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.     

Conformational epitope of the type III group B Streptococcus capsular polysaccharide.

The protective epitope of the type III group B streptococcal polysaccharide (GBSPIII) is length dependent and conformational. To obtain a more accurate characterization of the conformational epitope, ELISA inhibition and surface plasmon resonance studies were conducted on two GBSPIII-specific mAbs using a large panel of oligosaccharide probes. The results of the studies confirmed that 2 repeating units (RU) is the minimum binding unit and that, while increases in chain length from 2 RU to 7 RU caused further optimization of the epitope, it remained monovalent. A 3-fold increase in affinity was observed between 7 RU and 20 RU, which, by surface plasmon resonance studies on a Fab, was shown to be due to both further optimization of the individual epitope and the occurrence of multivalency of epitope. The data support our hypothesis that the conformational epitope is an extended helical segment of the GBSPIII. GBSPIII exists mainly in the random coil form, which structurally mimics short oligosaccharide self Ags, but it can infrequently and spontaneously form extended helices. Although not prevalent in GBSPIII, the immune system preferentially selects these helical epitopes because they are unique to the polysaccharide. Contrary to a previously proposed model of GBSPIII binding in which the binding of the first Ab propagates a continuum of helical epitopes, our binding kinetics are consistent only with the helical epitope's being discontinuous and infrequent.     

Analytical methods for identifying and quantitating deamidated sialic acid (2-keto-3-deoxy-D-glycero-D-galactonononic acid) and alpha 2----8-linked poly(oligo)nonulosonate residues in glycoconjugates.

In 1986 we reported the natural occurrence of deaminated neuraminic acid (2-keto-3-deoxy-D-glycero-D-galactonononic acid, KDN) in fish egg glycoprotein. Subsequently, we have shown that many types of sialic acid as well as KDN occur in polymeric chains, poly(oligo)-Sia and poly(oligo)KDN in nature. In this study we demonstrate that the conventional colorimetric and gas-liquid chromatographic methods used in the analysis of sialic acid can be applied to analysis of these new nonulosonate and poly(oligo)nonulosonates. We report that the thiobarbituric acid reaction can be used to analyze both free and bound KDN, but gives lower extinction values when applied to poly(oligo)KDN without prior hydrolysis. Further, the published hydrolytic and/or methanolytic procedures are suitable to release the terminal sialic acid residues, but are not appropriate for quantitative release of the nonulosonic acids from poly(oligo)nonulosonates. A new gas-liquid chromatographic procedure for the identification-quantitation of nonulosonates in poly(oligo)meric forms is described.     

Purification and characterization of a novel broad-specificity (alpha 1----2, alpha 1----3 and alpha 1----6) mannosidase from rat liver.

We have identified a mannosidase in rat liver that releases alpha 1----2, alpha 1----3 and alpha 1----6 linked manose residues from oligosaccharide substrates, MannGlcNAc where n = 4-9. The end product of the reaction is Man alpha 1----3[Man alpha 1----6]Man beta 1----4GlcNAc. The mannosidase has been purified to homogeneity from a rat liver microsomal fraction, after solubilization into the aqueous phase of Triton X-114, by anion-exchange, hydrophobic and hydroxyapatite chromatography followed by chromatofocusing. The purified enzyme is a dimer of a 110-kDa subunit, has a pH optimum between 6.1 and 6.5 and a Km of 65 microM and 110 microM for the Man5GlcNAc-oligosaccharide or Man9GlcNAc-oligosaccharide substrates, respectively. Enzyme activity is inhibited by EDTA, by Zn2+ and Cu2+, and to lesser extent by Fe2+ and is stabilized by Co2+. The pattern of release of mannose residues from a Man6GlcNAc substrate shows an ordered hydrolysis of the alpha 1----2 linked residue followed by hydrolysis of alpha 1----3 and alpha 1----6 linked residues. The purified enzyme shows no activity against p-nitrophenyl-alpha-mannoside nor the hybrid GlcNAc Man5GlcNAc oligosaccharide. The enzyme activity is inhibited by swainsonine and 1-deoxymannojirimycin at concentrations 50-500-fold higher than required for complete inhibition of Golgi-mannosidase II and mannosidase I, respectively. The data indicate strongly that the enzyme has novel activity and is distinct from previously described mannosidases.     

