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)

Gangliosides are sialylated glycosphingolipids whose biosynthesis is catalyzed by a series of endoplasmic reticulum (ER)- and Golgi-resident glycosyltransferases. Protein expression, processing, and subcellular localization of the key regulatory enzymes for ganglioside biosynthesis, sialyltransferase II (ST-II) and N-acetylgalactosaminyltransferase I (GalNAcT), were analyzed upon transient expression of the two enzymes in the neuroblastoma cell lines NG108-15 and F-11. The enzymes were endowed with a C-terminal epitope tag peptide (FLAG) for immunostaining and immunoaffinity purification using a FLAG-specific antibody. Mature ST-II-FLAG and GalNAcT-FLAG were expressed as N-glycoproteins with noncomplex oligosaccharides. ST-II-FLAG was distributed to the Golgi apparatus, whereas GalNAcT-FLAG was found in the ER and Golgi. Inhibition of early N-glycoprotein processing with castanospermine resulted in a distribution of ST-II-FLAG to the ER, whereas that of GalNAcT-FLAG remained unaltered. In contrast to GalNAcT, the activity of ST-II and the amount of immunostained enzyme were reduced concomitantly by 75% upon incubation with castanospermine. This was due to a fourfold increased turnover of ST-II-FLAG, which was not found with GalNAcT-FLAG. The ER retention and increased turnover of ST-II-FLAG were most likely due to its inability to bind to calnexin upon inhibition of early N-glycoprotein processing. Calnexin binding was not observed for GalNAcT-FLAG, indicating a differential effect of N-glycosylation on the turnover and subcellular localization of the two glycosyltransferases.
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PMID:Effect of N-glycosylation on turnover and subcellular distribution of N-acetylgalactosaminyltransferase I and sialyltransferase II in neuroblastoma cells. 1082 Jan 96

The sialyltransferase gene family is comprised of 16 cloned enzymes. All members contain two conserved protein domains, termed the S- and L-sialylmotifs, that participate in substrate binding. Of only six invariant amino acids, two are cysteines, with one found in each sialylmotif. Although the recombinant soluble form of ST6Gal I has six cysteines, quantitative analysis indicated the presence of only one disulfide linkage, and thiol reducing agents dithiothreitol and beta-mercaptoethanol inactivated the enzyme. Analysis of site-directed mutants showed that alanine or serine mutants of invariant Cys(181) or Cys(332) exhibit no detectable activity, either by direct assay or by staining of the transfected cells with Sambucus nigra agglutinin, which recognizes the product NeuAcalpha2,6Galbeta1,4GlcNAc on glycoproteins. In contrast, alanine mutations of charged residues adjacent to either cysteine showed little or no effect on enzyme activity. Immunofluorescence microscopy showed that although the wild type sialyltransferase is properly localized in the Golgi apparatus, the inactive cysteine mutants are retained in the endoplasmic reticulum. The results suggest that the invariant cysteine residues in the L- and S-sialylmotifs participate in the formation of an intradisulfide linkage that is essential for proper conformation and activity of ST6Gal I.
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PMID:Conserved cysteines in the sialyltransferase sialylmotifs form an essential disulfide bond. 1127 97

Previously, we demonstrated that beta 1,4galactosyltransferase (gal-T1) reversibly segregates from alpha 2,6sialyltransferase (ST6Gal) to swollen vesicles after monensin treatment of the cells. To further explore this phenomenon, we investigated the response to monensin of various Golgi proteins. Within 30 min of monensin treatment, gal-T1 moved from the Golgi apparatus, as defined by localization of giantin, to swollen vesicles whereas ST6Gal, alpha 2,3(N)sialyltransferase, mannosidase II, and N-acetylgalactosaminyltransferase 2 remained associated with the Golgi apparatus. Stably transfected CHO cells exhibited a similar phenomenon of monensin-induced displacement of recombinant gal-T1 to swollen vesicles while recombinant ST6Gal remained colocalized with endogenously expressed giantin. Gal-T1 and the cation-insensitive mannose 6-phosphate receptor colocalized in swollen vesicles as observed at both light and electron microscopic levels. When monensin was replaced by chloroquine, gal-T1 remained arrested in swollen vesicles. Brefeldin A treatment known to cause relocation of Golgi-associated gal-T1 to the endoplasmic reticulum had no effect on gal-T1 trapped in swollen vesicles. This evidence suggests that monensin blocks gal-T1 trafficking in post-Golgi structures and argues against swelling of gal-T1-containing trans Golgi cisternae as previously assumed.
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PMID:Colocalization of beta 1,4galactosyltransferase with mannose 6-phosphate receptor in monensin-induced TGN-derived structures. 1144 50

