Helical epitope of the group B meningococcal alpha(2-8)-linked sialic acid polysaccharide.

The immunological properties of the group B meningococcal alpha(2-8)-linked sialic acid polysaccharide have been rationalized in terms of a model where the random coil nature of the polymer can be described by the presence of local helices. The conformational versatility of the alpha NeuAc(2-8)alpha NeuAc linkage has been explored by NMR studies at 600 MHz in conjunction with potential energy calculations for colominic acid, an alpha(2-8)NeuAc polymer, and the trisaccharide alpha NeuAc(2-8)alpha NeuAc(2-8)beta NeuAc. Potential energy calculations were used to estimate the energetically favorable conformers and to describe the wide range of helices which the polymer can adopt. No unique conformer was found to satisfy all NMR constraints, and only ensemble averaged nuclear Overhauser enhancements could correctly simulate the experimental data. Conformational differences between the polymer and the trisaccharide could be best explained in terms of slight changes in the relative distribution of conformers in solution. Similar helical parameters for the alpha(2-8)NeuAc polymer and poly(A) were proposed as the basis for their cross-reactivity to a monoclonal antibody IgMNOV. The unusual length dependency for binding of oligosaccharide to group B specific antibodies was postulated to arise from the recognition of a high-order local helix with an extended conformation which was not highly populated in solution.     

Determinant specificities of the groups B and C polysaccharides of Neisseria meningitidis

A meningococcal group B-specific horse antiserum contains at least two distinct populations of antibodies with specificities for determinants on the group B capsular polysaccharide antigen. These two populations were differentiated on the basis of the ability of only one of them to be absorbed from the antiserum by the structurally related colominic acid. The nature of the colominic acid-specific determinant was elucidated by a radioimmunoassay inhibition technique with the use of a series of linear alpha-(2----8)-linked oligomers of sialic acid as inhibitors. Colominic acid was labeled by prior removal of its N-acetyl groups, followed by their replacement with the use of [3H]acetic anhydride. The conformational nature of the determinant was proposed because of the unusually large size (10 sialic acid residues) of the oligomer required to function as an efficient inhibitor. The structure of the determinant responsible for the second population of group B-specific antibodies has not been determined, but it is obviously based on an as yet undefined conformational or structural feature peculiar to the group B meningococcal polysaccharide. In contrast to the colominic acid-specific group B determinant, the determinant responsible for the group C polysaccharide-specific rabbit antibodies proved to be more conventional. Inhibitory properties of the alpha-(2----9)-linked oligomers maximized with those containing four or five sialic acid residues, which is consistent with the approximate estimated maximal size of an antibody site.     

A protein-sialyl polymer complex involved in colominic acid biosynthesis. Effect of tunicamycin.

A protein-NeuAc complex involved in colominic acid biosynthesis has been identified in membrane preparations of Escherichia coli K-235. This compound had an Mr (estimated by SDS/polyacrylamide-gel electrophoresis and autoradiography) of about 100,000 and played the role of an 'initiator' or 'primer' (endogenous acceptor) in the synthesis of the whole polymer. Incubations of E. coli membranes with CMP-[14C]NeuAc (CMP-N-[14C]acetylneuraminic acid) pointed to the existence of a protein fraction (primer acceptor) that linked residues of sialic acid (N-acetylneuraminic acid, NeuAc) up to a maximal size, later releasing them as low-Mr sialyl polymers (LMrS, Mr less than 10,000). In the presence of colominic acid (final acceptor) the radioactivity linked to the protein quickly decreased, appearing stoichiometrically bound to the whole polysaccharide. When membrane preparations were previously digested with Streptomyces proteinase or de-activated by heating (80 degrees C, 10 min), no incorporation of labelled NeuAc into trichloroacetic acid-insoluble material was detected. These results suggested that colominic acid molecules are synthesized while they are bound to a proteinaceous acceptor that is subsequently excised in the presence of colominic acid, generating the native protein. The antibiotic tunicamycin inhibited the biosynthesis of colominic acid, affecting the synthesis of this protein-(NeuAc)n intermediate. All these results are described here for the first time.     

