Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.1.53 (sialidase)
2,694 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The receptor protein for thyrotrophin (thyroid-stimulating hormone; TSH) is associated with a glycosphingolipid moiety. The protein belongs to the family of receptors that couple to guanine nucleotide binding proteins; the glycosphingolipid contains sialic acid and belongs to the family of gangliosides. This report defines the structure of the receptor ganglioside in the Fisher rat thyroid cell line (FRTL-5). Receptor protein was purified by TSH affinity chromatography from FRTL-5 cells, biosynthetically labelled with [3H]galactose and [3H]glucosamine, and resolved by SDS-PAGE. A single radiolabelled band of Mr approximately 80 kDa, corresponding to the predicted size of the cloned receptor, contained ganglioside. Gangliosides were extracted from unlabelled receptor protein after SDS-PAGE and probed on TLC plates with 125I-labelled Limax flavus agglutinin or the B subunit of cholera toxin, before and after digestion with Vibrio cholerae sialidase or beta-galactosidase. The TSH receptor (TSH-R) ganglioside belongs to the gangliotetraose family, having sialic acid attached to both galactose molecules. Its sialic acid is devoid of negative charge because of the formation of internal esterlactones. Its structure is lactonized N-acetylneuraminyl-(alpha 2-->3)galactosyl(beta 1-->3)-N-acetylgalactosaminyl(beta 1-->4)-[N-acetylneuraminyl(alpha 2-->3)]galactosyl(beta 1-->4)glucosyl(beta 1-->1)ceramide (GDla-lactone). Ganglioside lactones have not been previously described as components of thyroid cells. They are highly rigid and are more likely than their parent structures to serve as molecular recognition sites and elicit immunoreactivity. Identification of this unique ganglioside intimately associated with the TSH receptor implies that it has an integral role in receptor structure and function.
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PMID:Characterization of ganglioside associated with the thyrotrophin receptor. 773 42

In the measurement of total lipid-bound sialic acids involving periodic acid oxidation, as in the periodate-resorcinol assay, the inner sialic acids of disialoglycolipids (such as GD3 and GD2) are not involved because their alpha 2,8 ketosidic linkages are resistant to periodic acid oxidation, even after acid/enzyme hydrolysis or alkali pretreatment. However, the sialic acids from these glycolipids can be recovered completely after cleavage of alpha 2,8 linkages by V. cholerae sialidase in the presence of cholic acid, sodium dodecyl sulphate and calcium. Interestingly, removal of calcium or detergent(s) or both significantly minimizes the sialidase action on the disialyl residues of these gangliosides. Therefore, we recommend sialidase (Vibrio cholerae) pretreatment of the glycolipids in the presence of cholic acid, SDS and Ca2+ for complete recovery of sialic acids from di- and polysialogangliosides and for accurate measurement of total lipid-bound sialic acids by periodate-resorcinol assay.
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PMID:An improved method for the measurement of total lipid-bound sialic acids after cleavage of alpha 2,8 sialic acid linkage with Vibrio cholerae sialidase in the presence of cholic acid, SDS and Ca2+. 780 12

Factor IX and factor X have sialic acid in O-linked and N-linked oligosaccharides on their activation peptides, and a terminal sialic acid is found on a recently described O-linked tetrasaccharide at Ser-61 in the light chain of human factor IXa. In studies presented here, the potential role of sialic acid residues in mediating activity of human coagulation factors IX and X was tested after enzymatic removal of sialic acid residues. In contrast to previous reports, treatment of factor IX or factor IXa with recombinant sialidase did not decrease the rate of factor IX activation or proteolytic properties of human factor IXa. The activation rates of factor IX and desialated factor IX were indistinguishable when treated with factor XIa, with factor VIIa/tissue factor complex, and with the factor X activating enzyme from Russell's viper venom. Desialated human factor IXa showed full activity in the non-activated partial thromboplastin time assay and retained full "tenase" activity in a coupled amidolytic assay. Similar experiments with human factor X showed no detectable loss of clotting activity in the prothrombin time assay after desialation. Additionally, desialated human factor X was cleaved by the factor X activating enzyme from Russell's viper venom and intrinsic tenase at the same rate as untreated factor X when analyzed by SDS-polyacrylamide gel electrophoresis. These studies have shown that factor IX and factor X clotting activity are not dependent on sialic acid content. Further studies are needed to determine whether desialated factor IX binds to endothelial cells, and whether factors IX and X are more rapidly cleared from circulation or have altered susceptibility to proteolysis after enzymatic removal of sialic acid.
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PMID:Enzymatic removal of sialic acid from human factor IX and factor X has no effect on their coagulant activity. 789 89

