Controlled acid hydrolysis of an O-antigen fragment yields univalent heptasaccharide haptens containing one 3,6-dideoxyhexose epitope.
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Antibodies↗
Biomedical subjects
D R Bundle
Publications and source records attributed to D R Bundle.
Preliminary crystallographic analysis of a Fab specific for the O-antigen of Shigella flexneri cell surface lipopolysaccharide with and without bound saccharides.
The Fab of a monoclonal anti-carbohydrate antibody, SYA/J6 (IgG3, kappa, murine), raised against the O-polysaccharide antigen of the cell surface lipopolysaccharide of variant Y Shigella flexneri, a Gram negative bacterium, has been crystallized in the unliganded form and in complex with tri- and pentasaccharide antigens. The three crystal forms belong to the tetragonal space group P4(3)2(1)2, or P4(1)2(1)2, with very similar unit cell dimensions and an asymmetric unit that contains one molecule of about 50,000 Daltons, and a fourth crystal form belongs to monoclinic space group P2(1) that contains four molecules of Fab in an asymmetric unit. Whereas diffractions of these crystals on an area detector-rotating anode system extend to only about 3.5 A resolution, those measured using an imaging plate and synchrotron radiation at the Photon Factory facility extend to 2.5 A.
Antibodies, Monoclonal↗
The synthesis of chemically modified disaccharide derivatives of the Shigella flexneri Y polysaccharide antigen.
Disaccharide analogs related to the 2-acetamido-2-deoxy-3-O-(alpha-L- rhamnopyranosyl)-beta-D-glucopyranose element of the Shigella flexneri Y polysaccharide antigen have been synthesized and used to map the binding site of murine monoclonal antibodies GC-4 and SYA/J6 by solid-phase inhibition assays. N-Acetyl, N-trifluoroacetyl and N-benzyloxycarbonyl derivatives of methyl-2-amino-4,6-O-benzylidene-2-deoxy-beta-D-glucopyranoside 1, 3, and 4 were glycosylated by rhamnopyranosyl bromide and thioglycoside donors 5 and 6. These in turn provided access to a series of alpha-L-Rha p-(1-->3)-beta-D-GlcNp-(1-->O)-Me disaccharide glycosides with amino 13, N-acetyl 10, N-propionyl 14, N-pivaloyl 15, and N-trifluoroacetyl 11 functionalities. Congeners of the disaccharide 10 were synthesized with monodeoxy groups introduced at the C-4 and C-6 positions of the GlcNAc residues and at the C-4' position of the rhamnose unit. Chlorosulfation of the selectively protected disaccharide 24, followed by reduction of the 4-chloro-4-deoxy compound 25, was used to prepare the 4-deoxy congener 27, while the C-6 and C-4' deoxy derivatives 31 and 23 were assembled from their respective pre-functionalized monosaccharide building blocks 29 and 19.
Carbohydrate Sequence↗
Crystal structure to 2.45 A resolution of a monoclonal Fab specific for the Brucella A cell wall polysaccharide antigen.
The atomic structure of an antibody antigen-binding fragment (Fab) at 2.45 A resolution shows that polysaccharide antigen conformation and Fab structure dictated by combinatorial diversity and domain association are responsible for the fine specificity of the Brucella-specific antibody, YsT9.1. It discriminates the Brucella abortus A antigen from the nearly identical Brucella melitensis M antigen by forming a groove-type binding site, lined with tyrosine residues, that accommodates the rodlike A antigen but excludes the kinked structure of the M antigen, as envisioned by a model of the antigen built into the combining site. The variable-heavy (VH) and variable-light (VL) domains are derived from genes closely related to two used in previously solved structures, M603 and R19.9, respectively. These genes combine in YsT9.1 to form an antibody of totally different specificity. Comparison of this X-ray structure with a previously built model of the YsT9.1 combining site based on these homologies highlights the importance of VL:VH association as a determinant of specificity and suggests that small changes at the VL:VH interface, unanticipated in modeling, may cause significant modulation of binding-site properties.
Antibody Specificity↗
Recognition of a carbohydrate antigenic determinant of Salmonella by an antibody.
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Antibodies, Bacterial↗
Immunochemical characterization of polyclonal and monoclonal Streptococcus group A antibodies by chemically defined glycoconjugates and synthetic oligosaccharides.
