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)

The hepatic acute phase response is accompanied by increased levels of Gal beta 1-4GlcNAc alpha 2,6-sialyltransferase activity in liver and in circulation. Previous studies suggested that cytokines and glucocorticoids mediate the induction of this sialyltransferase activity. In this study the regulation of sialyltransferase expression by dexamethasone in H35 rat hepatoma cells is assessed by Northern hybridization and enzyme activity assays. Exposure of H35 cells to 1 microM dexamethasone for 24 h causes a 3-4-fold enrichment of sialyltransferase mRNA and a corresponding increase in enzymatic activity. The induction of sialyltransferase mRNA begins within 3 h of dexamethasone treatment and reaches a plateau within 24 h. Sialyltransferase mRNA induction is dose dependent; the minimum concentration of dexamethasone necessary for induction is 10(-8) M, and induction was maximal at 10(-6) M. Induction is sensitive to actinomycin D, suggesting that regulation may be exerted by altering the rate of mRNA synthesis. Puromycin and cycloheximide are ineffective in blocking induction, suggesting that de novo protein synthesis is not required for induction. Finally, dexamethasone alone is sufficient for maximum induction of sialyltransferase mRNA. In contrast, maximal induction of alpha 1-acid glycoprotein, a well studied hepatic acute phase reactant, requires both dexamethasone and cytokines, implying that different pathways exist for the induction of participants in the acute phase response.
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PMID:Regulation of beta-galactoside alpha 2,6-sialyltransferase gene expression by dexamethasone. 291 88

The Ia+ B cell lymphoma, AKTB-1b, fails to stimulate thymic lymphocytes in a one-way mixed lymphocyte reaction unless pretreated with sialidase or inhibitors of N-linked oligosaccharide processing. A comparison of different sialidases and sialyltransferases suggests that the removal of only a subset of total surface sialic acid, rather than net desialylation of the cell surface, is required. Three sialidases were compared, including Vibrio cholerae (VC) and Clostridium perfringens (CP), which will cleave alpha 2-3, alpha 2-6, and alpha 2-8, sialic acid linkages, and Newcastle Disease virus (NDV), which will remove only alpha 2-3 and alpha 2-8 linked sialic acid. When treated with equivalent units of sialidase, CP-, VC-, and NDV-treated cells were 24-fold, sixfold, and threefold better stimulators than untreated cells. In contrast, VC released 1.3-fold and 2.5-fold more sialic acid per cell than did CP or NDV, respectively. Furthermore, VC was superior in reducing the levels of binding of the sialic acid-specific lectin, Limulus polyphemus agglutinin, in exposing Gal beta 1-3GalNAc and Gal beta 1-4GlcNAc residues, and in desialylating gangliosides. Two-dimensional gel analysis indicated that VC and CP were both equal and superior to NDV in the desialylation of iodinatable cell-surface proteins, including H-2Kk, I-A beta k, and a highly sialylated 65,000 dalton protein of unknown identity. Maximal resialylation of CP-treated cells with exogenously added CMP-NANA and either the alpha 2-3(Gal beta 1-3GalNAc) or alpha 2-6(Gal beta 1-4GlcNAc) sialyltransferase did not reduce the stimulatory capacity of these cells. However, resialylation of VC-treated cells with just CMP-NANA alone resulted in 49% reversal of their stimulatory capacity, and no additional reversal could be achieved with either of the sialyltransferases. Although the alpha 2-6(Gal beta 1-4GlcNAc) sialyltransferase was capable of adding back approximately 10% of the sialic acid removed, the endogenous activity added back approximately 0.1% of the total sialic acid removed. SDS-PAGE gels of the sialylated cells indicated that the exogenously added sialyltransferase labeled many different proteins, whereas the endogenous activity labeled far fewer proteins, predominantly in 46,000 and 25,000 m.w. range. Both the desialylation and resialylation data suggest that the sialidase-dependent stimulation is due to the desialylation of specific membrane structures. Together with previous studies, these data suggest that the sialic acids involved are probably alpha 2-6 linked to N-linked glycosyl moieties.
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PMID:Cell surface sialic acid influences tumor cell recognition in the mixed lymphocyte reaction. 295 14