This paper reviews experiments from this lab that have tested the hypothesis that pH of the Golgi (pH(G)) of cystic fibrosis (CF) airway epithelial cells is alkaline compared to normal, that this altered pH affects sialyltransferase and other Golgi enzymes controlling biochemical composition of the plasma membrane and that altered surface biochemistry increases bacterial binding. We generated a plasmid encoding a modified green fluorescence protein-sialyltransferase (GFP-ST) chimera protein that was pH-sensitive and localized to the Golgi when transfected into HeLa cells and also CF and normal or cystic fibrosis transmembrane conductance regulator- (CFTR)-corrected airway epithelial cells. Digital imaging microscopy of these Golgi-localized probes showed that there was no correlation between pH(G) (6.4-7.0) and the presence of CFTR, whether cells were in HCO(3)(-)/CO(2)-containing or in HCO(3)(-)/CO(2)-free solutions. Activation of CFTR by raising cell [cAMP] had no effect on pH(G). Thus, CFTR seemed not to be involved in controlling pH(G). Experiments on HeLa cells using an avidin-sialyltransferase chimera in combination with a pH-sensitive fluorescent biotin indicated that even in cells that do not express CFTR, Cl(-) and K(+) conductances of the Golgi and other organelle membranes were large and that pH(G) was controlled solely by the H(+) v-ATPase countered by a H(+) leak. A mathematical model was applied to these and other published data to calculate passive H(+) permeability (P(H+)) of the Golgi, endoplasmic reticulum, trans-Golgi network, recycling endosomes and secrety granules from a variety of cells. An organelle's acidity was inversely correlated to its calculated P(H+). We conclude that the CFTR plays a minor role in organelle pH regulation because other (Cl(-) and K(+)) channels are present in sufficient numbers to shunt voltages generated during H(+) pumping. Acidity of the Golgi (and perhaps other organelles) appears to be determined by the activity of H(+) pumps countered by H(+) leaks.
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PMID:Cystic fibrosis transmembrane conductance regulator and H+ permeability in regulation of Golgi pH. 1187 64

ADP-ribosylation factors (Arf), a family of small GTP-binding proteins, play important roles in intracellular trafficking in animal and yeast cells. Here, we investigated the roles of two Arf homologs, Arf1 and Arf3 of Arabidopsis, in intracellular trafficking in plant cells. We generated dominant negative mutant forms of Arf 1 and Arf3 and examined their effect on trafficking of reporter proteins in protoplasts. Arf1[T31N] inhibited trafficking of H(+)-ATPase:green fluorescent protein (GFP) and sialyltransferase (ST):GFP to the plasma membrane and the Golgi apparatus. In addition, Arf1[T31N] caused relocalization of the Golgi reporter protein ST:GFP to the endoplasmic reticulum (ER). In protoplasts expressing Arf1[T31N], ST:red fluorescent protein remained in the ER, whereas H(+)-ATPase:GFP was mistargeted to another organelle. Also, expression of Arf1[T31N] in protoplasts resulted in profound changes in the morphology of the ER. The treatment of protoplasts with brefeldin A had exactly the same effect as Arf1[T31N] on various intracellular trafficking pathways. In contrast, Arf3[T31N] did not affect trafficking of any of these reporter proteins. Inhibition experiments using mutants with various domains swapped between Arf1 and Arf3 revealed that the N-terminal domain is interchangeable for trafficking inhibition. However, in addition to the T31N mutation, motifs in domains II, III, and IV of Arf1 were necessary for inhibition of trafficking of H(+)-ATPase:GFP. Together, these results strongly suggest that Arf1 plays a role in the intracellular trafficking of cargo proteins in Arabidopsis, and that Arf1 functions through a brefeldin A-sensitive factor.
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PMID:ADP-ribosylation factor 1 of Arabidopsis plays a critical role in intracellular trafficking and maintenance of endoplasmic reticulum morphology in Arabidopsis. 1217 64

