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Showing papers on "Sialic acid published in 1996"


Journal ArticleDOI
TL;DR: An ELISA assay is described for measuring the binding of influenza virus A-X31 to α-sialoside groups that are linked to biotin-labeled polyacrylamides, and the role of steric stabilization in the mechanism is shown to be directly related to the binding affinity of the polymers for the viral surface.
Abstract: An ELISA assay is described for measuring the binding of influenza virus A-X31 to α-sialoside groups that are linked to biotin-labeled polyacrylamides. The efficacy of these polymers in inhibiting the adhesion of influenza virus to erythrocytes (as measured by a hemagglutination assay) was shown to be directly related to the binding affinity of the polymers for the viral surface: the differences in inhibitory efficacy among the polymeric inhibitors and monomeric α-methyl sialoside, among fractions of a polymeric, polyvalent inhibitor with narrow molecular weight ranges, and among polymeric inhibitors prepared by copolymerization or modification of a preformed polymer chain, all correlated with differences in the affinity of the inhibitors for the surface of the virus. The polymeric inhibitors studied had affinities for the viral surface that ranged between 103 and >106 greater than α-methyl sialoside, on the basis of total sialic acid groups in solution. The role of steric stabilization in the mechanism ...

333 citations


Journal ArticleDOI
TL;DR: The core oligosaccharides of low-molecular-weight lipopolysaccharide (LPS) of pathogenic Neisseria spp.
Abstract: The core oligosaccharides of low-molecular-weight lipopolysaccharide (LPS), also termed lipooligosaccharide (LOS), of pathogenic Neisseria spp. mimic the carbohydrate moieties of glycosphingolipids present on human cells. Such mimicry may serve to camouflage the bacterial surface from the host. The LOS component is antigenically and/or chemically identical to lactoneoseries glycosphingolipids and can become sialylated in Neisseria gonorrhoeae when the bacterium is grown in the presence of cytidine 5′-monophospho-N-acetylneuraminic acid, the nucleotide sugar of sialic acid. Strains of Neisseria meningitidis and Haemophilus influenzae also express similarly sialylated LPS. Sialylation of the LOS influences susceptibility to bactericidal antibody, may decrease or prevent phagocytosis, cause down-regulation of complement activation, and decrease adherence to neutrophils and the subsequent oxidative burst response. The core oligosaccharides of LPS of Campylobacter jejuni serotypes which are associated with the development of the neurological disorder, Guillain-Barre syndrome (GBS), exhibit mimicry of gangliosides. Cross-reactive antibodies between C. jejuni LPS and gangliosides are considered to play an important role in GBS pathogenesis. In contrast, the O-chain of a number of Helicobacter pylori strains exhibit mimicry of Lewisx and Lewisy blood group antigens. The role of this mimicry remains to be investigated, but may play a role in bacterial camouflage, the induction of autoimmunity and immune suppression in H. pylori-associated disease.

303 citations


Journal ArticleDOI
TL;DR: It is reported that a limited set of structurally related gangliosides, known to be expressed on myelinated neurons in vivo, are ligands for MAG, consistent with the conclusion that MAG-mediated cell-cell interactions involve MAG-ganglioside recognition and binding.
Abstract: Nerve cells depend on specific interactions with glial cells for proper function. Myelinating glial cells are thought to associate with neuronal axons, in part, via the cell-surface adhesion protein, myelin-associated glycoprotein (MAG). MAG is also thought to be a major inhibitor of neurite outgrowth (axon regeneration) in the adult central nervous system. Primary structure and in vitro function place MAG in an immunoglobulin-related family of sialic acid-binding lactins. We report that a limited set of structurally related gangliosides, known to be expressed on myelinated neurons in vivo, are ligands for MAG. When major brain gangliosides were adsorbed as artificial membranes on plastic microwells, only GT1b and GD1a supported cell adhesion of MAG-transfected COS-1 cells. Furthermore, a quantitatively minor ganglioside expressed on cholinergic neurons, GQ1b alpha (also known as Chol-1 alpha-b), was much more potent than GT1b or GD1a in supporting MAG-mediated cell adhesion. Adhesion to either GT1b or GQ1b alpha was abolished by pretreatment of the adsorbed gangliosides with neuraminidase. On the basis of structure-function studies of 19 test glycosphingolipids, an alpha 2,3-N-acetylneuraminic acid residue on the terminal galactose of a gangliotetraose core is necessary for MAG binding, and additional sialic acid residues linked to the other neutral core saccharides [Gal(II) and GalNAc(III)] contribute significantly to binding affinity. MAG-mediated adhesion to gangliosides was blocked by pretreatment of the MAG-transfected COS-1 cells with anti-MAG monoclonal antibody 513, which is known to inhibit oligodendrocyte-neuron binding. These data are consistent with the conclusion that MAG-mediated cell-cell interactions involve MAG-ganglioside recognition and binding.

