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:2.4.99.10 (sialyltransferase)
1,547 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A 4.7-kb region of DNA sequence contained at the right end of the myxoma virus EcoRI-G2 fragment located 24 kb from the right end of the 163-kb genome has been determined. This region of the myxoma virus genome encodes homologs of the vaccinia virus genes A51R, A52R, A55R, A56R, and B1R; the myxoma virus gene equivalents have been given the prefix M. The MA55 gene encodes a protein belonging to the kelch family of actin-binding proteins, while the MA56 gene encodes a member of the immunoglobulin superfamily related to a variety of cellular receptors and adhesion molecules. A novel myxoma virus early gene, MST3N, is a member of the eukaryotic sialyltransferase gene family located between genes MA51 and MA52. Detergent lysates prepared from myxoma virus-infected cell cultures contained a virally encoded sialyltransferase activity that catalyzed the transfer of sialic acid (Sia) from CMP-Sia to an asialofetuin glycoprotein acceptor. Analysis of the in vitro-sialylated glycoprotein acceptor by digestion with N-glycosidase F and by lectin binding suggested that the MST3N gene encodes an enzyme with Galbeta1,3(4)GlcNAc alpha2,3-sialyltransferase specificity for the N-linked oligosaccharide of glycoprotein. Lectin binding assays demonstrated that alpha2,3-sialyltransferase activity is expressed by several known leporipoxviruses that naturally infect Sylvilagus rabbits. The sialyltransferase is nonessential for myxoma virus replication in cell culture; however, disruption of the MST3N gene caused attenuation in vivo. The possible implications of the myxoma virus-expressed sialyltransferase in terms of the host's defenses against infection are discussed.
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PMID:Myxoma virus encodes an alpha2,3-sialyltransferase that enhances virulence. 997 21

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

The mRNA expression of the sialyltransferase genes is regulated in a cell type specific manner. We show here the epithelium cell-specific transcriptional regulation of the human Galbeta1, 3GalNAc/Galbeta1, 4 GlcNAc alpha2,3-sialyltransferase gene (hST3Gal IV). Using a luciferase assay, we identified a functional DNA portion within hST3Gal IV genomic DNA that confers an epithelial cell line specific enhancer, located in nucleotide number (nt) -520 to -420 within the B3 promoter. This element contains two sequences similar to AP2 recognition motifs. Co-transfection with an AP2 expression vector stimulated the enhancer activity of nt -520 to -420 element eight-fold compared with that using parental vector. Site-directed mutagenesis of AP2 sites showed that two AP2 motifs are essential for enhancer activity in HeLa cells. These results suggest that AP2 plays a critical role in the epithelium-cell specific transcriptional regulation of the hST3Gal IV gene.
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PMID:Epithelial-cell-specific transcriptional regulation of human Galbeta1,3GalNAc/Galbeta1,4GlcNAc alpha2,3-sialyltransferase (hST3Gal IV) gene. 1019 43

We have previously shown that costimulation of endothelial cells with IL-1 + IL-4 markedly inhibits VCAM-1-dependent adhesion under flow conditions. We hypothesized that sialic acids on the costimulated cell surfaces may contribute to the inhibition. Northern blot analyses showed that Gal beta 1-4GlcNAc alpha 2, 6-sialyltransferase (ST6N) mRNA was up-regulated in cultured HUVEC by IL-1 or IL-4 alone, but that the expression was enhanced by costimulation, whereas the level of Gal beta 1-4GlcNAc/Gal beta 1-3GalNAc alpha2,3-sialyltransferase (ST3ON) mRNA was unchanged. Removing both alpha 2,6- and alpha 2,3-linked sialic acids from IL-1 + IL-4-costimulated HUVEC by sialidase significantly increased VCAM-1-dependent adhesion, whereas removing alpha 2,3-linked sialic acid alone had no effect; adenovirus-mediated overexpression of ST6N with costimulation almost abolished the adhesion, which was reversible by sialidase. The same treatments of IL-1-stimulated HUVEC had no effect. Lectin blotting showed that VCAM-1 is decorated with alpha 2,6- but not alpha 2,3-linked sialic acids. However, overexpression of alpha 2,6-sialyltransferase did not increase alpha 2,6-linked sialic acid on VCAM-1 but did increase alpha 2,6-linked sialic acids on other proteins that remain to be identified. These results suggest that alpha 2,6-linked sialic acids on a molecule(s) inducible by costimulation with IL-1 + IL-4 but not IL-1 alone down-regulates VCAM-1-dependent adhesion under flow conditions.
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PMID:Endothelial alpha 2,6-linked sialic acid inhibits VCAM-1-dependent adhesion under flow conditions. 1045 33

