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)

A simple preparation of the "core-II" N-acetylglucosaminyltransferase (UDP-D-GlcpNAc:beta-D-Galp-(1-->3)-alpha-D-GalpNAc (GlcNAc to GalNAc) beta-(1-->6)-GlcNAc-transferase, GlcNAcT, EC 2.4.1.102) from commercial mouse kidney acetone powder is reported. The enzyme obtained in a single step of affinity chromatography is suitable for use in preparative oligosaccharide synthesis. In conjunction with previously described preparations of beta-(1-->4)-galactosyltransferase (EC 2.4.1.22), alpha-(2-->3)-sialytransferase (EC 2.4.99.6) and alpha-(1-->3/4)-fucosyltransferase (EC 2.4.1.65), the GlcNAcT was used in the first step of a sequence which converted the disaccharide beta-D-Galp-(1-->3)-alpha-D-GalpNAc-OR into the sialyl-LeX-containing structure alpha-D-NeupAc-(2-->3)-beta-D-Galp- (1-->4)-[alpha-L-Fucp-(1-->3)]-beta-D-GlcpNAc-(1-->6)-[beta-D-Galp - (1-->3)]-alpha-D-GalpNAc-OR (5), where R = (CH2)8CO2Me. Hexasaccharide 5, thus assembled in only one week once the enzymes were prepared, was characterized by 1H and 13C NMR spectroscopy and fast-atom bombardment mass spectrometry, as were all intermediate oligosaccharides. The core II GlcNAcT thus joins the expanding repertoire of readily available reagents for the rapid assembly of oligosaccharides.
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PMID:Use of the "core-2"-N-acetylglucosaminyltransferase in the chemical-enzymatic synthesis of a sialyl-LeX-containing hexasaccharide found on O-linked glycoproteins. 810 68

To elucidate control mechanisms of O-glycan biosynthesis in leukemia and to develop biosynthetic inhibitors we have characterized core 2 UDP-GlcNAc:Gal beta 1-3GalNAc-R(GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase (EC 2.4.1.102; core 2 beta 6-GlcNAc-T) and CMP-sialic acid: Gal beta 1-3GalNAc-R alpha 3-sialyltransferase (EC 2.4.99.4; alpha 3-SA-T), two enzymes that are significantly increased in patients with chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML). We observed distinct tissue-specific kinetic differences for the core 2 beta 6-GlcNAc-T activity; core 2 beta 6-GlcNAc-T from mucin secreting tissue (named core 2 beta 6-GlcNAc-T M) is accompanied by activities that synthesize core 4 [GlcNAc beta 1-6(GlcNAc beta 1-3)GalNAc-R] and blood group I [GlcNAc beta 1-6(GlcNAc beta 1-3)Gal beta-R] branches; core 2 beta 6-GlcNAc-T in leukemic cells (named core 2 beta-GlcNAc-T L) is not accompanied by these two activities and has a more restricted specificity. Core 2 beta 6-GlcNAc-T M and L both have an absolute requirement for the 4- and 6-hydroxyls of N-acetylgalactosamine and the 6-hydroxyl of galactose of the Gal beta 1-3GalNAc alpha-benzyl substrate but the recognition of other substituents of the sugar rings varies, depending on the tissue. alpha 3-sialyltransferase from human placenta and from AML cells also showed distinct specificity differences, although the enzymes from both tissues have an absolute requirement for the 3-hydroxyl of the galactose residue of Gal beta 1-3GalNAc alpha-Bn. Gal beta 1-3(6-deoxy)GalNAc alpha-Bn and 3-deoxy-Gal beta 1-3GalNAc alpha-Bn competitively inhibited core 2 beta 6-GlcNAc-T and alpha 3-sialyltransferase activities, respectively.
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PMID:Processing O-glycan core 1, Gal beta 1-3GalNAc alpha-R. Specificities of core 2, UDP-GlcNAc: Gal beta 1-3 GalNAc-R(GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase and CMP-sialic acid: Gal beta 1-3GalNAc-R alpha 3-sialyltransferase. 829 5