290 citations


Journal Article
TL;DR: The finding that unusual gangliosides are expressed in breast tumors may provide the basis for their immunological diagnosis and vaccine therapy.
Abstract: Breast tumors that were histopathologically diagnosed as invasive ductal carcinoma were examined in relation to their abnormal expression of gangliosides. Total ganglioside levels that were expressed as lipid-bound sialic acids were significantly higher in breast tumor tissues than in normal mammary tissues. Two kinds of unusual gangliosides were found to be expressed in many cases of breast tumors. One was a group of O-acetylated gangliosides, such as O-acetyl-GD3 and O-acetyl-GT3. They are known as fetal gangliosides, which appear in fetal brains. The other was an N-glycolylneuraminic acid-containing ganglioside, N-glycolyl-GM3, which had not been previously found in normal human tissues. The finding that unusual gangliosides are expressed in breast tumors may provide the basis for their immunological diagnosis and vaccine therapy.

284 citations


Journal ArticleDOI
TL;DR: In this paper, the authors identified a full-length cDNA clone in the dbEST data base, of which the predicted amino acid sequence has extensive homology to other mammalian and bacterial neuraminidases, including the F(Y)RIP domain and Asp-boxes.
Abstract: Neuraminidases (sialidases) have an essential role in the removal of terminal sialic acid residues from sialoglycoconjugates and are distributed widely in nature. The human lysosomal enzyme occurs in complex with beta-galactosidase and protective protein/cathepsin A (PPCA), and is deficient in two genetic disorders: sialidosis, caused by a structural defect in the neuraminidase gene, and galactosialidosis, in which the loss of neuraminidase activity is secondary to a deficiency of PPCA. We identified a full-length cDNA clone in the dbEST data base, of which the predicted amino acid sequence has extensive homology to other mammalian and bacterial neuraminidases, including the F(Y)RIP domain and "Asp-boxes." In situ hybridization localized the human neuraminidase gene to chromosome band 6p21, a region known to contain the HLA locus. Transient expression of the cDNA in deficient human fibroblasts showed that the enzyme is compartmentalized in lysosomes and restored neuraminidase activity in a PPCA-dependent manner. The authenticity of the cDNA was verified by the identification of three independent mutations in the open reading frame of the mRNA from clinically distinct sialidosis patients. Coexpression of the mutant cDNAs with PPCA failed to generate neuraminidase activity, confirming the inactivating effect of the mutations. These results establish the molecular basis of sialidosis in these patients, and clearly identify the cDNA-encoded protein as lysosomal neuraminidase.

277 citations


Book ChapterDOI
TL;DR: This chapter examines humoral defense factors by focusing on lysozyme, complement, interferon, C-reactive protein, transferrin, and lectin, and it has been demonstrated that Pacific hagfish granulocytes show chemotactic migration in response to the human C5a and lipopolysaccharide (LPS)-activated hag fish plasma.
Abstract: This chapter examines humoral defense factors by focusing on lysozyme, complement, interferon, C-reactive protein, transferrin, and lectin. Lysozyme is found in a wide range of vertebrates and is one of the defensive factors against an invasion by microorganisms. It splits the β linkages between N -acetylmuramic acid and N -acetylglucosamine in the cell walls of Gram-positive bacteria, thus, preventing them from invading. In fishes, lysozyme is distributed mainly in tissues rich in leucocytes, such as the head kidney, at sites where the risk of bacterial invasion is high, such as the skin, the gills, and the alimentary tract, and in the eggs. It has been demonstrated that Pacific hagfish granulocytes show chemotactic migration in response to the human C5a and lipopolysaccharide (LPS)-activated hagfish plasma. This indicates that specific chemoattractant receptors are present on the surface of hagfish leukocytes and LPS activation of hagfish plasma generates a potent chemotactic product. It has been observed that the catfish alternative complement pathway (ACP) is an efficient defense mechanism against nonpathogenic Gram-negative bacteria that contain no sialic acid, but the catfish ACP is inhibited by the large amount of sialic acid contained in pathogenic Gram-negative bacteria, such as Aeromonas salmonicida and Flavobacterium columnaris , indicating that sialic acid is an important virulence factor for establishing an initial infection.