The substrate specificity of an alpha2,3-sialyltransferase (v-ST3Gal I) obtained from myxoma virus infected RK13 cells has been determined. Like mammalian sialyltransferase enzymes, the viral enzyme contains the characteristic L- and S-sialyl motif sequences in its catalytic domain. Analysis of the deduced amino acid sequences of cloned sialyltransferases suggests that v-ST3Gal I is closely related to mammalian ST3Gal IV. v-ST3Gal I catalyzes the transfer of sialic acid from CMP-NeuAc to Type I (Galbeta1-3GlcNAcbeta) II (Galbeta1-4GlcNAcbeta) and III (Galbeta1-3GalNAcbeta) acceptors. In addition, the viral enzyme also transfers sialic acid to the fucosylated acceptors Lewis(x) and Lewis(a). This substrate specificity is unlike any sialyltransferases described to date, though it is most comparable with those of mammalian ST3Gal IV enzymes. The products from reactions with fucosylated acceptors were characterized by capillary zone electrophoresis, (1)H-NMR spectroscopy and mass spectrometry. They were shown to be 2,3-sialylated Lewis(x) and 2,3-sialylated Lewis(a), respectively.
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PMID:A novel viral alpha2,3-sialyltransferase (v-ST3Gal I): transfer of sialic acid to fucosylated acceptors. 1070 30

The effect of the various glycosyltransferases on glycosphingolipids was examined, using transfected swine endothelial cell (SEC) lines. The reactivity of parental SEC to normal human serum (NHS) and Griffonia simplicifolia IB(4) (GSIB4) lectin, which binds to the Gal alpha1-3 Gal beta 1-4 GlcNAc-R (alpha-galactosyl epitope), was reduced by approximately 20% by the treatment with D-PDMP (D-threo-1-phenyl-2-decan- oylamino-3-morpholino-1-propanol), suggesting that glycosphingolipids contained by SEC have a considerable amount of the alpha-galactosyl epitope. The overexpression of two different types of glycosyltransferase, N-acetylglucosaminyl transferase III (GnT-III), as well as alpha2, 6-sialyltransferase (ST6Gal I), alpha2,3-sialyltransferase (ST3Gal III), and alpha1,2-fucosyltransferase (alpha1,2FT), suppresses the total antigenicity of SEC significantly. However, the reduction in reactivities toward NHS and GSIB4 lectin in the case of GnT-III transfectants was milder than those in other transfectants. Western blot analysis indicated that the glycoproteins in all transfectants had diminished reactivity to NHS and GSIB4 lectin to approximately the same extent. Therefore, the neutral glycosphingolipids of these transfectants were separated by thin layer chromatography, followed by immunostaining with NHS and GSIB4 lectin. The levels of the alpha-galactosyl epitope in glycosphingolipids were not decreased in the GnT-III transfectants but were in the ST6Gal I, ST3Gal III, and alpha1,2FT transfectants. These data indicate that ST6Gal I, ST3Gal III, and alpha1,2FT reduced the alpha-galactosyl epitope in both glycoproteins and glycosphingolipids, while GnT-III reduced them only in glycoproteins.
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PMID:Reduction of the major xenoantigen on glycosphingolipids of swine endothelial cells by various glycosyltransferases. 1091 Sep 78

