A novel strategy for the synthesis of neoglycoconjugates from deacylated deep rough lipopolysaccharides

Müller‐Loennies, Sven; Grimmecke, Dieter; Brade, Lore; Lindner, Buko; Kosma, Paul; Brade, Helmut

doi:10.1177/09680519020080040601

Cited by 13 publications

(2 citation statements)

References 27 publications

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“…For ELISA, such oligosaccharides (Fig. 1) were conjugated to BSA after dephosphorylation at the anomeric position of the lipid A and reaction with allylamine and isothiocyanate as published (Müller-Loennies et al 2002). The amount of ligand present in the conjugates was determined by measuring the amount of protein (Bradford assay, Bio-Rad) and Kdo (thiobarbiturate assay).…”

Section: Methodsmentioning

confidence: 99%

Analysis of cross-reactive and specific anti-carbohydrate antibodies against lipopolysaccharide from Chlamydophila psittaci

et al. 2009

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Chlamydiae contain a rough-type lipopolysaccharide (LPS) of 3-deoxy-alpha-d-manno-oct-2-ulopyranosonic acid residues (Kdo). Two Kdo trisaccharides, 2.8/2.4- and 2.4/2.4-linked, and a branched 2.4[2.8]2.4-linked Kdo tetrasaccharide occur in Chlamydiaceae. While the 2.8/2.4-linked trisaccharide contains a family-specific epitope, the branched Kdo oligosaccharide occurs only in Chlamydophila psittaci and antibodies against it will be useful in human and veterinarian diagnostics. To overcome the generation of cross-reactive antibodies that bind with high affinity to a dominant epitope formed by 2.4/2.4-linked Kdo, we immunized mice with a synthetic 2.4[2.8]-linked branched Kdo trisaccharide and used phage display of scFv to isolate recombinant antibody fragments (NH2240-31 and SAG506-01) that recognize the branched Kdo oligosaccharide with a K(D) of less than 10 nM. Importantly, although these antibodies used germline genes coding for an inherited Kdo recognition site, they were able clearly to distinguish between 2.4[2.8]2.4- and 2.4/2.4-linked Kdo. Sequence determination, binding data, and X-ray structural analysis revealed the basis for the improved discrimination between similar Kdo ligands and indicated that the alteration of a stacking interaction from a phenylalanine residue in the center of the combining site to a tyrosine residue facing away from the center favors recognition of branched 2.4[2.8]2.4-linked Kdo residues. Immunofluorescence tests of infected cell monolayers using this antibody show specific staining of C. psittaci elementary bodies that allow it to be distinguished from other pathogenic chlamydiae.

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Section: Methodsmentioning

confidence: 99%

Analysis of cross-reactive and specific anti-carbohydrate antibodies against lipopolysaccharide from Chlamydophila psittaci

et al. 2009

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“…BSA neoglycoconjugates of oligosaccharides isolated from recombinant bacteria 50,51 and of a synthetic origin 32,40,52−56 were prepared as described previously. 27,57 Monoclonal antibodies were derived from Balb/c mice and generated as described previously. 40 Isothermal Titration Calorimetry (ITC) and Enzyme-Linked Immunosorbent Assay (ELISA).…”

Section: ■ Experimental Methodsmentioning

confidence: 99%

Subtle Changes in the Combining Site of the Chlamydiaceae-Specific mAb S25-23 Increase the Antibody–Carbohydrate Binding Affinity by an Order of Magnitude

et al. 2018

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Murine antibodies S25-23, S25-26, and S25-5 derive from a common germ-line origin, and all bind the Chlamydiaceae family-specific epitope αKdo(2→8)αKdo(2→4)αKdo (where Kdo is 3-deoxy-α-d-manno-oct-2-ulosonic acid) with high affinity and specificity. These antibodies recognize the entire trisaccharide antigen in a linkage-dependent manner via a groove composed largely of germ-line residues. Despite sharing identical heavy and light chain genes, S25-23 binds the family-specific epitope with nanomolar affinity, which is an order of magnitude higher than that of S25-26, while S25-5 displays an affinity between those of S25-23 and S25-26. We determined the high-resolution crystal structures of S25-23 and S25-5 antigen binding fragments in complex with a pentasaccharide derived from the LPS of Chlamydia and measured the affinity of S25-5 for chlamydial LPS antigens using isothermal titration microcalorimetry. The 1.75 Å resolution structure of S25-23 shows how subtle conservative mutations Arg(L)-27E to lysine and Ser(H)-56 to threonine lead to an order of magnitude increase in affinity. Importantly, comparison between previous S25-26 structures and the 1.99 and 2.05 Å resolution liganded and unliganded structures of S25-5, respectively, shows how a Ser(L)-27E mutation results in an intermediate affinity due to the reduced enthalpic penalty associated with complex formation that would otherwise be required for arginine in this position. This strategy allows for subtle adjustments in the combining site via affinity maturation that have dramatic consequences for the affinity of an antibody for its antigen.

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Preparation of Glycoconjugate Vaccines

Zou

Jennings

2008

Carbohydrate‐Based Vaccines and Immunotherapies

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A novel strategy for the synthesis of neoglycoconjugates from deacylated deep rough lipopolysaccharides

Cited by 13 publications

References 27 publications

Analysis of cross-reactive and specific anti-carbohydrate antibodies against lipopolysaccharide from Chlamydophila psittaci

Analysis of cross-reactive and specific anti-carbohydrate antibodies against lipopolysaccharide from Chlamydophila psittaci

Subtle Changes in the Combining Site of the Chlamydiaceae-Specific mAb S25-23 Increase the Antibody–Carbohydrate Binding Affinity by an Order of Magnitude

Preparation of Glycoconjugate Vaccines

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