Identification of a new ganglioside from the starfish Asterias rubens.
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Animals↗
Biomedical subjects
R Schauer
Publications and source records attributed to R Schauer.
Solution conformations of GM3 gangliosides containing different sialic acid residues as revealed by NOE-based distance mapping, molecular mechanics, and molecular dynamics calculations.
The conformation of the GM3 ganglioside, Neu5Ac alpha 2-3Gal beta 1-4Glc beta 1-1 Cer, and its analogs containing the Neu5Gc or Neu4Ac5Gc residues (Gc = glycolyl, CH2OHCO) was investigated in Me2SO-d6 solution with the aid of a distance-mapping procedure based on rotating-frame NOE contacts, with hydroxyl protons being used as long-range sensors defining the distance constraints. A pronounced flexibility found for both the Neu-Gal and Gal-Glc linkages was confirmed by 1000-ps molecular dynamics simulations. Similar results, although based on a smaller number of NOE constraints, were obtained for GM3 gangliosides anchored in mixed D2O/dodecylphosphocholine-d38 micelles and for the Neu5Ac-, Neu5Gc-, and Neu5,9Ac2-sialyllactoses dissolved in D2O. No noteworthy differences in conformational behavior of the glycan chains of the three gangliosides or sialyllactoses were observed in either of the media.
Animals↗
Mouse liver cytidine-5'-monophosphate-N-acetylneuraminic acid hydroxylase. Catalytic function and regulation.
In this paper, we present the results of an investigation into the catalytic properties of CMP-Neu5Ac hydroxylase (Neu5Ac: N-acetylneuraminic acid) in high-speed supernatants of mouse liver. The enzyme was most active in Hepes/NaOH pH 7.4 and was markedly inhibited by relatively small increases in ionic strength, though the inhibition was abolished by desalting procedures. Several nonionic detergents could activate the hydroxylase to various degrees in a concentration-dependent manner. Ionic detergents and a number of phospholipids were, however, generally inert or inhibitory. The lack of inhibitory influence of a wide range of nucleotides revealed that CMP-Neu5Ac hydroxylase binds its sugar-nucleotide substrate with a high degree of specificity. Thus, even millimolar concentrations of several cytidine nucleotides elicited virtually negligible inhibition, though the reaction product, CMP-Neu5Gc (Neu5Gc: N-glycoloylneuraminic acid), was a weak inhibitor. The results also indicate that the enzyme is not regulated by any nucleotides or sugar-nucleotides. Dilution of high-speed supernatants with buffer gave rise to a decrease in the specific activity of the hydroxylase, implicating the involvement of more than one component in catalysis. Activity could be restored by the addition of a heat extract of the supernatant. The active principle in this extract was found to be a heat-stable protein with a molecular mass of about 17 kDa. Immunochemical studies allowed this protein to be identified as cytochrome b5 and it was shown that this electron carrier is essential for the activity of CMP-Neu5Ac hydroxylase. Inhibition studies using iron ligands and activation by exogenous iron salts suggest the involvement of a non-haem iron cofactor in the catalytic cycle of this hydroxylase. Cytochrome b5 may thus serve as an electron donor for this postulated cofactor.
1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
Occurrence of sialic acids in Drosophila melanogaster.
Sialylated oligosaccharides, which are cell type-specific and developmentally regulated, have been implicated in a variety of complex biological events. Their broad functional importance is reflected by their presence in a wide variety of phyla extending from Echinodermata through higher vertebrates. Here, sialic acids are detected throughout development in an insect, Drosophila. Homopolymers of alpha 2,8-linked sialic acid, polysialic acid, are developmentally regulated and only expressed during early Drosophila development.
Animals↗
Elucidation of the topological parameters of N-acetylneuraminic acid and some analogues involved in their interaction with the N-acetylneuraminate lyase from Clostridium perfringens.
A series of neuraminic acid derivatives modified in the side chain or at C-3, C-4 or C-5 were tested as substrates of inhibitors of N-acetylneuraminate lyase (EC 4.1.3.3) from Clostridium perfringens. The results, together with Km and Ki values reported previously, indicate that the region most important for the binding of sialic acids is an equatorial zone reaching from C-8 via the ring oxygen atom to C-4 of the sugar molecule, whereas the substituents at C-9 and C-5 may be varied to a higher extent without significantly disturbing enzyme action. It is shown that stereo-electronic factors are responsible for the immediate heterolytic fragmentation of the cyclic sialic acid into pyruvic acid and 2-acetamidomannose or a related C-6 sugar.
Clostridium perfringens↗
Use of influenza C virus for detection of 9-O-acetylated sialic acids on immobilized glycoconjugates by esterase activity.