Thomsen-Friedenreich (TF)-related blood group antigens, such as TF, Tn, and their sialylated variants, belong to a family of tumor-associated carbohydrates. The aim of the present study was to examine tumor-associated alterations of glycosyltransferases involved in the biosynthesis of the TF glycotope in colorectal carcinomas. To this end, glycosyltransferase expression was examined in 40 cases of colorectal carcinoma specimens classified according to the WHO/Union International Contre Cancer guidelines and in "normal" mucosa of the same patients. Occurrence of TF glycotope was examined by immunohistochemistry with the monoclonal antibody A78-G/A7. Expression of sialyltransferases CMP-sialic acid:Galbeta1,3GalNAc-R alpha3-sialyltransferase I and II (ST3Gal-I and ST3Gal-II) and CMP-sialic acid:Galbeta1,3GalNAc-R alpha6-sialyltransferase (ST6GalNAc-II) and of core 2 beta1,6-N-acetylglucosaminyltransferase was determined by reverse transcription-PCR in the same cryostat sections used for immunohistochemistry. Additionally, alpha2,3-sialyltransferase enzyme activity was studied in each of these tissues. The TF glycotope was detected in 7% of the normal mucosa, but in 57% of the carcinoma samples. Expression of alpha2,3-sialyltransferases ST3Gal-I, ST3Gal-II, and enzyme activity of alpha2,3-sialyltransferase was significantly increased (P < 0.001) in carcinoma specimens compared with normal mucosa. ST3Gal-I mRNA expression was significantly increased (P = 0.05) in cases showing invasion of lymph vessels. Expression of ST6GalNAc-II was significantly increased (P = 0.04) in cases with metastases to lymph nodes along the vascular trunk. Moreover, ST6GalNAc-II expression provides an prognostic factor for patient survival (log rank, P = 0.02). In an attempt to study the functional relevance of the glycosyltransferases for TF biosynthesis, SW480 colorectal cells were transfected with each of the enzymes, and cell surface expression of the TF glycotope was examined by flow cytometry. The presence of TF was not altered by transfection of the cells with either sialyltransferase ST3Gal-I or ST3Gal-II. However, successful transfection with core 2 beta1,6-N-acetylglucosaminyltransferase led to reduced expression of TF. In contrast, increased cell surface expression of TF was found after ST6GalNAc-II transfection. Thus, expression of TF on the cell surface of SW480 colorectal carcinoma cells depends on the ratio of core 2 beta1,6-N-acetylglucosaminyltransferase and ST6GalNAc-II. Earlier immunohistological studies demonstrated that TF is a prognostic factor for patient survival. Our results suggest that sialyltransferase ST6GalNAc-II is of crucial relevance for the prognostic significance of TF.
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PMID:Overexpression of sialyltransferase CMP-sialic acid:Galbeta1,3GalNAc-R alpha6-Sialyltransferase is related to poor patient survival in human colorectal carcinomas. 1138 97

Natural human interferon-gamma (hIFN-gamma) contains mainly biantennary complex-type sugar chains. We previously remodeled the branch structures of N-glycans on hIFN-gamma in Chinese hamster ovary (CHO) cells by overexpressing UDP-N-acetylglucosamine: alpha1,6-D-mannoside beta1,6-N-acetylglucosaminyltransferase (GnT-V). Normal CHO cells primarily produced hIFN-gamma having biantennary sugar chains, whereas a CHO clone, designated IM4/Vh, transfected with GnT-V, primarily produced hIFN-gamma having GlcNAcbeta1-6 branched triantennary sugar chains when sialylation was incomplete and an increase in poly-N-acetyllactosamine (Galbeta1-4GlcNAcbeta1-3)n was observed. In the present study, we introduced mouse Galbeta1-3/4GlcNAc-R alpha2,3-sialyltransferase (ST3Gal IV) and/or rat Galbeta1-4GlcNAc-R alpha2,6-sialyltransferase (ST6Gal I) cDNAs into the IM4/Vh cells to increase the extent of sialylation and to examine the effect of sialyltransferase (ST) type on the linkage of sialic acid. Furthermore, we speculated that sialylation extent might affect the level of poly-N-acetyllactosamine. We isolated four clones expressing different levels of alpha2,3-ST and/or alpha2,6-ST. The extent of sialylation of hIFN-gamma from the IM4/Vh clone was 61.2%, which increased to about 80% in every ST transfectant. The increase occurred regardless of the type of overexpressed ST, and the proportion of alpha2,3- and alpha2,6-sialic acid corresponded to the activity ratio of alpha2,3-ST to alpha2,6-ST. Furthermore, the proportion of N-glycans containing poly-N-acetyllactosamine was significantly reduced (less than 10%) in the ST transfectants compared with the parental IM4/Vh clone (22.9%). These results indicated that genetic engineering of STs is highly effective for regulating the terminal structures of sugar chains on recombinant proteins in CHO cells.
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PMID:Genetic engineering of CHO cells producing human interferon-gamma by transfection of sialyltransferases. 1151 14

