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:2.4.99.6 (sialyltransferase)
1,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

CMP-NeuAc: Gal beta 1,4GlcNAc alpha 2,6 sialyltransferase (alpha 2,6-ST) [EC 2.4.99.1] is developmentally regulated, shows a high degree of tissue specificity, and appears to play a role in oncogenic transformation and metastasis. In the present study, we have performed the first detailed analysis of the expression of alpha 2,6-ST and alpha 2,6-linked sialoglycoconjugates in human brain tumors. We used a polyclonal, monospecific anti-rat alpha 2,6-ST antibody and the alpha 2,6-linked sialic acid-specific lectin, Sambucus nigra agglutinin (SNA) for histochemical studies, and a human alpha 2,6-ST-specific cDNA probe for Northern analysis. Meningiomas, chordomas and craniopharyngiomas frequently expressed alpha 2,6-ST and alpha 2,6-linked sialoglycoconjugates. Among the different meningioma subtypes, meningothelial meningiomas stained more strongly with both anti-alpha 2,6-ST antibody and SNA than the fibroblastic and anaplastic meningiomas. On the other hand, all tumors of glial origin and medulloblastomas were virtually devoid of either alpha 2,6-ST or alpha 2,6-linked sialoglycoconjugate expression. Moreover, very weak to negligible expression of both alpha 2,6-ST and alpha 2,6-linked sialoglycoconjugates was observed in brain metastases. In conclusion, alpha 2,6-ST and alpha 2,6-linked sialoglycoconjugate expression is associated with non-neuroectodermal epithelial-like tumors.
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PMID:The expression of Gal beta 1,4GlcNAc alpha 2,6 sialyltransferase and alpha 2,6-linked sialoglycoconjugates in human brain tumors. 883 41

A bacterial sialyltransferase, named sialyltransferase 0160, was purified from a marine bacterium that had been isolated from seawater from Sagami Bay, Kanagawa. This strain has been identified as Photobacterium damsela, and named P. damsela JT0160. Sialyltransferase 0160 was purified 688-fold to homogeneity from the crude extract of the cells with a yield of 19% using a combination of anion exchange chromatography, hydroxyapatite chromatography, gel filtration chromatography, and affinity chromatography. The purified enzyme migrated as a single band (61 kDa) on sodium dodecyl sulfate-polyacrylamide gel. This sialyltransferase was found to be a beta-galactoside alpha 2,6-sialyltransferase [EC 2.4.99.1] which catalyzes the incorporation of NeuAc from CMP-NeuAc into the galactose residue of the carbohydrate chain at position 6 on the basis of an analysis of the enzymatic reaction products with HPLC, 1H-, 13C-NMR spectroscopy, and fast atom bombardment mass spectroscopy.
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PMID:Purification and characterization of a marine bacterial beta-galactoside alpha 2,6-sialyltransferase from Photobacterium damsela JT0160. 886 51

ST6Gal I (beta-galactoside alpha 2,6-sialyltransferase, SiaT-1, ST6N, EC 2.4.99.1) mediates the attachment of the alpha 2,6-sialyl linkage common on N-linked glycans. Previous work suggests substantial inter-species conservation in SIAT1, the gene encoding ST6Gal I. In human and in rat, hepatic-specific SIAT1 transcription is initiated at Exon I. Here we report a surprising departure in the structural organization of the murine ST6Gal I gene. By a combination of primer extension analysis, 5'-RACE analysis, and analysis of genomic sequences, we show that the murine hepatic ST6Gal I mRNA contains a novel region 5' of Exon I. This novel sequence is encoded on a discrete upstream exon, Exon H. In contrast to human and rat hepatic ST6Gal I, the murine mRNA is transcriptionally initiated at the start of Exon H. Differential mRNA blot analysis indicates that transcripts containing Exon H sequences are preferentially expressed in liver.
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PMID:Murine hepatic beta-galactoside alpha 2,6-sialyltransferase gene expression involves usage of a novel upstream exon region. 914 64

Rat serum was found to contain an inhibitor of pure alpha2-6 sialyltransferase (EC 2.4.99.1). The inhibitor was a high Mr protein isolated by molecular filtration on Sephadex G100, followed by anion exchange chromatography on Sephadex DEAE A25, then separation on Sepharose CL 4B, and finally by isoelectric focusing. Electrophoretic separation and subsequent N-terminal sequence analysis of the active inhibitor preparation showed the presence of two protein components, identified as rat C-reactive protein, and rat alpha1 macroglobulin. Pure rat CRP did not inhibit alpha2-6 sialyltransferase. Treatment of the inhibitor preparations with monospecific antibodies against rat alpha1 macroglobulin blocked inhibitory activity in a dose-dependent manner. The results present strong evidence that alpha1 macroglobulin is capable of acting as an inhibitor of alpha2-6 sialyltransferase. No inhibition of galactosyltransferase (EC 2.4.1.38) could be detected, indicating that the interaction with alpha1 macroglobulin may have specificity among the glycosyltransferases. The entrapment of alpha2-6 sialyltransferase by alpha1 macroglobulin presented here occurs in vitro and will require further in vivo investigations to determine the precise physiological significance. Independent of the physiologic significance is the finding that this interaction occurs in vitro, which, pending elucidation of the precise mechanism and specificity, may provide a new tool for investigations into the functional significance of sialylation, and potentially for use or design of new inhibitors of therapeutic value in physiologic conditions involving altered levels of sialylation.
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PMID:Identification of rat alpha1 macroglobulin as an inhibitor of rat Galbeta1-4GlcNAc alpha2-6 sialyltransferase. 937 81