An overlay and a solid-phase assay are presented which allow the specific detection of 9-O-acetylated sialic acids on sialoglycoconjugates immobilized on microtiter plates, nitrocellulose or separated on thin-layer chromatograms. The assay takes advantage of two different biological properties of influenza C virus, its high-affinity binding to 9-O-acetylated sialic acids and its sialate 9-O-acetylesterase that is used for detection of bound virus with fluorogenic or chromogenic substrates. Though simple and rapid, the assay is highly sensitive with a detection limit of 65 fmol 9-O-acetylated sialic acid in 9-O-acetylated ganglioside GD1a. Influenza C virus is able to bind to a wide spectrum of sialoglycoconjugates like mucins, serum glycoproteins or gangliosides containing naturally or synthetically O-acetylated sialic acids. 9-O-Acetyl-N-glycoloylneuraminic acid can also function as a high-affinity receptor determinant for influenza C virus. While the acetyl ester at the 9 position is essential for virus binding in all cases, a 4-O-acetyl group is not recognized. In addition to alpha(2.3) or alpha(2.6) bonds, 9-O-acetyl-N-acetylneuraminic acid in alpha(2.8) linkage to N-acetylneuraminic acid is also functionally active.
Acetylesterase↗
Structural analysis of a novel sialic-acid-containing trisaccharide from Rhodobacter capsulatus 37b4 lipopolysaccharide.
Sialic-acid-containing lipopolysaccharides from Rhodobacter capsulatus 37b4 (S-form lipopolysaccharide), KB-1 (R-type lipopolysaccharide) and Sp 18 (deep R-type lipopolysaccharide) were investigated for the linkage and substitution of sialic acids. Methylation analysis and behaviour towards acid and enzymic hydrolysis indicated a non-reducing terminal location of sialic acids in the R-type lipopolysaccharide of strain Sp 18, whereas an internal, chain-linked location of sialic acids was found in the lipopolysaccharides of strains 37b4 and KB-1. For these latter strains, methylation analysis revealed a substitution of sialic acids by other sugars at position 7 for strain 37b4 and positions 4 and 7 for strain KB-1. In accordance with the chain-linked position of sialic acids, mild hydrolysis of R. capsulatus 37b4 lipopolysaccharide with acetic acid released a trisaccharide with sialic acid at the reducing terminus. Structural investigation of this trisaccharide by methylation analysis, 1H- and 13C-NMR spectroscopy revealed the presence of the disaccharide Gal1-6Glc at the non-reducing end, probably with an alpha-anomeric configuration of the galactose residue, i.e. melibiose, beta-glycosidically linked to position 7 of sialic acid. Therefore the structure Gal alpha 1-6Glc beta 1-7Neu5Ac is proposed for this core oligosaccharide from R. capsulatus 37b4 lipopolysaccharide.
Carbohydrate Sequence↗
Analysis of carbohydrate composition and sialidase activity in oral secretions of patients with tumors in the upper aerodigestive tract.
Total saliva and secretions from parotid and submandibular glands of patients with carcinomas in the oral cavity, oropharynx or larynx and a control group of healthy individuals were analyzed for concentrations of glycosidically bound sugars and free N-acetylneuraminic acid as well as for sialidase activity. When compared to the data obtained for normal donors, the relative amounts of the individual monosaccharides fucose, galactose, mannose, N-acetylgalactosamine, and N-acetylglucosamine as components of glycoconjugates showed variable differences to the group of tumor patients depending on the type of secretion and the location of the tumor. The percentage of glycosidically linked N-acetylneuraminic acid, however, was always higher for the healthy donors. A significant difference was found in the amount of free sialic acid, with the exception of submandibular gland secretion from a patient with an oropharyngeal carcinoma, and sialidase activity which were increased for tumor patients, independent of the type of secretion and the location of the tumor. From these results it is concluded that free sialic acid and sialidase activity may be considered as markers for carcinomas in the upper aerodigestive tract.
Humans↗
Effects of site-specific mutations on the enzymatic properties of a sialidase from Clostridium perfringens.
Three site-specific mutations were performed in two regions of a sialidase gene from Clostridium perfringens which are known to be conserved in bacterial sialidases. The mutant enzymes were expressed in Escherichia coli and, when measured with MU-Neu5Ac as substrate, exhibited variations in enzymatic properties compared with the wild-type enzyme. The conservative substitution of Arg 37 by Lys, located in a short conserved region upstream from the four repeated sequences common in bacterial sialidase genes, was of special interest, as KM and Vmax, as well as K(i) measured with Neu5Ac2en, were dramatically changed. These data suggest that this residue may be involved in substrate binding. In addition to its low activity, this mutant enzyme has a lower temperature optimum and is active over a more limited pH range. This mutation also prevents the binding of an antibody able to inhibit the wild-type sialidase. The other mutations, located in one of the consensus sequences, were of lower influence on enzyme activity and recognition by antibodies.
