Molecular modeling of sialyloligosaccharide fragments into the active site of influenza virus N9 neuraminidase

Molecular modeling studies have been carried out to investigate the interactions between substrate sialyloligosaccharide (SOS) fragments bearing different glycosidic linkages and influenza virus N9 neuraminidase, a surface glycoprotein of influenza virus subtype N9. The studies revealed that the allowed orientation for sialic acid (SA) is less than 1% in the Eulerian space at the active site. The active site of this enzyme has enough space to accommodate various SOS fragments, NeuNAcalpha(2-3)Gal, NeuNAcalpha(2-6)Gal, NeuNAcalpha(2-8)NeuNAc and NeuNAcalpha(2-9)NeuNAc, but on specific conformations. In the bound conformation, among these substrates there exists a conformational similarity leading to a structural similarity, which may be an essential requirement for the cleavage activity of the neuraminidases irrespective of the type of glycosidic linkage.     

Antibodies against sialyloligosaccharides coupled to protein

The beta-(p-aminophenyl)ethylamine derivatives of sialyloligosaccharides can be coupled to proteins via their phenylisothiocyanate intermediates under conditions that preserve labile sugar linkages. Bovine serum albumin containing 10 to 40 mol of oligosaccharides/mol of protein and keyhole limpet hemocyanin containing 1,100 mol of oligosaccharide/mol of protein have been prepared with the following oligosaccharides: Neu-NAc alpha 2-3Gal beta 1-4Glc, NeuNAc alpha 2-6Gal beta 1-4Glc, Neu-NAc alpha 2-6Gal beta 1-4GlcNAc beta 1-4Glc, Gal beta 1-3[Neu-NAc alpha 2-6]GlcNAc beta 1-4Glc, and NeuNAc alpha 2-3Gal- beta 1-3[NeuNAc alpha 2-6]GlcNAc beta 1-4Glc. Rabbits immunized with these synthetic glycoproteins produce antibodies directed against the oligosaccharides. The specificities of these antibodies are determined by comparing inhibitory activities of structurally related oligosaccharides in radioimmunoassay and by double diffusion analysis in agarose gels using oligosaccharide-protein conjugates as precipitating antigens. The antibodies distinguish positional isomers of sialic acid.     

N-linked oligosaccharide changes with oncogenic transformation require sialylation of multiantennae

Glycopeptides derived from NIH 3T3 fibroblasts and these cells transformed by transfection with human DNA containing oncogene H-ras were analyzed by 500-MHz 1H-NMR spectroscopy and binding to immobilized lectins. The cells were metabolically labeled with D-[3H]glucosamine or L-[3H]fucose and the glycopeptides included in Bio-Gel P-10 (Mr 5000-3500) were separated into neutral and charged fractions on DEAE-cellulose. The major portion (80%) of these [3H]fucose glycopeptides from the non-transformed NIH 3T3 fibroblasts were neutral or contained one or two charged residues, whereas 90% of the glycopeptides from the transformed cells contained two or more charged residues. The structure of the predominant neutral glycopeptide from the non-transformed NIH 3T3 cells was determined by 1H-NMR spectroscopy to be tetraantennary containing terminal Gal alpha 1----3. (formula; see text) This structure was verified by binding to the immobilized alpha-Gal-specific lectin, Griffonia simplicifolia I and leukoagglutinating phytohemagglutinin from Phaseolus vulgaris (L-PHA), which binds certain tri- or tetraantennary glycopeptides. In contrast, the structure derived by NMR spectroscopy of one of the predominant charged glycopeptides from the transformed cells was triantennary containing terminal NeuNAc alpha 2----3 in addition to Gal alpha 1----3. (formula; see text) In attempting to verify this structure by lectin-binding properties it was found that removal of NeuNAc alpha 2----3 reduced the affinity to L-PHA - agarose. The other major glycopeptides of the transformed cells which were more charged also cotained NeuNAc alpha 2----3 but no NeuNAc alpha 2----6 or Gal alpha 1----3. A tentative structure was proposed for the major glycopeptide of the first charged class from NIH 3T3 cells on the basis of lectin-binding properties and the NMR spectrum which showed, in addition to NeuNAc alpha 2----3, the presence of NeuNAc alpha 2----6 and Gal alpha 1----3. On the basis of the NMR spectrum and other results, it is concluded that the presence of tetraantennary oligosaccharides are not sufficient for the transformed oligosaccharide phenotype. Rather, the tri- or tetraantennae must be sialylated in alpha 2----3 linkage, on more than one antennae, when properties of transformation are expressed in NIH 3T3 cells. Prior to transformation the tetraantennary oligosaccharides of these cells are terminated in alpha-Gal residues, whereas after transformation alpha-Gal residues appear to be replaced by NeuNAc alpha 2----3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of terminal NeuNAcalpha2-3Galbeta1-4GlcNAc sequence in lipooligosaccharides of Neisseria meningitidis