Quinic acid (4) was transformed into phosphitamides 6, 14, and 15, which could be readily linked to 5'-O-unprotected cytidine derivative 7; ensuing oxidation of the obtained phosphite triesters with tert-butylhydroperoxide furnished the corresponding phosphate triesters 8, 16, and 17, respectively. Hydrogenolytic debenzylation of the phosphate moiety, base catalysed removal of acetyl protective groups, and basic hydrolysis of the methylester of the quinic acid moiety furnished CMP-Neu5Ac analogues 1-3. In order to measure their inhibition of sialyltransferases, a nonradioactive sialyltransferase assay [employed for alpha(2-6)-sialyltransferase from rat liver (EC 2.4.99.1)] based on reversed-phase HPLC separation of UV-labelled acceptor 20 (p-nitrophenyl glycoside of N-acetyllactosamine) from the UV-labelled product 21 (p-nitrophenyl glycoside of sialyl alpha(2-6')-N-acetyllactosamine) and p-nitrophenylalanine as internal standard was developed. The assay reproduced the reported K(M) values for CMP-Neu5Ac and N-acetyllactosamine and the Ki values for CDP. 1 and 2 turned out to be potent sialyltransferase inhibitors.
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PMID:New sialyltransferase inhibitors based on CMP-quinic acid: development of a new sialyltransferase assay. 961 21

Hamster cell lines are common hosts for recombinant protein production, e.g. erythropoietin (Epo). Terminal sialylation of native human proteins is characteristically in both alpha-2,3 and alpha-2,6 linkage to galactose at the termini of N-linked oligosaccharides but only in alpha-2,3 linkage in recombinant proteins expressed in hamster cells which do not express alpha-2, 6-sialyltransferase (ST6GalI) (EC 2.4.99.1). This difference could alter the bioactivity of certain recombinant proteins. Chinese hamster ovary (CHO) cells stably transfected with human ST6GalI cDNA linked to the hamster metallothionein II promoter expressed highly inducible authentic ST6GalI activity. Untransfected CHO cells and CHO cells stably expressing ST6GalI cDNA when transfected with a human Epo cDNA expression cassette secreted immunoreactive Epo. Human Epo from singly transfected Pro-5 CHO cells induced significant reticulocytosis (7.00+/-1.58%; mean+/-S.D. % reticulocytes; control conditioned medium 3.04+/-1.29%; P<0.0024), whereas Epo from Pro-5 cells coexpressing ST6GalI elicited a more modest reticulocytosis (4.62+/-1.02%). Thus for recombinant human Epo, engineering CHO cells to express ST6GalI activity does not enhance Epo bioactivity in vivo in mice. The availability of CHO cells that express high levels of ST6GalI activity now enables systematic studies to determine the functional requirement for this form of sialylation in recombinant human proteins.
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PMID:Stable expression of human alpha-2,6-sialyltransferase in Chinese hamster ovary cells: functional consequences for human erythropoietin expression and bioactivity. 983 8

The cDNAs encoding soluble forms of human beta-1, 4-galactosyltransferase I (EC 2.4.1.22), alpha-2,6-sialyltransferase (EC 2.4.99.1), and alpha-1,3-fucosyltransferase VI (EC 2.4.1.65), respectively, have been expressed in the methylotrophic yeast Pichia pastoris. The vector pPIC9 was used, which contains the N-terminal signal sequence of Saccharomyces cerevisiae alpha-factor to allow entry into the secretory pathway. The recombinant enzymes had similar kinetic properties as their native counterparts. Their identity was confirmed by Western blotting. Recombinant enzymes may be used for in vitro synthesis of oligosaccharides.
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PMID:Expression of functional soluble forms of human beta-1, 4-galactosyltransferase I, alpha-2,6-sialyltransferase, and alpha-1, 3-fucosyltransferase VI in the methylotrophic yeast Pichia pastoris. 1062 93