Synthetic oligosaccharides of increasing complexity that represent different epitopes of the Streptococcus Group A cell-wall polysaccharide were used as haptens and glycoconjugates of bovine serum albumin (BSA) and horse hemoglobin (HHb) to characterize polyclonal and monoclonal antibodies. Rabbits were immunized with the BSA glycoconjugates of a linear trisaccharide, branched trisaccharide, and branched pentasaccharide. The binding specificities of the polyclonal antisera were determined by a series of inhibition ELISA studies in which disaccharide through pentasaccharide haptens were used as inhibitors of antibody-glycoconjugate binding. Monoclonal antibodies derived from mice immunized with a killed bacterial vaccine were selected for their binding to native polysaccharide antigen coupled to BSA and the BSA glycoconjugates of the di- and linear tri-saccharides. Polyclonal antibodies were moderately specific for the oligosaccharide epitope of the immunizing glycoconjugate and only those antibodies raised to the branched pentasaccharide antigen showed cross-reaction with the bacterial antigen. The behaviour of selected monoclonal antibodies parallels the binding profile of polyclonal antibodies in that the two highest-titre antibodies were directed toward an epitope displayed by the branched pentasaccharide.
Animals↗
Thermodynamics of oligosaccharide binding to a monoclonal antibody specific for a Salmonella O-antigen point to hydrophobic interactions in the binding site.
The thermodynamic characteristics of oligosaccharide binding to an antibody binding site that is dominated by aromatic amino acids suggest that the hydrophobic effect contributes substantially to complex formation as well as hydrogen bonding and van der Waals interactions. A detailed titration microcalorimetric study on the temperature dependence of the binding of a trisaccharide, representing the epitope of a Salmonella O-antigen, showed that its maximum binding to the monoclonal antibody Se155-4 occurs just below room temperature and both enthalpy and entropy changes are strongly dependent on temperature in a mutually compensating manner. The heat capacity change also shows an unusually strong temperature dependence being large and negative above room temperature and positive below. van't Hoff analysis of the temperature dependence of the binding constant yielded a biphasic curve with two apparent intrinsic enthalpy estimations (approximately -100 kJ mol-1 above 18 degrees C and approximately +100 kJ mol-1 below), each very different from the calorimetrically determined enthalpies (ranging from about -60 kJ mol-1 to -20 kJ mol-1). This was interpreted as being due to large enthalpy contributions from concomitant reactions, most notably changes in solvation. Linear plots, -delta H0 versus -T delta S0, observed for temperature-dependent measurements mirror the behavior seen for a series of functional group replacements, suggesting that the molecular and physical origin of these phenomena are closely related and linked to the role of water in complex formation. The thermodynamic results are compared to the mode of binding determined from a 2.05-A resolution structure of the Fab-oligosaccharide complex, and with literature data for the heat capacities of sugars in aqueous solution and for the thermodynamics of carbohydrate binding to transport proteins and lectins.
Antibodies, Monoclonal↗
Bacterial expression and secretion of various single-chain Fv genes encoding proteins specific for a Salmonella serotype B O-antigen.
Active single-chain Fv molecules encoded by synthetic genes have been expressed and secreted to the periplasm of Escherichia coli using the ompA secretory signal. Four different constructs were developed to investigate the effects of peptide linker design and VL-VH orientation on expression, secretion, and binding to a Salmonella O-polysaccharide antigen. Peptide linker sequences derived from the elbow regions of the Fab molecule were used alone or in combination with the flexible (GGGGS)2 sequence. VL and VH domain order in the single chain molecules had a profound effect on the level of secretion but hardly influenced total expression levels, which were approximately 50 mg/liter, chiefly in the form of inclusion bodies. With VL in the NH2-terminal position, the amount of secreted product obtained was 2.4 mg/liter, but when VH occupied this position the yield was less than 5% of this value. Enzyme immunoassays of the four products showed domain order and linker sequence affected antigen binding by less than an order of magnitude. Attempts to express active Fv from dicistronic DNA were unsuccessful, but active Fv was obtained from single-chain Fv by enzymic cleavage at a site in the elbow linker peptide. The thermodynamic binding parameters of intact and cleaved single-chain Fvs determined by titration microcalorimetry were similar to those of bacterially produced Fab and mouse IgG.
Amino Acid Sequence↗
The synthesis of antibody binding-site probes: a hexasaccharide and two pentasaccharides related to the Brucella A antigen and prepared by in situ activation of thioglycosides with bromine.
Two pentasaccharide analogues and a hexasaccharide fragment of the Brucella A antigen [----2)-alpha-D-Rhap4NFo-(1----]n have been prepared as their methyl glycosides. The pentasaccharide analogues each have two formamido groups replaced by hydroxyl groups. Protected derivatives of the three oligosaccharides were prepared by in situ activation with bromine of mono- and di-saccharide thioglycosides of D-rhamnose and 4-azido-4,6-dideoxy-D-mannose in the presence of a glycosyl acceptor and silver triflate as promoter. Reduction of the azido groups with hydrogen sulfide, N-formylation with ethyl formate, and hydrogenolysis then gave the target pentasaccharide glycosides.
