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:2.4.99.7 (sialyltransferase)
1,534 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two mouse L cell variant lines (CL 3 and CL 6) selected for resistance to the toxic plant lectin ricin were restricted in their ability to replicate the two alphaviruses Sindbis virus and Semliki Forest virus. CL 3 cells have been shown to exhibit increased CMP-sialic acid:glycoprotein sialyltransferase and GM3 synthetase activities, whereas CL 6 cells have been shown to contain decreased UDPgalactose:glycoprotein galactosyltransferase and UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase activities. The adsorption of Sindbis virus to CL 6 cells was considerably reduced, suggesting that the loss or inaccessibility of the receptors for Sindbis virus accounted for a major defect in virus production in these cells. In contrast, CL 3 synthesized Sindbis viral RNA and proteins but were unable to convert the precursor glycoprotein PE2 to the structural protein E2. The cleavage of PE2 to E2 was also blocked in both CL 3 and CL 6 cells infected with Semliki Forest virus.
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PMID:Restricted replication of two alphaviruses in ricin-resistant mouse L cells with altered glycosyltransferase activities. 21 29

Two variant mouse L cell lines (termed CL 3 and CL 6) have been selected for resistant to ricin, a galactose-binding lectin with potent cytotoxic activity. The resistant lines exhibit a 50 to 70% decrease in ricin binding and a 300- to 500-fold increase in resistance to the toxic effects of ricin. Crude membrane preparations of CL 3 cells have increased sialic acid content (200% of control), while the galactose, mannose, and hexosamine content is within normal limits. Both the glycoproteins and glycolipids of CL 3 cells have increased sialic acid, with the GM3:lactosylceramide ratios for parent L and CL 3 cells being 0.29 and 1.5, respectively. In contrast, the membranes of CL 6 cells have a decrease in sialic acid, galactose, and hexosamine content with mannose being normal. Both cell lines have specific alterations in glycosyltransferase activities which can account for the observed membrane sugar changes. CL 3 cells have increased CMP-sialic acid:glycoprotein sialyltransferase and GM3 synthetase activities, while CL 6 cells have decrease UDP-GlcNAc:glycoproteinN-acetylglucosaminyltransferase and DPU-galactose:glycoprotein galactosyltransferase activities. The increased sialic acid content of CL 3 cells serves to mask ricin binding sites, since neuraminidase treatment of this cell line restores ricin binding to essentially normal levels. However, the fact that neuraminidase-treated CL 3 cells are still 45-fold resistant to ricin indicates that either a special class of productive ricin binding sites is not being exposed or that the cell line has a second mechanism for ricin resistance.
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PMID:Isolation and characterization of two mouse L cell lines resistant to the toxic lectin ricin. 100 11

We have previously reported that ganglioside GM3 was remarkably increased during monocytoid differentiation of human myelogenous leukemia cell line HL-60 cells and that neolacto series gangliosides (NeuAc-nLc) were enriched during granulocytoid differentiation. In addition, HL-60 was differentiated into monocytic lineage by exogenous GM3 and into granulocytoid by NeuAc-nLc. In the present report, the enzymatic bases of glycosphingolipid biosynthesis in HL-60 during differentiation induced by 12-O-tetradecanoylphorbol-13-acetate and all-trans-retinoic acid were investigated. The following results were of particular interest. (i) Lactosylceramide alpha 2-->3 sialyltransferase (GM3 synthase) was remarkably up-regulated during monocyte differentiation, while the GM3 synthase level did not change in granulocytic differentiation. (ii) By contrast, lactosylceramide beta 1-->3N-acetylglucosaminyltransferase (Lc3Cer synthase) was down-regulated during monocytic differentiation, while the activity of Lc3Cer synthase was found to increase in granulocytic differentiation. (iii) The activities of four downstream glycosyltransferases (for synthesis of NeuAc-nLc) were found to increase or to remain unchanged during monocytic and granulocytic differentiation. These results strongly suggested the following. The dramatic GM3 increase and the decrease of NeuAc-nLc during monocytic differentiation are the consequences of the up-regulation of GM3 synthase and the down-regulation of Lc3Cer synthase, although the downstream enzymes are ready to catalyze their enzyme reactions. The notable increase of NeuAc-nLc and the relative decrease of GM3 during granulocytic differentiation are the results of the unchanged level of GM3 synthase and the up-regulation of Lc3Cer synthase together with the activation of the downstream glycosyltransferases. These results suggest that these two key upstream glycosyltransferases, GM3 synthase and Lc3Cer synthase, play critical roles in regulating the glycosphingolipid biosynthesis in HL-60 cells during differentiation. This switching mechanism of these two glycosyltransferases, together with our previous findings, might be one of the most important parts of the determining system of differentiation direction in human myeloid cells into monocytic or granulocytic lineages.
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PMID:Total metabolic flow of glycosphingolipid biosynthesis is regulated by UDP-GlcNAc:lactosylceramide beta 1-->3N-acetylglucosaminyltransferase and CMP-NeuAc:lactosylceramide alpha 2-->3 sialyltransferase in human hematopoietic cell line HL-60 during differentiation. 142 95