Amino Acid Sequence↗
Purification and characterization of a novel sialidase found in procyclic culture forms of Trypanosoma brucei.
A membrane-bound sialidase (EC 3.2.1.18) was found in procyclic trypomastigotes of Trypanosoma brucei. The mammalian stage bloodstream form, however, displayed no sialidase activity. This sialidase is an integral surface protein, linked to the membrane via a glycosylphosphatidylinositol anchor. After osmotic lysis and solubilization with Triton CF-54, the enzyme was purified 1900-fold by gel filtration and ion exchange chromatography. Its size, as determined by conventional and high-performance liquid gel chromatography, is 67 kDa. The sialidase is active over a broad pH and temperature range with optima at pH 6.9 and 35 degrees C, respectively. No loss of activity is observed after 4 freeze-thaw cycles. T. brucei sialidase activity is inhibited by N-(4-nitrophenyl)oxamic acid and 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, the latter, however, being less effective. N-Acetylneuraminic acid shows no inhibitory effect, whereas a variety of metal ions are potent inhibitors. The sialidase is activated by di- and tricarboxylic acids, but inhibited by chloride. Relative hydrolysis rates of various sialic acid-containing compounds reveal that de-O-acetylated bovine submandibular gland mucin is the preferred substrate and that alpha(2-3)-linkages are hydrolyzed faster than alpha(2-6)-linkages.
Animals↗
Binding of Plasmodium falciparum 175-kilodalton erythrocyte binding antigen and invasion of murine erythrocytes requires N-acetylneuraminic acid but not its O-acetylated form.
Sialic acid on human erythrocytes is involved in invasion by the human malaria parasite, Plasmodium falciparum. Mouse erythrocytes were used as a reagent to explore the question of whether erythrocyte sialic acid functions as a nonspecific negative charge or whether the sialic acid is a necessary structural part of the receptor for merozoites. Human erythrocytes contain N-acetylneuraminic acid (Neu5Ac), whereas mouse erythrocytes, which are also invaded by P. falciparum merozoites, contain 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) and N-glycoloylneuraminic acid (Neu5Gc), in addition to Neu5Ac. We compared the effects of sialidase and influenza C virus esterase treatments of mouse erythrocytes on invasion and the binding of a 175-kDa P. falciparum protein (EBA-175), a sialic acid-dependent malaria ligand implicated in the invasion process. Sialidase-treated mouse erythrocytes were refractory to invasion by P. falciparum merozoites and failed to bind EBA-175. Influenza C virus esterase, which converts Neu5,9Ac2 to Neu5Ac, increased both invasion efficiency and EBA-175 binding to mouse erythrocytes. Thus, the parasite and EBA-175 discriminate between Neu5Ac and Neu5,9Ac2, that is, the C-9 acetyl group interferes with EBA-175 binding and invasion by P. falciparum merozoites. This indicates that sialic acid is part of a receptor for invasion.
Animals↗
Nature and biosynthesis of sialic acids in the starfish Asterias rubens. Identification of sialo-oligomers and detection of S-adenosyl-L-methionine: N-acylneuraminate 8-O-methyltransferase and CMP-N-acetylneuraminate monooxygenase activities.
Mass spectrometric and NMR spectroscopic analyses of bound sialic acids from the starfish Asterias rubens revealed the presence of N-acetylneuraminic acid (4%), N-acetyl-8-O-methylneuraminic acid (12%), N-acetyl-9-O-acetyl-8-O-methylneuraminic acid (less than 1%), N-glycoloylneuraminic acid (19%), N-glycoloyl-8-O-methylneuraminic acid (47%), and N-glycoloyl-9-O-acetyl-8-O-methylneuraminic acid (18%). Analysis of sialo-oligomeric material, obtained after mild acid hydrolysis, demonstrated that N-glycoloyl-8-O-methylneuraminic acid can occur as di- and tri-oligomers, linked through the anomeric center and the N-glycoloyl moiety, Neu5Gc8Me-alpha(2----O5)-Neu5Gc8Me and Neu5Gc8Me-alpha(2----O5)-Neu5Gc8Me-alpha (2----O5)-Neu5Gc8Me. Studies on the biosynthesis of N-acyl-8-O-methylneuraminic acid in A rubens, using the tracer S-adenosyl-L-[methyl-14C]methionine, showed that N-acylneuraminate 8-O-methyltransferase activity was present predominantly in the membrane fraction. CMP-N-acetylneuraminic acid monooxygenase activity was found in the soluble protein fraction, in agreement with investigations on the corresponding vertebrate enzyme.