Mammalian spermatozoa must undergo maturational changes between the events of mating and fertilization. These biochemical and functional alterations, collectively termed capacitation, take place as spermatozoa traverse the female reproductive tract. The preparatory biochemical changes include removal, modification, and reorganization of sperm surface molecules. Although details of all the changes are not known, lectin binding studies have provided evidence suggesting that carbohydrate moieties of sperm surface glycoproteins are modified during capacitation. In an attempt to gain insight into the potential modifications of sperm plasma membrane glycoproteins, we quantified glycoprotein-modifying enzyme activities in the uterine and oviductal fluid of the hamster during the 4 days of the estrous cycle. These enzymes are known to modify existing glycoproteins, either by adding sugar residues (glycosyltransferases) or by removing terminal sugar residues (glycosidases). Data from these studies showed that 1) levels of all glycosyltransferase activities assayed (sialyltransferase, fucosyltransferase, galactosyltransferase, and N-acetylglucosaminyltransferase) were negligible in the uterine fluid at the onset of ovulation (Day 1) but sharply increased preceding ovulation (Day 4); 2) levels of the four glycosyltransferase activities assayed were higher in the oviductal fluid at the onset of ovulation (Day 1) and then gradually decreased through the remainder of the estrous cycle (Day 2 to Day 4); 3) levels of all glycohydrolase activities (acidic alpha-D-mannosidase, beta-D-galactosidase, beta-D-glucuronidase, beta-D-glucosaminidase, and alpha-L-fucosidase) and protein in the uterine and oviductal fluids did not vary widely during the 4 days of the cycle. These results demonstrate a temporal surge of glycosyltransferase activities in the genital tract fluids of the hamster. The temporal changes in the glycoprotein-modifying enzymes may have an effect on the glycosylation of sperm plasma membrane and zona pellucida glycoproteins at the site of fertilization or may alter the surface glycoproteins of the fertilized egg in the uterus prior to implantation.
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PMID:Temporal surge of glycosyltransferase activities in the genital tract of the hamster during the estrous cycle. 872 23

Mucin type O-glycans with core 2 branches are distinct from nonbranched O-glycans, and the amount of core 2 branched O-glycans changes dramatically during T cell differentiation. This oligosaccharide is synthesized only when core 2 beta-1, 6-N-acetylglucosaminyltransferase (C2GnT) is present, and the expression of this glycosyltransferase is highly regulated. To understand how O-glycan synthesis is regulated by the orderly appearance of glycosyltransferases that form core 2 branched O-glycans, the subcellular localization of C2GnT was determined by using antibodies generated that are specific to C2GnT. The studies using confocal light microscopy demonstrated that C2GnT was localized mainly in cis to medial-cisternae of the Golgi. We then converted C2GnT to a trans-Golgi enzyme by replacing its Golgi retention signal with that of alpha-2,6-sialyltransferase, which resides in trans-Golgi. Chinese hamster ovary cells expressing wild type C2GnT and the chimeric C2GnT were then subjected to oligosaccharide analysis. The results obtained clearly indicate that the conversion of C2GnT into a trans-Golgi enzyme resulted in a substantial decrease of core 2 branched oligosaccharides. These results, taken together, strongly suggest that the predominance of core 2 branched oligosaccharides in those cells expressing C2GnT is due to the fact that C2GnT is located earlier in the Golgi than alpha-2,3-sialyltransferase that competes with C2GnT for the common substrate. Furthermore, alteration of Golgi localization renders the chimeric C2GnT much less efficient in synthesizing core 2 branched oligosaccharides, indicating the critical role of orderly subcellular localization of glycosyltransferases.
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PMID:Altered Golgi localization of core 2 beta-1,6-N-acetylglucosaminyltransferase leads to decreased synthesis of branched O-glycans. 927 27

Carbohydrates on cell surfaces are important biomolecules in various biological recognition processes. Elucidation of the biological roles of complex oligosaccharides necessitates an efficient methodology to synthesize these compounds and their analogs. Enzymatic synthesis renders itself to be useful in the construction of an oligosaccharide structure owing to its mild reaction condition, high regio- and stereoselectivity. This review article focuses on the recent progress in oligosaccharide syntheses catalyzed by glycosyltransferases, namely sialyltransferase, galactosyltransferase, fucosyltransferase, and N-acetylglucosaminyltransferase. A survey of the latest patent and literature related to this field is also included.
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PMID:Utilization of glycosyltransferases to change oligosaccharide structures. 937 27