247 citations


Journal ArticleDOI
TL;DR: A method involving their conversion into methyl esters has been developed that produces strong positive-ion signals from N-linked oligosaccharides containing sialic acid and from gangliosides, which are stable, even in the presence of alpha-cyano-4-hydroxycinnamic acid.
Abstract: Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of oligosaccharides and gangliosides normally causes loss of sialic acid, particularly when α-cyano-4-hydroxycinnamic acid is used as the matrix. In addition, the potential signal is split because both positive and, to a greater extent, negative ions are formed while signals are frequently complicated as the result of partial alkali-salt formation. In order to stabilize the sialic acid moieties under MALDI conditions and to divert all of the signal into the positive-ion mode, a method involving their conversion into methyl esters has been developed. The method is relatively rapid and produces strong positive-ion signals from N-linked oligosaccharides containing sialic acid and from gangliosides. The latter compounds are stable, even in the presence of α-cyano-4-hydroxycinnamic acid. They give abundant molecular (MNa+) ions, but with sufficient residual in-source fragmentation to allow the sequence of the sugar chain to be determined. The sialic acid residue is stable after methylation, irrespective of its linkage to the parent molecule.

238 citations


Journal ArticleDOI
TL;DR: It is demonstrated that encapsulation hinders the primary event in the development of the disease, but the spontaneous switching of encapsulated wild‐type bacteria to a capsule‐negative phenotype promotes meningococcal adherence and invasion into mucosal epithelial cells.
Abstract: Cell surface-located sialic acids of the capsule and the lipooligosaccharide (LOS) are both pivotal virulence factors in Neisseria meningitidis, promoting survival and dissemination of this pathogen which can cause both sepsis and meningitis. With the aid of a unique set of isogenic meningococcal mutants defective in the expression of cell surface-located sialic acids, we have demonstrated that encapsulation hinders the primary event in the development of the disease, but the spontaneous switching of encapsulated wild-type bacteria to a capsule-negative phenotype promotes meningococcal adherence and invasion into mucosal epithelial cells. Genetic analysis of the capsule-negative, invasive bacteria revealed a unique mechanism for modulation of capsule expression based on the reversible inactivation of an essential sialic acid biosynthesis gene, siaA, by insertion/excision of a naturally occurring insertion sequence element, IS1301. Inactivation of siaA regulates both capsule expression and endogenous LOS sialylation. This is the first example of an insertion sequence element-based genetic switch mechanism in the pathogenic bacterium and is an important step in the understanding of bacterial virulence.

215 citations


Journal ArticleDOI
TL;DR: The nature of the glycosidic linkages appears to be the principal determinant of specificity, rather than the position of particular hydroxyl groups, in the P16 virus, consistent with the interactions seen in the two complexes.

181 citations


Journal ArticleDOI
TL;DR: Using mutants in genes affecting LAH serorecognition of flagellin it was demonstrated that sialic acid alone is not the LAH epitope, rather, the epitope(s) is complex, probably involving multiple glycosyl and/or amino acid residues.
Abstract: The flagellins of Campylobacter spp. differ antigenically. In variants of C. coli strain VC167, two antigenic flagellin types determined by sero-specific antibodies have been described (termed T1 and T2). Post-translational modification has been suggested to be responsible for T1 and T2 epitopes, and, using mild periodate treatment and biotin hydrazide labelling, flagellin from both VC167-T1 and T2 were shown to be glycosylated. Glycosylation was also shown to be present on other Campylobacter flagellins. The ability to label all Campylobacter flagellins examined with the lectin LFA demonstrated the presence of a terminal sialic acid moiety. Furthermore, mild periodate treatment of the flagellins of VC167 eliminated reactivity with T1 and T2 specific antibodies LAH1 and LAH2, respectively, and LFA could also compete with LAH1 and LAH2 antibodies for binding to their respective flagellins. These data implicate terminal sialic acid as part of the LAH strain-specific epitopes. However, using mutants in genes affecting LAH serorecognition of flagellin it was demonstrated that sialic acid alone is not the LAH epitope. Rather, the epitope(s) is complex, probably involving multiple glycosyl and/or amino acid residues.