Antibodies, Bacterial↗
Recognition of a cell-surface oligosaccharide of pathogenic Salmonella by an antibody Fab fragment.
The 2.05 angstrom (A) resolution crystal structure of a dodecasaccharide-Fab complex revealed an unusual carbohydrate recognition site, defined by aromatic amino acids and a structured water molecule, rather than the carboxylic acid and amide side chains and a structured water molecule, rather than the carboxylic acid and amide side chains that are features of transport and other carbohydrate binding proteins. A trisaccharide epitope of a branched bacterial lipopolysaccharide fills this hydrophobic pocket (8 A deep by 7 A wide) in an entropy-assisted association (association constant = 2.05 x 10(5) liters per mole, enthalpy = -20.5 +/- 1.7 kilojoules per mole, and temperature times entropy = +10.0 +/- 2.9 kilojoules per mole). The requirement for the complementarity of van der Waals surfaces and the requirements of saccharide-saccharide and protein-saccharide hydrogen-bonding networks determine the antigen conformation adopted in the bound state.
Amino Acid Sequence↗
Sensitive titration microcalorimetric study of the binding of Salmonella O-antigenic oligosaccharides by a monoclonal antibody.
The binding of several oligosaccharide haptens by a monoclonal antibody, Se155-4, specific for Salmonella serogroup B O-antigen was studied by titration microcalorimetry. In the software developed by Wiseman et al. [Wiseman, T., Williston, S. & Brandts, J.F. (1989) Anal. Biochem. 17, 131-137] the number of binding sites/macromolecule is one of the optional regression parameters in the non-linear least-squares analysis of the calorimetric data. Instead, an approach was adopted in which the concentration of binding sites was treated as a regression parameter, obviating the requirement for precise values of antibody absorption coefficients and minimizing effects due to partially inactive antibody preparations. Furthermore, performing the least-squares analysis in two steps, first using a differential heat mode and then an integral heat mode, was shown to yield the most accurate results. The technique gave accurate results using not more than 1-2 mumol ligand and less than 7 mg antibody. Haptens 2-5 were oligomers of the O-antigenic repeating unit varying in chain length by 2-5 repeating units and a trisaccharide glycoside 1, which filled the binding site. The latter hapten exhibited a favourable entropy contribution to binding (delta Go = -31 kJ.mol-1; delta Ho = -21 kJ.mol-1 and -T delta So -10 kJ.mol-1), while all four oligomers 2-5 showed a constant binding energy delta Go = -33 kJ.mol-1, composed of increasingly stronger enthalpy forces compensated by an increasingly unfavourable entropy contribution. These observations are compared with results from enzyme immunoassays and a high-resolution crystal structure for the dodecasaccharide 3 bound to the Fab derived from Se155-4.
Antibodies, Monoclonal↗
The design and synthesis of antibody binding site probes: three pentasaccharide analogues of the Brucella A antigen prepared by activation in situ of thioglycosides with bromine.
Three pentasaccharide analogues of the Brucella A antigen [----2)-alpha-D-Rhap4NFo-(1----], each with one formamido group replaced by a hydroxyl group, have been prepared as their methyl glycosides. Mono- and di-saccharide thioglycosides of D-rhamnose and 4-azido-4,6-dideoxy-D-mannose were used as glycosyl donors for the preparation of protected pentasaccharide derivatives with trisaccharides as intermediates. Glycosylations were performed by activation in situ of the thioglycosides with bromine in the presence of a glycosyl acceptor and silver triflate as promoter. Reduction of the azido groups with hydrogen sulfide. N-formylation with ethyl formate, and hydrogenolysis then gave the target pentasaccharides.
Antigens, Bacterial↗
Access to fluorescent probes via allyl glycosides: the synthesis of a Brucella trisaccharide epitope linked to a coumarin.
Oligosaccharide allyl glycosides are demonstrated to provide a route to fluorescent probes and simple inhibitors. Ethyl 2-O-acetyl-4-azido-3-O-benzoyl-4,6-dideoxy-1-thio-alpha-D-mannopyranosid e (6) was used as glycosyl donor in the preparation of the trisaccharide [alpha-D-Rha p4NFo-(1----2)-]2-alpha-D-Rha p4NFo-O-allyl (16). Thioglycoside 6 was activated with N-iodosuccinimide and triflic acid or by bromine in the glycosylations and the inhibitor 16 was obtained after deprotection by transesterification, reduction of the azido groups with hydrogen sulfide, and N-formylation with ethyl formate. Ozonolysis of the allyl glycoside in 16 and reductive amination with 7-amino-4-methylcoumarin then gave the target fluorescent trisaccharide conjugate.
