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 enzyme activities involved in O-glycosylation have been studied in three insect cell lines, Spodoptera frugiperda (Sf-9), Mamestra brassicae (Mb) and Trichoplusia ni (Tn) cultured in two different serum-free media. The structural features of O-glycoproteins in these insect cells were investigated using a panel of lectins and the glycosyltransferase activities involved in O-glycan biosynthesis of insect cells were measured (i.e., UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, UDP-Gal:core-1 beta1, 3-galactosyltransferase, CMP-NeuAc:Galbeta1-3GalNAc alpha2, 3-sialyltransferase, and UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase activities). First, we show that O-glycosylation potential depends on cell type. All three lepidopteran cell lines express GalNAcalpha-O-Ser/Thr antigen, which is recognized by soy bean agglutinin and reflects high UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity. Capillary electrophoresis and mass spectrometry studies revealed the presence of at least two different UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases in these insect cells. Only some O-linked GalNAc residues are further processed by the addition of beta1,3-linked Gal residues to form T-antigen, as shown by the binding of peanut agglutinin. This reflects relative low levels of UDP-Gal:core-1 beta1,3-galactosyltransferase in insect cells, as compared to those observed in mammalian control cells. In addition, we detected strong binding of Bandeiraea simplicifolia lectin-I isolectin B4 to Mamestra brassicae endogenous glycoproteins, which suggests a high activity of a UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase. This explains the absence of PNA binding to Mamestra brassicae glycoproteins. Furthermore, our results substantiated that there is no sialyltransferase activity and, therefore, no terminal sialic acid production by these cell lines. Finally, we found that the culture medium influences the O-glycosylation potential of each cell line.
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PMID:O-glycosylation potential of lepidopteran insect cell lines. 1008 87

The amyloid beta precursor protein can exist as both a membrane-bound and a secreted protein, with the former having the potential to generate the amyloid beta peptide present in the neuritic plaques which are characteristic of Alzheimer's disease. In this study, we have used a clone of the AtT20 mouse pituitary cell line which expresses high levels of the amyloid beta precursor protein to characterize the glycosylation state of the secreted and membrane-bound forms of the protein and to examine the role of post-translational modifications in protein processing. Lectin blot analysis of immunoprecipitated amyloid beta precursor protein demonstrated that the soluble form of the protein contains significant amounts of sialic acid, with the lectin staining being reduced in the particulate cellular fractions. Treatment of the cells with mannosidase inhibitors to interfere with the formation of complex-type N-linked glycans resulted in a decrease in secreted amyloid beta precursor protein and an increase in the level of the cellular form of the protein. The increase in amyloid beta precursor protein levels in the cellular fraction was accompanied by an increase in perinuclear staining. Furthermore, cells overexpressing the alpha2,6(N)-sialyltransferase enzyme also demonstrated an increase in amyloid beta precursor protein secretion. These results suggest that the presence of terminal sialic acid residues on complex-type N-glycans may be required for the optimal transport of the amyloid beta precursor protein from the Golgi to the cell membrane with the subsequent cleavage to generate the secreted form of the protein.
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PMID:The role of the protein glycosylation state in the control of cellular transport of the amyloid beta precursor protein. 1018 30

The effect of remodeling of a glycoantigen such as the alpha-Gal epitope, Galalpha1,3Galbeta1,4GlcNAc-R, by the introduction of glycosyltransferase genes on natural killer (NK) cell-mediated direct cytotoxicity was investigated using human peripheral blood mononuclear cells (PBMC) or an NK-like cell line, YT cells, as an effector, and swine endothelial cells (SEC) as a target. Several SEC transfectants were established by transfection with the genes for beta1,4-N-acetylglucosaminyltransferase III, alpha2, 3-sialyltransferase and alpha1,2-fucosyltransferase. These transfections led to dramatic reductions in both direct and indirect NK cell-mediated cytotoxicity, by 72-94% in the case of PBMC and 27-72% in that of YT cells, in addition to an effective reduction in xenoantigenicity, which is substantially caused by the alpha-Gal epitope, to human natural antibodies. The NK cell-mediated direct cytotoxicity was remarkably blocked by an anti-alpha-Gal epitope monoclonal antibody or GSI lectin which preferentially binds to the epitope. Furthermore, treatment of the parental cells with alpha-galactosidase resulted in a significant reduction in cytotoxicity. These results suggest that the alpha-Gal epitope is involved not only in hyperacute rejection and acute vascular rejection, but also in NK cell-mediated direct cytotoxicity. Thus, the genetic remodeling of the alpha-Gal epitope and probably other glycoantigens as well can be expected to represent a new approach for overcoming not only indirect but also direct immunity to xenografts.
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PMID:Regulation of natural killer cell-mediated swine endothelial cell lysis through genetic remodeling of a glycoantigen. 1057 58