Pig brain cytosolic sialidase purified to homogeneity, showed a single protein band on SDS-PAGE under non-reducing conditions, and three bands using reducing conditions, suggesting a complex of different units. The sialidase complex (molecular mass, M(r), 180 kDa) was resolved into a catalytic unit (M(r) 30 kDa), active but very liable upon storage at 4 degrees C and freezing and thawing, and two protective units (66 kDa and 42 kDa), inactive, but capable to stabilize the catalytic unit. Recombination of the catalytic and protective units (optimal ratio, 1:1, by weight) gave rise to a stable active complex. Using GD1a as substrate, the catalytic unit showed a Michaelis-Menten kinetics, and the complex a sigmoid-shaped kinetics, whereas a Michaelis-Menten kinetics was exhibited with MU-NeuAc in both cases. The apparent Vmax and Km values of the catalytic unit for MU-NeuAc and GD1a were 105.1 and 110.0 mU/mg protein, and 4.2 x 10(-5) and 1.6 x 10(-5) M, respectively. The model we propose for cytosolic sialidase complex is one of each protective units and 2-3 catalytic units. The sialidase complex and protective units did not display any beta-D-galactosidase, beta-D-N- acetylglucosaminidase, alpha-L-fucosidase, alpha-D-glucosidase and carboxypeptidase activities.
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PMID:Cytosolic sialidase from pig brain: a 'protein complex' containing catalytic and protective units. 794 53

Mammalian oocytes are surrounded by an extracellular glycocalyx, the zona pellucida (ZP). In the mouse, the ZP is composed of three glycoproteins, designated mZP1, mZP2, and mZP3. Extensive studies in this species have resulted in the identification of primary (mZP3) and secondary (mZP2) receptors for spermatozoa. In this paper we present evidence for the occurrence of poly-N-acetyllactosaminyl glycans and an O-linked trisaccharide on mZP2 and mZP3. When exhaustively digested with endo-beta-galactosidase, an enzyme known to cleave repeating units of acetyllactosamine (3Gal beta 1, 4GlcNAc beta 1), mZP2 and mZP3 showed an apparent reduction in size by 23 kDa and 16 kDa, respectively. Experimental evidence included in this report indicates that polylactosaminyl glycans are present on N-linked sugar chains. In addition, O-linked sugar chains of mZP3 have been characterized. First, treatment of de-N-glycosylated mZP3 with O-glycanase in the presence of exo-glycosidases (sialidase, alpha-L-fucosidase, and N-acetylglucosaminidase) caused an apparent reduction in its size by 2-3 kDa as determined by SDS-PAGE. Second, treatment of the de-N-glycosylated mZP3 with mild alkali in the presence of 1 M NaB3H4 released radiolabeled oligosaccharide (OS) that eluted from a high-resolution Bio-Gel P-4 column at the position of a trisaccharide. The radiolabeled OS had the following structure: GlcNAc-->Gal beta 1,3GalNAcol. The structure was established by sizing on the Bio-Gel P-4 column, followed by examination of the susceptibility of the OS to exo-glycosidases and by its absorbability to immobilized lectin (PNA). Potential roles of N-linked and O-linked sugar chains in sperm-egg interaction are herein discussed.
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PMID:O-linked trisaccharide and N-linked poly-N-acetyllactosaminyl glycans are present on mouse ZP2 and ZP3. 794 82