168 citations


Journal ArticleDOI
TL;DR: A possible role for the trisaccharide in the etiology of neuropathies is indicated, and a difference for distinguishing neuropathic strains from nonneuropathic strains may be the presence of a sialyltransferase required for the synthesis of this trisACcharide.
Abstract: A Campylobacter jejuni strain of serotype O:10 was isolated from a patient who had Miller-Fisher syndrome. In its biochemical reactions and cellular morphology, the isolate was characteristic of typical C. jejuni. Antibodies against extracted lipopolysaccharide (LPS) were detected by passive hemagglutination in the acute- and convalescent-phase patient sera. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with the O:10 antiserum, it was demonstrated that the strain possessed both low- and high-molecular-weight molecules. Chemical analysis of the LPS revealed that the core oligosaccharide has a terminal trisaccharide epitope consisting of two molecules of sialic acid linked to galactose, a structure reflecting the terminal region of human ganglioside GD3. As this trisaccharide is also present in LPS cores of serotype O:19 strains from patients with Guillain-Barre syndrome but not in cores of nonneuropathic C. jejuni, a possible role for the trisaccharide in the etiology of neuropathies is indicated, and a difference for distinguishing neuropathic strains from nonneuropathic strains may be the presence of a sialyltransferase required for the synthesis of this trisaccharide.

Journal ArticleDOI
TL;DR: The results provide further evidence that immunoglobulin superfamily cell adhesion molecules use the GFCC′C″ β-sheet of membrane-distal V-set domains to bind structurally diverse ligands, suggesting that this surface is favored for cell-cell recognition.

Journal ArticleDOI
TL;DR: The hemagglutinating activity of transmissible gastroenteritis virus (TGEV), an enteric porcine coronavirus, was analyzed and found to be dependent on the presence of alpha-2,3-linked sialic acid on the erythrocyte surface, suggesting that the sIALic acid binding site is blocked by virus-associated competitive inhibitors.
Abstract: The hemagglutinating activity of transmissible gastroenteritis virus (TGEV), an enteric porcine coronavirus, was analyzed and found to be dependent on the presence of alpha-2,3-linked sialic acid on the erythrocyte surface. N-Glycolylneuraminic acid was recognized more efficiently by TGEV than was N-acetylneuraminic acid. For an efficient hemagglutination reaction the virions had to be treated with sialidase. This result suggests that the sialic acid binding site is blocked by virus-associated competitive inhibitors. Porcine respiratory coronavirus (PRCV), which is serologically related to TGEV but not enteropathogenic, was found to be unable to agglutinate erythrocytes. Incubation with sialidase did not induce a hemagglutinating activity of PRCV, indicating that the lack of this activity is an intrinsic property of the virus and not due to the presence of competitive inhibitors. Only monoclonal antibodies to an antigenic site that is absent from the S protein of PRCV were able to prevent TGEV from agglutinating erythrocytes. The epitope recognized by these antibodies is located within a stretch of 224 amino acids that is missing in the S protein of PRCV. Our results indicate that the sialic acid binding activity is also located in that portion of the S protein. The presence of a hemagglutinating activity in TGEV and its absence in PRCV open the possibility that the sialic acid binding activity contributes to the enterotropism of TGEV.

