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)

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

Electron microscopic observations showed that the fungal metabolite brefeldin A caused disassembly of the Golgi complex in human choriocarcinoma cells and accumulation of alkaline phosphatase (ALP) in the endoplasmic reticulum (ER) and nuclear envelope, where ALP was not apparently detectable in control cells. Pulse/chase experiments with [35S]methionine demonstrated that in the control cells, ALP synthesized as a 63-kDa precursor form was rapidly converted to a 66-kDa form, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type, which was expressed on the cell surface after 30 min of chase. In contrast, in the brefeldin-A-treated cells the precursor was gradually converted to a 65-kDa form, slightly smaller than the control mature form, which was not expressed on the cell surface even after a prolonged time of chase. Kinetics of the ALP processing in the brefeldin-A-treated cells demonstrated that the precursor was initially converted to an intermediate form, partially sensitive to endo-beta-N-acetylglucosaminidase H (endo H), then to an endo-H-resistant 65-kDa form. In addition, this form was found to be sensitive to neuraminidase digestion, though its sialylation was not so complete as that of the control mature form. Taken together, these results suggest that under disassembly of the Golgi complex caused by brefeldin A, oligosaccharide-processing enzymes including sialyltransferase, an enzyme in the trans Golgi cisterna(e) and/or the trans Golgi network, might be redistributed into the ER and involved in processing of the oligosaccharides of ALP accumulating there.
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PMID:Intracellular accumulation and oligosaccharide processing of alkaline phosphatase under disassembly of the Golgi complex caused by brefeldin A. 226 2

Purified beta-N-acetylglucosaminide beta(1-4)galactosyltransferase and partially purified beta-galactoside alpha(2-6)-sialyltransferase were used to elongate and terminate glycan chains of agalacto-ovalbumin and endo-beta-N-acetylglucosaminidase H-treated yeast invertase in vitro. In the presence of both transferases, 0.1 mol sialic acid was incorporated per mol agalacto-ovalbumin within 24 h. Evidence is presented to show that purification of the galactosylated intermediate increases the efficiency of sialylation. Incorporation of sialic acid into endo-beta-N-acetylglucosaminidase H-treated oligomannose glycoproteins may be useful for in vivo stabilization of these glycoproteins by preventing uptake in liver or reticuloendothelial cells.
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PMID:Galactosyltransferase-dependent sialylation of complex and endo-N-acetylglucosaminidase H-treated core N-glycans in vitro. 308 81

A method for the modification of the oligosaccharide moiety of even small amounts of purified glycoproteins by enzymatic glycosylation and deglycosylation is described. The method includes noncovalent immobilization of the glycoproteins onto the polystyrene surface of the wells of microtiter plates used as reaction tubes, deglycosylation or glycosylation by incubation either with exoglycosidases or endoglycosidases or with glycosyltransferases, and the characterization of the modified glycan structures by probing them with lectins. Placental transferrin receptor employed as a model glycoprotein was modified in amounts of as little as 100 ng removing sialic acid residues, hybrid-type glycans or all types of N-glycans with neuraminidase, endo-beta-N-acetylglucosaminidase H or peptide-N4-(acetyl-beta-glucosaminyl) asparagine amidase. Asialotransferrin receptor was alpha-2,6-sialylated with alpha-2,6-sialyltransferase from rat liver, but could not be alpha-2,3-sialylated with alpha-2,3-sialyltransferase from porcine liver. Changes in the structure and in the relative amount of the oligosaccharides could be monitored semiquantitatively with high sensitivity by the binding of digoxigenin-labeled lectins and anti-digoxigenin Fab fragments. The method is easy to use, does not require immobilization of the enzymes employed, offers simple separation of the enzymes and the product, and leaves the protein intact for further studies.
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PMID:Enzymatic modeling of the oligosaccharide chains of glycoproteins immobilized onto polystyrene surfaces. 750 10