The CMP-Neu5Ac:Galbeta1-3GalNAc alpha2,3-sialyltransferase (ST3Gal I, EC 2.4.99.4) is a Golgi membrane-bound type II glycoprotein that catalyses the transfer of sialic acid residues to Galbeta1-3GalNAc disaccharide structures found on O-glycans and glycolipids. In order to gain further insight into the structure/function of this sialyltransferase, we studied protein expression, N-glycan processing and enzymatic activity upon transient expression in the COS-7 cell line of various constructs deleted in the N-terminal portion of the protein sequence. The expressed soluble polypeptides were detected within the cell and in the cell culture media using a specific hST3Gal I monoclonal antibody. The soluble forms of the protein consisting of amino acids 26-340 (hST3-Delta25) and 57-340 (hST3-Delta56) were efficiently secreted and active. In contrast, further deletion of the N-terminal region leading to hST3-Delta76 and hST3-Delta105 gave also rise to various polypeptides that were not active within the transfected cells and not secreted in the cell culture media. The kinetic parameters of the active secreted forms were determined and shown to be in close agreement with those of the recombinant enzyme already described (H. Kitagawa, J.C. Paulson, J. Biol. Chem. 269 (1994)). In addition, the present study demonstrates that the recombinant hST3Gal I polypeptides transiently expressed in COS-7 cells are glycosylated with complex and high mannose type glycans on each of the five potential N-glycosylation sites.
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PMID:Delineation of the minimal catalytic domain of human Galbeta1-3GalNAc alpha2,3-sialyltransferase (hST3Gal I). 1169 Jun 53

An Escherichia coli strain expressing three recombinant enzymes, i.e., cytidine 5'-monophosphate (CMP) kinase, sialic acid aldolase and cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase, was utilized as a biocatalyst for the production of CMP-NeuAc. Both recombinant E. coli extract and whole cells catalyzed the production of CMP-NeuAc from CMP (20 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetylphosphate (60 mM), resulting in 90% conversion yield based on initial CMP concentration used. It was confirmed that endogenous acetate kinase can catalyze not only the ATP regeneration in the conversion of CMP to CDP but also the conversion of CDP to CTP. On the other hand, endogenous pyruvate kinase and polyphosphate kinase could not regenerate ATP efficiently. The addition of exogenous acetate kinase to the reaction mixture containing the cell extract increased the conversion rate of CMP to CMP-NeuAc by about 1.5-fold, but the addition of exogenous inorganic pyrophosphatase had no influence on the reaction. This E. coli strain could also be employed as an enzyme source for in situ regeneration of CMP-NeuAc in a sialyltransferase catalyzed reaction. About 90% conversion yield of alpha2,3-sialyl-N-acetyllactosamine was obtained from N-acetyllactosamine (20 mM), CMP (2 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetyl phosphate (80 mM) using the recombinant E. coli extract and alpha2,3-sialyltransferase.
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PMID:Production of cytidine 5'-monophosphate N-acetylneuraminic acid using recombinant Escherichia coli as a biocatalyst. 1235 62


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