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)

A membrane-associated ganglioside-hydrolyzing sialidase was purified to apparent homogeneity from bovine brain. The enzyme was solubilized with Triton X-100 plus sodium cholate from the particulate fraction and purified over 100,000-fold by sequential chromatography on DEAE-cellulose, octyl-Sepharose, heparin-Sepharose, Sephacryl S-200, MonoQ, RCA-agarose, thiol-activated Sepharose, and ganglioside-affinity Sepharose. The final enzyme preparation exhibited a specific activity of 4,851.3 micromol/h/mg protein and an apparent molecular mass of 52 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme preferentially hydrolyzed gangliosides other than GM1 and GM2 but demonstrated hardly any activity against glycoproteins and oligosaccharides. Gangliosides GD3, GD1a, and GT1b were much better substrates than GM3 and GD1b in the presence of Triton X-100, but the latter became more sensitive to the sialidase with addition of sodium cholate. The enzyme was activated by dithiothreitol, strongly inhibited by 4-hydroxy-mercuribenzoate, and firmly adsorbed to thiol-activated Sepharose, indicating that free sulfhydryl groups are essential for its catalytic activity. Subcellular fractionation experiments revealed that the enzyme is mainly located in the synaptosomal fraction.
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PMID:Purification and characterization of a membrane-associated ganglioside sialidase from bovine brain. 956 23

Gangliosides are sialo-glycosphingolipids that play important roles in the interaction of cells with their environment and are thus involved in the regulation of many cellular events. Sialic acid residues are important for the conformation of a glycomolecule, their structural stability and their functions. Although decreased brain ganglioside sialic acid has been previously reported as a result of chronic ethanol treatment in rats, no reports are available on the sialylation of specific gangliosides and/or the mechanism leading to depletion of their sialic acid residues. Therefore, in this investigation, we have examined the effects of chronic ethanol treatment on (1) incorporation of [4,5-3H]N-acetylmannosamine (ManNAc) into specific rat brain gangliosides, GD3, GD1a, GT1a, and GT1b; and (2) enzymatic activities of brain sialyltransferase and sialidase at specific subcellular levels. The experiments were done in male Wistar rats pair-fed with either ethanol or control liquid diets for a period of 8 weeks. The rats were intracerebroventricularly injected with labeled ManNAc (30 microCi/rat) and killed after 90 min. Radioactivity was determined in respective ganglioside bands separated on a thin layer chromatography system. Specific activities of sialyltransferase and sialidase were assessed using GM3 and GD3 as substrates, respectively. The results showed significant decreases of 57.7% (p < 0.001) and 68.9% (p < 0.001), respectively, in the labeled ManNAc incorporation into GD3 and GD1a fractions in rats of the ethanol group, compared with rats of the control group. No significant changes were noted in the incorporation of labeled ManNAc into GT1a or GT1b ganglioside fractions between the ethanol and control groups. Concomitantly, compared with control rats, a decrease of 18.9% (p < 0.05), 20.6% (p < 0.05), and 15.8% (p < 0.001) was found in the sialyltransferase activity, respectively, at the whole brain, and brain Golgi and synaptosomal levels. However, dramatic increases of 32.4% (p < 0.05), 105% (p < 0.001), and 150% (p < 0.001) in sialidase activity were found, respectively, at the whole brain and brain cytosol and synaptosomal fractions of rat treated chronically with ethanol. Thus, we conclude that the deleterious actions of ethanol on the sialylation of rat brain gangliosides is specific, and the reduced sialic acid label found in GD3 and GD1a in this study is mainly due to increased activity of brain sialidase. Furthermore, the study reaffirms our tenet that, regardless of whether it is the liver or the brain, glycosylation cascade is one of the main target of the deleterious attacks of ethanol.
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PMID:Long-term ethanol consumption selectively impairs ganglioside pathway in rat brain. 975 36

