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 found that rhabdomyosarcoma (RMS) subcellular membranes contain sialyltransferase activities for LcOse4Cer and GgOse4Cer acceptors. Chromatographic analyses and neuraminidase lability of the sialyltransferase products indicated that the principal site of sialylation was the non-reducing terminal galactosyl moiety. In order to control for the effects of cell density in culture, metastatic S4T18 RMS cells and nonmetastatic F9-4/21 RMS cells were harvested at 2 X 10(4) to 6 X 10(4) per cm2 prior to analyses. Irrespective of metastatic potential, we found that sialyltransferase-specific activities were influenced by cell densities. F9-4/21 cells, for example, at a density of 6 X 10(4), produced membranes with sialyltransferase-specific activities to LcOse4Cer 1.9-fold higher than cells at 2.1 X 10(4)/cm2. Metastatic potential (predetermined in vivo) appeared to be correlated with an accelerated effect of cell density on the sialyltransferase activity to LcOse4Cer. Metastatic S4T18 cells at 6.3 X 10(4)/cm2 yielded membranes with sialyltransferase-specific activities 5.4-fold higher than membranes from cells at 1.9 X 10(4)/cm2. Conversely, fucosyltransferase activities in the presence of LcOse4Cer were highest in non-metastatic F9-4/21 cells at low cell densities. Quantitative analyses of monosialoganglioside fractions of RMS cells were in agreement with the sialyl-transferase studies. HPLC and HPTLC analyses demonstrated the presence of glucosamine-containing monosialoganglioside with Rf identical with the radioactive products of LcOse4Cer sialylation, which increased 4.5-fold on a per mg protein basis as cell densities increased in S4T18 cells in culture from 1.9 X 10(4)/cm2 to 6.3 X 10(4)/cm2. Plasma membrane marker Na+, K+, ATPase-specific activity also increased in RMS metastatic cells in a manner comparable to that described for the sialyl-transferase activity to LcOse4Cer. Our results suggest that metastatic potential is expressed in the rate of sialylation at specific membrane sites of RMS intercellular contact. We propose a process of selection for metastasis whereby specific cell surface non-reducing galactosyl termini are recognized by intercellular transferases and lectins in the primary tumor, and the corresponding labile sialylated sites (on disseminated cells) are recognized by host neuraminidases.
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PMID:Monosialoganglioside biosynthesis by subcellular membranes of rhabdomyosarcoma cell lines differing in metastatic potential. 233

We are interested in determining whether carbohydrates are important regulatory determinants in the intracellular transport and secretion of glycoproteins. In the present study, we have used swainsonine, an indolizidine alkaloid, to modify the structure of N-glycosidically linked complex oligosaccharides. By inhibiting Golgi mannosidase II, swainsonine prevents the trimming of GlcNAc(Man)5(GlcNAc)2 to GlcNAc-(Man)3(GlcNAc)2, resulting in the formation of hybrid-type oligosaccharides. We find, from pulse-chase experiments using [35S]methionine and immunoprecipitation of individual proteins from culture media, that swainsonine treatment (1 microgram/ml) accelerated the secretion of glycoproteins (transferrin, ceruloplasmin, alpha 2-macroglobulin, and alpha 1-antitrypsin) by decreasing the lag period by 10-15 min relative to untreated cultures. The enhanced secretion was specific for glycoproteins since the secretion of albumin, a nonglycoprotein, was unaffected. When alpha 1-antitrypsin was immunoprecipitated from the cell lysates, sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorographic analysis demonstrated that the conversion of the high-mannose precursor to the hybrid form in swainsonine-treated cells occurred more rapidly (by about 10 min) than the conversion to the complex form in control cells. Since both the hybrid and complex forms of alpha 1-antitrypsin are terminally sialylated by sialyltransferase in the trans-Golgi, these results suggest that swainsonine-modified glycoproteins traverse the Golgi more rapidly than their normal counterparts. Therefore, accelerated transport within this organelle may account for the decreased lag period of glycoprotein secretion in the swainsonine-treated cultures.
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PMID:Swainsonine treatment accelerates intracellular transport and secretion of glycoproteins in human hepatoma cells. 257 69