Group B and C Neisseria meningitidis are the major cause of meningococcal disease in the United States and in Europe. N . meningitidis lipooligosaccharide (LOS), a major surface antigen, can be divided into 12 immunotypes of which L1 through L8 were found among Group B and C organisms. Groups B and C but not Group A may sialylate their LOSs with N-acetylneuraminic acid (NeuNAc) at the nonreducing end because they synthesize CMP-NeuNAc. Using sialic acid-galactose binding lectins as probes in an ELISA format, six of the eight LOS immunotypes (L2, L3, L4, L5, L7, and L8) in Groups B and C bound specifically to Maackia amurensis leukoagglutinin (MAL), which recognizes NeuNAcalpha2- 3Galbeta1-4GlcNAc/Glc sequence, but not to Sambucus nigra agglutinin, which binds NeuNAcalpha2-6Gal sequence. The combination of SDS-PAGE and MAL-blot analyses revealed that these six LOSs contained only the NeuNAcalpha2-3Galbeta1-4GlcNAc trisaccharide sequence in their 4.1 kDa LOS components, which have a common terminal lacto-N-neotetraose (LNnT, Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) structure when nonsialylated as shown by previous studies. The LOS-lectin binding was abolished when the LOSs were treated with Newcastle disease viral neuraminidase which cleaves alpha2-->3 linked sialic acid. Methylation analysis of a representative LOS (L2) confirmed that NeuNAc is 2-->3 linked to Gal. Thus, these LOSs structurally mimic certain glycolipids, i.e., paragloboside (LNnT-ceramide) and sialylparagloboside and some glycoproteins in having LNnT and N-acetyllactosamine sequences, respectively, with or without alpha2-->3 linked NeuNAc. The molecular mimicry of the LOSs may play a role in the pathogenesis of N.meningitidis by assisting the organism to evade host immune defenses in man.      

Embryonal lactosaminoglycan. The structure of branched lactosaminoglycans with novel disialosyl (sialyl alpha 2----9 sialyl) terminals isolated from PA1 human embryonal carcinoma cells

Lactosaminoglycan glycopeptides were isolated from human PA1 embryonal carcinoma cells and their structures were elucidated. The glycopeptides were digested by Escherichia freundii endo-beta-galactosidase before and after the modifications by exoglycosidases. The core glycopeptides and oligosaccharides thus obtained and the intact glycopeptides were analyzed by methylation, fast atom bombardment-mass spectrometry, and high-performance liquid chromatography. Based on these experiments, the structures of PA1 lactosaminoglycans were found to have the following unique features. 1) Three lactosaminoglycan fractions of different molecular weights were isolated by Sephadex G-50 gel filtration. Lactosaminoglycans of the highest molecular weight (GpI) have tetra-antennary cores, those of intermediate molecular weight (GpII) have triantennary cores and those of low molecular weight (GpIII) have triantennary and tetra-antennary cores. 2) GpI is composed of 22-26 lactosaminyl units and 7-9 branched galactose residues, GpII is composed of 16-22 lactosaminyl units and 5-7 branched galactose residues, and GpIII is composed of 12-16 lactosaminyl units and 3-4 branched galactose residues. 3) Each branch is short and is composed of the Gal beta 1----4GlcNAc beta 1----6 structure. 4) Sialic acid is preferentially linked to nonreducing terminal regions and a significant amount of the novel disialosyl structure, NeuNAc alpha 2----9NeuNAc alpha 2----3/6Gal, is present at the terminals of the longer polylactosaminyl side chains. 5) These lactosaminoglycans are carried by cell surface glycoproteins of Mr = 80,000 approximately 120,000, as evidenced by lectin-agarose chromatography.     