Structural and conformational analysis of sialyloligosaccharides using carbon-13 nuclear magnetic resonance spectroscopy

The analysis of the carbon-13 chemical shift data of NeuAc alpha (2----3)Gal beta (1----4)Glc and NeuAc alpha (2----3)Gla beta-(1----4)GlcNAc and their respective NeuAc alpha (2----6) isomers established distinct and different conformations of the sialic acid residue, depending on the type of anomeric linkage [alpha-(2----3) vs. alpha (2----6)]. Interactions between the NeuAc residue and the Glc or GlcNAc residue are particularly strong in the case of the alpha (2----6) isomers. Similar effects are observed for the larger oligosaccharides [II3(NeuAc)2Lac and IV6NeuAcLcOse4] and even in intact glycoproteins and polysaccharides. It is proposed that the NeuAc alpha (2----3) isomers assume an extended conformation with the sialic residue at the end (terminal) of the oligosaccharide chain or branch. The NeuAc alpha (2----6) isomers are assumed to be folded back toward the inner core sugar residues.     

Structural determination of the sialic acid polysaccharide antigens of Neisseria meningitidis serogroups B and C with carbon 13 nuclear magnetic resonance.

The application of 13-C nuclear magnetic resonance to the analysis of some sialic acid-containing meningococcal polysaccharide antigens is described. Complete assignments of the spectra of both the native serogroup B and the de-O-acetylated serogroup C polysaccharides have been made. These assignments were based on the corresponding data for some related monomers (sialic acid and its alpha-and beta-methylglycosides) and on supportive chemical evidence. The data indicate that the serogroup B polysaccharide is a 2 yields 8-alpha-linked homopolymer of sialic acid, identical in structure with colominic acid from Escherichia coli, whereas the de-O-acetylated serogroup C polysaccharide is a 2 yield 9-alpha-linked homopolymer. The native serogroup C polysaccharide is O-acetylated (1.16 mol of O-acetyl per sialic acid residue), all the O-acetyl substituents being located only at C-7 and C-8 of the sialic acid residues, and in addition contains unacetylated residues (24%). The polysaccharide contains di-O-acetylated residues (O-acetyl on C-7 and C-8), and at least one of the possible monoacetylated residues at C-7 or C-8.     

Structural studies on the sialic acid polysaccharide antigen of Escherichia coli strain Bos-12.

A polysaccharide, antigenically related to group C meningococcus, has been isolated from Escherichia coli strain Bos-12 (016; K92; NM). Like groups B and C meningococcal polysaccharide, the Bos-12 antigen is a pure polymer of sialic acid. 13C NMR studies on the meningococcal group B and C polysaccharides have indicated that the former consists of sialic acid units linked 2 leads to 8- alpha, whereas the latter contains the sialic acid residues linked 2 leads to 9-alpha (Bhattacharjee, A.K., Jennings, H.J., Kenny, C.P., Martin, A., and Smith, I.C.P. (1975), J. Biol. Chem. 250, 1926). Comparison of natural abundance 13C NMR spectra of the Bos-12 polysaccharide with group B and C meningococcal polysaccharides established that Bos-12 was either (a) an equimolar mixture of 2 leads to 8-alpha linked sialic acid homopolymers or (b) a 2 leads to 8-alpha/2 leads to 9-alpha heteropolymer. These possibilities were distinguished in the following manner. The fact that Bos-12 polysaccharide precipitated with anti-group C serum but not with anti-group B serum would seem to exclude a. Further, chemical studies (periodate oxidation followed by tritiated NaBH4 reduction) gave saccharides with a radioactive-labeling pattern expected for alternating 2 leads to 8-alpha/2 leads to 9-alpha sialic acid linkages. Bos-12 is thus an 2 leads to 8/2 lead to 9-alpha heteropolymer.     

Cell-free biosynthesis of the O-acetylated N-acetylneuraminic acid capsular polysaccharide of group C meningococci.

