Showing papers on "Sialic acid published in 1989"

Hemagglutinins from Two Influenza Virus Variants Bind to Sialic Acid Derivatives with Millimolar Dissociation Constants: A 500 MHz Proton Nuclear Magnetic Resonance Study

Abstract: The equilibrium binding of influenza virus hemagglutinin to derivatives of its cell-surface ligand, sialic acid, was measured by nuclear magnetic resonance (NMR) spectroscopy. Binding was quantified by observing perturbations of sialic acid resonances in the presence of protein. The major perturbation observed was a chemical shift of the N-acetyl methyl resonance, presumably due to the proximity of the methyl group to tryptophan 153. X-31 hemagglutinin binds to the methyl alpha-glycoside of sialic acid with a dissociation constant of 2.8 mM and does not bind to the methyl beta-glycoside. Replacing the 4-hydroxyl group of sialic acid with an acetyl group has little effect, while replacing the 7-hydroxyl group with an acetyl prevents binding. Experiments with sialylated oligosaccharides confirm literature reports that mutations at amino acid 226 change the specificity of hemagglutinin for alpha(2,6) and alpha(2,3) glycosidic linkages. The NMR line broadening of sialyloligosaccharides suggests that sialic acid is the only component that contacts the protein. Saccharides containing two sialic acid residues appear to have two separate binding modes. Hemagglutinin that has undergone a low pH induced conformational change retains the ability to bind sialic acid.

Mouse macrophage hemagglutinin (sheep erythrocyte receptor) with specificity for sialylated glycoconjugates characterized by a monoclonal antibody.

Abstract: An inhibitory rat mAb, SER-4, has been raised to the mouse macrophage (M phi)-restricted hemagglutinin, sheep erythrocyte receptor (SER), which binds unopsonized sheep erythrocytes through recognition of sialylated glycoconjugates. This receptor was originally defined on mouse resident bone marrow M phi where it was implicated in adhesive interactions of these cells with proliferating hematopoietic cells. In the present study using mouse serum-induced thioglycollate-elicited peritoneal M phi (TPM) as a model system for SER expression, mAb SER-4 IgG2a completely blocked rosette formation at 1 microgram/ml. The inhibition was likely to be via steric hindrance rather than through a direct interaction with the putative sialic acid binding site of SER because F(ab')2 and Fab fragments of mAb SER-4 gave a maximum inhibition of 50-60% and 0% respectively, despite binding effectively to the SER-4 antigen (Ag). Immunoprecipitation and Western blotting experiments with cultured M phi or tissue extracts demonstrated that the Ag recognized by SER-4 mAb is a single chain molecule with an apparent Mr by SDS-PAGE of 185 x 10(3) (reduced) or 170 x 10(3) (non-reduced) and is distinct from members of the leukocyte common Ag family. Expression of SER and SER-4 Ag in culture were closely correlated and depended on the presence of mouse serum for optimal induction. Further evidence that the SER-4 Ag is functionally equivalent to SER was provided by immunocytochemistry in which the overall pattern of staining in tissues was consistent with previous rosetting experiments. In the bone marrow, expression of the SER-4 Ag was restricted to the resident bone marrow M phi population with no expression on monocytes. High expression was also observed on stromal M phi within the subcapsular sinus and medullary cords in lymph nodes and on marginal metallophils in the spleen. These results therefore confirm that SER is a novel M phi-restricted receptor whose distribution and properties indicate a role in cellular interactions in hematopoietic and lymphoid tissues.

Definition of a bacterial virulence factor: sialylation of the group B streptococcal capsule.

Abstract: Sialylation of bacterial capsules has been proposed as an important virulence factor for several species of encapsulated pathogens, including group B Streptococcus. We have constructed a transposon mutant strain of type III group B Streptococcus that expresses a capsular polysaccharide differing from the wild type only in that the mutant strain's capsule lacks sialic acid. The mutant polysaccharide is antigenically identical to the capsular polysaccharide of type 14 Streptococcus pneumoniae, as predicted by the structures of the type III group B Streptococcus and S. pneumoniae polysaccharides. Loss of capsular sialic acid was associated with loss of virulence in the mutant strain in a neonatal rat model of lethal group B Streptococcus infection. These studies demonstrate directly that capsular sialic acid is a critical virulence determinant for type III group B Streptococcus and support the general hypothesis that surface sialylation aids pathogenic microorganisms in evading host defenses.