Sialidase L releases 2,7-anhydro-NeuAc from sialoglycoconjugates (Li, Y.-T., Nakagawa, H., Ross, S. A., Hansson, G., and Li, S.-C. (1990) J. Biol. Chem. 265, 21629-21633). This enzyme has been purified more than 10,000-fold from Macrobdella leech. The final preparation gives a single protein band on SDS-polyacrylamide gel electrophoresis with the molecular mass of 84 kDa. The pI is determined to be 6.0 using isoelectric focusing. With 4-methylumbelliferyl-alpha-NeuAc as substrate, the pH optimum is between pH 5.5-7.0. Unlike regular sialidases, sialidase L is not inhibited by 2-deoxy-2,3-dehydro-NeuAc. Two of the seven tryptic peptides derived from sialidase L contain the consensus repeat S-X-D-X-G-X-T-W that has been found in the regular sialidases. Among various sialoglycoconjugates tested, sialidase L cleaves only the NeuAc alpha 2-->3Gal linkage. NeuAc alpha 2-->6Gal, NeuAc alpha 2-->6GalNAc, NeuAc alpha 2-->6GlcNAc, NeuAc alpha 2-->8-NeuAc, and NeuAc alpha 2-->9NeuAc linkages are not hydrolyzed. At pH 7.0, sialidase L and Clostridial sialidase release 46 and 92% of sialic acid, respectively, from bovine fetuin, indicating that sialidase L selectively cleaves NeuAc alpha 2-->3Gal linkages in fetuin. Sialidase L is the first sialidase found to exhibit a strict specificity toward the hydrolysis of the NeuAc alpha 2-->3Gal linkage, and it should become useful for the selective cleavage of NeuAc alpha 2-->3Gal linkages in sialoglycoconjugates without destroying other sialosyl linkages.
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PMID:Purification and characterization of sialidase L, a NeuAc alpha 2-->3Gal-specific sialidase. 803 34

The target molecules on the cell surface of Trypanosoma cruzi trypomastigotes reacting with lytic anti-alpha-galactosyl antibodies from chronic patients with Chagas' disease (Ch anti-Gal) have been purified by solvent extraction and identified as glycoconjugates migrating in the 74-96-kDa range (F2 antigen) and in the 120-200-kDa range (F3 antigen) on SDS-PAGE. The F3 antigen was tested for binding to Ch and normal human serum (NHS) anti-Gal and to MoAb 3C9. We observed that Ch anti-Gal and MoAb 3C9, but not NHS anti-Gal, bind strongly to the trypomastigote glycoconjugates. These antibodies, however, did not compete with each other for binding to F3 molecules, indicating that they are recognizing different epitopes. Binding of Ch anti-Gal to F3 antigen is abolished by treatment of these molecules with alpha- but not beta-galactosidase. Binding of 3C9 MoAb is abolished by treatment of F3 with sialidase. F2/F3 antigens absorbed Ch anti-Gal as well as lytic antibodies from total chagasic sera. These antigens also specifically discriminate between the serum reactivity of patients with active Chagas' disease and those of sera from cured patients, drug-treated patients with dissociated serology (positive conventional serology, negative trypanolytic activity), healthy individuals, and patients with several other infectious diseases. We also observed that F2/F3 antigens are anchored to the parasite membrane via glycosylphosphatidylinositol (GPI). The alpha-galactosyl epitopes recognized by Ch anti-Gal are present in a series of O-linked oligosaccharide chains in the mucin-like glycoprotein component of the complex.
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PMID:GPI-anchored glycoconjugates from Trypanosoma cruzi trypomastigotes are recognized by lytic anti-alpha-galactosyl antibodies isolated from patients with chronic Chagas' disease. 808 Dec 63

Sialidase activities of rabbit blood cells and serum were measured. The leucocyte particulate fraction showed the highest specific activity of sialidase towards mixed gangliosides and sialyllactose, and the cytosolic fraction showed for fetuin. Predominant sialidase activity in the blood was detected in erythrocyte particulate fraction when mixed gangliosides were used as substrate. The sialidase for ganglioside was solubilized from the erythrocyte ghosts by using Triton X-100. The solubilized sialidase was purified 1886-fold by sequential chromatographies on DEAE-cellulose, EAH-Sepharose 4B, Octyl-Sepharose CL-4B, Sephadex G-100, concanavalin-A--Sepharose, N-(p-aminophenyl)oxamic acid-agarose and Heparin-Sepharose CL-6B. The optimum pH of purified sialidase was 4.5 for ganglioside mixture, and this enzyme exhibited M(r) = 48,000 by gel filtration. When the purified sialidase was subjected to SDS/PAGE, a major sialidase-active protein band at M(r) = 54,000 and another fainter inactive protein band with M(r) = 115,000 were observed. The purified enzyme was active towards oligosaccharides, gangliosides, fetuin glycopeptide and 4-methylumbelliferyl alpha-D-N-acetylneuraminic acid except for glycoproteins tested. Fe2+, Fe3+ and dithiothreitol significantly inhibited the enzyme activity, while Triton X-100 activated the enzyme. Inside-out vesicles and unsealed ghosts of rabbit erythrocyte showed the sialidase activity for mixed gangliosides but not for resealed ghosts or intact erythrocytes. These results indicate that the active site of this sialidase is oriented mainly on the inside of the erythrocyte membrane and not on the outside. Treatment of rabbit erythrocyte unsealed ghosts with phosphatidylinositol-specific phospholipase C liberated no sialidase activity toward mixed gangliosides from the ghosts.
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PMID:Sialidase in rabbit blood. Characterization of sialidase purified from rabbit erythrocyte membrane. 817 46