Structural variations of O-linked oligosaccharides present in leukosialin isolated from erythroid, myeloid, and T-lymphoid cell lines

Structures of O-linked oligosaccharides of leukosialin isolated from K562 erythroid, HL-60 promyelocytic, and HSB-2 T-lymphoid cell lines were examined. Leukosialin was isolated by specific immunoprecipitation from cells which were metabolically labeled with [3H]glucosamine, and glycopeptides were isolated after Pronase digestion. O-Linked oligosaccharides were released by alkaline borohydride treatment, and the structures of purified oligosaccharides were elucidated by specific exoglycosidase digestion, Smith degradation, and methylation anaylsis. Oligosaccharides from K562 cells were found to be GalNAcOH, Gal beta 1----3GalNAcOH, NeuNAc alpha 2----6GalNAcOH, NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH. On the other hand, oligosaccharides from HL-60 and HSB-2 cells were found to be NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAcOH, Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3)Gal beta 1----3)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3Gal beta 1----3)GalNAcOH. These results clearly indicate that leukosialin can be differently glycosylated with O-linked chains, and each erythroid or myeloid (and T-lymphoid) cell line expresses a characteristic set of O-linked oligosaccharides which differ in core structures as well as in sialylation.     

Disialogangliosides and Their Interaction with Cholera Toxin—Investigation by Molecular Modeling,Molecular Mechanics and Molecular Dynamics

Abstract

Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface disialogangliosides (GD1A, GD1B and GD3) in aqueous environment. The molecular mechanics calculation reveals that water mediated hydrogen bonding network plays a significant role in the structural stabilization of GD1A, GD1B and GD3. These water mediated hydrogen bonds not only exist between neighboring residues but also exist between residues that are separated by 2 to 3 residues in between. The conformational energy difference between different conformational states of gangliosides correlates very well with the number of water mediated and direct hydrogen bonds. The spatial flexibility of NeuNAc of gangliosides at the binding site of cholera toxin is worked out. The NeuNAc has a limited allowed eulerian space at the binding site of Cholera Toxin (2.4%). The molecular modeling, molecular mechanics and molecular dynamics of disialo- ganglioside-cholera toxin complex reveal that cholera toxin can accommodate the disialo- ganglioside GD1A in three different modes. A single mode of binding is permissible for GD1B and GD3. Direct and water mediated hydrogen bonding interactions stabilizes these binding modes and play an essential role in defining the order of specificity for different disialogangliosides towards cholera toxin. This study not only provides models for the disialoganglioside-cholera toxin complexes but also identifies the NeuNAc binding site as a site for design of inhibitors that can restrict the pathogenic activity of cholera toxin.     

Isolation and NMR spectroscopic characterization of sialic acid compounds and hemofiltrate of chronic uremia patients

Eight sialyloligosaccharides have been isolated from the hemofiltrate of a patient with end stage renal disease using reverse osmosis, gel filtration, ion-exchange and high-performance liquid chromatography. The structures were predominantly elucidated by one- and two-dimensional 1H- and 13C-NMR spectroscopy: 1 NeuAc alpha 2-3Gal beta 1-4Glc; 2 NeuAc alpha 2-6Gal beta 1-4Glc; 3 NeuAc alpha 2-3Gal beta 1-4GlcNAc; 4 NeuAc-alpha 2-6Gal beta 1-4GlcNAc; 5 NeuAc alpha 2-3Gal beta 1-4-GlcNAc alpha 1-P; 6 NeuAc alpha 2-6Gal beta 1-4GlcNAc alpha 1-P; 7 NeuAc alpha 2-3Gal beta 1-3GalNAc alpha 1-P; 8 NeuAc alpha 2-8NeuAc. While compounds 1-7 are also components of normal human urine, di-N-acetyl-D-neuraminic acid (8) could be isolated for the first time from biological material. The origin and possible clinical relevance of these compounds have to be proved in further investigations.     

Structures of novel sialylated O-linked oligosaccharides isolated from human erythrocyte glycophorins

The O-linked oligosaccharides attached to human erythrocyte glycophorins were extensively characterized. In addition to the previously described disialylated tetrasaccharide, NeuNAc alpha 2----3Gal beta 1----3 (Neu-NAc alpha 2----6)GalNAcOH and monosialylated trisaccharide, NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, novel trisialylated oligosaccharides were isolated. Methylation analysis, fast atom bombardment-mass spectrometry, and enzymatic degradation were used to elucidate the following novel structures: formula; see text: These results suggest that O-linked oligosaccharides with a disialosyl group, NeuNAc alpha 2----8NeuNAc alpha 2----, may be present in various tissues.     