The activation of human T-lymphocytes by anti-CD3 antibodies and interleukin-2 results in a marked increase in apparent molecular weight of the major cell-surface sialoglycoprotein. Both forms of the sialoglycoprotein were identified as leukosialin by a monospecific antiserum, and the differences in molecular weight were found to be due to changes in the carbohydrate structures. Our results suggest that resting T-lymphocytes express on leukosialin the disialotetrasaccharides NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)Gal-NAc-Ser/Thr, whereas activated human T-cells carry on leukosialin exclusively the more complex structures NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc-Ser/Thr. The radical shift in the biosynthetic pathway of O-glycans in activated T-lymphocytes compared to resting cells is apparently caused by a decrease of alpha 2----6 sialyltransferase activity and by the parallel dramatic stimulation of the beta 1----6GlcNAc-transferase. Since both enzymes compete for the same precursor substrate, the coordinate changes in their activities are most likely responsible for the complete change of the carbohydrate structures on leukosialin during the activation of human T-lymphocytes.
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PMID:Human T-lymphocyte activation is associated with changes in O-glycan biosynthesis. 297 63

Sialyltransferase (Gal beta 1,4GlcNAc alpha 2,6 sialyltransferase) was localized by immunoelectron microscopy in rat liver hepatocytes using affinity-purified antibodies. Immunoreactivity for sialyltransferase was found in the Golgi apparatus, where it was restricted to an interconnected system consisting of the trans-cisternae and the trans-tubular network. This region of the Golgi apparatus exhibited both TPPase and CMPase activity and was the intracellular site where sialic acid residues bound to glycoprotein were detected using the Limax flavus lectin. Sialyltransferase and sialic acid residues were not detected in medial and cis-cisternae of the Golgi apparatus. These findings suggest that in rat hepatocytes sialylation of N-linked glycoproteins occurs in the complex formed by the trans-cisternae and the trans-tubular network of Golgi apparatus.
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PMID:Demonstration of an extensive trans-tubular network continuous with the Golgi apparatus stack that may function in glycosylation. 300 Jun 3

Purified lactotetraosylceramide (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc1-Cer) was tested for its ability to accept [14C]sialic acid from CMP-[14C]sialic into monosialoganglioside fractions in the presence of membrane fractions purified from human colorectal carcinoma cells (SW1116). Membrane fractions were isolated by three different methods: sucrose density centrifugation, CMP-agarose gel column chromatography, and LcOse4 gel chromatography. We optimized the incubation conditions for detergent dependency (taurocholate), pH (6.3), and acceptor concentration. The sialyltransferase activity was dependent on membrane protein and linear for time up to at least 4 h. The LcOse4 affinity chromatography of the crude microsomal membrane pellet from these cells yielded a membrane fraction that was 136-fold enriched in LcOse4 acceptor specific activity compared to cell homogenates. The apparent Km for the sialyltransferase activity with LcOse4Cer acceptor in the presence of affinity-purified membranes was 20 microM and the Vmax was 7 pmol h-1 (100 micrograms of protein)-1. Acceptor capabilities of other core structures were 5-20-fold lower: LcOse4Cer much greater than GgOse4Cer greater than nLcOse4Cer much greater than GbOse4Cer. The enzymatic activity was purified further (900-fold) by a combination of LcOse4 and CMP affinity gels. SDS-PAGE electrophoresis of this material showed a major set of closely migrating bands of Mr 58,000-54,000 compared to authentic proteins, as well as a minor band at 27,000. We analyzed picomole quantities of the radioactive product by convenient controlled short-term hydrolyses with an endoglycoceramidase and sialidases (from four different sources) in comparison to sialylated tetrasaccharides of known structure.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and characterization of a CMP-sialic:LcOse4Cer sialyltransferase from human colorectal carcinoma cell membranes. 306 17

