy(a)/GPA bound anti-Fy(a) antibody. However, binding of IL-8 to the recombinant proteins was not detected, which indicated that an N-terminal domain of the Duffy antigen is not sufficient for an effective chemokine binding. The lack of the chemokine binding was not likely to be due to the lack of glycosylation of the Fy/GPA, since IL-8 was effectively bound to de-N-glycosylated erythrocytes.

Structural studies of the O-specific polysaccharide from Plesiomonas shigelloides strain CNCTC 113/92.

The structure of the O-specific side chain of the lipopolysaccharide (LPS) of Plesiomonas shigelloides, strain CNCTC 113/92 has been investigated by NMR spectroscopy, matrix-assisted laser desorption/ionization time of flight mass spectrometry and sugar and methylation analysis. It was concluded that the polysaccharide is composed of a hexasaccharide repeating unit with the following structure: in which D-beta-D-Hepp is Dglycero-beta-Dmanno-heptopyranose and 6d-beta-D-Hep is 6-deoxy-beta-Dmanno-heptopyranose. This structure represents a novel hexasaccharide repeating unit of bacterial O-antigen that is characteristic and unique to the Plesiomonas shigelloides strain. Using the high-resolution magic angle spinning technique, 1H-NMR spectra were also obtained for the O-polysaccharide components of isolated LPS and in their original form directly on the surface of bacterial cells.

Structures of the O-specific polysaccharides from Yokenella regensburgei (Koserella trabulsii) strains PCM 2476, 2477, 2478, and 2494: high-resolution magic-angle spinning NMR investigation of the O-specific polysaccharides in native lipopolysaccharides and directly on the surface of living bacteria.

The structures of the carbohydrate O-specific side-chain moiety of the lipopolysaccharides (LPS) of Yokenella regensburgei, strains PCM 2476, 2477, 2478, and 2494, have been investigated by (1)H and (13)C NMR, fast atom bombardment tandem mass spectrometry (FAB-MSMS), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, methylation analysis, partial acid hydrolysis, and immunological methods. It was concluded that the O-specific polysaccharides of strains 2476, 2477, 2478, and 2494 are composed of the same basic trisaccharide repeating unit having the structure -->3)-alpha-D-FucpNAc-(1-->2)-L-alpha-D-Hepp-(1-->3)-6-deoxy -alpha-L- Talp-(1-->, in which L-alpha-D-Hepp is L-glycero-alpha-D-manno-heptopyranose. The detailed analysis revealed, however, differences in O-acetylation patterns of the 6-deoxy-L-talose residue, with 2- and 4-O-acetyl disubstituted -->3)-6-deoxy-alpha-L-Talp-(1--> in strain PCM 2476 and a 2-O-acetylated residue in strains 2477, 2478, and 2494. These structures represent novel, trisaccharide repeating units of bacterial O-antigens that are characteristic and unique to the Y. regensburgeispecies. By use of the high-resolution magic-angle spinning (HR-MAS) technique, (1)H NMR spectra of the O-polysaccharides directly in isolated LPS were obtained. This allowed for almost full assignment and structural determination of the polysaccharide. By this technique the O-polysaccharide components were also observed in their original form directly on the surface of living bacterial cells.

Structural studies of the O-specific polysaccharide of Hafnia alvei strain PCM 1207 lipopolysaccharide.

The structure of the O-specific side-chain of the Hafnia alvei strain PCM 1207 lipopolysaccharide (LPS) has been investigated. Methylation analysis, partial acid hydrolysis, matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) MS, fast atom bombardment (FAB)-MS/MS and 1H- and 13C-NMR spectroscopy were the principal methods used. Glycerol phosphate was identified as a constituent in the polysaccharide and the following structure of a pentasaccharide repeating unit was established: The polysaccharide is partially (approximately 10%) substituted with O-acetyl groups. The lipopolysaccharide was also subjected to high resolution magic angle spinning (HR-MAS) NMR analysis, which showed both the signals of the O-specific polysaccharide as well as several signals from unsubstituted core oligosaccharides. This confirmed the presence of the described structure in the native LPS.

Non-typical lipopolysaccharide core regions of some Hafnia alvei strains: structural and serological studies.

The lipopolysaccharides of Hafnia alvei strains 23, 1222 and 39 were found to have non-typical core region. On the basis of sugar and methylation analyses, 1H-nuclear magnetic resonance spectra and matrix-assisted laser-desorption ionization-time of flight mass spectrometry, it was concluded that the core oligosaccharide of strains 23 and 1222 has the same structure as Escherichia coli R4 core region, and the core oligosaccharide of strain 39 has the structure of Salmonella Ra core. Using the serological methods (passive hemagglutination, enzyme-linked immunosorbent assay and immunoblotting) and the anti-conjugate sera directed against E. coli R4 and Salmonella Ra core oligosaccharides we have confirmed the structural results presented above.