Murine monoclonal antibodies directed to the human histo-blood group A transferase (UDP-GalNAc:Fuc alpha 1----2Gal alpha 1----3-N-acetylgalactosaminyltransferase) and the presence therein of N-linked histo-blood group A determinant

Mouse MAbs (WKH-1 through -3) to the human histo-blood group A glycosyltransferase (Fuc alpha 1----2Gal alpha 1----3 galactosaminyltransferase) were established by immunization with the purified native A transferase protein. Hybridomas were selected on the basis of solid-phase reactivity with the purified native A transferase, cell immunofluorescence and immunoprecipitation of transferase activity, and absence of reactivity with blood group ABH carbohydrate determinants. Three MAbs, thus selected, were found most likely to react with the protein epitopes unrelated to carbohydrate epitopes of purified A transferase. The MAbs reacted with cells having high A transferase activity and immunoprecipitated the A transferase activity as well as the 40,000 MW iodinated transferase protein. The antibodies were shown, however, to immunoprecipitate and partially inhibit not only A1 and A2 but also B transferase activity from plasma and A transferase from human lung, and to react with B cells expressing B transferase, thus indicating a cross-reactivity with B transferase. In contrast, they showed no reactivity with various cells having the O phenotype and did not immunoprecipitate the A transferase from porcine submaxillary glands or the alpha 1----2fucosyltransferase from Colo205 cells. The purified A glycosyltransferase was found to carry blood group A carbohydrate determinants by immunochemical detection with a panel of anti-carbohydrate MAbs. These determinants are believed to be N-linked, since treatment of the purified A transferase with N-glycanase removed activity. Immunohistological studies of three epithelial tissues showed that the antibodies stained the Golgi area of cells in epithelia from A and B, but not O, individuals.     

Synthesis of oligosaccharide fragments of O-specific Shigella flexneri polysaccharides. II. Synthesis of trisaccharide Glc alpha1----3RHa alpha1----2Rha alpha1----OMe and tetrasaccharide GlcNAc beta1----2(Llc alpha1----3)Rha alpha1----2Rh alpha1----OMe

Methyl glycosides of the title linear trisaccharide and branched tetrasaccharide were synthesized by stepwise glycosylation. These oligosaccharides represent the fragments of O-antigenic polysaccharides of Shigella flexneri serotypes 2b, 3a, 5b, and X.     

Cloning and characterization of DNA complementary to human UDP-GalNAc: Fuc alpha 1----2Gal alpha 1----3GalNAc transferase (histo-blood group A transferase) mRNA

Based on the partial amino acid sequence, the cDNA encoding UDP-GalNAc:Fuc alpha 1----2Gal alpha 1----3GalNAc transferase, the specific primary gene product of histo-blood group A gene (A transferase), was cloned and sequenced. Poly(A)+ RNA from human stomach cancer cell line MKN45, expressing high levels of A antigen, was used for construction of a lambda gt10 cDNA library. Degenerate synthetic oligodeoxynucleotides were used for polymerase chain reactions to detect the presence of the sequence of interest in cDNA (presence test) and to identify the correct clones (identification test) after screening the library with a radiolabeled polymerase chain reaction amplified fragment. Nucleotide sequence analysis revealed a coding region of 1062 base pairs encoding a protein of 41 kDa. Hydrophobicity plot analysis shows the existence of three domains: N-terminal short stretch, transmembranous hydrophobic region, and a long C-terminal domain (a feature common to all glycosyltransferases cloned so far). Southern hybridization analysis has shown that this DNA does not represent a multigene family. No restriction fragment length polymorphism was found to correlate with ABO blood group type. Bands were detected in Northern hybridization of mRNAs from cell lines expressing A, B, AB, or H antigens. These results suggest that sequences of ABO genes are essentially very similar (with minimal differences), and the inability of the O gene to encode A or B transferases is probably due to structural differences rather than A or B transferase expression failure.     

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.     

Comparison of the conformational dynamics of the (1----4)- and (1----6)-linked alpha-D-glucans using 13C-NMR relaxation.

The conformational dynamics of alpha-(1----4)- and alpha-(1----6)-glucan homooligomers in the nanosecond time domain have been compared by measuring the 13C-nmr longitudinal relaxation times T1 for carbons of the terminal and interior sugar residues. Measurements are reported on monomeric glucose and on oligomers containing up to ten glucose residues at room temperature in aqueous solution at concentrations of 3 and 20 g/dL. The carbons of terminal residues display longer relaxation times than do those of interior residues, presumably as a consequence of a greater degree of conformational mobility of the chain ends. The T1s of the reducing terminal residues of all oligomers are significantly longer than those of the corresponding nonreducing termini, a phenomenon that we associate tentatively with the anomeric equilibrium at the reducing end. Carbons of the reducing terminal residues in the beta-anomeric form relax more slowly than their alpha-anomeric counterparts. At 20 g/dL the mean T1s for carbons of the terminal and interior residues attain asymptotic behavior with increasing chain length at a chain length of about six residues, and carbons of the alpha-(1----4)-linked maltooligomers relax significantly more slowly than those of the corresponding alpha-(1----6)-linked isomaltooligomers. The T1s of both glucan series increase with decreasing concentration. This concentration dependence disappears below 3 g/dL, where the T1s of the two series of homoligomers are no longer distinguishable. This suggests that in dilute aqueous solution at room temperature viscous damping effects predominate over contributions to the T1-sensitive conformational dynamics from structural differences in the glycosidic linkage region. At 3 g/dL the approach to long chain-length asymptotic behavior is more protracted than at 20 g/dL, and the T1s of carbons of interior oligomeric residues appear to match the corresponding high-polymer behavior at a chain length of eight and greater.     