We have addressed the question of whether or not Golgi fragmentation, as exemplified by that occurring during drug-induced microtubule depolymerization, is accompanied by the separation of Golgi subcompartments one from another. Scattering kinetics of Golgi subcompartments during microtubule disassembly and reassembly following reversible nocodazole exposure was inferred from multimarker analysis of protein distribution. Stably expressed alpha-2,6-sialyltransferase and N-acetylglucosaminyltransferase-I (NAGT-I), both C-terminally tagged with the myc epitope, provided markers for the trans-Golgi/trans-Golgi network (TGN) and medial-Golgi, respectively, in Vero cells. Using immunogold labeling, the chimeric proteins were polarized within the Golgi stack. Total cellular distributions of recombinant proteins were assessed by immunofluorescence (anti-myc monoclonal antibody) with respect to the endogenous protein, beta-1,4-galactosyltransferase (GalT, trans-Golgi/TGN, polyclonal antibody). ERGIC-53 served as a marker for the intermediate compartment). In HeLa cells, distribution of endogenous GalT was compared with transfected rat alpha-mannosidase II (medial-Golgi, polyclonal antibody). After a 1-h nocodazole treatment, Vero alpha-2,6-sialyltransferase and GalT were found in scattered cytoplasmic patches that increased in number over time. Initially these structures were often negative for NAGT-I, but over a two- to threefold slower time course, NAGT-I colocalized with alpha-2,6-sialyltransferase and GalT. Scattered Golgi elements were located in proximity to ERGIC-53-positive structures. Similar trans-first scattering kinetics was seen with the HeLa GalT/alpha-mannosidase II pairing. Following nocodazole removal, all cisternal markers accumulated at the same rate in a juxtanuclear Golgi. Accumulation of cisternal proteins in scattered Golgi elements was not blocked by microinjected GTPgammaS at a concentration sufficient to inhibit secretory processes. Redistribution of Golgi proteins from endoplasmic reticulum to scattered structures following brefeldin A removal in the presence of nocodazole was not blocked by GTPgammaS. We conclude that Golgi subcompartments can separate one from the other. We discuss how direct trafficking of Golgi proteins from the TGN/trans-Golgi to endoplasmic reticulum may explain the observed trans-first scattering of Golgi transferases in response to microtubule depolymerization.
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PMID:Scattered Golgi elements during microtubule disruption are initially enriched in trans-Golgi proteins. 943

Sialyl-Lex (sLex) antigen expression recognized by KM93 monoclonal antibody was significantly down-regulated during differentiation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in human pre-B lymphocytic leukemia cell line KM3. The sLex determinants were almost exclusively expressed on O-linked oligosaccharide chains of an O-glycosylated 150-kDa glycoprotein (gp150). A low shear force cell adhesion assay showed that TPA treatment significantly inhibited E-selectin-mediated cell adhesion. Transcript and/or enzyme activity levels of alpha1-->3-fucosyltransferase, alpha2-->3-sialyltransferase, beta1-->4-galactosyltransferase, and elongation beta1-->3-N-acetylglucosaminyltransferase did not correlate with sLex expression levels. However, transcript and enzyme activity levels of core 2 GlcNAc-transferase (C2GnT) were significantly down-regulated during TPA treatment. Following transfection and constitutive expression of full-length exogenous C2GnT transcript, C2GnT enzyme activities were maintained at high levels even after TPA treatment and down-regulation of cell surface sLex antigen expression by TPA was completely abolished. Furthermore, in the transfected cells, the KM93 reactivity of gp150 was not reduced by TPA treatment, and the inhibition of cell adhesion by TPA was also blocked. These results suggest that sLex expression is critically regulated by a single glycosyltransferase, C2GnT, during differentiation of KM3 cells.
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PMID:Single glycosyltransferase, core 2 beta1-->6-N-acetylglucosaminyltransferase, regulates cell surface sialyl-Lex expression level in human pre-B lymphocytic leukemia cell line KM3 treated with phorbolester. 975 22

Retinoic acid (RA) plays an important role in differentiation stage in which it also influences glycoconjugate metabolism. Previous work in our laboratory has shown that treatment with RA modifies glycolipid synthesis and distribution in total Xenopus embryos during development. In this study we have investigated the activity of the following anabolic enzymes involved in glycolipid biosynthesis: sialyltransferase-1 (SAT-1), GM3(beta1, 4)-N-acetylgalactosaminyltransferase (GalNAcT-1) and LacCer(beta1, 3)N-acetylglucosaminyltransferase (GlcNAcT-1). These enzymes are located at the branching point of lactosylceramide (Lc(2)) metabolism. Enzyme activities were assayed after treatment with different doses of RA added exogenously to the medium during the first 7 days of Xenopus embryo development. Our results show that RA activates GlcNAcT-1, the enzyme that drives Lc(2)to the glycolipids of the lacto-series, and SAT-1 that inserts Lc(2)in the ganglio-series pathway. These data support our previous analysis of glycolipid pattern in Xenopus embryos after RA treatment (Rizzo et al., 1995;Cell Biol Int19: 895-901) indicating a possible correlation between the distribution of glycolipids and the enzymes involved in their metabolism.
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PMID:Glycolipid glycosyltransferase activities during early development of Xenopus: effect of retinoic acid. 1056 Nov 17

