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)

The sialyltransferase and galactosyltransferase activities of the Golgi-rich fraction from rat liver were enhanced by the binding of wheat germ agglutinin (WGA). The sialytransferase was more sensitive than the galactosyltransferase to the WGA. Maximal stimulation of the galactosyltransferase activity resulted from the binding of 60--80 micrograms WGA to the Golgi membrane, while only 40 micrograms of WGA produced a maximal enhancement in the sialyltransferase activity. Within 5 min of WGA binding, the Golgi sialytransferase activity was doubled. After the initial binding of WGA to the Golgi fraction, the galactosyltransferase activity was decreased by 30%. However, in 15 min the activity was doubled by the binding of WGA. The activities of both enzymes were further enhanced by incubation for up to 90 min. The stimulation of both sialyltransferase and galactosyltransferase activities by WGA was reversed by N-acetyl-D-glucosamine (GlcNAc), the specific inhibitor of agglutination by WGA. Complete reversal of the enhanced activity was observed after 20--30 min in the presence of 1 micromol GlcNAc. The association constant for the binding of WGA to the Golgi membranes was calculated to be 4.16 X 10(-6) M from a Steck-Wallach plot. The 'n' value or mean binding sites was calculated as 5.26 X 10(-5) M/mg of Golgi membrane protein.
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PMID:The effect of wheat germ agglutinin on sialyl and galactosyltransferases of rat liver Golgi membranes. 48 41

We investigated biosynthesis, intracellular transport and release of beta-galactoside alpha-2,6-sialyltransferase in a dexamethasone-inducible rat hepatoma cell line. Confluent cells were induced by 10 microM dexamethasone for 24 h, and metabolically labelled with [35S]methionine/cysteine, followed by immunoprecipitation of sialyltransferase and electrophoretic/fluorographic analysis. The 35S-labelled enzyme was synthesized as a 46-kDa precursor, converted to an intermediate 47-kDa form after 1 h, and gradually to a mature form of 48 kDa within the following 3 h. By means of either tunicamycin inhibition of N-glycosylation or cleavage of N-glycans from isolated sialyltransferase using N-glycosidase F, the sizes of the precursor and the mature form were reduced to 41 kDa and 43 kDa, respectively. After a 4-h chase, treatment with endoglycosidase H revealed two distinct molecular forms of sialyltransferase, bearing either two N-acetyllactosamine-type or one oligomannose-type and one N-acetyllactosamine-type N-linked sugar chain. In addition, sialyltransferase became sensitive to neuraminidase digestion after a 4-h chase. The half-life of intracellular [35S]sialyltransferase was estimated at 3 h. A soluble form was detectable in the supernatant, 2 h after the pulse. Only 12% of the initially labelled sialyltransferase was found in the medium after 12 h, while 73% of the enzyme was degraded intracellularly. To characterize a possible intracellular degradation site, we studied intracellular transport in the presence of either secretion-blocking or acidotropic agents or protease inhibitors. Degradation was significantly delayed by all treatments. Our results show that sialyltransferase follows the secretory pathway as a membrane protein and is retained at a late Golgi stage. We suggest that the bulk of sialyltransferase in rat hepatoma cells is diverted to a post-Golgi degradation pathway. This route contrasts with the post-Golgi trafficking of beta-1,4-galactosyltransferase in HeLa cells, which is constitutively secreted [Strous, G. J. A. M. & Berger, E. G. (1982) J. Biol. Chem. 257, 7623-7628].
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PMID:Biosynthesis and intracellular transport of alpha-2,6-sialyltransferase in rat hepatoma cells. 152 30

The beta-galactoside alpha 2,6-sialyltransferase has been localized to the trans cisternae of the Golgi apparatus and the trans Golgi network where it transfers sialic acid residues to terminal positions on N-linked oligosaccharides. It is a type II transmembrane protein possessing a 9-amino acid amino-terminal cytoplasmic tail, a 17-amino acid signal anchor domain, and a 35-amino acid stem region which tethers the large luminal catalytic domain to the membrane anchor. Previous work has demonstrated that the soluble sialytransferase catalytic domain is rapidly secreted from Chinese hamster ovary cells. These results suggest that the signals for Golgi apparatus localization do not reside in the catalytic domain of the enzyme but must reside in the cytoplasmic tail, signal anchor domain, and/or stem region. To determine which amino-terminal regions are required for Golgi apparatus localization, mutant sialyltransferase proteins were constructed by in vitro oligonucleotide-directed mutagenesis, expressed in Cos-1 cells, and localized by indirect immunofluorescence microscopy. Signal cleavage-sialyltransferase mutants which consist of only the stem and catalytic domain of the enzyme are not rapidly secreted but are retained intracellularly and predominantly localized to the Golgi apparatus. However, deletion of either the stem region or the cytoplasmic tail of the membrane-bound sialyltransferase does not alter its Golgi apparatus localization. In addition, sequential replacement of the amino acids of the sialyltransferase signal anchor domain with amino acids from the signal anchor domain of a plasma membrane protein, the influenza virus neuraminidase does not alter the Golgi apparatus localization of the sialyltransferase. These observations suggest that sequences in the signal anchor region and stem region allow the Golgi apparatus localization of the membrane-bound and soluble forms of the sialytransferase, respectively, and that both regions may contain Golgi apparatus localization signals.
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PMID:The signal anchor and stem regions of the beta-galactoside alpha 2,6-sialyltransferase may each act to localize the enzyme to the Golgi apparatus. 156 12