Molecular characterization and expression in Escherichia coli of the gene complex encoding the polysaccharide capsule of Neisseria meningitidis group B

Abstract: The gene complex encoding all determinants of the biosynthesis pathway of the capsule of group B meningococci (cps) has been cloned in Escherichia coli. A 24-kilobase large chromosomal fragment is necessary for capsule expression on the E. coli surface. By transposon and deletion mutagenesis, two separate steps in transport of the polysaccharide from the cytoplasm to the periplasm and further to the cell surface became evident. Mutants were also isolated that accumulate soluble poly(sialic acid) in the cytoplasm. The cloned cps complex conferred to E. coli strain GC6 sensitivity for E. coli K1-specific phages; phage sensitivity was enhanced in two distinct classes of cps mutants. Southern blot experiments revealed homology to some or all other Neisseria meningitidis capsular types and other Neisseria species, depending on the fragment of the cps complex used as probe.

Gangliosides in normal human serum. Concentration, pattern and transport by lipoproteins.

Abstract: The total content and pattern of gangliosides were determined in the unfractionated sera of 11 healthy human adults and in isolated lipoproteins. The total content of lipid-bound sialic acid was 10.5 +/- 3.2 nmol/ml serum. The ganglioside profile consisted of more than ten different components. The major ganglioside was GM3, followed by GD3, GD1a, GM2, GT1b, MG-3 (sialosyllactoneotetraosylceramide), GD1b and GQ1b. Traces of four additional gangliosides could not be quantified reliably. Ganglioside patterns did not vary in sera taken from healthy adults of different age and sex. Approximately 98% of human serum gangliosides were transported by serum lipoproteins, predominantly by LDL (66%), followed by HDL (25%) and VLDL (7%). The quantitative distribution of individual gangliosides in VLDL and LDL was almost the same as that in the unfractionated serum; some differences existed with the ganglioside profile in HDL.

Heterogeneous distribution and transmembrane signaling properties of lymphocyte function-associated antigen (LFA-1) in human lymphocyte subsets.

Abstract: The role of LFA-1, a member of the integrin supergene family, in intercellular adhesion, including lymphocyte-endothelial cell (EC) binding, has been established We now demonstrate that differences in LFA-1 cell surface density are responsible for the variable adhesion efficiency of lymphocyte subsets to EC Electrophoretic analysis revealed multiple glycosylated isoforms of both alpha and beta subunits, largely as a result of different degrees of sialylation, with variable expression among different lymphocyte subsets Neuraminidase digestion before EC adhesion increased the binding efficiency of all lymphocyte subsets, although the relative increase in each subset was proportional to the initial LFA-1 sialic acid content LFA-1 cross-linking resulted in phosphoinositide hydrolysis and a rise in [Ca2+]i when using anti-alpha but not anti-beta subunit antibodies These findings indicate that the density of LFA-1 on lymphocyte subsets controls their adhesive properties, and that the LFA-1 alpha subunit has transmembrane signaling properties that may result in activation events after interaction with its natural ligands

Structure and Localization of Gangliosides

Abstract: Gangliosides are sialic acid-containing glycosphingolipids (GSLs) found primarily in the plasma membrane of virtually all vertebrate tissues and are particularly abundant in the nervous system. They constitute part of the glycocalyx network surrounding the cell surface and are crucial in determining the properties and functions of cells. Structurally, they contain a hydrophobic ceramide chain to which a hydrophilic oligosaccharide is glycosidically linked. Some representative brain gangliosides are shown in Figure 1.

Binding of pertussis toxin to eucaryotic cells and glycoproteins.

Abstract: The binding of pertussis toxin and its subunits to cell surface receptors and purified glycoproteins was examined. The interaction of pertussis toxin with components of two variant Chinese hamster ovary (CHO) cell lines was studied. These cell lines are deficient in either sialic acid residues (LEC 2) or sialic acid and galactose residues (LEC 8) on cell surface macromolecules. The binding of pertussis toxin to components of these cells differed from the binding of the toxin to wild-type components. Although the toxin bound to a 165,000-dalton glycoprotein found in N-octylglucoside extracts of wild-type cells, it did not bind to components found in extracts of LEC 2 cells. In contrast, the toxin bound to components found in extracts of LEC 8 cells, which are variant cells that contain increased amounts of terminal N-acetylglucosamine residues on cell surface macromolecules. These results suggest that the receptor for pertussis toxin on CHO cells contains terminal acetamido-containing sugars. The cytopathic effect of the toxin on both types of variant cells was much reduced compared with its effects on wild-type cells. Thus, optimal functional binding of pertussis toxin appears to require a complete sialyllactosamine (NeuAc----Gal beta 4GlcNAc) sequence on surface macromolecules. In addition to studying the nature of the eucaryotic receptor for pertussis toxin, we examined corresponding binding sites for glycoproteins on the toxin molecule. Binding of both S2-S4 and S3-S4 dimers of the toxin to cellular components and purified glycoproteins was observed. The two dimers bound to a number of glycoproteins containing N-linked oligosaccharides but not O-linked oligosaccharides, and differences in the binding of the two dimers to some glycoproteins was noted. These data indicate that the holotoxin molecule contains at least two glycoprotein-binding sites which may have slightly different specificities for glycoproteins. Images

Genetic analysis of chromosomal mutations in the polysialic acid gene cluster of Escherichia coli K1.