A cell-free system was established to study the biosynthesis of group C meningococcal capsular polysaccharide, an alpha-2 leads to 9-linked N-acetylneuraminic acid (NeuAc) homopolymer containing O-acetyl groups at either C7 or C8. Sialyltransferase activity, isolated from group C meningococcus strain C-11, catalyzed incorporation of [14C]NeuAc from CMP (CMP--[14C]NeuAc) into polymeric form. This sialyltransferase was stimulated by addition of meningococcus group C and Escherichia coli K92 capsular polysaccharides, the latter being an alpha-2 leads to 8- and alpha-2 leads to 9-linked NeuAc heteropolymer. Group C meningococcal sialyltransferase did not require divalent ions but was stimulated by Mn2+. Attempts to demonstrate a lipid-soluble intermediate in the biosynthesis of this NeuAc polymer were unsuccessful. Meningococcal group C sialyltransferase incorporated NeuAc into a membrane-associated product. The polysaccharide can be extracted from the membrane-bound fraction with Triton X-100. The newly synthesized polysaccharide coprecipitates with authentic group C antigen in meningococcal group C antiserum and is degraded by sodium metaperiodate, indicating that the NeuAc polymer synthesized by the cell-free system consists of alpha-2 leads to 9 linkage. Meningococcal group C spheroplast membranes contain an O-acetylase that can catalyze the transfer of acetyl groups from acetyl coenzyme A to the in vitro-synthesized polysaccharide.     

Newcastle disease virus contains a linkage-specific glycoprotein sialidase. Application to the localization of sialic acid residues in N-linked oligosaccharides of alpha 1-acid glycoprotein

Newcastle disease virus sialidase was found to exhibit strict specificity for hydrolysis of the NeuAc alpha 2 leads to 3Gal linkage contained in glycoprotein oligosaccharides both N-linked to asparagine and O-linked to threonine or serine under conditions that left oligosaccharides containing the NeuAc alpha 2 leads to 2 leads to 6Gal and NeuAc alpha 2 leads to 6GallNAc linkages intact. This was determined, in part, by examining the viral sialidase for its ability to hydrolyze glycoprotein oligosaccharides derivatized with purified sialyltransferases to contain the [14C]NeuAc alpha 2 leads to 3Gal, [14C]NeuAc alpha 2 leads to 6GalNAc, and [14C]NeuAc alpha 2 leads to 6Gal linkages. The viral sialidase was also tested for hydrolysis of the NeuAc alpha 2 leads to 3Gal and NeuAc alpha 2 leads to 6Gal linkages on the N-linked oligosaccharides of alpha 1-acid glycoprotein. Selective hydrolysis of the NeuAc alpha 2 leads to 3Gal linkage was shown by periodate oxidation and by 500-MHz 1H-NMR spectroscopy of native and sialidase-treated glycopeptides. The NMR spectra, together with composition data, further indicated that the NeuAc alpha 2 leads to 3Gal and NeuAc alpha 2 leads to 6Gal linkages were localized to specific branches of the major tri- and tetraantennary oligosaccharides of alpha 1-acid glycoprotein. The results indicate that the Newcastle disease virus sialidase can initiate the selective degradation of N-linked oligosaccharide branches containing the NeuAc alpha 2 leads to 3Gal linkage.     

Substrate specificity of two bacteriophage-associated endo-N-acetylneuraminidases.

For Escherichia coli Bos12 (O16:K92:H-), a bacteriophage (phi 92) has been isolated which carries a depolymerase active on the K92 capsular polysaccharide. As seen under the electron microscope, phi 92 belongs to Bradley's morphology group A and is different from the phage phi 1.2 previously described (Kwiatkowski et al., J. Virol. 43:697-704, 1982), which grows on E. coli K235 (O1:K1:H-), depolymerizes colominic acid, and belongs to morphology group C. The specificity of the phi 1.2- and phi 92-associated endo-N-acetylneuraminidases has been studied with respect to the following substrates (all alkali treated, and where NeuNAc represents N-acetylneuraminic acid): (i) [-alpha-NeuNAc-(2 leads to 8)-]n (colominic acid), (ii) [-alpha-NeuNAc-(2 leads to 8)-alpha-NeuNAc-(2 leads to 9)-]n (E. coli K92 polysaccharide), and (iii) [-alpha-NeuNAc-(2 leads to 9)-]n (Neisseria meningitidis type C capsular polysaccharide). The increase in periodate consumption of these glycans upon incubation with purified phi 1.2 or phi 92 particles was measured, and the split products obtained from all substrates after exhaustive degradation were analyzed by gel chromatography. It was found that the Neisseria polysaccharide is not appreciably affected by either virus enzyme and that phi 1.2 only depolymerizes a small fraction of the K92 glycan. Colominic acid, however, is completely degraded by both agents, phi 92 yielding smaller fragments (one to six NeuNAc residues) than phi 1.2 (two to seven). Phage phi 92 additionally depolymerizes the K92 glycan, essentially to oligosaccharides of two, four, and six residues. The size distribution of these K92 oligosaccharides indicates that the phi 92 enzyme predominantly cleaves the alpha(2 leads to 8) linkages in this polymer.     