Structural studies of the O-specific chains of Hafnia alvei strains 744, PCM 1194 and PCM 1210 lipopolysaccharides.

The structures of the O-specific chains of the Hafnia alvei strain 744 and PCM strains 1194 and 1210 lipopolysaccharides have been investigated. Methylation analysis, dephosphorylation, NMR spectroscopy, matrix-assisted laser-desorption ionisation time-of-flight mass spectrometry and fast-atom-bombardment mass spectrometry were the principal methods used. It was concluded that the polysaccharides of strains 744 and PCM 1194 are composed of the same pentasaccharide repeating unit having the following structure: [structure in text] and that the polysaccharide of strain PCM 1210 is composed of a pentasaccharide repeating unit having the following structure: [structure in text].

Structural studies of the O-specific chain of Hafnia alvei strain PCM 1190 lipopolysaccharide.

The structure of the O-specific side-chain of the lipopolysaccharide of Hafnia alvei strain PCM 1190 has been investigated. Methylation analysis, partial acid hydrolysis, Smith degradation, NMR spectroscopy, MALDI-TOF, and FAB mass spectrometry in combination with collision-induced-decomposition MS/MS were the principal methods used. It was concluded that the polysaccharide is composed of heptasaccharide repeating units having the following structure: [formula: see text] Only 80% of the repeating units are complete, whereas 20% of them are lacking the alpha-D-glucopyranosyl group.

Structural studies of the O-specific polysaccharide of Hafnia alvei strain PCM 1206 lipopolysaccharide containing D-allothreonine.

The structure of the O-specific side-chain of the Hafnia alvei strain PCM 1206 lipopolysaccharide has been investigated. Methylation analysis, partial acid hydrolysis, FAB-MS/MS and 1H-NMR and 13C-NMR spectroscopy were the principal methods used. D-Allothreonine (D-aThr), amide-linked to the D-galacturonic acid, was identified as a constituent in the polysaccharide and the following structure of a pentasaccharide repeating unit was established: [structure: see text].

Serological characterisation of anti-endotoxin sera directed against the conjugates of oligosaccharide core of Escherichia coli type R1, R2, R3, J5 and Salmonella Ra with tetanus toxoid.

The covalent conjugates of oligosaccharide core: Escherichia coli type R1, R2, R3, J5 and Salmonella Ra with tetanus toxoid have been prepared using reaction of reductive amination. The neoglycoconjugates were good immunogens in rabbits yielding a high level of anti-lipopolysaccharide antibodies of IgG class. The antibodies were used to examine the possibility of their reactions with smooth lipopolysaccharides. We have found that all antisera were able to react with the lipopolysaccharide molecules of identical or related core type, possessing core oligosaccharides substituted with O-specific chains. These reactions were shown in both the ELISA assay and the immunoblotting test.

Anti-endotoxin antibodies directed against Escherichia coli R-1 oligosaccharide core-tetanus toxoid conjugate bind to smooth, live bacteria and smooth lipopolysaccharides and attenuate their tumor necrosis factor stimulating activity.

The reactivity of anti-OS R1-tetanus toxoid serum with various smooth and rough lipopolysaccharides have been determined in enzyme-linked immunosorbent assay (ELISA) and with intact, live smooth bacterial cells in flow cytometry. All tested smooth lipopolysaccharides of R1 core type belonging to nine different O-serotypes reacted strongly with anti-conjugate antibodies. Flow cytometry showed that anti-core antibodies labelled more than 95% of bacterial cells with high intensity of fluorescence. The anti-conjugate serum was able to react with free form of smooth lipopolysaccharides and to inhibit their TNF stimulation activity in in vitro and in vivo models.

Structural studies of the O-specific chain of Hafnia alvei strain 32 lipopolysaccharide.

The structure of the O-specific side chain of the Hafnia alvei strain 32 lipopolysaccharide has been investigated. Methylation analysis, partial acid hydrolysis, Smith degradations, NMR spectroscopy, MALDI-TOF and FAB mass spectrometry in combination with collision-induced decomposition MS/MS were the principal methods used. It is concluded that the polysaccharide is composed of pentasaccharide repeating units having the following structure which is partially O-acetylated in the 2- (20%) and 3- (50%) position of the-->4)-alpha-D-GalpA-(1-->residue. [sequence :see text] A MALDI-TOF mass spectrum of the O-specific chains indicated that they consisted of up to 16 repeating units.

Structural studies of the O-specific polysaccharide of Hafnia alvei strain 1209 lipopolysaccharide.