Polymers having (1----4)- and (1----6)-linked alpha-D-glucopyranosyl groups as acceptors in the glycogen synthase reaction.

Insoluble, light-sensitive polymers linked to maltose, maltotriose, a glycogen-branch point trisaccharide, and panose were synthesized and served in a comparative study as acceptors in the glycogen synthase (UDP-D-glucose:glycogen 4-alpha-D-glucosyltransferase, EC 2.4.1.11) reaction. The highest transfer rate was observed with the maltotrio polymer. Extending the acceptor linearly with (1----4)-linked alpha-D-glucopyranosyl residues improved the transfer, whereas (1----6)-linked alpha-D-glucopyranosyl branches decreased it.     

A bisubstrate analog inhibitor for alpha(1----2)-fucosyltransferase

Porcine submaxillary beta-galactoside alpha(1----2)-fucosyltransferase is known to transfer a fucosyl residue from guanosine 5'-diphosphofucose (GDP-fucose) to the 2-OH group of beta-D-galactopyranosides with inversion of configuration at the fucopyranosyl anomeric carbon. A bisubstrate analog (1) of the postulated transition-state for this reaction, which has O-2 of phenyl beta-D-galactopyranoside attached to the terminal phosphorous of GDP through a flexible ethylene bridge, has been chemically synthesized and evaluated as an inhibitor of this enzyme. Compound 1 was found to be a competitive inhibitor with respect to both GDP-fucose and phenyl beta-D-galactopyranoside for both the membrane-bound and soluble forms of the fucosyltransferase. It was also a competitive inhibitor with respect to the alternate acceptor beta DGal(1----3)beta DGlcNAcO(CH2)8-COOMe. The Ki values were in the range 2.3-16 microM. Compound 1 is the first example of a bisubstrate analog inhibitor for a glycosyltransferase which binds to both the acceptor and donor recognition sites of the enzyme. The potential of a bisubstrate analog strategy for the production of specific glycosyltransferase inhibitors is discussed.     

An immunochemical study of the combining site specificities of C57BL/6J monoclonal antibodies to alpha (1----6)-linked dextran B512

This is the first report of an immunochemical study of the combining site specificities of a set of monoclonal antibodies to dextran B512 from C57BL/6J mice. The results confirm previous observations on antidextran combining sites and reveal specificities not seen earlier extending the observed repertoire of antibody combining sites to the single alpha (1----6)-linked glucosyl antigenic determinant. Eight C57BL/6J anti-dextran B512 hybridomas, four IgM,kappa and four IgA,kappa, were produced by PEG fusion of immune spleen cells with the nonproducer myeloma cell line P3X63Ag8 6.5.3. Antibody combining site specificities were determined by quantitative precipitin assays with 14 dextrans. Native dextrans with high percentages of linear alpha (1----6)-linked glucoses, similar to the immunogen B512, were the best precipitinogens; dextrans with alternating alpha (1----3), alpha (1----6) linkages, and highly branched dextrans were less effective. All antibodies precipitated with a synthetic, unbranched alpha (1----6)-linked dextran, suggesting their combining sites were "groove-like" and directed toward internal sequences of alpha (1----6)-linked residues, rather than "cavity-like" and directed toward a nonreducing terminal glucose. Two of the IgA hybridomas gave biphasic precipitin curves with dextran B512; this was shown to be due to differences in the precipitability of IgA monomers and polymers. Differences were observed in the reactivities of several dextrans considered previously to be structurally similar, and a newly proposed structural model of dextran B1299S was assessed. Quantitative precipitin inhibition studies with alpha (1----6)-linked isomaltosyl (IM) oligosaccharides, IM2 to IM9, showed that maximum inhibition was reached with IM6 or IM7, consistent with earlier estimates of the upper limit for the sizes of anti-B512 combining sites. Two IgM hybridomas showed a unique pattern, with inhibition being obtained only with IM5 or larger IM oligosaccharides. Association constants of the antidextrans for dextran B512 and for IM7, determined by affinity gel electrophoresis, ranged from 10(2) to 10(4) ml/g, comparable to earlier findings with antidextrans and other anticarbohydrate antibodies.     