Polysialic acid-containing glycoproteins consisting of extended chains of at least 55 sialyl residues (DP55, where DP represents degree of polymerization) are expressed on human neuroblastoma cells, CHP-134. The strategy used for detecting these unique carbohydrate structures was based on the use of two highly specific prokaryotic-derived enzyme systems and an anti-polysialosyl antibody (H.46). These probes were developed for the detection of polysialic acid on neural cell adhesion molecules (Troy, F. A., Hallenbeck, P. C., McCoy, R. D., and Vimr, E. R. (1987) Methods Enzymol. 138, 169-185). Proof for the presence of long chain multimers of sialic acid was based on two types of experiments which utilized: 1) a glycopeptide fraction of CHP-134 cells, labeled metabolically with D-[3H]GlcN and 2) a membrane fraction from CHP-134 cells which served as an exogenous acceptor of [14C] NeuNAc residues in an Escherichia coli K1 sialyltransferase assay. In vitro, this enzyme CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase catalyzes the transfer of [14C]NeuNAc from CMP-[14C]NeuNAc to exogenous acceptors containing at least 3 sialyl residues. In the first series of experiments, endo-N-acetylneuraminidase (Endo-N), a bacteriophage-derived enzyme specific for hydrolyzing poly-alpha-2,8-sialosyl chains containing a minimum of 5 sialyl residues was used. Limit Endo-N digestion of the 3H-glycopeptides from the [3H] GlcN-labeled cells released short [3H]sialyl oligomers [( 3H]DP1-6) which were degraded to [3H]NeuNAc by exosialidase. Partial Endo-N digestion released a series of [3H]sialyl oligomers extending up to DP55. The longer (DP20-55) and intermediate sized (DP10-20) oligomers were isolated and converted to short oligomers ((3H]DP1-6) by retreating with Endo-N, thus confirming their identity as homo-oligomers of alpha-2,8-linked [3H]NeuNAc residues. In the second series of experiments, a membrane fraction of CHP-134 cells was radiolabeled in vitro with [14C]NeuNAc by E. coli K1 sialyltransferase. The membrane fraction had a major portion of radioactivity that was high Mr and polydisperse (Mr 100,000-250,000) as demonstrated in sodium dodecyl sulfate-polyacrylamide gels. Using Western blotting, pre-existing material of similar size was shown to react with antibody H.46.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Extended polysialic acid chains (n greater than 55) in glycoproteins from human neuroblastoma cells. 328 35

Numerous investigations suggest that cell surface glycoconjugates, and in particular sialic acids, are directly involved in determining the metastatic phenotype. To further evaluate this hypothesis, we have used a variety of techniques to probe the cell surfaces of several metastatic variants of the murine B16 melanoma that were selected for experimental lung-colonizing ability (Fidler, I. (1973) Nature 242, 148-149) or for their ability to spontaneously metastasize from the site of a subcutaneous injection (Stackpole, C. W., Alterman, A. L., and Fornabaio, D. M. (1985) Invasion & Metastasis 5, 125-142). Using a highly sensitive high performance liquid chromatography sialic acid assay in conjunction with Vibrio cholerae sialidase, we find that none of these metastatic variants differ significantly in their overall levels of cell surface sialic acid. Using highly purified, linkage-specific sialyltransferases, in conjunction with specific glycosidases, to probe the cell surface saccharide topography of specific penultimate oligosaccharides, we also find no significant differences between the efficient lung-colonizing variant, B16-F10 and the poorly-colonizing B16-F1 or B16-Flr variants. In contrast, the spontaneously metastatic variants examined contain substantially different levels of specific penultimate sialylation sites. The tumorigenic but nonmetastatic B16-LM3/G3.26 variant contains 4-fold more penultimate Gal beta 1-3GalNAc sialylation sites than the tumorigenic and highly metastatic B16-LM3/G3.12 variant when CMP[3H]NeuAc and the alpha 2-3Gal beta 1-3GalNAc sialyltransferase are used to probe the melanoma cell surfaces. Several prominent glycoconjugates of apparent Mr 43,000, 40,000, and 30,000 are especially evident upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the nonmetastatic cells. The nonmetastatic variant also contains 2-fold more Gal beta 1-4GlcNAc sialylation sites than the metastatic variant when the alpha 2-6Gal beta 1-4GlcNAc sialyltransferase is used as a cell surface probe. In this case, glycoconjugates of apparent Mr 74,000, 45,000, and 43,000 are more prominently observed on the cell surfaces of the nonmetastatic variant. These data indicate that the differences in lung-colonizing abilities of B16 melanoma metastatic variants do not correlate with the numbers or sialylation states of specific penultimate oligosaccharide structures on their surfaces. However, the relative levels of specific penultimate saccharide structures do correlate with the ability of the cells to undergo spontaneous metastasis from a subcutaneous tumor.
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PMID:Cell surface sialylation and tumor metastasis. Metastatic potential of B16 melanoma variants correlates with their relative numbers of specific penultimate oligosaccharide structures. 337 1