The normal chronological changes in the ganglioside composition of human milk during lactation were examined by means of a high-performance thin-layer chromatography (HPTLC) micro-method with 1 ml of milk from each lactation. Six human milk ganglioside compositions were found, which were designated as GM3, GD3, GX1, GX2, GX3 and GX4. GX1-GX4, which had not been described previously, were tentatively assumed to be gangliosides of the c-series because they did not react to the GA1 antibody after sialidase treatment. GD3 was the major composition of the colostrum (GD3, 42-56%; GM3, 2.22-6.5%). GM3 increased sharply at eight days postpartum (GD3, 32.22%; GM3, 27.79%) and then increased gradually after eight days until examined at seven weeks postpartum (GM3/GD3, 0.84-2.67). The newly found GX1-GX4 showed some variability in the percentage composition between individuals, and there were no distinct differences between the colostrum and the later milk. The drastic compositional changes in GM3 and GD3 during lactation might have some biological significance, such as in immunological activity, somatic growth and the nervous system.
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PMID:Chronological changes in the ganglioside composition of human milk during lactation. 1036 77

Gangliosides of eye lenses from normal and experimentally induced diabetic rats were investigated by methods including glycolipid-overlay techniques. Adult rat eye lens showed a complex ganglioside pattern that consisted of six major ganglioside components. These gangliosides were identified as GM3, GD3, GD1a, GD1b, GT1b, and GQ1b based upon their reactivity to anti-GM1 antibody after in situ sialidase treatment and mobility on thin-layer chromatography (TLC). Gangliosides in eye lens were further characterized by TLC-immunostaining with A2B5, a specific monoclonal antibody directed toward c-series gangliosides. Eye lens contained GT3 as the main c-series ganglioside component. Unexpectedly, the relative concentration of GT3 in total gangliosides of eye lens was highest among neural and extra-neural tissues examined. Administration of streptozotocin to rats caused a severe reduction in the GT3 content in eye lenses as early as day 3 without apparent changes in the composition of major gangliosides. Alloxan failed to produce such an effect despite producing similar hyperglycemic conditions. These results suggest that rat eye lens probably contains a streptozotocin-susceptible cell type(s), which is highly enriched with c-series gangliosides.
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PMID:Gangliosides of rat eye lens: a severe reduction in the content of C-series gangliosides following streptozotocin treatment. 1104 11

Gangliosides are constituents of the cell membrane and are known to have important functions in neuronal differentiation. We employed an embryonal carcinoma stem cell line P19 as an in vitro model to investigate the expression of gangliosides during neuronal development. After treatment with retinoic acid, these cells differentiate synchronously into neuron-like cells by a series of well-defined events of development. We examined several aspects of ganglioside metabolism, including the changes of ganglioside pattern, the activities and gene expression of several enzymes at different stages of differentiation, and the distribution of gangliosides in differentiating neurons. Undifferentiated P19 cells express mainly GM3 and GD3. After P19 cells were committed to differentiation, the synthesis of complex gangliosides was elevated more than 20-fold, coinciding with the stage of neurite outgrowth. During the maturation of differentiated cells, the expression of c-series gangliosides was downregulated concomitantly with upregulation of the expression of a- and b-series gangliosides. We also examined the distribution of gangliosides in differentiating neurons by confocal and transmission electron microscopy after cholera toxin B subunit and sialidase treatment. Confocal microscopic studies showed that gangliosides were distributed on the growth cones and exhibited a punctate localization on neurites and soma. Electron microscopic studies indicated that they also are enriched on the plasma membranes of neurites and the filopodia as well as on the lamellipodia of growth cones during the early stage of neurite outgrowth. Our data demonstrate that the expression of gangliosides in P19 cells during RA-induced neuronal differentiation resembles that of the in vivo development of the vertebrate brain, and hence validates it as an in vitro model for investigating the function of gangliosides in neuronal development.
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PMID:Expression of gangliosides in neuronal development of P19 embryonal carcinoma stem cells. 1105 5