Conformational aspects critical to the immunospecificity of the type III group B streptococcal polysaccharide

Immunization of rabbits with group B type III streptococcus organisms induces two distinct populations of antibodies with a specificity for determinants on the native capsular polysaccharide antigen of these organisms. Some of the structural and conformational features of the two determinants responsible for the formation of these antibodies were elucidated by (13)C NMR and serological studies on the native type III polysaccharide and some of its structurally modified analogues. The specificity of the determinant corresponding to the major population of antibodies is dependent of the presence of sialic acid residues on the native type III antigen, and although these residues are not an integral part of the determinant, they exert conformational control over it. The carboxylate groups of the sialic acid residues are an important factor in this control mechanism which could possibly involve intramolecular hydrogen bonding. The terminal sialic acid residues control the orientation of the penultimate beta-d-galactopyranose residues with respect to the backbone of the native antigen. The orientation of these residues is critical to the determinant because the determinant is probably small and is located precisely at the junction of the same beta-d-galactopyranose residues with the backbone of the native type III antigen. The determinant corresponding to the other population of antibodies is not sialic acid dependent. This determinant is located on the backbone of the native antigen in the vicinity of the other determinant but on the opposite side to the oligosaccharide branches. In this position, its conformation is unaffected even by the removal of the oligosaccharide branches from the native antigen.     

Structure and immunochemistry of an oligosaccharide repeating unit of the capsular polysaccharide of type III group B Streptococcus. A revised structure for the type III group B streptococcal polysaccharide antigen

We have derived oligosaccharides from the capsular polysaccharide of type III group B Streptococcus by enzymatic hydrolysis of a specific backbone glycosidic bond utilizing an endo-beta-galactosidase from Flavobacterium keratolyticus. Enzymatic digestion of the polysaccharide produced oligosaccharide fragments of one or more pentasaccharide repeating units. On the basis of 13C NMR, 1H NMR, and methylation analyses, it was established that the smallest digestion fragment was alpha-D-NeupNAc-(2----3)-beta-D-Galp-(1----4)-[beta-D-Glcp-(1----6 )]- beta-D-GlcpNAc-(1----3)-beta-D-Gal. The isolation of this oligosaccharide is consistent with the susceptibility of the beta-D-Galp-(1----4)-beta-D-Glcp linkage in the backbone of the type III group B streptococcal polysaccharide and confirms that the polysaccharide is composed of a pentasaccharide repeating unit. High resolution 13C NMR spectroscopic studies indicated that, as in the case of the pentasaccharide, the terminal sialic acid residues of the type III group B streptococcal polysaccharide were linked to O-3 and not to O-6 of its branch beta-D-galactopyranosyl residues as had been previously reported (Jennings, H. J., Rosell, K.-G., and Kasper, D. L. (1980) Can. J. Chem. 58, 112-120). This linkage was confirmed in an independent methylation analysis of the type III group B streptococcal polysaccharide. Thin layer chromatogram binding assay and radioactive antigen binding assays with radiolabeled oligosaccharides demonstrated the single repeating unit pentasaccharide oligosaccharide to be poorly antigenic. Increasing oligosaccharide size to a decasaccharide consisting of two repeating units resulted in an 8-fold increase in antigen binding in the direct radioactive antigen binding assay. The results suggest that a region of the immunodeterminant site critical for antibody binding is located in the backbone of the polysaccharide and involves the beta-D-galactopyranose-(1----4) beta-D-glucopyranose bond.     