Previous studies have shown that treatment of S91-C2 murine melanoma cells with beta-all-trans-retinoic acid (RA) results in growth inhibition, enhanced activity of sialyltransferase, and increased glycosylation of a Mr 160,000 cell surface sialoglycoprotein (gp160). None of these effects could be detected in mutant clones (e.g., S91-C154) selected from the S91-C2 cells for resistance to RA-induced growth inhibition. These findings suggest that modulation by RA of gp160 might be related causally to growth inhibition. In this study we examined the possible role of gp160 in growth regulation using specific antibodies to this glycoprotein. Metabolic labeling of S91-C2 cells with either [3H]glucosamine or [35S]methionine revealed that the cells shed into the growth medium a gp160-like glycoprotein, in addition to several other macromolecules. The gp160-like glycoprotein was isolated from concentrated conditioned medium after preparative polyacrylamide slab gel electrophoresis in the presence of sodium dodecylsulfate by excision of the corresponding protein band. Rabbits were immunized with this material and immunoblotting revealed that their sera contained antibodies that bound specifically to gp160 in extracts of untreated or RA-treated S91-C2 cells. Indirect immunofluorescence staining followed by fluorescence-activated cell sorter analysis demonstrated that the anti-gp160 antibodies bound to the surface of both untreated and RA-treated S91-C2 cells and that the treated cells bound more of the antibodies than untreated ones. In contrast, these antibodies bound to the same extent to untreated and RA-treated resistant S91-C154 cells. The growth of S91-C2 cells in the presence of anti-gp160 antibodies in semisolid medium as well as in monolayer cultures was inhibited in a dose-dependent fashion. Fifty % growth inhibition was obtained at an immunoglobulin concentration of 10 micrograms/ml. The growth of cells exposed concurrently to RA and anti-gp160 antibodies was also inhibited strongly in semisolid medium, but the antibodies caused only a small increase in the inhibitory effect of RA in monolayer cultures. No inhibition by the antibodies of either anchorage-independent growth or anchorage-dependent growth of S91-C154 cells, grown in the absence or presence of RA, was observed. These results support the suggestion that cell surface gp160 might be involved in growth regulation in the S91-C2 cells.
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PMID:Growth inhibition of murine melanoma cells by antibodies to a cell surface glycoprotein implicated in retinoic acid action. 355 69

An assay method for glycosphingolipid glycosyltransferase activity using simple Sephadex G-50 gel permeation chromatography in an aqueous solvent has been developed. An acceptor glycosphingolipid and a donor radioactive nucleotide sugar were incubated with an enzyme source. The reaction mixture was loaded onto a Sephadex G-50 column previously equilibrated with 0.3% (w/v) Triton X-100, 0.1 M sodium chloride, and 0.02% (w/v) sodium azide. The radiolabeled reaction product was eluted by the same solvent in the excluded volume and was collected directly into a liquid scintillation vial, separated from other radioactive compounds. This assay method was utilized to determine the activity of cytidine 5'-monophosphate-N-acetylneuraminic acid:GM3 ganglioside sialyltransferase, which catalyzes the synthesis of GD3 ganglioside, and proved to be as reliable and sensitive as previously published assay procedures. In addition, this assay can be carried out in less time and is simpler than previously reported procedures.
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PMID:Glycosphingolipid glycosyltransferase assay using sephadex G-50 chromatography in aqueous phase. 367 12

Because alterations in cell membrane sialoglycoconjugates can affect the behavior of neoplastic cells, we investigated the effects of in vitro treatment with antimetabolites used in cancer therapy on the expression of membrane sialic acid in cultured HL-60 leukemic cells. In these studies, cells were incubated with Vibrio cholerae neuraminidase to remove surface sialic acid. Reappearance of membrane sialic acid during drug treatment was followed (a) by measuring changes in radioactive surface labeling of viable cells with sodium metaperiodate-sodium[3H]-borohydride, (b) by measuring the decline in accessible surface galactosyl receptor sites which occurred coincident with membrane sialic acid replacement, and (c) by measuring the incorporation of [3H]glucosamine into membrane-associated neuraminidase-labile sialic acid. We were especially interested in learning whether drugs that affect intracellular pools of cytidine triphosphate (CTP), an important nucleotide intermediate in sialylation reactions, could inhibit regeneration of membrane sialic acid. 3-Deazauridine, a competitive inhibitor of CTP synthetase, depleted CTP pools and curtailed surface membrane resialylation with little or no effect on synthesis of de novo sialic acid from precursor sugars. The addition of cytidine restored CTP pools and sialic acid regeneration. Acivicin, a glutamine antagonist, also depleted CTP pools and curtailed surface membrane resialylation. In addition, it retarded de novo synthesis of sialic acid. The addition of cytidine restored intracellular CTP pools and sialic acid regeneration. However, both cytidine and guanosine were required to restore sialic acid synthesis from precursor sugars. 1-beta-D-Arabinofuranosylcytosine, a competitive inhibitor of sialic acid synthetase and of sialyltransferase, inhibited both de novo sialic acid synthesis and membrane resialylation. Only the latter effect was reversed by the addition of exogenous cytidine. Hydroxyurea, an agent shown previously to inhibit glycoconjugate production in hamster fibroblasts, curtailed membrane resialylation and de novo synthesis of sialic acid without depleting CTP pools. Doxorubicin, at levels that caused marked arrest of cell proliferation, had no effect on sialic acid synthesis or expression on the membrane surface. These data suggest that antimetabolites, apart from their cytotoxic effects or effects on cellular growth, may directly inhibit the expression of membrane sialic acid.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Effects of pyrimidine antagonists on sialic acid regeneration in HL-60 cells. 385 65