The use of porcine liver (2----3)-alpha-sialyltransferase in the large-scale synthesis of alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-D-GlcpNAc, the epitope of the tumor-associated carbohydrate antigen CA 50.

alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-D-GlcpNAc (2) and, alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-beta-D-GlcpNAcOMBn+ ++ were prepared on a large scale by the action of beta-D-Galp-(1----3)-D-GalpNAc (2----3)-alpha-sialyltransferase (partially purified from porcine liver) on beta-D-Galp-(1----3)-D-GlcpNAc and beta-D-Galp-(1----3)-beta-D-GlcpNAcOMBn, respectively. The trisaccharide 2 is the epitope of the tumor-associated carbohydrate antigen CA 50, highly expressed in human pancreatic adenocarcinoma.     

A comparison of the IgG isotype expression in the so-called thymus-independent immune responses to alpha(1----3) and alpha(1----6) dextran

This paper presents data on the IgG antibody response against two "thymus-independent" dextran (Dex) antigens from Leuconostoc mesenteroides, alpha(1----3) Dex B 1355S and alpha(1----6) Dex B 512F in BALB/c and C57BL/6 mice, which are considered to be responders or low responders to the respective antigen. The data point toward three common rules governing the two anti-Dex responses despite immunogenetic and antigenic disparities: (1) age dependency of the IgG isotype regulation of the response; (2) down-regulation of IgG isotype expression by T cells; and (3) individually determined preposition for IgG isotype formation in a given animal.     

Localization of alpha 1----3-linked mannoses in the N-linked oligosaccharides of Saccharomyces cerevisiae mnn mutants

Neutral and phosphorylated N-linked oligosaccharides were isolated from Saccharomyces cerevisiae mnn9 and mnn9 gls1 mutant mannoproteins and separated into homologues that differed in the number of terminal alpha 1----3-linked mannoses. In each type of oligosaccharide, the addition of such mannose was shown to occur in an ordered rather than a random fashion. The results confirm and extend an earlier report that dealt with the N-linked oligosaccharides from yeast invertase [Trimble, R.B., & Atkinson, P.H. (1986) J. Biol. Chem. 261, 9815-9824], and they suggest that the postulated processing pathway can be generalized to include phosphorylated and glucose-containing N-linked oligomannosides. We conclude that this processing pathway is identical for the analogous oligosaccharides from the mnn9 and wild-type strains of S. cerevisiae. Analysis of the mnn2 mnn10 mannoprotein revealed that a similar modification occurred at the branched terminus of the outer chain as well as in the core in this mutant.     

Total synthesis of the mollu-series glycosyl ceramides alpha-D-Manp-(1----3)-beta-D-Manp-(1----4)-beta-D-Glcp-(1----1)-Cer and alpha-D-Manp-(1----3)-[beta-D-Xylp-(1----2)]-beta-D-Manp-(1----4)-beta- D-Glcp-(1----1)-Cer

The mollu-series glycosphingolipids, O-alpha-D-mannopyranosyl-(1----3)-O-beta-D-mannopyranosyl-(1----4)-O-bet a-D-glucopyranosyl-(1----1)-2-N-tetracosanoyl-(4E)-sphingeni ne and O-alpha-D-mannopyranosyl-(1----3)-O-[beta-D-xylopyranosyl-(1----2])-O- beta-D-mannopyranosyl-(1----4)-O-beta-D-glucopyranosyl-(1----1)-2-N- tetracosanoyl-(4E)-sphingenine, were synthesized for the first time by using 2,3,4-tri-O-acetyl-D-xylopyranosyl trichloroacetimidate, methyl 2,3,4,6-tetra-O-acetyl-1-thio-alpha-D-mannopyranoside, benzyl O-(4,6-di-O-benzyl-beta-D-mannopyranosyl)-(1----4)-2,3,6-tri-O-benzyl-be ta-D- glucopyranoside 9, and (2S,3R,4E)-2-azido-3-O-(tert-butyldiphenylsilyl)-4-octade cene-1,3-diol 6 as the key intermediates. The hexa-O-benzyl disaccharide 9 was prepared by coupling two monosaccharide synthons, namely, 2,3-di-O-allyl-4,6-di-O-benzyl-alpha-D-mannopyranosyl bromide and benzyl 2,3,6-tri-O-benzyl-beta-D-glucopyranoside. It was demonstrated that azide 6 was highly efficient as a synthon for the ceramide part in the coupling with both glycotriaosyl and glycotetraosyl donors, particularly in the presence of trimethylsilyl triflate.