Although most glycosphingolipids (GSLs) are thought to be located in the outer leaflet of the plasma membrane, recent evidence indicates that GSLs and their precursor, ceramide, are also associated with intracellular organelles and, particularly, mitochondria. GSL biosynthesis starts with the formation of ceramide in the endoplasmic reticulum (ER), which is transported by controversial mechanisms to the Golgi apparatus, where stepwise addition of monosaccharides on to ceramides takes place. We now report the presence of GSL-biosynthetic enzymes in a subcompartment of the ER previously characterized and termed 'mitochondria-associated membrane' (MAM). MAM is a membrane bridge between the ER and mitochondria that is involved in the biosynthesis and trafficking of phospholipids between the two organelles. Using exogenous acceptors coated on silica gel, we demonstrate the presence of ceramide glucosyltransferase (Cer-Glc-T), glucosylceramide galactosyltransferase and sialyltransferase (SAT) activities in the MAM. Estimation of the marker-enzyme activities showed that glycosyltransferase activities could not be ascribed to cross-contamination of MAM by Golgi membranes. Cer-Glc-T was found to have a marked preference for ceramide bearing phytosphingosine as sphingoid base. SAT activities in MAM led to the synthesis of G(M3) ganglioside and small amounts of G(D3). G(M1) was also synthesized along with G(M3) upon incubation of the fraction with exogenous unlabelled G(M3), underlying the presence of other sphingolipid-specific glycosyltransferases in MAM. On the basis of our results, we propose MAM as a privileged compartment in providing GSLs for mitochondria.
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PMID:The mitochondria-associated endoplasmic-reticulum subcompartment (MAM fraction) of rat liver contains highly active sphingolipid-specific glycosyltransferases. 1257 62

Actin filaments are thought to play an important role in intracellular trafficking in various eukaryotic cells. However, their involvement in intracellular trafficking in plant cells has not been clearly demonstrated. Here, we investigated the roles actin filaments play in intracellular trafficking in plant cells using latrunculin B (Lat B), an inhibitor of actin filament assembly, or actin mutants that disrupt actin filaments when overexpressed. Lat B and actin2 mutant overexpression inhibited the trafficking of two vacuolar reporter proteins, sporamin:green fluorescent protein (GFP) and Arabidopsis thaliana aleurain-like protein:GFP, to the central vacuole; instead, a punctate staining pattern was observed. Colocalization experiments with various marker proteins indicated that these punctate stains corresponded to the Golgi complex. The A. thaliana vacuolar sorting receptor VSR-At, which mainly localizes to the prevacuolar compartment, also accumulated at the Golgi complex in the presence of Lat B. However, Lat B had no effect on the endoplasmic reticulum (ER) to Golgi trafficking of sialyltransferase or retrograde Golgi to ER trafficking. Lat B also failed to influence the Golgi to plasma membrane trafficking of H+-ATPase:GFP or the secretion of invertase:GFP. Based on these observations, we propose that actin filaments play a critical role in the trafficking of proteins from the Golgi complex to the central vacuole.
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PMID:Actin filaments play a critical role in vacuolar trafficking at the Golgi complex in plant cells. 1572 71

The negative effects of ammonium on recombinant protein productivity and glycosylation have been well documented, but the interaction of ammonium on glycosylation genes has not been completely elucidated. In this study, the effects of elevated ammonium on 12 glycosylation related genes in Chinese hamster ovary cells were evaluated by quantitative real time reverse transcriptase polymerase chain reaction. Numerous cytosol and endoplasmic reticulum (ER) localized genes associated with early glycosylation steps were insensitive to the ammonium condition. The initial expression of uridine diphosphate (UDP)-galactose transporter was higher for the ammonium-treated culture, while the initial expressions of cytosine monophosphate (CMP)-sialic acid transporter, beta(1,4)-galactosyltransferase, and UDP-glucose pyrophosphorylase were higher for the control culture. alpha(2,3)-sialyltransferase was observed to have lower expression level under the elevated ammonium condition compared to the control culture. This study indicates that galactosylation and sialylation inhibition is mainly due to decreased gene expression of galactosyltransferase, sialyltransferase, and CMP-sialic acid transporter and not due to sialidase. These unbalanced initial glycosylation and branching steps can explain the higher molecular heterogeneity under ammonium stress. Moreover, this study indicates that elevated ammonium has limited effects on the glycosylation genes associated with the ER and cytosol compared to the genes associated with the Golgi.
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PMID:Effects of elevated ammonium on glycosylation gene expression in CHO cells. 1638 Feb 82