A soluble sialidase that can degrade recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells has been isolated and purified to near homogeneity from the cell culture fluid of this host. Purification of approximately 34,000-fold was carried out using conventional purification techniques including sequential DEAE-Sepharose and S-Sepharose ion-exchange chromatography, followed by hydrophobic interaction chromatography with Phenyl-Toyopearl. Final purification was achieved by heparin-agarose and chromatofocusing chromatography. The minimum molecular weight of the sialidase on SDS-PAGE was approximately 43,000 Da. When the final preparation was examined under non-denaturing conditions, two major (pI = 6.8 and 7.0) and five minor electrophoretic forms with different isoelectric points were identified. The basis for the electrophoretic heterogeneity is not known, but it was not due to carbohydrate diversity since no carbohydrates were detected on the purified protein. The enzyme degraded a variety of sialyl-conjugate substrates, at a pH optimum of 5.9, including intact glycoproteins, oligosaccharides and gangliosides with a 4-fold preference for 2,3- versus 2,6-linked sialic acid residues. With ganglioside substrates, internally linked sialic acid residues were not cleaved by the enzyme. Delineation of this enzyme from the lysosomal and plasma membrane sialidases was made using inhibition studies with C-9 substituted 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2- enonic acid derivatives. The enzyme was identified in several CHO cell lines by immunoblotting using antiserum raised against a synthetic peptide based on amino acid sequence of a fragment derived by trypsin digestion of the purified sialidase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation and properties of a soluble sialidase from the culture fluid of Chinese hamster ovary cells. 828 58

Cell surface carbohydrates have been shown to be altered during cellular differentiation. Alveolar type II (ATII) cells in culture gradually lose their differentiated phenotype. Therefore, the aim of this study was: (1) to characterize changes in terminal carbohydrates of cell surface glycoproteins of rat ATII cells cultured for 1 to 5 days on plastic, and (2) to assess the concomitant changes in sialidase and sialyltransferase activity of ATII cell homogenates. Cells were surface-labeled with potassium-[3H]-borohydride after oxidation by sodium periodate at millimolar concentrations, galactose oxidase or neuraminidase plus galactose oxidase, allowing for the specific labeling of terminal sialic acids, terminal galactose/N-acetylgalactosamine (Gal/GalNAc), or terminal an penultimate Gal/GalNAc residues, respectively. Glycoproteins were separated by SDS-PAGE. On day 1, cells were heavily coated with sialic acids, since no labeling could be introduced with galactose oxidase alone. From day 1 to day 5, we observed a selective and progressive desialylation of two glycoproteins (200 and 165 kD). At the same time, the ATII cells' sialidase activity (pH 4.2) exhibited an 8-fold increase (60.3 +/- 4.0 pmol/min/mg protein on day 1 versus 406.9 +/- 3.7 pmol/min/mg protein on day 5), whereas the sialyltransferase activity increased 2-fold (212 +/- 8 fmol/min/mg protein on day 1 versus 395 +/- 82 fmol/min/mg protein on day 5) and the supernatant sialidase activity was unchanged (2.8 +/- 0.7 pmol/min/ml on day 5). Thus, the phenotypic changes of ATII cells in primary culture are accompanied by a partial cell surface desialylation and an increase in intracellular sialidase activity.
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PMID:Cell surface carbohydrates of rat alveolar type II cells in primary culture. 842 6


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