Conformational dynamics of sialyl Lewisx in aqueous solution and its interaction with selectinE. A study by molecular dynamics

Three dimensional structures of sialyl Lewis(x) (SLe(x)) in aqueous solution and bound to selectinE are described based on an exhaustive conformational analysis and several long molecular dynamics simulations using different glycosidic regions as starting conformations. It appears from this study that when the oligosaccharide is free in solution the NeuNAcalpha(2-3)Gal segment favors glycosidic conformation in three different regions in the (Phi,Psi) plane with propensity of populations in the ratio 1:8:1. Each one of these structures is characteristically stabilized by specific hydrogen bonding interaction between NeuNAc and Gal. On the other hand, the Gal-GlcNAc-Fuc segment can exist in four different conformational states. Based on the topology of SLe(x) we are able to predict that out of all the allowed conformations in solution only two of these structures possess a geometry that would fit without steric clashes into the binding location of selectinE. In both of these binding modes, segment Gal-GlcNAc-Fuc adopts a unique conformation. The only difference between the two SLe(x) conformers that can successfully bind to selectinE is given by two possible regions in glycosidic space in the fragment NeuNAcalpha(2-3)Gal. A large conformational departure from the crystallographic data is observed for two lysine residues at the binding site of selectinE. These two residues play an important role when SLe(x) binds selectinE in aqueous solution. These findings help reconcile the X-ray data, in which these residues appear to be 1 nm away from SLe(x), with recent liquid NMR data reporting couplings between these protein residues and the sugar.     

Disialogangliosides and their interaction with cholera toxin - investigation by molecular modeling, molecular mechanics and molecular dynamics

Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface disialogangliosides (GD1A, GD1B and GD3) in aqueous environment. The molecular mechanics calculation reveals that water mediated hydrogen bonding network plays a significant role in the structural stabilization of GD1A, GD1B and GD3. These water mediated hydrogen bonds not only exist between neighboring residues but also exist between residues that are separated by 2 to 3 residues in between. The conformational energy difference between different conformational states of gangliosides correlates very well with the number of water mediated and direct hydrogen bonds. The spatial flexibility of NeuNAc of gangliosides at the binding site of cholera toxin is worked out. The NeuNAc has a limited allowed eulerian space at the binding site of Cholera Toxin (2.4%). The molecular modeling, molecular mechanics and molecular dynamics of disialoganglioside-cholera toxin complex reveal that cholera toxin can accommodate the disialoganglioside GD1A in three different modes. A single mode of binding is permissible for GD1B and GD3. Direct and water mediated hydrogen bonding interactions stabilizes these binding modes and play an essential role in defining the order of specificity for different disialogangliosides towards cholera toxin. This study not only provides models for the disialoganglioside-cholera toxin complexes but also identifies the NeuNAc binding site as a site for design of inhibitors that can restrict the pathogenic activity of cholera toxin.     

Isolation and characterization of novel phosphate-containing sialyloligosaccharides from normal human urine

Three phosphate-containing sialyloligosaccharides were isolated from normal human urine using charcoal adsorption, gel-filtration chromatography, ion-exchange chromatography and paper chromatography. Studies including gas-liquid chromatography of monosaccharide and disaccharide derivatives, methylation analysis, phosphate determination, ion-exchange chromatography and glycosidase and phosphatase treatments indicated the following three structures for the compounds isolated: NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(alpha)-P; NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc(alpha)-P; NeuAc(alpha 2-3)Gal(beta 1-3)GalNAc(alpha)-P. These sialyloligosaccharide 1-phosphates represent a novel class of oligosaccharides. Their oligosaccharide chains are identical with the common sialyloligosaccharide end groups of glycoproteins and glycolipids. The excretion of these compounds in normal human urine may indicate the existence of a novel, as yet unrevealed pathway in the metabolism of complex carbohydrates.     

Wheat germ agglutinin dimers bind sialyloligosaccharides at four sites in solution: proton nuclear magnetic resonance temperature studies at 360 MHz

Equilibrium binding studies have been performed over a range of temperatures from 25.4 to 47.3 degrees C between wheat germ agglutinin isolectin I (WGA I) and the alpha 2-3 isomer of (N-acetylneuraminyl)lactose (NeuNAc alpha 2-3Gal beta 1-4G1c). Proton nuclear magnetic resonance spectroscopy at 360 MHz has been used to monitor titrations in this system under conditions where the fraction of total ligand which is bound is small, yet the fractional occupation of sites covers a wide range. Several of the ligand resonances, including the N-acetyl methyl and the axial and equatorial hydrogens at carbon 3 of the NeuNAc residue, are shifted and broadened in the presence of WGA due to chemical exchange between the free and bound environments. The lifetime broadening of the N-acetyl resonance at room temperature of a series of related sialyloligosaccharides has been previously used by us to measure binding affinities to two WGA isolectins [Kronis, K.A., & Carver, J.P. (1982) Biochemistry 21, 3050-3057]. In this paper we report the temperature dependence of the apparent bound shifts and the apparent bound line widths of the N-acetyl, H3a, and H3e peaks. The true bound shifts for the three resonances have been obtained from these data by using the equations derived by Swift and Connick [Swift, T.J., & Connick, R.E. (1962) J. Chem. Phys. 37, 307-320]. The total bound shifts, per monomer, were found to be -1.98, -4.0, and -0.8 ppm for the N-acetyl, the H3a, and the H3e resonances, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two positional isomers of sialylheptasaccharides isolated from the urine of a patient with sialidosis