Severe neurological deficits and mental retardation are frequently associated with disrupted ganglioside metabolism in a variety of gangliosidoses and lysosomal storage disorders. Accumulation of glycosphingolipids (GSLs) in the central nervous system (CNS) of humans and animals affected with several types of mucopolysaccharidoses (MPS) also correlates with the severity of neurological dysfunction. Mucopolysaccharidosis type IIID (MPS IIID) is characterized by deficiency in lysosomal N-acetylglucosamine 6-sulfatase activity and the accumulation and excretion of heparan sulfates and N-acetylglucosamine 6-sulfate. We investigated the metabolism of GSLs in the prenatal, neonatal, and adult MPS IIID caprine brains and an MPS experimental cell culture model. The amounts of total glycolipids in prenatal, neonatal, and adult MPS IIID caprine brains were about 2-fold higher than those in control samples. GM3, GD3, and lactosyl ceramide were the principal GSLs which abnormally accumulated in caprine MPS IIID brains. These changes may be, in part, due to the reduction of sialidase and UDP-N-acetylgalactosamine:GM3 N-acetylgalactosaminyltransferase (GalNAc-T) activities in MPS IIID caprine brain. To further examine the possible mechanism of GSL accumulation in MPS IIID brains, we employed a cell culture model using suramin-treated neuronal cultures of differentiated P19 cells. HPTLC analysis showed elevated GSLs in suramin-treated cells. Metabolic pulse-chase labeling study revealed that the GSL accumulation in suramin-treated cells may be attributed to both disturbed biosynthesis and significantly slower degradation of GSLs. In addition, the consistency of observations in the cell culture and caprine models supports the cell culture system as a means of evaluating GSL metabolic perturbations.
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PMID:Metabolic studies of glycosphingolipid accumulation in mucopolysaccharidosis IIID. 1124 30

Unlike microbial sialidases, mammalian sialidases possess strict substrate specificity, for example the human membrane-associated sialidase, which hydrolyzes only gangliosides. To cast light on the molecular basis of this narrow substrate preference, predicted active site amino-acid residues of the human membrane sialidase were altered by site-directed mutagenesis. When compared with the active site amino-acid residues proposed for Salmonella typhimurium sialidase, only five out of 13 residues were found to be different to the human enzyme, these being located upstream of the putative transmembrane region. Alteration of seven residues, including these five, was followed by transient expression of the mutant enzymes in COS-1 cells and characterization of their kinetic properties using various substrates. Substitution of glutamic acid (at position 51) by aspartic acid and of arginine (at position 114) by glutamine or alanine resulted in retention of good catalytic efficiency toward ganglioside substrates, whereas other substitutions caused a marked reduction. The mutant enzyme E51D exhibited an increase in hydrolytic activity towards GM2 as well as sialyllactose (which are poor substrates for the wild-type) with change to a lower Km and a higher Vmax. R114Q demonstrated a substrate specificity shift in the same direction as E51D, whereas R114A enhanced the preference for gangliosides GD3 and GD1a that are effectively hydrolyzed by the wild-type. The inhibition experiments using 2-deoxy-2,3-didehydro-N-acetylneuraminic acid were consistent with the results in the alteration of substrate specificity. The findings suggest that putative active-site residues of the human membrane sialidase contribute to its substrate specificity.
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PMID:Site-directed mutagenesis of human membrane-associated ganglioside sialidase: identification of amino-acid residues contributing to substrate specificity. 1129 36

To examine the specificity of monoclonal antibody A2B5, four A2B5-reactive gangliosides (designated as G-1, G-2, G-3 and G-4) were purified from bonito fish brain. Ganglioside-1, -2, and -3 migrated above GD1b, below GQ1b, and far below GQ1b on thin-layer chromatography. Ganglioside-4 had the slowest chromatographic mobility and migrated below G-3. The structures of these gangliosides were characterized by overlay analysis with glycolipid-specific ligands, product analysis after sialidase or mild acid treatment, and electrospray ionization-mass spectrometry (ESI-MS). Accordingly, G-1, G-2 and G-3 were identified to be GT3, GQ1c and GP1c, respectively. The ganglioside G-4 was shown to have the following structure: NeuAc-NeuAc-NeuAc-Galbeta1-3Gal NAcbeta1-4(NeuAc-NeuAc-NeuAcalpha2-3)Galbeta1-4Glcbeta1-1'Cer. The antibody A2B5 reacted with these c-series gangliosides, but not with GD3 and other gangliosides and neutral glycosphingolipids. The antigenic epitope for A2B5 was assumed to include the trisialosyl residue connected to the inner galactose of the hemato- or ganglio-type oligosaccharide structure of gangliosides. Phylogenetic analysis of brain gangliosides using the A2B5 preparation demonstrated that c-series gangliosides are enriched in lower animals, especially bony fish of different species. The monoclonal antibody A2B5 would be a useful tool for examining the distribution and function of c-series gangliosides.
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PMID:The specificity of monoclonal antibody A2B5 to c-series gangliosides. 1143 74