Endo-N-acetylneuraminidase associated with bacteriophage particles.

A bacteriophage (phi 1.2) has been isolated for Escherichia coli K235 (O1:K1:H-). phi 1.2 is specific for the host capsular polysaccharide (colominic acid). The phage forms plaques with acapsular halos and thus carries a glycanase activity for colominic acid, a homopolymer of alpha (2 leads to 8)-linked N-acetylneuraminic acid (NeuNAc) residues. Upon incubation with purified phi 1.2 particles, a solution of K1 polysaccharide loses viscosity and consumes increasing amounts of periodate. Also, by gel filtration, the production of colominic oligosaccharides (down to a size of two to three NeuNAc residues) can be demonstrated. No NeuNAc monomers, however, are formed. The capsules of E. coli strains with the K92 antigen, which consists of NeuNAc residues linked by alternating alpha (2 leads to 8) and alpha (2 leads to 9) bonds, are also depolymerized by the phi 1.2 enzyme. Under the electron microscope, phage phi 1.2 is seen to belong to Bradley's morphology group C (D. E. Bradley, Bacteriol. Rev. 31:230-314, 1967); it has an isometric head, carrying a baseplate with six spikes. By analogy to other virus particles with host capsule depolymerase activity, it is probable that the phi 1.2 endo-N-acetylneuraminidase activity is associated with these spikes.     

Structures of asparagine-linked oligosaccharides of the glycoprotein fetuin having sialic acid linked to N-acetylglucosamine

In the accompanying paper (Bendiak et al., 1989), the separation of a series of oligosaccharides released from asparagine residues of fetuin was described. A series of NMR experiments, which included one- and two-dimensional nuclear Overhauser enhancement, two-dimensional correlation spectroscopy, and two-dimensional relayed-coherence spectroscopy, as well as permethylation analyses, established a Gal beta 1----3(NeuAc alpha 2----6)GlcNAc beta 1----4Man unit common to a series of purified structures. These oligosaccharides contained either three, four, or five glycosidically linked sialic acid residues. The NeuAc residue in alpha 2----6 linkage to GlcNAc gives rise to diagnostic chemical shift perturbations of particular proton signals in the oligosaccharides.     

Heavy metal binding to heparin disaccharides. II. First evidence for zinc chelation.

To map out the heavy metal binding sites of iduronic acid containing oligosaccharides isolated from human kidneys, we studied Zn(II) binding by nuclear magnetic resonance (NMR) and molecular modeling to two disaccharides isolated after nitrous acid depolymerization of heparin and two synthetic disaccharides representative of the heparin structure, namely, IdopA2S (alpha 1,4)AnManOH, 1 alpha, IdopA2S (alpha 1,4)AnManOH6S, 1b, IdopA2S-(alpha 1,4)GlcNS alpha Me, 2a, and IdopA2S (alpha 1,4)GlcNS6S alpha Me, 2b (see previous article in this series). A conformational analysis of the metal free and metal bound solutions was made by comparing calculated [(NOE)]s, [T1]s, and [J]s to experimental values. The 1C4, 4C1, and 2S0 conformations of the L-idopyranosiduronate ring and the 4E and 4T3 of the anhydro-D-mannitol ring are evaluated as are rotations about the C5-C6 hydroxymethylene of the AnManOH(6S) or GlcNS (6S) residues. The NOE between IdopA2S H1 and H3 and the known NOE between H2 and H5, as well as the T1 of IdopA2S H3, are introduced as NMR observables sensitive to the IdopA2S ring conformation. Similarly, a NOE between IdopA2S H5 and AnManOH(6S) or GlcNS(6S) H3 was observed that directly restricts the allowed interglycosidic conformational space. For all disaccharides, the Zn(II) bound spectral data are consistent with models in which these motions are partially "frozen" such that the 1C4 conformation of the IdopA2S is stabilized along with the 4T3 conformation of the AnManOH(6S) ring. The interglycosidic conformation is also stabilized in one of two minima. Electrostatic potential energy calculations gave the best overall agreement with experiment and suggest metal binding conformations with the carboxylate and ring oxygen of the IdopA2S residues (1C4 conformation) and either O3 of the GlcNS(6S) residues or the sulfate oxygens of the 6-sulphate for 2b providing additional chelating sites. These chelation models concur with the observation of marked 13C and 1H NMR chemical shifts for the IdopA2S resonances and of GlcNS H3 for 2 alpha and GlcNS6S C6 for 2b. This study of model compounds implicates the IdopA2S(alpha 1,4)GlcNS6S group as part of the heavy metal binding site in biologically important acidic oligosaccharides such as heparin.     