The role of acidic glycosphingolipids in cell growth and differentiation was investigated using the multipotent leukemia cell line K562. When GM3 was added to cell culture media, the growth of K562 cells was remarkably inhibited and the cells were shown to have megakaryocytoid morphology. Ultrastructural study demonstrated that K562 cells treated with GM3 had platelet peroxidase-positive structures, which were considered to be the specific marker of megakaryocyte. Furthermore, AP-3 directed against an epitope present on membrane glycoprotein IIIa reacted with the GM3-treated cells. Free N-acetylneuraminic acid, GM1, GM2, GD1a, and a mixture of bovine brain gangliosides containing GD1a and GT1b did not affect growth of K562 cells or show morphological changes. According to chemical analyses, GM3 content increased in megakaryocytoid differentiation induced by tetradecanoylphorbol-13-acetate, whereas GM3 decreased in erythroid differentiation induced by hemin. Enzymatic analysis showed that the GM3 increase during megakaryocytoid differentiation was a result of the sialyltransferase activation. These results indicated that exogenous GM3 induced differentiation of K562 cells into a "GM3-rich" lineage, i.e., mainly megakaryocytoid lineage, and that GM3 accumulation in the GM3-rich lineage was the result of the activation of GM3 synthase. These findings strongly suggested that GM3 ganglioside, a minor membrane component, has a crucial role in not only the differentiation induction but also the determination of the differentiation direction in pluripotent K562 cells.
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PMID:Ganglioside GM3 can induce megakaryocytoid differentiation of human leukemia cell line K562 cells. 200 80

A CMP-sialic acid: GM3 sialyltransferase (GD3 synthase) and a CMP-sialic acid: LacCer sialyltransferase (GM3 synthase) have been purified 10,000- and 3,000-fold, respectively, from the Triton X-100 extract of rat brain. The two enzymes were purified and resolved by affinity chromatography on two successive CDP-Sepharose columns by NaCl gradient elution. Final purification of GD3 synthase was achieved by specific elution from a 'GM3 acid'-Sepharose column with buffer containing GM3. Sodium dodecylsulfate-gel electrophoresis of GD3 synthase revealed a single major protein band with an apparent molecular weight of 55,000.
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PMID:Purification to homogeneity of GD3 synthase and partial purification of GM3 synthase from rat brain. 230 11

Six naturally occurring and three synthetic molecular species of lactosylceramide (LacCer) were used to examine the molecular species specificity of CMP-N-acetylneuraminate:lactosylceramide alpha 2,3-sialyltransferase in a Golgi-rich fraction of rat liver. The enzyme molecular species specificity was determined either in the presence of nonspecific lipid transfer protein or in the presence of detergents. Assays performed in the presence of transfer protein showed that for those lactosylceramide molecular species with either d18:1 or d18:0 long chain base the enzyme activity decreased linearly as the effective carbon number of the fatty acid increased. An increase in the carbon number of the long chain base decreased the activity of the enzyme twice as much as a corresponding increase in the carbon number of the fatty acid. On the other hand, when the enzyme activity was assayed in the presence of detergents, there was no significant difference in activity among the various molecular species of lactosylceramide based upon the carbon number of the fatty acid or on the presence of a double bond in the long chain base. However, the decrease in enzyme activity with an increase in the carbon number of the long chain base persisted. These results demonstrate that sialyltransferase has binding specificity with respect to the long chain base, but not the fatty acid. The apparent molecular species towards the fatty acid is related to the aqueous solubility of the various LacCer molecular species.
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PMID:Lactosylceramide molecular species specificity of rat liver CMP-N-acetylneuraminate:lactosylceramide sialyltransferase. 261 78

CMP-N-acetylneuraminate:lactosylceramide alpha-2,3-sialyltransferase is tightly associated with the luminal side of the Golgi membrane as is its lipid substrate, lactosylceramide. In order to understand the kinetics, properties, and regulation of this enzyme, it is necessary to alter the amount and type of substrate in the membrane while minimizing changes in the membrane environment or in the conformation of the enzyme. Therefore, nonspecific lipid transfer protein, which accelerates the transfer of phospholipids, cholesterol, and glycosphingolipids between membranes was used to study the properties and kinetics of rat liver CMP-N-acetylneuraminate:lactosylceramide sialyltransferase. These results are compared to those obtained in parallel experiments using detergent-solubilized substrate. Enzyme activity was increased four- to fivefold by transfer protein and was consistently higher than the activity measured in the presence of detergents. In contrast to the results obtained with detergents, the enzyme activity increased linearly with both Golgi protein and with incubation time for up to 60 min. The Km values for the water-soluble substrate, CMP-neuraminic acid, were virtually identical when determined in the presence of transfer protein (0.23 mM) or detergents (0.27 mM). On the other hand, the apparent Km values for the lipophilic substrate, lactosylceramide, were markedly different when determined in the presence of transfer protein (47.9 microM) or in the presence of detergents (1.2 microM). These observations suggest that transfer protein is a useful tool to study the properties and kinetics of membrane-bound enzymes when both the enzyme and substrate are components of the same membrane.
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PMID:Nonspecific lipid transfer protein in the assay of a membrane-bound enzyme CMP-N-acetyl-neuraminate:lactosylceramide sialyltransferase. 335 52