Abstract: The kps gene cluster of Escherichia coli K1 encodes functions for sialic acid synthesis, activation, polymerization, and possibly translocation of polymer to the cell surface. The size and complexity of this membrane polysaccharide biosynthetic cluster have hindered genetic mapping and functional descriptions of the kps genes. To begin a detailed investigation of the polysialic acid synthetic mechanism, acapsular mutants were characterized to determine their probable defects in polymer synthesis. The mutants were tested for complementation with kps fragments subcloned from two separately isolated, functionally intact kps gene clusters. Complementation was assayed by immunological and biochemical methods and by sensitivity to the K1-specific bacteriophage K1F. The kps cluster consisted of a central 5.8-kilobase region that contained at least two genes coding for sialic acid synthetic enzymes, a gene encoding the sialic acid-activating enzyme, and a gene encoding the sialic acid polymerase. This biosynthetic region is flanked on one side by an approximately 2.8-kilobase region that contains a potential regulatory locus and at least one structural gene for a polypeptide that appears to function in polysialic acid assembly. Flanking the biosynthetic region on the opposite side is a 6- to 8.4-kilobase region that codes for at least three proteins which may also function in polymer assembly and possibly in translocating polymer to the outer cell surface. Results of transduction crosses supported these conclusions and indicated that some of the kps genes flanking the central biosynthetic region may not function directly in transporting polymer to the cell surface. The results also demonstrate that the map position and probable function of most of the kps cluster genes have been identified. Images

Structure of the major concanavalin A reactive oligosaccharides of the extracellular matrix component laminin

Abstract: Laminin, a high molecular weight (1,000,000) glycoprotein component of basement membranes, was isolated from the EHS murine tumor as a noncovalent complex with entactin by lectin affinity chromatography using the alpha-D-galactosyl binding lectin Griffonia simplicifolia I (GS I). Entactin was removed from this complex by passage over Sephacryl S-1000 in the presence of SDS. Compositional analysis showed that the affinity-purified laminin contained 25-30% carbohydrate by weight. Methylation analysis revealed that the oligosaccharides of laminin contained bi- and triantennary chains, the blood group I structure, and repeating sequences of 3Gal beta 1,4GlcNAc beta 1 units. Free oligosaccharides were derived from the asparagine-linked glycans of affinity-purified laminin by hydrazinolysis, re-N-acetylation, and reduction with NaB3H4. When fractionated by affinity chromatography on concanavalin A (Con A)-Sepharose, 80% of the oligosaccharides passed through the column unretarded and a single peak corresponding to 20% of the oligosaccharides was adsorbed and specifically eluted with a linear gradient of 0-30 mM methyl alpha-D-glucopyranoside. Further fractionation of the Con A reactive oligosaccharides on GS I-Sepharose demonstrated that 70% of these oligosaccharides possess at least one terminal nonreducing alpha-D-galactopyranosyl unit. The Con A reactive oligosaccharides were subjected to sequential digestion with endo- and exoglycosidases, and the reaction products were analyzed by gel filtration chromatography on a column of Bio-Gel P4. We thereby obtained evidence for a variety of structures not previously reported to exist on murine laminin including hybrid biantennary complex and biantennary complex structures containing poly(lactosaminyl) repeating units. The poly(lactosaminyl) units occur either on one or on both branches of the biantennary chains, as well as in more highly branched blood group I poly(lactosamine) structures. All sialic acid is present as N-acetylneuraminic acid linked alpha 2,3 to galactose.

Biosynthesis and processing of human immunodeficiency virus type 1 envelope glycoproteins: effects of monensin on glycosylation and transport.