We describe the structures of two positional isomers of sialylheptasaccharide isolated from the urine of a patient with sialidosis with partial deficiency of beta-galactosidase. Based on structural studies including compositional sugar analysis, exoglycosidase digestion, chemical ionization mass spectrometry, proton nuclear magnetic resonance spectrometry, and methylation analysis, their structures were deduced to be as follows: AcNeu alpha 2----6Gal beta 1----4GlcNac beta 1----2Man alpha 1----3(Man alpha 1----6)Man beta 1----4GlcNac; AcNeu alpha 2----6Gal beta 1----4GlcNac beta 1----2Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNac. Sialyloligosaccharide 1 has previously been found in the urine and liver of patients with mucolipidosis I and II and sialidosis, but sialyloligosaccharide 2 has not been found yet in human urine. These two sialyloligosaccharides could not be completely separated by any chromatographic procedures tested. The analytical techniques, including methylation study and NMR spectroscopy, could not clearly detect the differences between them. However, alpha-mannosidase treatment gave important information for the structural analyses of these sialyloligosaccharides.     

Theoretical conformation analysis of specific O-antigenic polysaccharides. Polysaccharide of Shigella dysenteriae of serotype 2

Theoretical conformational analysis of Shigella dysenteriae serotype 2 polysaccharide containing repeating unit: -4Gal beta 1-3GalNAc alpha 1-3(GlcNAc alpha 1-4)GalNAc alpha 1-4Glc alpha 1- has been carried out. It is shown that spatial forms of the polymer are determined by conformational states of Glc alpha 1-4Gal links which form the internal bends of the chain. The structures of low energy are shown to be left three-fold helices with an axially projected repeat of approximately 12 A. N-Acetamide groups of side residues alpha-D-GlcNAc are the most accessible for intermolecular interactions and may determine immunological specificity of the polysaccharide.     

Poly-N-acetyllactosaminyl O-glycans attached to leukosialin. The presence of sialyl Le(x) structures in O-glycans.

Poly-N-acetyllactosamine extension has been found in O-glycans in addition to N-glycans and glycosphingolipids. Attempts were made in HL-60 and K562 cells to determine the amount of poly-N-acetyllactosaminyl O-glycans in the major sialoglycoprotein, leukosialin. Leukosialin was immunoprecipitated from [3H]glucosamine-labeled HL-60 and K562 cells. Glycopeptides were prepared by Pronase digestion, and O-glycan-containing glycopeptides were isolated by affinity chromatography using Jacalin-agarose. The glycopeptides bound to Jacalin-agarose and those unbound were treated with alkaline borohydride, and the released O-glycans were fractionated by Bio-Gel P-4 filtration. Sequential glycosidase digestion of the O-glycans, with or without pretreatment by fucosidase or neuraminidase, revealed the following conclusions. 1) Leukosialin from HL-60 cells contains about 1-2 poly-N-acetyllactosaminyl O-glycan chains/molecule. 2) About 50% of these poly-N-acetyllactosaminyl O-glycans contain sialyl Le(x) termini, NeuNAc alpha 2-->3Gal beta 1-->4 (Fuc alpha 1-->3)GlcNAc beta 1-->R. The amount of sialyl Le(x) structure in leukosialin is roughly equivalent to that on cell surfaces of HL-60 cells. 3) Leukosialin from K562 cells, on the other hand, contains no detectable amount of poly-N-acetyllactosaminyl O-glycans. 4) The presence of poly-N-acetyllactosamine in O-glycans is dependent on the core 2 beta 1,6-N-acetylglucosaminyl transferase. 5) Jacalin-agarose binds to sialylated small oligosaccharides such as NeuNAc alpha 2-->3Gal beta 1-->3(NeuNAc alpha 2-->6) GalNAc but not the hexasaccharide NeuNAc alpha 2-->3Gal beta 1-->3(NeuNAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->6) GalNAc. These results indicate that the formation of polylactosaminyl O-glycans and sialyl Le(x) structure in O-glycans is dependent on the core 2 formation.     