Journal ArticleDOI
TL;DR: Results indicate that a specific high-mannose type oligosaccharide linked to the major outer membrane protein of C. trachomatis mediates attachment and infectivity of the organism to HeLa cells.
Abstract: The structure of the carbohydrate of the 40-kD major outer membrane component of Chlamydia trachomatis and its role in defining infectivity of the organism were investigated. The oligosaccharides were released from the glycoprotein by N-glycanase digestion, coupled to a 2-aminopyridyl residue, and subjected to two-dimensional sugar mapping technique. The major fractions consisted of "high-mannose type" oligosaccharides containing 8-9 mannose residues. Bi- and tri-antennary "complex type" oligosaccharides having terminal galactose were detected as minor components. These oligosaccharides were N-linked and contained no sialic acid. This structural profile is consistent with our previous characterization based on lectin-binding and glycosidase digestion. Functional specificity of identified chlamydial oligosaccharides was analyzed using glycopeptides fractionated from ovalbumin and structurally defined oligosaccharides from other sources. The glycopeptide fraction having high-mannose type oligosaccharide, as compared to those having complex or hybrid-type, showed a stronger inhibitory effect on attachment and infectivity of chlamydial organisms to HeLa cells. Among high-mannose type oligosaccharides, the strongest inhibition was observed with mannose 8 as compared with mannose 6, 7, or 9. These results indicate that a specific high-mannose type oligosaccharide linked to the major outer membrane protein of C. trachomatis mediates attachment and infectivity of the organism to HeLa cells.

Journal ArticleDOI
TL;DR: Results show that peptides based on conserved and dimorphic regions of MSP‐1, interact with human red blood cells (RBCs), and showed that the RBC receptors are not sialic acid dependent and appear to be proteic in nature.
Abstract: To determine amino acid sequences of the Plasmodium falciparum MSP-1 protein that interact with red blood cell membranes in a specific receptor-ligand interaction, 78 sequential peptides, 20 amino acids long and spanning the entire length of the molecule, were synthesized and analysed with a specific binding assay developed for this purpose. Results show that peptides based on conserved and dimorphic regions of MSP-1, interact with human red blood cells (RBCs). This interaction occurs predominantly with peptides contained within the MSP-1 proteolytic fragments of 83 kDa, 38 kDa, 33 kDa and 19 kDa. Affinity constants of these peptides were between 140 and 250 nM. Peptide-RBC binding post enzyme treatment showed that the RBC receptors are not sialic acid dependent and appear to be proteic in nature. Some of these peptides inhibited merozoite invasion of RBCs yet did not inhibit intra-erthrocytic development. These peptides, in conjunction with those from other merozoite surface proteins, may be used to rationally design a second generation of synthetic peptide-based malaria vaccines.

Journal ArticleDOI
TL;DR: In this review, the properties, carbohydrate specificities and potential biological functions of the Sialoadhesin family of sialic acid-dependent adhesion molecules, associated with diverse biological processes, are reviewed.
Abstract: For many years evidence has accumulated that sialic acids function in cellular interactions either by masking or as a recognition site. However, receptors or adhesion molecules mediating such functions between eukaryotic cells were unknown until about 5 years ago, when it was found that the members of the Selectin family mediate adhesion of leukocytes to specific endothelia through binding to sialylated glycans like sialyl Lewis. More recently, the Sialoadhesin family of sialic acid-dependent adhesion molecules was defined within the superfamily of immunoglobulin-like molecules. So far, it has been shown that sialoadhesin (Sn), CD22, CD33, the myelin-associated glycoprotein (MAG) and the Schwann cell myelin protein (SMP) belong to this family. In contrast to the Selectins, these proteins are associated with diverse biological processes, i.e. hemopoiesis, neuronal development and immunity. In this review their properties, carbohydrate specificities and potential biological functions are discussed. Finally, we provide perspectives with respect to the nature of ligands, implications of sialic acid modifications and future research.

Journal ArticleDOI
15 Mar 1996-Virology
TL;DR: A sensitive microscale binding assay is developed to study the interaction between influenza hemagglutinin and its cell surface receptor sialic acid using real-time surface plasmon resonance to quantitate the tight binding achieved through the multivalent interaction between BHA rosettes and the fetuin-derivitized sensor surface.