Abstract: When human immunodeficiency virus type 1 envelope glycoproteins were expressed in 293 cells by using a recombinant adenovirus expression vector, the envelope precursor (gp160) was initially glycosylated by cotranslational addition of N-linked high-mannose oligosaccharide units to the protein backbone and then cleaved to gp120 and gp41. The subunits gp120 and gp41 were then further modified by the addition of fucose, galactose, and sialic acid, resulting in glycoproteins containing a mixture of hybrid and complex oligosaccharide side chains. A fraction of glycosylated gp160 that escaped cleavage was further modified by the terminal addition of fucose and galactose, but the addition of sialic acid did not occur, consistent with the notion that it is compartmentalized separately from the gp120 envelope protein. Processing and transport of gp160 were blocked by the monovalent ionophore monensin, which at high concentrations (25 microM and above) was a potent inhibitor of the endoproteolytic cleavage of gp160; at lower concentrations (1 to 10 microM), it selectively blocked the secondary glycosylation steps so that smaller products were produced. Monensin (1 microM) treatment also resulted in a reduction in syncytium formation, which was observed when recombinant infected cells were cocultivated with CD4-bearing HeLa cells. The infectivity of human immunodeficiency virus type 1 was also reduced by monensin treatment, a decrease that may be due to incompletely glycosylated forms of gp120 that have a lower affinity for the CD4 receptor.

Structures of the sugar chains of interferon-gamma produced by human myelomonocyte cell line HBL-38.

Abstract: Interferon-gamma produced by the human myelomonocyte cell line HBL-38 contained galactose, mannose, fucose, N-acetylglucosamine, and N-acetylneuraminic acid as sugar components. Sugar chains were liberated from interferon-gamma by hydrazinolysis. Free amino groups of the sugar chains were acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine under new reaction conditions in which no sialic acid residue was hydrolyzed. The pyridylamino (PA-) derivatives of the sugar chains thus obtained were purified by gel filtration and reversed-phase HPLC. Seven major PA-sugar chains were isolated and the structure of each purified PA-sugar chain was identified by stepwise exoglycosidase digestion and 500-mHz 1H-NMR spectroscopy. The results indicated that the structures of the major PA-sugar chains were of the biantennary type, to which 0 to 2 mol of fucose and 1 to 2 mol of N-acetylneuraminic acid were linked as shown below. (formula; see text)

Gangliosides interact directly with plasminogen and urokinase and may mediate binding of these fibrinolytic components to cells

Abstract: Receptors for the fibrinolytic molecules plasminogen and urokinase are expressed at high capacity on a wide variety of peripheral blood cells and transformed cell lines. We have considered whether gangliosides, components of the outer leaflets of cell membranes, may modulate the interactions of these fibrinolytic ligands with cells. Radiolabeled plasminogen and urokinase bound directly to insolubilized gangliosides. The interactions were saturable and were 50% inhibited by 2.2 microM unlabeled plasminogen or 12 nM unlabeled urokinase, respectively. A panel of gangliosides inhibited binding of both ligands to U937 monocytoid cells, and the order of decreasing inhibitory effectiveness was GD1a greater than GM1 greater than GT1b greater than GM2, while GM3 was minimally effective. The individual components of gangliosides, hexoses, hexosamines, sialic acid, GM1 pentasaccharide, ceramides, and glucocerebrosides were ineffective in in inhibiting the binding of plasminogen and urokinase either to cells or to insolubilized gangliosides. Binding of both ligands to endothelial cells and granulocytes and binding of plasminogen to platelets were also inhibited by gangliosides. U937 cells were cultured with gangliosides to allow incorporation of these glycolipids into the cell membranes. After 3 days of culture, both urokinase binding and plasminogen binding to the cells became enhanced. These results suggest that gangliosides can directly bind to these fibrinolytic components and may mediate or modulate the interactions of plasminogen and urokinase with a variety of cell types.

A photoreactive derivative of radiolabeled GM1 ganglioside: preparation and use to establish the involvement of specific proteins in GM1 uptake by human fibroblasts in culture.

Abstract: A new procedure was used to synthesize a derivative of ganglioside GM1 containing a photoreactive nitrophenyl azide group at the end of the fatty acyl moiety, using deAc-deAcyl-GM1 obtained by deacetylation of the sialic acid and deacylation of the ceramide portion of GM1. This deAc-deAcyl-GM1 was first acylated at the long chain base amino group with 12-aminododecanoic acid, which has the amino group protected by a fluorenyl residue, and tritium labeled at the sialic acid amino group with ({sup 3}H)acetic anhydride of very high specific radioactivity. Cultured human fibroblasts were exposed to mixtures of radioactive photolabeled GM1 for different times and then illuminated and the radioactive protein patterns studied by SDS-PAGE. After 2 h of exposure, the photolabeled GM1 was stably associated to the cells and underwent almost no metabolic processing, behaving exactly as the underivatized natural GM1. Under these conditions very few proteins became radioactive. Thus, it is evident that the ganglioside binding to fibroblasts and insertion into the outer layer of the plasma membrane involve few individual proteins. When the incubation was prolonged to 24 h, photolabeled GM1 underwent extensive metabolic processing and gave origins to the corresponding ganglioside derivatives of GM2, GM3, and GD1a. Under these conditionsmore » many proteins became radioactive, a consequence of GM1 transfer from the surface to the interior or the cell and of the ready availability of interaction of GM1 and its metabolites.« less

Intravenously injected sialidase inactivates attachment sites for lymphocytes on high endothelial venules.