Sialyltransferases responsible for the formation of sugar chains in glycoproteins were studied in rat hepatoma in comparison with rat liver. Hepatoma induced by feeding Wistar rats with 3'-methyl-4-dimethylaminoazobenzene (MeDAB) was more active than Wistar liver in sialylating asialo-orosomucoid, and this was due to an increased activity of Gal(beta 1----4)GlcNAc (alpha 2----6) sialyltransferase, the major sialyltransferase in these tissues. Gal(beta 1----3,4)GlcNAc (alpha 2----3) sialyltransferase and the sialyltransferase acting on asialo-bovine submaxillary mucin were, however, decreased in the hepatoma. A similar pattern of sialyltransferase alterations was observed in regenerating liver and other tumors such as AH-109A hepatoma and Sato lung cancer, both of which had been inoculated into Donryu rats. In contrast to these sialyltransferases, the activities of the sialyltransferases responsible for the formation of gangliosides were markedly different even between Wistar and Donryu livers. When compared with Wistar liver, MeDAB-induced hepatoma was higher in lactosylceramide- and lower in GM3-sialyltransferase activity, but these two activities were both lower in AH-109A compared with Donryu liver.
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PMID:Comparative study of the levels of sialyltransferases responsible for the formation of sugar chains in glycoproteins and gangliosides in rat liver and hepatomas. 313 1

Golgi-membrane-bound Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase (CMP-N-acetylneuraminate:beta-galactoside alpha 2-6-sialyltransferase, EC 2.4.99.1) behaves as an acute-phase reactant increasing about 5-fold in serum in rats suffering from inflammation. The mechanism of release from the Golgi membrane is not understood. In the present study it was found that sialyltransferase could be released from the membrane by treatment with ultrasonic vibration (sonication) followed by incubation at reduced pH. Maximum release occurred at pH 5.6, and membranes from inflamed rats released more enzyme than did membranes from controls. Galactosyltransferase (UDP-galactose:N-acetylglucosamine galactosyltransferase; EC 2.4.1.38), another Golgi-located enzyme, which does not behave as an acute-phase reactant, remained bound to the membranes under the same conditions. Release of the alpha 2-6-sialyltransferase from Golgi membranes was substantially inhibited by pepstatin A, a potent inhibitor of cathepsin D-like proteinases. Inhibition of release of the sialyltransferase also occurred after preincubation of sonicated Golgi membranes with antiserum raised against rat liver lysosomal cathepsin D. Addition of bovine spleen cathepsin D to incubation mixtures of sonicated Golgi membranes caused enhanced release of the sialyltransferase. Intact Golgi membranes were incubated at lowered pH in presence of pepstatin A to inhibit any proteinase activity at the cytosolic face; subsequent sonication showed that the sialyltransferase had been released, suggesting that the proteinase was active at the luminal face of the Golgi. Golgi membranes contained a low level of cathepsin D activity (EC 3.4.23.5); the enzyme was mainly membrane-bound, since it could only be released by extraction with Triton X-100 or incubation of sonicated Golgi membranes with 5 mM-mannose 6-phosphate. Immunoblot analysis showed that the transferase released from sonicated Golgi membranes at lowered pH had an apparent Mr of about 42,000 compared with one of about 49,000 for the membrane-bound enzyme. Values of Km for the bound and released enzyme activities were comparable and were similar to values reported previously for liver and serum enzymes. The work suggests that a major portion of sialyltransferase containing the catalytic site is released from a membrane anchor by a cathepsin D-like proteinase located at the luminal face of the Golgi and that this explains the acute-phase behaviour of this enzyme.
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PMID:The role of a cathepsin D-like activity in the release of Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase from rat liver Golgi membranes during the acute-phase response. 314 77

Rat peritoneal leukocytes (PEC) were fractionated on Percoll gradients to prepare populations of monocytes/lymphocytes and polymorphonuclear leukocytes; adherent (monocyte enriched) and non-adherent (lymphocytes/polymorphonuclear leukocytes) cells were also isolated from PEC. Cytokines were prepared from PEC and subfractions and injected into rats to induce the acute phase reactants serum alpha 1-acid glycoprotein and sialyltransferase; negative acute phase parameters serum albumin and liver hexosaminidase were also assayed. Monocyte derived cytokines (monokines) mimicked the acute phase response of all four parameters in vivo. The sialyltransferase isoenzyme that responded to monokine was identified as the Gal beta 1----4GlcNAc alpha 2----6 isoenzyme.
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PMID:Studies of monokines as mediators of the acute phase response: effects on sialyltransferase, albumin, alpha 1-acid glycoprotein and beta-N-acetylhexosaminidase. 316 99