Journal ArticleDOI
TL;DR: VAP-1 naturally exists as a 170-kD sialoglycoprotein that uses sialic acid residues to interact with its counter-receptors on lymphocytes under nonstatic conditions and extends the role of carbohydrate- mediated binding in lymphocyte-endothelial cell interactions beyond the known selectins.
Abstract: The regulated interactions of leukocytes with vascular endothelial cells are crucial in controlling leukocyte traffic between blood and tissues. Vascular adhesion protein-1 (VAP-1) is a novel, human endothelial cell molecule that mediates tissue-selective lymphocyte binding. Two species (90 and 170 kD) of VAP-1 exist in lymphoid tissues. Glycosidase digestions revealed that the mature 170-kD form of VAP-1 expressed on the lumenal surfaces of vessels is a heavily sialylated glycoprotein. The sialic acids are indispensable for the function of VAP-1, since the desialylated form of VAP-1 no longer mediates lymphocyte binding. We also show that L-selectin is not required for binding of activated lymphocytes to VAP-1 under conditions of shear stress. The 90-kD form of VAP-1 was only seen in an organ culture model, and may represent a monomeric or proteolytic form of the larger species. These data indicate that L-selectin negative lymphocytes can bind to tonsillar venules via the VAP- 1-mediated pathway. Moreover, our findings extend the role of carbohydrate-mediated binding in lymphocyte-endothelial cell interactions beyond the known selectins. In conclusion, VAP-1 naturally exists as a 170-kD sialoglycoprotein that uses sialic acid residues to interact with its counter-receptors on lymphocytes under nonstatic conditions.

Journal ArticleDOI
TL;DR: The expression of secretory MUC1 glycoforms was inconsistent based on the decreasing contents of sialic acid and on the concomitant increase of immunodetectable TF antigen and the buoyant densities measured in CsCl gradients revealed heterogeneity of the physicochemical species and a significant reduction of their carbohydrate contents compared to M UC1 from skim milk.
Abstract: A highly immunogenic peptide motif within the tandem repeat domain of MUC1 mucin is assumed to be exposed during development of breast cancer due to altered O-glycosylation. To elucidate the structural aspects of these changes, we have isolated and analysed the integrated or secretory MUC1 glycoforms from carcinoma cell lines or solid tumors and from human milk. The buoyant densities measured in CsCl gradients for MUC1 glycoforms from cancer cells revealed heterogeneity of the physicochemical species and a significant reduction of their carbohydrate contents compared to MUC1 from skim milk. Immunoreactivity patterns of MUC1 glycoforms from tumor or T47D cells exhibited a lack of fucosylated Lewis blood-group-related antigens and the appearance of core-type antigen sialyl-(NeuG1)-TF, Galβ1–3(NeuGlα2–6)GalNAc. Structural chemistry of MUC1 oligosaccharides demonstrated that the cancer-associated glycoforms carry mainly sialylated trisaccharides NeuAcα2–3Galβ1–3GalNAc or NeuAcα2–6(Galβ1–3)GalNAc, exhibit a concomitant decrease in the ratio of GlcNAc/GalNAc, a reduction or disappearance of l-fucose, and a partial substitution of N -acetylneuraminic acid by the N-glycolylated variant. On comparison to the secretory MUC1 in human milk, the glycoforms on human milk fat globule membranes showed apparently identical patterns of O-linked oligosaccharides with a preponderance of neutral polylactosamino-glycans. During serum-free cultivation of T47D cells over 4 weeks, the expression of secretory MUC1 glycoforms was inconsistent based on the decreasing contents of sialic acid and on the concomitant increase of immunodetectable TF antigen.

Journal ArticleDOI
TL;DR: In a rabbit model the ptmA mutant showed a reduced ability to elicit protection against subsequent challenge with heterologous strains of the same Lior serotype compared to the parental wild‐type strain, suggesting that the surface‐exposed post‐translational modifications may play a significant role in the protective immune response.
Abstract: Two genes have been identified in Campylobacter coli VC167 which are required for the biosynthesis of post-translational modifications on flagellin proteins. The ptmA gene encodes a protein of predicted M(r) 28,486 which shows significant homology to a family of alcohol dehydrogenases from a variety of bacteria. The ptmB gene encodes a protein of predicted M(r) 26,598 with significant homology to CMP-N-acetylneuraminic acid synthetase enzymes involved in sialic acid capsular biosynthesis in Neisseria meninigitidis and Escherichia coli K1. Site-specific mutation of either ptmA or ptmB caused loss of reactivity with antisera specific to the post-translational modifications and a change in the isoelectric focusing fingerprints relative to the parent strains. Mutation of ptmB, but not of ptmA, caused a change in apparent M(r) of the flagellin subunit in SDS-PAGE gels. The ptmA and ptmB genes are present in other strains of Campylobacter. In a rabbit model the ptmA mutant showed a reduced ability to elicit protection against subsequent challenge with heterologous strains of the same Lior serotype compared to the parental wild-type strain. This suggests that the surface-exposed post-translational modifications may play a significant role in the protective immune response.