Rate, mechanism, and immunochemical studies of lactonisation in serogroup B and C polysaccharides of Neisseria meningitidis

Meningococcal Serogroup B polysaccharide and colominic acid, which are (2----8)-alpha-linked homopolymers of sialic acid, undergo lactonisation at low pH at a rate which is dependent upon the molecular size and upon the salt form (Na+ or Ca2+). Meningococcal Serogroup C polysaccharide, a (2----9)-alpha-linked homopolymer of sialic acid with acetyl groups present at O-7 and/or O-8, reacts with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to give an O-acylisourea. The rate of formation of O-acylisourea does not differ substantially between O-acetylated (O-Ac+) C, non-O-acetylated (O-Ac-) C, and B polysaccharide. 13C-N.m.r. spectroscopy shows that, in the absence of O-acetyl groups, the majority of the activated carboxyl groups of C polysaccharide condense with an adjacent HO-8 to form a delta-lactone. Immunochemical studies show that the antigenicity of B polysaccharide is markedly reduced on lactonisation of less than 20%, as measured by a radioimmunoassay using an anti-B monoclonal antibody, and that low-molecular-weight colominic acid is poorly antigenic both before and after lactonisation, suggesting the presence of conformational determinants on B polysaccharide. In contrast, lactonisation and/or formation of O-acylisourea groups in the (O-Ac+)-C polysaccharide does not cause a significant decrease in the antigenicity, which is consistent with a sequential (structural) determinant on the molecule.     

Cleavage of the polysialosyl units of brain glycoproteins by a bacteriophage endosialidase. Involvement of a long oligosaccharide segment in molecular interactions of polysialic acid

Polysialosyl chains containing alpha 2-8-linked N-acetylneuraminic acid have been suggested to modulate the biological activity of a neural cell adhesion molecule. Polysialosyl glycopeptides isolated from developing brain were incubated with a bacteriophage containing endosialidase. Sialic acid oligomers up to 7 residues long were liberated both from the glycopeptides and colominic acid. The substrate specificity of the endosialidase was studied with sialic acid oligomers of different sizes prepared from colominic acid. It was found that the endosialidase required the simultaneous presence adjacent to the site of cleavage a minimum of 3 sialic acid residues on the distal side and a minimum of 5 sialic acid residues on the proximal (reducing end) side. From the fragments liberated by the enzyme the existence of polysialic acid chains up to at least 12 residues long in the glycopeptides were concluded. This was also supported by the interaction of the glycopeptides with a meningococcal group B polysaccharide antiserum, which was found to require 10 residues or more for binding. The results indicate that the brain polysialosyl glycopeptides contain a long polysialic acid segment, which is also specifically needed for certain molecular interactions. The implications of the findings for the biological properties of the neural cell adhesion molecule are discussed.     

Structure of the cell wall proteogalactomannan from Neurospora crassa. II. Structural analysis of the polysaccharide part

The native proteoheteroglycan (PHG) from mycelia of Neurospora crassa contain two kinds of carbohydrate chains differing structure. The oligosaccharides containing mannose and galactofuranose are attached by O-glycosidic linkages to serine or threonine residues in the protein (J. Biochem. 96, 1005-1011, 1984). The second kind of carbohydrate chain is a polysaccharide containing mannose and galactofuranose as the main sugar components. The results of structural studies with methylation and NMR analyses on the native PHG and some of its specifically degraded products obtained on partial acid hydrolysis and acetolysis indicate that the polysaccharide moiety of the PHG has an (alpha 1-6) linked mannan backbone with mainly (alpha 1-2) linked side chains, each of which consists of 2 to 5 mannose units, and most of the mannosyl side chains bear beta-galactofuranosyl residues linked to the 2 positions of the mannosyl nonreducing terminals. The galactofuranose residues are linked with each other by (beta 1-5) bonds.     