The biosynthesis of N-glycoloylneuraminic acid in fractionated porcine submandibular glands was investigated. The following substrates: [3H]N-acetylmannosamine, free [14C]N-acetylneuraminic acid, CMP-[14C]N-acetylneuraminic acid, [14C]N-acetylneuraminic acid linked alpha(2----3) to galactose residues, or alpha(2----6) to Gal-beta(1----4)-GlcNAc residues of porcine submandibular mucin and [14C]N-acetylneuraminic acid linked alpha(2----6) to GalNAc residues of ovine submandibular gland mucin were incubated, in the presence of cofactors, with the soluble protein, heavy membrane and microsomal fractions of porcine submandibular glands. Radio thin-layer chromatographic analysis revealed that only one substrate, CMP-[14C]N-acetylneuraminic acid, was hydroxylated. The product was identified as CMP-[14C]N-glycoloylneuraminic acid by (i) co-chromatography with non-radioactive CMP-N-glycoloylneuraminic acid standard, (ii) acid hydrolysis to free [14C]N-glycoloylneuraminic acid, (iii) alkaline hydrolysis to yield N-glycoloylneuraminic acid and 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid and (iv) transfer of [14C]N-glycoloylneuraminic acid to asialo-fetuin by sialyltransferase. 85% of CMP-N-acetylneuraminic acid hydroxylase activity was present in the soluble protein fraction, with small amounts of activity in the two particulate fractions. The CMP-N-acetylneuraminic acid hydroxylase in the soluble protein fraction had an absolute requirement for Fe2+ ions and a reducing cofactor. NADPH and NADH were by far the most effective cofactors, smaller amounts of hydroxylation could, however, be supported by ascorbic acid and 6,7-dimethyl-5,6,7,8-tetrahydrobiopterin.
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PMID:The biosynthesis of N-glycoloylneuraminic acid occurs by hydroxylation of the CMP-glycoside of N-acetylneuraminic acid. 320 54

Rat liver beta-galactoside alpha-2,6-sialyltransferase and Vibrio cholerae sialidase were used, in conjunction with CMP-N-acetyl-[3H]neuraminic acid, to probe the glycoconjugate distribution, sialylation state, and level of penultimate Gal beta 1-4GlcNAc residues on the surfaces of murine thymic lymphocytes. We report a detailed characterization of this sialyltransferase-mediated labeling system. Exogenous sialylation of intact cells is dependent on transferase, sugar nucleotide donor, cell number, and incubation time. Additionally, we have demonstrated that the system labeling the cell surface is noncytotoxic and nonmetabolic and is interacting with the entire cell population. Analysis of the exosialylated structures indicates that the sialyltransferase specifically produces an alpha 2-6 linkage on N-linked oligosaccharides. Using this labeling system, we have probed the cell surface saccharide structures of murine thymocytes and demonstrated that most Gal beta 1-4GlcNAc residues are sialylated in the native state. However, one antigen, T200 (Ly-5), is strikingly undersialylated when compared to other cell surface glycoproteins (e.g., Thy 1.2). Upon analysis of exogenously sialylated oligosaccharides, labeled sialic acid was found almost exclusively on monosialylated structures with the remainder on bisialylated oligosaccharides. This suggests that the purified sialyltransferase is very precise in its recognition of oligosaccharides present on the surface of living thymic lymphocytes. This paper illustrates the combined uses of specific glycosidases and glycosyltransferases and how they can be employed in the detailed study of selected cell surface saccharide structures on living nucleated cells.
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PMID:Sialyltransferases as specific cell surface probes of terminal and penultimate saccharide structures on living cells. 330 6


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