Journal ArticleDOI
TL;DR: Income and binding assays indicate that gene 4 encodes the rotavirus protein which mediates attachment to cells in culture for both sialic acid-dependent and -independent strains, and VP4 appears to function as the cell attachment protein in vivo as well as in vitro.
Abstract: To identify the rotavirus protein which mediates attachment to cells in culture, viral reassortants between the simian rotavirus strain RRV and the murine strains EHP and EW or between the simian strain SA-11 and the human strain DS-1 were isolated. These parental strains differ in the requirement for sialic acid to bind and infect cells in culture. Infectivity and binding assays with the parental and reassortant rotaviruses indicate that gene 4 encodes the rotavirus protein which mediates attachment to cells in culture for both sialic acid-dependent and -independent strains. Using ligated intestinal segments of newborn mice and reassortants obtained between the murine strain EW and RRV, we developed an in vivo infectivity assay. In this system, the infectivity of EW was not affected by prior treatment of the enterocytes with neuraminidase, while neuraminidase treatment reduced the infectivity of a reassortant carrying gene 4 from RRV on an EW background more than 80% relative to the controls. Thus, VP4 appears to function as the cell attachment protein in vivo as well as in vitro.

Journal ArticleDOI
TL;DR: It is concluded that in the infant rat model of meningococcal infection both forms of sialic acid on the bacterial cell surface are indispensable for systemic survival.
Abstract: We investigated the contribution of the polysialic acid capsule and of terminal lipooligosaccharide (LOS) sialylation to the pathogenicity of Neisseria meningitidis in vivo using a set of defined isogenic mutants of the N. meningitidis strain B 1940 deficient in either capsule synthesis or LOS sialylation. Furthermore a spontaneous capsule-deficient variant was investigated, which was capable of switching on the capsule synthesis at a frequency of 3 x 10(-3) in vitro. Infection of infant rats with the wild-type strain revealed a high potential to cause bacteremia. This potential was attenuated in the capsule-phase variable mutant (LOS sialylation+). However, using a mutant irreversibly deficient in capsule synthesis, but expressing a sialylated LOS, bacteremia could only be achieved using 10(6) times higher numbers of bacteria when compared to the wild-type. The unencapsulated bacteria were located extracellularly upon examination of blood smears, suggesting that defense mechanisms, i.e. phagocytosis, directed against unencapsulated meningococci were exhausted using very high infecting doses. Interestingly, when infant rats were infected with encapsulated meningococci which were unable to sialylate the LOS, bacteremia could never be achieved, even with an infective dose as high as 10(8) colony forming units (CFU). Despite the presence of capsular polysaccharide this mutant was phagocytosed by peritoneal phagocytes, as was the unencapsulated, LOS-sialylated mutant, suggesting that the inability to cause bacteremia was due to a higher susceptibility to the action of the complement system, which is virtually unsaturable. We conclude that in the infant rat model of meningococcal infection both forms of sialic acid on the bacterial cell surface are indispensable for systemic survival.

Journal ArticleDOI
TL;DR: It is demonstrated that cell surface 9-O-acetylation can affect a variety of biological recognition phenomena and provide a system for further exploration of the specific molecular mechanisms involved.