Allyl Compounds↗
Synthesis and expression in Escherichia coli of cistronic DNA encoding an antibody fragment specific for a Salmonella serotype B O-antigen.
A 1460-bp DNA encoding the two chains of the antigen-binding fragment (Fab) portion of a monoclonal antibody have been chemically synthesized and expressed in Escherichia coli. The antibody, Se155-4, is specific for a Salmonella serogroup B O-antigen and its crystal structure is under investigation. The genes were synthesized according to a strategy that allows for easy manipulation in genetic engineering studies of the Fab-binding site. Each gene is preceded by the ompA secretory signal and a ribosome-binding site, and has been expressed from the two-cistron DNA under the control of the lac promoter. Active Fab of 50 kDa with an inter-chain disulfide bond has been isolated from the periplasm of E. coli in a one-step affinity purification in high yield (2 micrograms/ml of cells). The bacterially produced Fab is as active as purified mouse Fab in antigen-binding and competitive immunoassays. This is the first example of a completely synthetic Fab gene and provides an ideal system to probe the nature of antigen binding by anti-carbohydrate antibodies.
Amino Acid Sequence↗
Molecular modeling of antibody-antigen complexes between the Brucella abortus O-chain polysaccharide and a specific monoclonal antibody.
A molecular model of the binding site of an anti-carbohydrate antibody (YsT9.1) has been developed using computer-assisted modeling techniques and molecular dynamics calculations. Sequence homologies among YsT9.1 and the Fv regions of McPC603, J539 and human Bence--Jones protein REI, all of which have solved crystal structures, provided the basis for the modeling. The groove-type combining site model had a topography which was complementary to low energy conformers of the polysaccharide, a Brucella O-antigen, and the site could be almost completely filled by a pentasaccharide epitope in either of two docking modes. Putative interactions between this epitope and the antibody are consistent with the known structural requirements for binding and lead to the design of oligosaccharide inhibitors that probe the veracity of the modeled docked complex. Ultimately both the Fv model and the docked complex will be compared with independent crystal structures of YsT9.1 Fab with and without pentasaccharide inhibitor, currently at the stage of refinement.
Amino Acid Sequence↗
Preliminary crystal structure analysis of an Fab specific for a Salmonella O-polysaccharide antigen.
The Fab from an IgG1, lambda murine monoclonal antibody with specificity for the O-polysaccharide antigen of Salmonella typhimurium has been crystallized in the absence and presence of hapten. The conditions for crystal growth were vapor diffusion equilibration with 16 to 23% polyethylene glycol 8000 solutions. Data have been collected from crystals of the complex in space group P212121, a = 60.6 A, b = 111.3 A, c = 61.1 A, and refinement of a molecular replacement solution is underway.
Animals↗
O-antigen biotin conjugates. Preparation and use in direct competitive enzyme immunoassays.
Bacterial O-antigens coupled to biotin were shown to function well as labelled antigens in direct enzyme immunoassay. O-polysaccharides released from lipopolysaccharides by mild acid hydrolysis were oxidized by sodium periodate at sites located within the lipopolysaccharide inner core region and the generated aldehyde groups were subjected to reductive amination with 1,3-diaminopropane to yield per-aminated O-polysaccharide derivatives. Biotin was coupled to the introduced amino groups by way of a N-hydroxy-succinimide ester derivative. Biotinylated polysaccharides were used in direct enzyme immunoassays, that employed monoclonal antibody coated microtitre plates and detection of bound biotinylated antigen by streptavidin/horseradish peroxidase reagent. The assay format was designed for rapid estimation of the affinity constants of monoclonal antibodies for small oligosaccharide inhibitors, but the assay is also well suited to fast and sensitive detection of bacterial O-polysaccharides at concentrations within the range 1-10 ng/ml.
Antibodies, Monoclonal↗
Carbohydrate-enzyme conjugates for competitive EIA.
Enzyme glycoconjugates prepared from oligosaccharides or polysaccharides are efficient and durable reagents for direct, competitive enzyme immunoassays based on solid phase antibody. This assay format facilitates simple measurement of association constants and relative inhibitory power of oligosaccharides, while also providing a sensitive quantitative assay of bacterial polysaccharide antigens. Enzyme-saccharide conjugates were prepared by established glycoconjugate methodologies, and protocols for their optimal use were established by investigating the effects of solid phase antibody concentration on the kinetic equilibrium of binding for different antigen-enzyme conjugate concentrations, in the presence and absence of inhibitor. Four discrete monoclonal antibody-carbohydrate antigen systems were studied and it is demonstrated for each, that the relative inhibitory power of oligosaccharides may be readily measured in a convenient assay format, and that association constants can be extrapolated from the same assay data.
Alkaline Phosphatase↗