Identification of N-acetylneuraminyl alpha 2-->3 poly-N-acetyllactosamine glycans as the receptors of sialic acid-binding Streptococcus suis strains.

Streptococcus suis is a common cause of sepsis, meningitis, and other serious infections in young piglets and also causes meningitis in humans. The cell-binding specificity of sialic acid-recognizing strains of Streptococcus suis was investigated. Treatment of human erythrocytes with sialidase or mild periodate abolished hemagglutination. Hemagglutination inhibition experiments with sialyl oligosaccharides indicated that the adhesin preferred the sequence NeuNAc alpha 2-3Gal beta 1-4Glc(NAc). Resialylation of desialylated erythrocytes with Gal beta 1-3(4)GlcNAc alpha 2-3-sialyltransferase induced a strong hemagglutination, whereas no or only weak hemagglutination was obtained with cells resialylated with two other sialyltransferases. Binding of radiolabeled bacteria to blots of erythrocyte membrane proteins revealed binding to the poly-N-acetyllactosamine-containing components Band 3, Band 4.5, and polyglycosyl ceramides and to glycophorin A. The involvement of glycophorin A as a major ligand was excluded by the strong hemagglutination of trypsin-treated erythrocytes and En(a-) erythrocytes defective in glycophorin A. Sensitivity of the hemagglutination toward endo-beta-galactosidase treatment of erythrocytes and inhibition by purified poly-N-acetyllactosaminyl glycopeptides indicated that the adhesin bound to glycans containing the following structure: NeuNAc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-.     

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.     

A serum test for cystic fibrosis using monoclonal antibody 19-9

Monoclonal antibody 19-9 detects a sialosylated Lea antigen with the following sugar sequence: NeuNAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal. . . . This antigen is detected as a mucin in the sera of many patients with gastrointestinal and pancreatic cancer. Elevated levels of sialosylated Lea antigen are also detected in serum from 14 of 16 patients with cystic fibrosis (87%). One of the two negative patients belongs to the Le(a-b-) blood group and so is unable to synthesize the sialosylated Lea antigen. The high percentage of cystic fibrosis patients with elevated sialosylated Lea antigen suggests that the 19-9 antibody may be useful for diagnosis of cystic fibrosis. Antibodies to other sialosylated carbohydrates in mucins may also be useful for detection of cystic fibrosis and may allow diagnosis of patients belonging to the Le(a-b-) blood group.     

Purification and characterization of the epitectin from human laryngeal carcinoma cells

The purification and partial characterization of epitectin (previously called Ca antigen) from a human cancer cell line is described. This glycoprotein, which is expressed on a wide range of human tumors and certain specialized normal epithelia, can be detected using monoclonal antibodies, Ca1, Ca2, and Ca3. The purified glycoprotein had a high density (1.40 g/ml) on isopycnic centrifugation indicating a high carbohydrate content. The molecular mass of epitectin as determined by size-exclusion chromatography ranged from 1.0 to 1.5 x 10(6) daltons. However, the purified epitectin gave two bands of apparent molecular weight 390,000 and 350,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The isoelectric points of epitectin and asialoepitectin were found to be 5.3-5.4 and 6.8, respectively. The oligosaccharides were isolated from metabolically labeled epitectin by alkaline borohydride treatment and their structures established based on high performance liquid chromatography and paper electrophoretic migration, sugar composition, the results of sequential exoglycosidase treatment, periodate oxidation, and methylation analysis. The structures of the three major fractions, which together account for about 80% of the radioactivity, were assigned as NeuNAc alpha 2----3Gal beta 1----(NeuNAc alpha 2----6)3GalNAc(OH), NeuNAc alpha 2----3Gal beta 1----3GalNAc(OH), and Gal beta 1----3 GalNAc(OH). The structures of the minor fractions were tentatively assigned as NeuNAc----Gal(NeuNAc----Gal----GlcNAc)----GalNAc(OH), Gal beta 1----(NeuNAc alpha 2----6)3GalNAc(OH), NeuNAc alpha 2----6GalNAc(OH), and GalNAc(OH). It is proposed that the protein sequence and/or the distribution of the saccharides on the protein core are the determinants on epitectin that are recognized by the Ca antibodies.