The glycosyltransferase alpha-2,6-sialyltransferase (ST) is a Type II membrane protein localized to the Golgi apparatus. The first 44 amino acids of this protein were able to specify Golgi retention of a fused marker protein, lysozyme. This section of ST contains a transmembrane segment which serves as a non-cleaved signal anchor. When lysozyme was fused to an equivalent region of a cell surface protein it now appeared on the cell surface. Analysis of chimeras between the two proteins revealed that the transmembrane segment of ST specifies Golgi retention. Furthermore, altering this segment in full-length ST results in the protein accumulating on the cell surface. However, the retaining effect of the transmembrane domain of ST is augmented by the presence of adjacent lumenal and cytoplasmic sequences from ST. If these sequences are spaced apart by a transmembrane domain of the same length as that of ST they too can specify Golgi retention. Thus retention in the Golgi of ST appears to involve recognition of an extended region of the protein within and on both sides of the bilayer.
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PMID:Sequences within and adjacent to the transmembrane segment of alpha-2,6-sialyltransferase specify Golgi retention. 193 90

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

As described previously (I. Kijima-Suda et al., Cancer Res., 46: 858-862, 1986), a sialyltransferase inhibitor, 5-fluoro-2',3'-isopropylidene-5'-O-(4-N-acetyl-2,4-dideoxy-3,6,7,8-tetra -O- acetyl-1-methoxycarbonyl-D-glycero-alpha-D-galactooctapyranosyl)ur idine (KI-8110), inhibits pulmonary metastasis of murine colon adenocarcinoma 26 sublines of high (NL-17) and low (NL-44) metastatic potential. To investigate the mechanism of this inhibition, the effect of KI-8110 on the metastatic cascade, especially on the interaction between tumor cells and platelets which may play a crucial role in tumor cell metastasis, was examined. NL-17 cells induced irreversible platelet aggregation in heparinized human platelet-rich plasma in vitro. This activity was reduced by pretreatment of the tumor cells with KI-8110. Inhibition of aggregation was also induced by the treatment of tumor cells with neuraminidase or Limax flavus agglutinin, a lectin specific for sialic acid. Sialic acid, fucose, sialyllactose, and bovine submaxillary mucin inhibited this tumor cell-induced platelet aggregation, while galactose, mannose, lactose, alpha 1-acid glycoprotein, fetuin, and asialo-bovine submaxillary mucin did not. KI-8110 also inhibited platelet-derived growth factor-dependent growth of NL-17 cells, but showed no effect on insulin or epidermal growth factor-dependent growth of the tumor cells. Platelet-derived growth factor-induced phosphorylation of membrane protein was reduced by treatment of NL-17 cells with KI-8110. The same result was obtained in the neuraminidase-treated membrane fraction of NL-17 cells. These results suggest that KI-8110 inhibits experimental tumor cell metastasis by inhibiting the interaction between tumor cells and host platelets in at least two pathways, and this may be due to a reduction of sialic acid contents of the membrane surface of tumor cells.
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PMID:Possible mechanism of inhibition of experimental pulmonary metastasis of mouse colon adenocarcinoma 26 sublines by a sialic acid: nucleoside conjugate. 328 33

Golgi vesicles were isolated and purified from rat liver, in which the specific activities of glycosyltransferases (e.g. GM3:CMP-NeuAc sialyltransferase, GD3 synthase; GM3:UDP-GalNAc galactosaminyltransferase, GM2 synthase) were 50-60-times enriched relative to microsomes or total homogenate. Synthesis of gangliosides GM2 and GM1 in such Golgi vesicles is, in the absence of any detergents, stimulated 6-fold and 20-fold respectively by phosphatidylglycerol. Other phospholipids like phosphatidylethanolamine and phosphatidylserine are also significantly stimulatory. With 50 micrograms Golgi protein and 1 nmol UDP-GalNAc, optimal stimulation of GM2 synthase was obtained with 20 micrograms of phosphatidylglycerol and 7.5 nmol of the lipid acceptor GM3. Under the same experimental conditions this stimulation exceeds (by about 40%) that obtained with optimal amount (200 micrograms) of the detergent octylglucoside. Phosphatidylglycerol, on the other hand, has virtually no stimulatory activity on the synthesis of ganglioside GD3 either in the presence of Mg2+ or Mn2+, indicating that facilitation by phospholipid of GM3 transport into Golgi vesicles was not the basis of stimulation of GM2 synthesis. Tunicamycin inhibits the synthesis of gangliosides GM2 and GM1 in isolated Golgi vesicles, but only in the absence of detergents. In the presence of phosphatidylglycerol, GM2 synthesis, for example, was inhibited by 60% by 2 micrograms tunicamycin and more than 85% by 10 micrograms tunicamycin, per 50 micrograms Golgi membrane protein. The inhibition was stronger on GM1 synthesis: 85% with 2.5 micrograms of the antibiotic. The dependence on phosphatidylglycerol and the degree of inhibition by tunicamycin of the synthetic activities are strictly dependent on the intactness of the Golgi vesicles: both phenomena become increasingly less evident when the vesicles are pelleted, and frozen and thawed several times, and completely disappear when the vesicles are solubilized by detergents or disrupted by ultrasonication. Furthermore, tunicamycin inhibition is reversible by increased concentration of phosphatidylglycerol. All these results indicate that phosphatidylglycerol does not stimulate, and tunicamycin does not inhibit, the transferases themselves; rather, the two opposing effects might relate to carrier-mediated transport, e.g. of nucleotide sugars, across Golgi vesicles.
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PMID:Ganglioside biosynthesis in Golgi apparatus of rat liver. Stimulation by phosphatidylglycerol and inhibition by tunicamycin. 686 62