Megalomicin (MGM) is a macrolide antibiotic which has been demonstrated previously to cause an anomalous glycosylation of viral proteins. Here we show that MGM produces profound alterations on Golgi morphology and function. The addition of MGM at 50 microM for 1 h caused a dilation of the Golgi detected by immunofluorescence staining for medial- and trans-Golgi markers. The effect of MGM was clearly more intense on the trans-side of the Golgi, as evidenced in electron microscope preparations. The effect on Golgi morphology was reversible and correlated with an impairment of glycoprotein processing in the trans-Golgi. Thus, although the vesicular stomatitis virus G protein was processed in the presence of MGM to an endoglycosidase H-resistant form, it was poorly sialylated. The sialylation of cellular proteins was also inhibited, resulting in cells with low level of sialylation on the cell surface. However MGM did not inhibit the activities of the galactosyl- or sialyltransferase as measured in vitro. MGM inhibited cis- to medial-, and more strongly, medial- to trans-Golgi transport of vesicular stomatitis virus G protein in an in vitro system, suggesting that the impairment in glycoprotein maturation observed in vivo is the result of intra-Golgi transport inhibition.
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PMID:Intra-Golgi transport inhibition by megalomicin. 863 86

The major alpha1,3fucosyltransferase activity in plasma, liver, and kidney is related to fucosyltransferase VI which is encoded by the FUT6 gene. Here we demonstrate the presence of alpha1, 3fucosyltransferase VI (alpha3-FucT VI) in the human HepG2 hepatoma cell line by specific activity assays, detection of transcripts, and the use of specific antibodies. First, FucT activity in HepG2 cell lysates was shown to prefer sialyl-N-acetyllactosamine as acceptor substrate indicating expression of alpha3-FucT VI. RT-PCR analysis further confirmed the exclusive presence of the alpha3-FucT VI transcripts among the five human alpha3-FucTs cloned to date. alpha3-FucT VI was colocalized with beta1,4galactosyltransferase I (beta4-GalT I) to the Golgi apparatus by dual confocal immunostaining. Pulse/chase analysis of metabolically labeled alpha3-FucT VI showed maturation of alpha3-FucT VI from the early 43 kDa form to the mature, endoglycosidase H-resistant form of 47 kDa which was detected after 2 h of chase. alpha3-FucT VI was released to the medium and accounted for 50% of overall cell-associated and released enzyme activity. Release occurred by proteolytical cleavage which produced a soluble form of 43 kDa. Monensin treatment segregated alpha3-FucT VI from the Golgi apparatus to swollen peripheral vesicles where it was colocalized with beta4-GalT I while alpha2,6(N)sialyltransferase remained associated with the Golgi apparatus. Both constitutive secretion of alpha3-FucT VI and its monensin-induced relocation to vesicles analogous to beta4-GalT I suggest a similar post-Golgi pathway of both alpha3-FucT VI and beta4-GalT I.
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PMID:alpha1,3Fucosyltransferase VI is expressed in HepG2 cells and codistributed with beta1,4galactosyltransferase I in the golgi apparatus and monensin-induced swollen vesicles. 1053 43

Pmel17 is a melanocyte/melanoma-specific protein that is essential for the maturation of melanosomes to form mature, fibrillar, and pigmented organelles. Recently, we reported that the less glycosylated form of Pmel17 (termed iPmel17) is sorted via the plasma membrane in a manner distinct from mature Pmel17 (termed mPmel17), which is sorted directly to melanosomes. To clarify the mechanism(s) underlying the distinct processing and sorting of Pmel17, we generated a highly specific antibody (termed alphaPEP25h) against an epitope within the repeat domain of Pmel17 that is sensitive to changes in O-glycosylation. alphaPEP25h recognizes only iPmel17 and allows analysis of the processing and sorting of iPmel17 when compared with alphaPEP13h, an antibody that recognizes both iPmel17 and mPmel17. Our novel findings using alphaPEP25h demonstrate that iPmel17 differs from mPmel17 not only in its sensitivity to endoglycosidase H, but also in the content of core 1 O-glycans modified with sialic acid. This evidence reveals that iPmel17 is glycosylated differently in the Golgi and that it is sorted through the secretory pathway. Analysis of Pmel17 processing in glycosylation-deficient mutant cells reveals that Pmel17 lacking the correct addition of sialic acid and galactose loses the ability to form fibrils. Furthermore, we show that addition of sialic acid affects the stability and sorting of Pmel17 and reduces pigmentation. Alterations in sialyltransferase activity and substrates differ between normal and transformed melanocytes and may represent a critical change during malignant transformation.
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PMID:Sialylated core 1 O-glycans influence the sorting of Pmel17/gp100 and determine its capacity to form fibrils. 1730 71