Abstract: Blood-borne lymphocytes initiate entry into secondary lymphoid organs, such as peripheral lymph nodes (PN) and gut-associated Peyer's patches (PP), by a highly specific adhesive interaction between the lymphocytes and the endothelium of specialized blood vessels known as a high endothelial venules (HEV). The selectivity with which functional subpopulations of lymphocytes migrate into particular lymphoid organs is believed to be regulated by the expression of cell adhesion receptors and complementary ligands on lymphocytes and HEV, respectively. The entry of lymphocytes into PN and PP has clearly been shown to involve distinct receptor-ligand pairs. Employing the Stamper-Woodruff in vitro adhesion assay, which measures lymphocyte attachment to HEV in cryostat-cut sections of lymphoid organs, we have previously shown that treatment of PN sections with two different sialidases inactivates HEV-adhesive ligands, whereas treatment of PP tissue sections has no effect on HEV-adhesive function. We now report that in vivo exposure of HEV to sialidase (after i.v. injection of the enzyme) also selectively prevents subsequent in vitro attachment of lymphocytes to PN HEV but not to PP HEV. Consistent with this organ-selective impairment of HEV-adhesive function by sialidase, i.v. injection of the enzyme is shown to prevent short term lymphocyte accumulation within peripheral lymph nodes while having no significant effect on accumulation in PP, blood, or nonlymphoid organs. Histologic examination with the sialic acid-specific lectin from Limax flavus verified that i.v. injected sialidase effectively removes stainable sialic acid moieties from HEV in both PN and PP. This study confirms that sialic acid is required for the adhesive function of PN HEV-ligands. A role for sialic acid as either a recognition determinant or as a regulatory molecule can be envisioned. In view of the fact that many pathogens release sialidase and cause substantially elevated serum levels of this enzyme, the present observations may have pathophysiologic significance. One mechanism by which such pathogens may avoid destruction is to inactivate susceptible HEV-ligands and disrupt the entry of lymphocytes into lymphoid organs where immune responses against the pathogens would normally be initiated.

Transfer of synthetic sialic acid analogues to N- and O-linked glycoprotein glycans using four different mammalian sialyltransferases.

Abstract: This paper presents kinetic properties of the transfer of several synthetic 9-substituted sialic acid analogues onto N- or O-linked glycoprotein glycans by four purified mammalian sialyltransferases: Gal beta 1,4GlcNac alpha 2,6sialyltransferase, Gal beta-1,4(3)GlcNAc alpha 2,3-sialyltransferase, Gal beta 1,3GalNAc alpha 2,3sialyltransferase, and GalNAc alpha 2,6sialyltransferase. The substituents at C-9 of the sialic acid analogues introduce special biochemical characteristics: 9-Amino-NeuAc represents, up to the present, the first derivative that is resistant toward bacterial, viral, and mammalian sialidases but is transferred by a sialyltransferase. 9-Acetamido-NeuAc, 9-benzamido-NeuAc, and 9-hexanoylamido-NeuAc differ in size and hydrophobic character from each other and from parent NeuAc. 9-Azido-NeuAc may be used to introduce a photoreactive label. The kinetic properties of the four sialyltransferases with regard to the donor CMP-glycosides differed distinctly depending on the structure of the substituent at C-9. CMP-9-amino-NeuAc was only accepted as donor substrate by Gal beta 1,4GlcNAc alpha 2,6sialyltransferase (rat liver), but the Km value was 14-fold higher than that of parent CMP-NeuAc. In contrast, 9-azido-NeuAc was readily transferred by each of these four enzymes. 9-Acetamido-NeuAc, which is a receptor analogue for influenza C virus, 9-benzamido-NeuAc, and 9-hexanoylamido-NeuAc were also accepted by each sialyltransferase, but incorporation values differed significantly depending on the enzyme used. For the first time, the resialylation of asialo-alpha 1-acid glycoprotein with 9-substituted sialic acid analogues by Gal beta 1,4GlcNAc alpha 2,6sialyltransferase is demonstrated.