Three sialyltransferase activities involved in ganglioside biosynthesis were studied in Golgi-enriched preparations of rat liver: the formation of GM3, GD3 and GD1a. The conditions for the quantitative assays of these enzymatic reactions were standardized and optimized, with Triton X-100 being used as detergent. The apparent Km values of each sialyltransferase for N-acetyl-2-(5'-cytidylyl)neuraminic acid (1.5 mM with GM3 synthase, 0.2 mM with GD3 synthase, and 0.5 mM with GD1a synthase) and the respective glycolipid substrates (0.08 mM for lactosylceramide, 0.1 mM for GM3, and 0.5 mM for GM1) were determined. Competition experiments showed that the three sialyltransferase activities are three individual catalytic entities. Moreover, evidence was found that product inhibition may play a role in the regulation of the activity of sialyltransferases.
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PMID:Ganglioside biosynthesis in rat liver. Characterization of three sialyltransferases. 376 20

It has previously been shown that when the molecular species specificity of rat liver Golgi CMP-N-acetylneuraminate:lactosylceramide alpha 2,3-sialyltransferase was determined, using as the substrate lactosylceramide (LacCer) incorporated into liposomes prepared with rat liver Golgi lipids, the enzyme showed a pronounced variation in activity towards the various molecular species of LacCer (J. Lipid Res. 1989. 30: 1789-1797). In this paper, -the LacCer molecular species specificity of sialyltransferase from neuroblastoma NB2a cells was examined using five naturally occurring and three synthetic molecular species of LacCer. The enzyme activity was determined by following the formation of [14C]GM3 from CMP-[14C]neuraminic acid and individual molecular species of LacCer incorporated into liposomes. Nonspecific lipid transfer protein was included in the enzyme assay to facilitate the transfer of LacCer and other lipids between the liposomes and the membrane where sialyltransferase is located. In these enzyme assays the liposomes contained approximately 10 times more lipid phosphorus than either the microsomal fraction of NB2a cells or the Golgi fraction of rat liver. Thus, in the presence of nonspecific lipid transfer protein, the lipid composition of the membrane where sialyltransferase is located was modified to resemble the lipid composition of the liposomes. When the molecular species specificity of NB2a cell sialyltransferase was determined with LacCer incorporated into liposomes prepared with NB2a cell lipids, the enzyme showed no specificity towards the various molecular species of LacCer. However, when the molecular species specificity of NB2a cell sialyltransferase was determined with LacCer incorporated into liposomes prepared with rat liver Golgi lipids, the enzyme showed a variation in activity towards the various LacCer molecular species similar to that observed with the liver Golgi enzyme using liposomes prepared with liver Golgi lipids. Likewise, when the molecular species specificity of rat liver Golgi sialyltransferase was determined with LacCer incorporated into liposomes prepared with NB2a cell lipids, the liver enzyme then showed no specificity towards the various molecular species of LacCer. These results indicate that the lipid environment of the membrane can alter the molecular species specificity of sialyltransferase towards its lipid substrate, LacCer.
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PMID:Effect of membrane lipids on the lactosylceramide molecular species specificity of CMP-N-acetylneuraminate:lactosylceramide sialyltransferase. 835 56

Expression cloning of a cDNA for the alpha2,3-sialyltransferase (GM3 synthase) (EC 2.4.99.-) gene was performed using a GM3-lacking mouse fibroblast line L cell and anti-GM3 monoclonal antibody. Plasmids from a cDNA library generated with poly(A)+ RNA of a mouse fibrosarcoma line CMS5j and pdl3027 (polyoma T antigen) were co-transfected into L cells. The isolated cDNA clone pM3T-7 predicted a type II membrane protein with 13 amino acids of cytoplasmic domain, 17 amino acids of transmembrane region, and a large catalytic domain with 329 amino acids. Introduction of the cDNA clone into L cells resulted in the neo-synthesis of GM3 and high activity of alpha2,3-sialyltransferase. Among glycosphingolipids, only lactosylceramide showed significant activity as an acceptor, indicating that this gene product is a sialyltransferase specific for the synthesis of GM3. An amino acid sequence deduced from the cloned cDNA showed the typical sialyl motif with common features among alpha2,3-sialyltransferases. Among various mouse tissues, brain, liver, and testis showed relatively high expression of a 2.3-kilobase mRNA, whereas all tissues, more or less, expressed this gene.
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PMID:Expression cloning of mouse cDNA of CMP-NeuAc:Lactosylceramide alpha2,3-sialyltransferase, an enzyme that initiates the synthesis of gangliosides. 1009 2


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