The beta 1,6 N-acetylglucosaminyltransferase (C2GnT) has been recently mapped to the cis/medial-Golgi compartment. To analyze the Golgi-targeting determinants of C2GnT, we constructed various deletion mutants of the enzyme fused to the enhanced green fluorescent protein (EGFP) and localized these proteins by fluorescence microscopy in living cells. We found that the N-terminal peptide encompassing amino acids 1 to 32 represents the minimal Golgi-targeting signal sufficient to localize EGFP to the same compartment as the full-length C2GnT. This peptide makes up the cytoplasmic and the transmembrane domains of the enzyme and was referred to as CTd (cytoplasmic and transmembrane domains). We compared the Golgi-targeting efficiency of the C2GnT-derived CTd with its homologous domains from other glycosyltransferases, including the H-type alpha(1,2)-fucosyltransferase (FucTI), the polypeptide N-acetylgalactosaminyltransferase-I (GalNAcT-I), the alpha(1,3)-fucosyltransferase VII (FucTVII), and the alpha(2,6)-sialyltransferase (ST6Gal-I) and found that the Golgi-targeting determinants of these glycosyltransferases were also composed of their cytosolic and transmembrane domains. To investigate whether the CTd of C2GnT could serve as a cis to medial Golgi-specific signal, we tested its ability to mislocalize two late-Golgi acting glycosyltransferases FucTI and FucTVII. We show that fusing the C2GnT-derived CTd with the catalytic domain of FucTVII resulted in a complete mislocalization of the enzyme to the C2GnT compartment, with a parallel alteration of sialyl-Lewis x synthesis and P-selectin binding. The intracellular distribution and activity of FucTI, however, were not affected. Thus, CTds of either early or late-Golgi acting glycosyltransferases represent the Golgi-targeting domains of these enzymes. In addition, we show that C2GnT-derived CTd can function as a cis/medial Golgi-targeting determinant.
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PMID:The cytosolic and transmembrane domains of the beta 1,6 N-acetylglucosaminyltransferase (C2GnT) function as a cis to medial/Golgi-targeting determinant. 1182 83

1. Rat liver microsomal preparations incubated in 1% Triton X-100 at 37 degrees C for 1h released about 60% of the membrane-bound UDP-galactose-glycoprotein galactosyltransferase (EC 2.4.1.22) into a high-speed supernatant. The supernatant galactosyltransferase which was solubilized but not purified by this treatment had a higher molecular weight than the serum enzyme as shown by Sephadex G-100 column chromatography. 2. The galactosyltransferase present in the high-speed supernatant was purified 680-fold by an affinity-column-chromatographic technique by using a column of activated Sepharose 4B coupled with alpha-lactalbumin. The galactosyltransferase ran as a single band on polyacrylamide gels and contained no sialyltransferase, N-acetylglucosaminyltransferase or UDP-galactose pyrophosphatase activities. 3. The purified membrane enzyme had properties similar to serum galactosyltransferase. It had an absolute requirement for Mn(2+) that could not be replaced by Ca(2+), Mg(2+), Zn(2+) or Co(2+), and was active over a wide pH range (6-8) with a pH optimum of 6.5. The apparent K(m) for UDP-galactose was 10.8mum. The protein alpha-lactalbumin modified the enzyme to a lactose synthetase by increasing substrate specificity for glucose in preference to N-acetylglucosamine and fetuin depleted of sialic acid and galactose. 4. The molecular weight of the membrane enzyme was 65000-70000, similar to that of the purified serum enzyme. Amino acid analyses of the two proteins were similar but not identical. 5. Sephadex G-100 column chromatography of the purified membrane enzyme showed a small peak (2-5%) of higher molecular weight than the purified serum enzyme. Inclusion of 1mm-epsilon-aminohexanoic acid in the isolation procedures increased this peak to as much as 30% of the total enzyme recovered. Increasing the epsilon-aminohexanoic acid concentration to 100mm resulted in no further increase in this high-molecular-weight fraction.
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PMID:Purification of membrane-bound galactosyltransferase from rat liver microsomal fractions. 1674 49


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