The addition of sialic acid to glycoproteins and glycolipids requires Golgi sialyltransferases to have access to their glycoconjugate substrates and nucleotide sugar donor, CMP-sialic acid. CMP-sialic acid is transported into the lumen of the Golgi complex through the CMP-sialic acid transporter, an antiporter that also functions to transport CMP into the cytosol. We localized the transporter using immunofluorescence and deconvolution microscopy to test the prediction that it is broadly distributed across the Golgi stack to serve the many sialyltransferases involved in glycoconjugate sialylation. The transporter co-localized with ST6GalI in the medial and trans Golgi, showed partial overlap with a medial Golgi marker and little overlap with early Golgi or trans Golgi network markers. Endoplasmic reticulum-retained forms of sialyltransferases did not redistribute the transporter from the Golgi to the endoplasmic reticulum, suggesting that transporter-sialyltransferase complexes are not involved in transporter localization. Next we evaluated the role of the transporter's N- and C-terminal cytoplasmic tails in its trafficking and localization. The N-tail was not required for either endoplasmic reticulum export or Golgi localization. The C-tail was required for endoplasmic reticulum export and contained di-Ile and terminal Val motifs at its very C terminus that function as independent endoplasmic reticulum export signals. Deletion of the last four amino acids of the C-tail (IIGV) eliminated these export signals and prevented endoplasmic reticulum export of the transporter. This form of the transporter supplied limited amounts of CMP-sialic acid to Golgi sialyltransferases but was unable to completely rescue the transporter defect of Lec2 Chinese hamster ovary cells.
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PMID:The CMP-sialic acid transporter is localized in the medial-trans Golgi and possesses two specific endoplasmic reticulum export motifs in its carboxyl-terminal cytoplasmic tail. 1692 16

Ganglioside glycosyltransferases organize as multienzyme complexes that localize in different sub-Golgi compartments. Here we studied whether in CHO-K1 cells lacking CMP-NeuAc: GM3 sialyltransferase (SialT2), the sub-Golgi localization of UDP-Gal:glucosylceramide beta-1,4-galactosyltransferase (GalT1) and CMP-NeuAc:lactosylceramide sialyltransferase (SialT1) complex is affected when SialT2, another member of this complex, is coexpressed. GalT1 and SialT1 sub-Golgi localization was determined by studying the effect of brefeldin A (BFA) and monensin on the synthesis of glycolipids and on the sub-Golgi localization of GalT1(1-52)-CFP (cyan fluorescent protein) and SialT1(1-54)-YFP (yellow fluorescent protein) chimeras by single cell fluorescence microscopy and by isopycnic subfractionation. We found that BFA, and also monensin, impair the synthesis of glycolipids beyond GM3 ganglioside in wild type (WT) cells but beyond GlcCer in SialT2(+) cells. Although BFA redistributed GalT1-CFP and SialT1-YFP to the endoplasmic reticulum in WT cells, a fraction of these chimeras remained associated with a distal Golgi compartment, enriched in trans Golgi network, and recycling endosome markers in SialT2(+) cells. In BFA-treated cells, the percentage of GalT1-CFP and SialT1-YFP associated with Golgi-like membrane fractions separated by isopycnic subfractionation was higher in SialT2(+) cells than in WT cells. These effects were reverted by knocking down the expression of SialT2 with specific siRNA. Results indicate that sub-Golgi localization of glycosyltransferase complexes may change according to the relative levels of the expression of participating enzymes and reveal a capacity of the organelle to adapt the topology of the glycolipid synthesis machinery to functional states of the cell.
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PMID:Modulation of GalT1 and SialT1 sub-Golgi localization by SialT2 expression reveals an organellar level of glycolipid synthesis control. 1695 Jul 84


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