Prokaryotic derived probes that specifically recognize alpha-2,8-ketosidically linked polysialosyl units were developed to identify and study the temporal expression of these unique carbohydrate moieties in developing neural tissue (Vimr, E. R., McCoy, R. D., Vollger, H. F., Wilkison, N. C., and Troy, F. A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1971-1975). These polysialosyl units cap N-linked oligosaccharides of the complex-type on neural cell adhesion molecules (N-CAM). A Golgi-enriched fraction from 20-day-old fetal rat brain contains a membrane-associated sialyltransferase that catalyzes the incorporation of [14C]N-acetylneuraminic acid [( 14C]NeuNAc) from CMP-[14C] NeuNAc into polymeric products. At pH 6.0, 84 pmol of NeuNAc mg of protein-1 h-1 were incorporated. In sodium dodecyl sulfate-polyacrylamide gels, the major radiolabeled species migrated with a mobility expected for N-CAM. A bacteriophage-derived endoneuraminidase specific for polysialic acid was used to demonstrate that at least 20-30% of the [14C]NeuNAc was incorporated into alpha-2,8-linked polysialosyl units. This was confirmed by structural studies which showed that the endoneuraminidase-sensitive brain material consisted of multimers of sialic acid. The addition of a partially purified preparation of chick N-CAM to the membranous sialyltransferase stimulated sialic acid incorporation 3-fold. The product of this reaction was also sensitive to endoneuraminidase and contained alpha-2,8-linked polysialosyl chains, thus showing that N-CAM can serve as an exogenous acceptor for sialylation in vitro. Sialic acid incorporated into adult rat brain membranes was resistant to endoneuraminidase, indicating that the poly-alpha-2,8-sialosyl sialyltransferase activity is restricted to an early developmental epoch. It is recommended that the enzyme described here be designated CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase and the trivial name poly-alpha-2,8-sialosyl sialyltransferase be adopted.
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PMID:CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase and the biosynthesis of polysialosyl units in neural cell adhesion molecules. 404 5

Platelet sialyltransferase activity was determined in four patients with primary platelet release disorder. Basal enzyme activity was significantly reduced in all. Moreover, enzyme activity in response to stimulation by collagen or sodium arachidonate was also reduced. Platelet total and surface sialic-acid content was normal. The results indicate that defective membrane sialytransferase may be involved in the pathogenesis of primary platelet release disorder.
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PMID:Reduced platelet sialyltransferase activity in patients with primary release disorder. 610 96

Lines of KB cells resistant to Sendai virus-induced cytolysis have been isolated and characterized (Toyama, S., Toyama, Su., and Uetake, H. (1977) Virology 76, 503-515). This study is concerned with the nature of this mutation. Plasma membrane fractions from Sil cells were found to have decreased amount of sialic acid and the same amount of galactose as compared to the membranes from parental KB cells. Sil cells exhibited an increase in sensitivity to toxic effects of ricin and a decrease in sensitivity to wheat germ agglutinin. Binding of wheat germ agglutinin to Sil cells was markedly decreased. Several membrane glycoproteins of Sil cells migrated slightly faster than the corresponding bands of wild type membrane when examined by gel electrophoresis in sodium dodecyl sulfate. Sil cells had decreased sialyltransferase activity that catalyzed the transfer of sialic acid residues from CMP-N-acetylneuraminic acid to glycoprotein acceptors containing Gal beta 1 leads to 3GalNAc alpha 1 leads to O-Ser(Thr) chain. The decreased enzyme activity could not be accounted for by the presence of inhibitors, altered pH optimum, or increased sialidase or CMP-sialic acid hydrolase activities. These results indicate that a molecular basis for the Sil cell phenotype might be the deficiency of sialyltransferase.
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PMID:Deficient cytidine monophospho-N-acetylneuraminic acid: glycoprotein sialyltransferase activity in a clone of KB cells with altered cell fusion ability. 640 1


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