Journal ArticleDOI
TL;DR: Porcine endothelial cells differ from human endothelial Cells by expression of glycosphingolipids that are absent in man: two Galα1–3Gal-terminated glycolipids recognized by human natural antibodies, and twoN-glycolylneuraminic acid- terminated gangliosides which are potent immunogens.
Abstract: Glycosphingolipids were isolated from primary cultures of porcine endothelial cells labelled with 14C-galactose or 14C-glucosamine. They were characterized by their mobility on thin layer chromatogram, their sensitivity to exoglycosidases, and their labelling with antibodies. In addition to the major glycosphingolipids, globotetra- and globotriaosylceramide, minor ones were identified as penta- and heptaglycosylceramide of the neolactoseries terminated by either Gal alpha 1-3Gal- (xenoreactive epitope) or Fuc alpha 1-2Gal- (H determinant). Two gangliosides were found, GM3 and GD3, and N-glycolylneuraminic acid was their major sialic acid. Therefore, porcine endothelial cells differ from human endothelial cells by expression of glycosphingolipids that are absent in man: two Gal alpha 1-3Gal-terminated glycolipids recognized by human natural antibodies, and two N-glycolylneuraminic acid-terminated gangliosides which are potent immunogens.

Journal Article
TL;DR: This animal model seems to mimic minimal change disease in children, diabetic nephropathy, and the renal effects of some bacterial infections.

Journal ArticleDOI
TL;DR: The data suggest that sialic acid addition modifies Kv1.1 channel function, possibly by influencing the local electric field detected by its voltage sensor, but that these carbohydrates are not required for cell surface expression.

Journal ArticleDOI
TL;DR: The site of cleavage between the diradylglycerol phosphate and inositol suggests that a mammalian phospholipase D could be involved in the solubilization of GPI-anchored proteins and demonstrates that theospholipid tail is needed for the full functional activity of CD59.
Abstract: CD59 (protectin) is a glycophosphoinositol (GPI)-anchored inhibitor of the membrane attack complex of complement found on blood cells, endothelia and epithelial cells. In addition to the lipid-tailed CD59, soluble lipid-free forms of CD59 are present in human body fluids. We have investigated the detailed structural composition of the naturally occurring soluble urinary CD59 (CD59u) using peptide mapping, anion-exchange chromatography, sequential exoglycosidase digestion and matrix-assisted laser-desorption mass spectrometry (MALDI-MS). CD59u exhibited an average M(r) of 12444 in MALDI-MS. Mass analysis of the isolated C-terminal peptide (T9) indicated that a GPI-anchor (at Asn-77) without an inositol-associated phospholipid was present in soluble CD59u. By using residue-specific exoglycosidases, chemical modification and MALDI-MS structures of seven different GPI-anchor variants were determined. Variant forms of the anchor had deletions and/or extensions of one or more monosaccharide units. Sialic acid linked to an N-acetylhexosamine-galactose arm was found in two GPI-anchor variants. The N-linked carbohydrate side chain of CD59u (at Asn-18) also displayed considerable heterogeneity. The predominant oligosaccharide chains were fucosylated biantennary and triantennary complexes with variable sialylation. Mono Q anion-exchange chromatography resolved urinary CD59 into nine different fractions that bound equally well to the terminal complement SC5b-8 complexes. Despite binding to C5b-8, soluble CD59u inhibited complement lysis at an approx. 200-fold lower efficiency than erythrocyte CD59. These results document the structural heterogeneity of both the GPI anchor and N-linked oligosaccharide of CD59 and demonstrate that the phospholipid tail is needed for the full functional activity of CD59. The site of cleavage between the diradylglycerol phosphate and inositol suggests that a mammalian phospholipase D could be involved in the solubilization of GPI-anchored proteins.

Journal ArticleDOI
TL;DR: In this paper, the authors examined the pattern of expression in the human thymus of two additional sialyltransferases, the Galβ1,4GlcNAc α2,6-sialyl transferase (ST6N) and the Gal β1, 3GalNAcα2,3/4GlncNAc (ST3N), and found that the level of ST3N mRNA expression was correlated with cell-surface glycosylation.

Journal ArticleDOI
TL;DR: Results indicate that C seriespolysialogangliosides are synthesized by a ganglioside-specific polysialyltransferase, GD3/GT3ST, that is specifically expressed in neural tissues.

Journal ArticleDOI
TL;DR: This study provides an example of synergy between two ligands directed toward the active sites of two different proteins, and reinforces the conclusion that steric stabilization is important for the activity of polyvalent inhibitors.