Development and evaluation of a rapid multianalyte particle-based flow cytometric assay for the quantification of meningococcal serogroup B-specific IgM antibodies in sera for nonculture case confirmation

Serogroup-specific antibody has been shown to be present in the sera of patients recovering from meningococcal disease, and thus the detection of such antibodies may aid in the confirmation of disease. There are currently no standard methods for measuring meningococcal serogroup B-specific antibody in sera. Here, we report the development of a microsphere-based immunoassay which utilizes colominic acid from Escherichia coli 07:K1 (L):NM to detect immunoglobulin M directed against serogroup B polysaccharide. The serogroup B assay was incorporated into a multiplex assay which also detects serogroup-specific immunoglobulin M for meningococcal serogroups A, C, Y and W-135. Using the method of cross-standardization, serogroup B-specific immunoglobulin M concentrations were assigned to the standard serum CDC 1992. The assay is able to detect increases in specific immunoglobulin M concentrations from acute to convalescent phase serum from serogroup B cases, and can be utilized in conjunction with the previously developed tetraplex immunoglobulin G detection assay for serogroups A, C, Y and W-135.     

Conformational and dynamic differences between N. meningitidis serogroup B and C polysaccharides, using n.m.r. spectroscopy and molecular mechanics calculations

1H-N.m.r. spectroscopy has been used to determine the conformation in aqueous solution of the sialic acid residues of the N. meningitidis serogroup B and non-O-acetylated (O-Ac-)-C polysaccharides, and of N-acetylneuraminic acid (NeuNAc). In all cases, the sugar adopts the 2C5 conformation. The side-chain of NeuNAc adopts a conformation such that H-7 and H-8 are approximately anti-periplanar. This conformation is also found in the (O-Ac-)-C polysaccharide, whereas H-7 and H-8 are gauche in the B polysaccharide. Molecular mechanics calculations have been used to probe the conformational preferences of the variously linked sialic acid residues, and the results are in general agreement with those based on the 1H-n.m.r. data. The 13C-n.m.r. spin-lattice relaxation-times have been interpreted in terms of the molecular dynamics of the B and (O-Ac-)-C polysaccharides. Molecular correlation times have been calculated and details of internal rotational or segmental motion elucidated. The C polysaccharide is characterised by internal or segmental motion in the C-7 to C-9 side-chain of the sialic acid repeating-unit, whereas the B polysaccharide has little or no such movement and tumbles in solution as a rigid species with internal rotation of only the pendant C-9 group. The conformational differences suggest a substantially different three-dimensional structure in solution for these polysaccharides.     

NMR studies of carbohydrates and carbohydrate-mimetic peptides recognized by an anti-group B Streptococcus antibody

As part of a program to investigate the origins of peptide-carbohydrate mimicry, the conformational preferences of peptides that mimic the group B streptococcal type III capsular polysaccharide have been investigated by NMR spectroscopy. Detailed studies of a dodecapeptide, FDTGAFDPDWPA, a molecular mimic of the polysaccharide antigen, and two new analogs, indicated a propensity for beta-turn formation. Different beta-turn types were found to be present in the trans and cis (Trp-10-Pro-11) isomers of the peptide: the trans isomer favored a type I beta-turn from residues Asp-7-Trp-10, whereas the cis isomer exhibited a type VI beta-turn from residues Asp-9-Ala-12. The interaction of the dodecapeptide FDTGAFDPDWPA with a protective anti-group B Streptococcus monoclonal antibody has also been investigated, by transferred nuclear Overhauser effect NMR spectroscopy and saturation-transfer difference NMR spectroscopy (STD-NMR). The peptide was found to adopt a type I beta-turn conformation on binding to the antibody; the peptide residues (Asp-7-Trp-10) forming this turn are recognized by the antibody, as demonstrated by STD-NMR experiments. STD-NMR studies of the interactions of oligosaccharide fragments of the capsular polysaccharide have also been performed and provide evidence for the existence of a conformational epitope.