The structure of the O-specific side chains of the Hafnia alvei strain 1209 lipopolysaccharide has been investigated. Methylation analysis and 1H-NMR and 13C-NMR spectroscopy were the principal methods used. It is concluded that the polysaccharide is composed of pentasaccharide repeating units that have the following structure: -->3)-beta-D-Galp-(1-->4)-alpha-D-Glcp-(1-->4)-beta-D-GlepA-(1--> 3)-beta-D-GalpNAc-(1 --> 4 increases 1 alpha-L-Rhap The relative intensity of the signals from the terminal repeating unit in the 1H-NMR spectrum, the amount of 2,3,6-tri-O-methylgalactose in the methylation analysis, and the matrix-assisted laser-desorption ionisation time-of-flight (MALDI-TOF) mass spectrum of the O-polysaccharide indicated that the structure is also the biological repeating unit and that the O-chains mainly consisted of 8-11 repeating units and, on average, ten repeating units.

Structural and serological studies of lipopolysaccharides of Citrobacter O35 and O38 antigenically related to Salmonella.

Structural analysis using 13C NMR spectroscopy and methylation showed that lipopolysaccharides (LPSs) of Citrobacter freundii O35 and Salmonella arizonae O59 have structurally identical O-specific polysaccharide chains, and those of C. freundii O38 and Salmonella kentucky differ only in the presence of O-acetyl groups in the former. Serological relationships between the structurally similar LPSs were demonstrated using inhibition of ELISA, rocket immunoelectrophoresis, double gel diffusion, and immunoblotting. The O-acetyl groups present in C. freundii O38 LPS are of little importance for its serological specificity. A cross-reaction was observed in immunoblotting between O-antisera to C. freundii O35 and S. arizonae O59 and a structurally related LPS of Pseudomonas aeruginosa O11a, 11b (Lányi-Bergan classification).

[Bacterial endotoxins--methods of structural analysis]. / Endotoksyny bakteryjne--metody analizy strukturalnej.

The modern structural methods used in the determination of lipopolysaccharides chemical structures were described. The combination of nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MALDI-TOF, FAB and EI) applied to structural analysis of lipopolysaccharides, with a few chosen examples of characteristic original spectra were presented.

Structural studies of the O-specific chain and a core hexasaccharide of Hafnia alvei strain 1192 lipopolysaccharide.

The structure of the O-specific side-chain and a core hexasaccharide of the Hafnia alvei strain 1192 lipopolysaccharide has been investigated. Methylation analysis, NMR spectroscopy, MALDI-TOF spectrometry, and various specific chemical degradations were the principal methods used. It is concluded that the polysaccharide is composed of hexasaccharide repeating-units having the following structure which is partially O-acetylated in the 2-position of the --> 4)-alpha-D-Glc pA-(1-->(70%) and on different positions of the L-Rha residues (50%). [Formula: see text] The core hexasaccharide was found to have the following structure: [Formula: see text]

Lipopolysaccharide core region of Hafnia alvei: serological characterization.

Covalent glycoconjugates containing, as a ligand lipopolysaccharide core, oligosaccharides of Hafnia alvei standard strain ATCC 13337 and R mutant 1 M were used to produce anti-H. alvei core antibodies. The sera obtained were tested in rocket immunoelectrophoresis, immunoblotting and ELISA using H. alvei lipopolysaccharides of various strains. The experiments were carried out to study the antigenic relationships between lipopolysaccharide core regions in the H. alvei genus.

[Protective properties of antibodies raised against conjugates of endotoxin core oligosaccharides with proteins]. / Wlasciwosci ochronne przeciwcial przeciw koniugatom oligocukrów rdzenia endotoksyn z bialkami.

Endotoxins are responsible for initiation of septic shock which increases the number of fatalities in Gram-negative bacteremia among hospital patients. The mortality from septic shock is still high despite recent developments in antibiotic therapy. These substances are unable to decrease the level of free lipopolysaccharides in the bloodstream. Another approach to the treatment and prevention of septicaemia involves stimulation of an immune response against LPS. It was found that immunization with core structures of endotoxin conjugated with proteins protected animals against infections and endotoxic shock. Anticonjugate sera are of great interest because they are directed against common parts of LPS and therefore could have cross-reactive and cross-protective potencies towards many Gram-negative rods.

Structural and serological characterization of Hafnia alvei lipopolysaccharide core region.

The structures and serological activities of core oligosaccharide of Hafnia alvei strains have been investigated. Methylation analysis, NMR spectroscopy and various specific degradation procedures were the principal methods used. It is concluded that, core hexasaccharides are identical in the lipopolysaccharides tested and are built of two glucose, three heptose and one 2-keto-3-deoxyoctulosonic acid residues. The antiserum raised against the ATCC13337 oligosaccharide core-tetanus toxoid conjugate cross-reacted strongly with all lipopolysaccharides used as antigens in ELISA test, suggesting that this core region is the common structure in the Hafnia genus.