Animals↗
Loss of sialic acid O-acetylation in human colorectal cancer cells.
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Acetyl Coenzyme A↗
The influence of alpha- and beta-galactose residues and sialic acid O-acetyl groups of rat erythrocytes on the interaction with peritoneal macrophages.
The significance of glycoconjugates on the surface of rat erythrocytes was studied in the interaction of these cells with homologous peritoneal macrophages. The erythrocytes exposing terminal alpha-galactose and thus of B blood group specificity, as well as sialic acid are not bound by the macrophages. beta-Galactose residues exposed by sialidase induced strong binding and additional alpha-galactosidase treatment enhanced the binding. beta-Galactose exposed on glycolipids after pronase and alpha-galactosidase treatment induced no binding. An intact protein core of the glycoproteins on the erythrocyte surface was necessary for interaction with macrophages. Partial de-O-acetylation of sialic acids prior to sialidase treatment stimulated subsequent binding of the erythrocytes.
Acetylation↗
Interaction of rat peritoneal macrophages with homologous sialidase-treated thrombocytes in vitro: biochemical and morphological studies. Detection of N-(O-acetyl)glycoloylneuraminic acid.
Sialidase treatment of rat thrombocytes led to an increased binding of these cells to homologous peritoneal macrophages, but had no significant effect on the rate of phagocytosis during the experimental time. As revealed by electron microscopy, the partially desialylated thrombocytes adhere to macrophages predominantly via a small part of the membrane in a way that the discoidal cells adopt a vertical position with regard to the macrophage surface. One adherent macrophage was able to bind up to 55 sialidase-treated thrombocytes. Maximum binding was already reached after release of 13% of sialic acids. This interaction could be inhibited by free D-galactose and compounds with terminal D-galactose residues. Bound thrombocytes were released from the macrophages by treatment with lactose or EDTA. These experiments suggest that the interaction is mediated by a galactose-specific receptor on the macrophage surface and that galactose on thrombocytes is not recognized if it is masked by terminal sialic acid residues. The total sialic acid amount of the thrombocytes studied was about 70 micrograms sialic acid/10(10) cells being composed of 78% N-glycoloylneuraminic acid, 17% N-acetylneuraminic acid and 5% of the novel sialic acid N-(O-acetyl)glycoloylneuraminic acid, which was identified by mass spectrometry. Sixty-two percent of these sialic acids were susceptible to enzymic hydrolysis with Vibrio cholerae sialidase.
Animals↗
2,3-Didehydro-2-deoxysialic acids structurally varied at C-5 and their behaviour towards the sialidase from Vibrio cholerae.
2,3-Didehydro-2-deoxy-N-trifluoroacetylneuraminic acid (5-trifluoroacetyl-Neu2en) (3) has been synthesised from Neu5Ac2en (1) by hydrazinolysis, to give Neu2en (2), followed by N-trifluoroacetylation. 2,3-Didehydro-2,3-dideoxy-D-glycero-D-galacto-2-nonulopyranoson ic acid (Kdn2en, 8) and 5-azido-2,3-didehydro-2,3,5-trideoxy-D-glycero-D-galacto-2-nonu lopyranosonic acid (5-azido-5-deoxy-Kdn2en, 9) have been prepared from the acetylated methyl esters of Kdn (4) and 5-azido-5-deoxy-Kdn (5) via Zemplén saponification. The behaviour of the above 2,3-didehydro-2-deoxysialic acids towards Vibrio cholerae sialidase has been investigated.
Kinetics↗
Reversible binding of sialidase-treated rat lymphocytes by homologous peritoneal macrophages.
After sialidase treatment, lymphocytes disappear from the blood stream, but reappear after a few hours. The behavior of sialidase-treated rat lymphocytes was investigated by in vitro binding studies with homologous peritoneal macrophages. A lymphocyte mixture from thymus and spleen was treated with sialidase and cultured up to 55 h, and at various times, the binding of the lymphocytes to glass-adherent macrophages was studied by light and electron microscopy; vital lymphocytes were only bound but not phagocytosed, and the interaction with macrophages was inhibited by D-galactose. During culture of lymphocytes, either separately or with macrophages, the binding was more and more reduced, and a second sialidase treatment of cultured lymphocytes led again to increased binding which could be inhibited by D-galactose. This change did not occur in the presence of N-acetyl-2,3-didehydro-2-deoxyneuraminic acid, an inhibitor of sialidases, showing the sialic acid specificity of this phenomenon. Thus, the reversibility of lymphocyte binding could be explained by resynthesis of cell surface sialic acids.
Animals↗
Differences in the amount of N-acetyl- and N-glycoloyl-neuraminic acid, as well as O-acylated sialic acids, of fetal and adult bovine tissues.
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Acylation↗