Two novel assay systems for the determination of sialyltransferase activity using a tetramethylrhodamine-labeled disaccharide Galbeta1-4GlcNAc (2) as the acceptor are described. The TMR-labeled disaccharide 2 was synthesized by directly coupling Galbeta1-4GlcNAc-O-(CH(2))(6)NH(2) (1) with 5-tetramethylrhodamine N-hydroxysuccinimide ester. The K(m) value for compound 2 obtained with alpha-2,6-sialyltransferase from rat liver (EC 2.4.99.1) was 160 +/- 20 microM. After incubation of compound 2 with sialyltransferase the product and the unreacted acceptor substrate were separated either by thin-layer chromatography (TLC) on C-18 silica gel plates or by polyacrylamide gel electrophoresis (PAGE). The density of the spots on the TLC plates and the fluorescence of the bands on the gel were quantified. The assay conditions were optimized using crude bovine colostrum extract and also alpha-2, 6-sialyltransferase from rat liver. The detection limits for the TLC and PAGE assays were 1 and 0.4 microU of the rat liver enzyme, respectively. Either assay allows the parallel investigation of several samples at a time and is useful for the testing of fractions during enzyme purification.
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PMID:Thin-layer chromatography and polyacrylamide gel electrophoresis-based assays for sialyltransferases using tetramethylrhodamine-labeled acceptors. 1099 67

Sialyltransferase activity has traditionally been studied by determining the rate at which the enzyme transfers a labeled donor sugar to an acceptor substrate. These types of assays can be difficult to quantitate, and the separation of untransfered donor sugar from the sialylated acceptor is time-consuming. The biosensor-based method described here is both rapid and semi-automated. The NeuAc-alpha2-6Gal-R-specific lectin Sambucus nigra agglutinin (SNA) immobilized to the carboxymethyl dextran surface of a BIAcore sensor chip was used to detect and measure the formation of the NeuAc-alpha2-6Gal-R moieties. The sialyltransferase assays were carried out using modified protocols based on the method described in Rearick, J.I., Sadler, J.E., Paulson, J.C., and Hill, R.L. (1979) Enzymatic characterization of betaD-galactoside alpha2-3 sialyltransferase from porcine submaxillary gland. J. Biol. Chem., 254, 4444-4451. The complete assay mixture was simply diluted before injection into the instrument. All injections were performed automatically using the robotics of the BIAcore instrument. Using this technique it is possible to detect product from 0.4 microU of commercial Galbeta1-4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) (ST6Gal I). One unit of sialyltransferase is defined as the quantity that will transfer 1 micromol of N-acetylneuraminic acid from cytidine monophosphate (CMP)-N-acetylneuraminic acid to asialofetuin per min at pH 6.5 and 37 degrees C. The method described here requires as little as 10 microl total assay volume, thus reducing the consumption of reagents. In addition, the sample is completely recoverable from the sensor chip surface, which allows for downstream analysis of the reaction product if desired. This method eliminates the need for labeled donor and acceptor molecules and does not require the separation of the substrates from the product before analysis. Although some kinetic properties of the enzyme can be estimated using this method, further development and validation is required. The method is most useful in determining qualitative estimates of ST6Gal I activity in tissue extracts and in characterizing the production of enzymes in cultured cell systems. The use of a microtiter plate assay format enables the rapid screening of multiple fractions for sialyltransferase activity.
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PMID:A rapid, semi-automated method for detection of Galbeta1-4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) activity using the lectin Sambucus nigra agglutinin. 1144 35

BLAST analysis of the human and mouse genome sequence databases using the sequence of the human CMP-sialic acid:beta-galactoside alpha-2,6-sialyltransferase cDNA (hST6Gal I, EC2.4.99.1) as a probe allowed us to identify a putative sialyltransferase gene on chromosome 2. The sequence of the corresponding cDNA was also found as an expressed sequence tag of human brain. This gene contained a 1590 bp open reading frame divided in five exons and the deduced amino-acid sequence didn't correspond to any sialyltransferase already known in other species. Multiple sequence alignment and subsequent phylogenic analysis showed that this new enzyme belonged to the ST6Gal subfamily and shared 48% identity with hST6Gal-I. Consequently, we named this new sialyltransferase ST6Gal II. A construction in pFlag vector transfected in COS-7 cells gave raise to a soluble active form of ST6Gal II. Enzymatic assays indicate that the best acceptor substrate of ST6Gal II was the free disaccharide Galbeta1-4GlcNAc structure whereas ST6Gal I preferred Galbeta1-4GlcNAc-R disaccharide sequence linked to a protein. The alpha2,6-linkage was confirmed by the increase of Sambucus nigra agglutinin-lectin binding to the cell surface of CHO transfected with the cDNA encoding ST6Gal II and by specific sialidases treatment. In addition, the ST6Gal II gene showed a very tissue specific pattern of expression because it was found essentially in brain whereas ST6Gal I gene is ubiquitously expressed.
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PMID:Identification and functional expression of a second human beta-galactoside alpha2,6-sialyltransferase, ST6Gal II. 1260 28


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