Sodium butyrate and dimethylsulfoxide (DMSO) have marked effects on the growth, morphology, and biochemistry of two human colonic adenocarcinoma cell lines in culture. Doubling times were increased between 18% and 660% while cell viability was unaffected. Both cell lines formed colonies in soft agar in the absence of butyrate of DMSO, but no colonies were observed in the presence of these agents. However, no differences in in vivo tumorigenicities, when cells were implanted in athymic mice, were seen following treatment. Gross morphological alterations including cell enlargement, process formation, and cellular flattening occurred during culture in butyrate or DMSO. Acrylamide gel electrophoresis in sodium dodecyl sulfate revealed no change in membrane protein constituents, but autoradiographic analysis of membrane glycoproteins demonstrated differences between treated and untreated cells. Ganglioside compositions were altered, and a sialyltransferase required for the synthesis of GM3 ganglioside was elevated by butyrate. Although cytoplasmic aminooligopeptidase remained unaffected by butyrate or DMSO, brush border-associated activity was enhanced by butyrate. Alkaline phosphatase also rose dramiatically during culture in butyrate but was not enhanced by DMSO.
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PMID:Effects of sodium butyrate and dimethylsulfoxide on biochemical properties of human colon cancer cells. 735 11

The single transmembrane domains (TMDs) of the resident glycosylation enzymes of the Golgi apparatus are involved in preventing these proteins moving beyond the Golgi. It has been proposed that either the TMDs associate, resulting in the formation of large oligomers of Golgi enzymes, or that they mediate the lateral segregation of the enzymes between lipid microdomains. Evidence for either type of interaction has been sought by examining the retention of sialyltransferase (ST), an enzyme of the mammalian trans Golgi. No evidence could be obtained for specific interactions or 'kin recognition' between ST and other proteins of the trans Golgi. Moreover, it is shown that the previously described kin recognition between enzymes of the medial Golgi involves the lumenal portions of these proteins rather than their TMDs. To investigate further the role of the ST TMD, the effects on Golgi retention of various alterations in the TMD were examined. The addition or removal of residues showed that the efficiency of retention of ST is related to TMD length. Moreover, when a type I plasma membrane protein was expressed with a synthetic TMD of 23 leucines it appeared on the cell surface, but when the TMD was shortened to 17 leucines accumulation in the Golgi was observed. These observations are more consistent with lipid-based sorting of ST TMD, but they also allow for reconciliation with the kin recognition model which appears to act on sequences outside of the TMD.
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PMID:An investigation of the role of transmembrane domains in Golgi protein retention. 758 99

Previous studies indicate that sialylation of lipopolysaccharide (LPS) by host CMP-N-acetylneuraminic acid (CMP-NANA) catalyzed by bacterial sialyltransferase rendered gonococci resistant to killing by phagocytes, to entry into epithelial cell lines, to killing by immune serum and complement, and to absorption of complement component C3. These results have been confirmed by comparing a sialyltransferase-deficient mutant (strain JB1) with its parent (strain F62) in appropriate tests. In contrast to F62, JB1 was very susceptible to killing by human polymorphonuclear phagocytes in opsonophagocytosis tests and incubation with CMP-NANA did not decrease the level of killing. The inherent resistance of F62 in these tests was probably due to LPS sialylation by CMP-NANA and lactate present in the phagocytes. A JB1 variant expressing the invasion-associated Opa protein was as able to enter Chang human conjunctiva epithelial cells as an Opa-positive variant of F62, suggesting that the sialyltransferase is not required for Opa-mediated entry. After incubation with CMP-NANA, the number of F62 variant gonococci entering cells but not that of JB1 variant gonococci was drastically reduced. Both JB1 and F62 were killed by incubation with rabbit antibody to gonococcal major outer membrane protein, protein I, and human complement, but only F62 was rendered resistant to the killing by incubation with CMP-NANA. Finally, both JB1 and F62 absorbed similar amounts of complement component C3 and the binding was decreased by incubation with CMP-NANA only for the wild type, F62.
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PMID:Functional characterization of a sialyltransferase-deficient mutant of Neisseria gonorrhoeae. 875 78


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