In earlier studies we have found that incubation of low density lipoprotein (LDL) with autologous blood plasma-derived serum leads to a loss of sialic acid from lipoprotein particles. In this study we demonstrated that sialic acid removed from LDL was transferred to glycoconjugates of lipoproteins, glycoproteins and sphingolipids of human serum. This showed that human serum contained the trans-sialidase activity. Gel-filtration chromatography of human blood serum demonstrated the presence of trans-sialidase activity in lipoprotein subfractions as well as in lipoprotein-deficient serum. Trans-sialidase (about 65 kDa) was isolated from lipoprotein-deficient serum using affinity chromatography carried out with Neu5Acalpha2-8Neu5Ac-sepharose FF-6. Optimal pH values for the trans-sialidase were 3.0, 5.0 and 7.0. Calcium and magnesium ions stimulated the enzyme activity at millimolar concentrations. Isolated enzyme can remove sialic acid from LDL, IDL, VLDL, and HDL particles (in decreasing rate order). Serum trans-sialidase transferred sialic acid from glycoconjugates of plasma proteins (fetuin, transferrin) and gangliosides (GM3, GD3, GM1, GD1a, GD1b). Sialylated glycoconjugates of human blood erythrocytes also served as substrate for serum trans-sialidase. We have found that sialic acid can also be removed from N- and O-linked glycans, sialylated Le(x) and Le(a), oligosialic acids, and sphingolipid carbohydrate chains. The rate of sialic acid release decreased in the following order: alpha2,6>alpha2,3>>alpha2,8. Transferred molecule of sialic acid can form alpha2,6, alpha2,3 and to a lesser degree alpha2,8 linkage with galactose, N-acetyl-galactosamine or sialic acid of acceptors. The glycoconjugates of erythrocytes, lipoprotein particles, plasma proteins, neutral sphingolipids and gangliosides may serve as acceptors of transferred sialic acid. Trans-sialidase-treated native LDL becomes desialylated and then can induce cholesteryl ester accumulation in human aortic intimal smooth muscle cells. Thus, trans-sialidase may be involved in the early stages of atherogenesis characterized by foam cell formation.
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PMID:Human plasma trans-sialidase causes atherogenic modification of low density lipoprotein. 1168 12

Earlier we have isolated from human plasma desialylated low density lipoproteins (dLDL) and showed that, first, dLDL induce cholesterol esters accumulation--the main process accompanying atherosclerosis development. Second, the process of lipoprotein desialylation took place in plasma, and, finally, sialic acids removed from LDL are transferred to other serum glycoconjugates. In this study we have isolated from human plasma an enzyme transferring sialic acid residues (trans-sialidase) by affinity chromatography and studied its donor and acceptor specificity. Isolated enzyme in the presence of saccharide-acceptor can remove sialic acids from different lipoproteins, glycoproteins (fetuin, transferrin), and gangliosides (GM3, GD3, GM1, GD1a, GD1b). Plasma enzyme translocates alpha2-6, alpha2-3 and to a lower extent alpha2-8 bonded sialic acids. Sialoglycoconjugates of human serum erythrocytes, serum lipoproteins, glycoproteins, and gangliosides can serve as donors of sialic acid for trans-sialidase. Desialylated lipoproteins, especially dLDL, are more preferable sialic acid acceptors. Transferred sialic acid is found to be alpha2-6, alpha2-3, and alpha2-8 connected.
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PMID:Human plasma trans-sialidase donor and acceptor specificity. 1222 90


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