The intracellular parasite Trypanosoma cruzi, the etiological agent of Chagas disease, sheds a developmentally regulated surface trans-sialidase, which is involved in key aspects of parasite-host cell interactions. Although it shares a common active site architecture with bacterial neuraminidases, the T.cruzi enzyme behaves as a highly efficient sialyltransferase. Here we report the crystal structure of the closely related Trypanosoma rangeli sialidase and its complex with inhibitor. The enzyme folds into two distinct domains: a catalytic beta-propeller fold tightly associated with a lectin-like domain. Comparison with the modeled structure of T.cruzi trans-sialidase and mutagenesis experiments allowed the identification of amino acid substitutions within the active site cleft that modulate sialyltransferase activity and suggest the presence of a distinct binding site for the acceptor carbohydrate. The structures of the Trypanosoma enzymes illustrate how a glycosidase scaffold can achieve efficient glycosyltransferase activity and provide a framework for structure-based drug design.
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PMID:Structural basis of sialyltransferase activity in trypanosomal sialidases. 1061 40

The effect of the various glycosyltransferases on glycosphingolipids was examined, using transfected swine endothelial cell (SEC) lines. The reactivity of parental SEC to normal human serum (NHS) and Griffonia simplicifolia IB(4) (GSIB4) lectin, which binds to the Gal alpha1-3 Gal beta 1-4 GlcNAc-R (alpha-galactosyl epitope), was reduced by approximately 20% by the treatment with D-PDMP (D-threo-1-phenyl-2-decan- oylamino-3-morpholino-1-propanol), suggesting that glycosphingolipids contained by SEC have a considerable amount of the alpha-galactosyl epitope. The overexpression of two different types of glycosyltransferase, N-acetylglucosaminyl transferase III (GnT-III), as well as alpha2, 6-sialyltransferase (ST6Gal I), alpha2,3-sialyltransferase (ST3Gal III), and alpha1,2-fucosyltransferase (alpha1,2FT), suppresses the total antigenicity of SEC significantly. However, the reduction in reactivities toward NHS and GSIB4 lectin in the case of GnT-III transfectants was milder than those in other transfectants. Western blot analysis indicated that the glycoproteins in all transfectants had diminished reactivity to NHS and GSIB4 lectin to approximately the same extent. Therefore, the neutral glycosphingolipids of these transfectants were separated by thin layer chromatography, followed by immunostaining with NHS and GSIB4 lectin. The levels of the alpha-galactosyl epitope in glycosphingolipids were not decreased in the GnT-III transfectants but were in the ST6Gal I, ST3Gal III, and alpha1,2FT transfectants. These data indicate that ST6Gal I, ST3Gal III, and alpha1,2FT reduced the alpha-galactosyl epitope in both glycoproteins and glycosphingolipids, while GnT-III reduced them only in glycoproteins.
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PMID:Reduction of the major xenoantigen on glycosphingolipids of swine endothelial cells by various glycosyltransferases. 1091 Sep 78

Colon cancer tissues display an increased activity of beta-galactoside alpha2,6 sialyltransferase (ST6Gal.I) and an increased reactivity with the lectin from Sambucus nigra (SNA), specific for alpha2,6-sialyl-linkages. Experimental and clinical studies indicate a contribution of these alterations to tumor progression, but their molecular bases are largely unknown. In many tissues, ST6Gal.I is transcriptionally regulated through the usage of different promoters that originate mRNAs diverging in the 5;-untranslated regions. RT-PCR analysis of 14 carcinoma samples, all expressing an increased ST6Gal.I enzyme activity, and of the corresponding normal mucosa revealed the presence of at least 2 transcripts. One, containing the 5;-untranslated exons, Y+Z, is thought to represent the "housekeeping" expression, and another previously described in hepatic tissues. Both the Y+Z and the hepatic transcripts were detectable in normal and cancer tissues but that latter form had a marked tendency to accumulate in cancer. The extent of alpha2,6-sialylation of glycoconjugates, as determined by SNA-dot blot analysis, was markedly enhanced in all cancer specimens, but the level of reactivity only partially correlated with the level of enzyme expression. Western blot analysis revealed a strikingly heterogeneous pattern of SNA reactivity among cancer tissues. These data indicate that: i) during neoplastic transformation of colonic cells, ST6Gal.I expression may be modulated through a differential promoter usage; ii) the extent of alpha2,6-sialylation of cancer cell membranes is not a direct function of the ST6Gal.I activity, strongly suggesting the existence of other, more complex mechanisms of regulation.
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PMID:Beta-galactoside alpha2,6 sialyltransferase in human colon cancer: contribution of multiple transcripts to regulation of enzyme activity and reactivity with Sambucus nigra agglutinin. 1096 40

Glycosylation is key posttranslational modification for membrane-bound and secreted proteins that can influence both the secondary structure and the function of the protein backbone. In order to investigate the effect of altered cellular glycosylation potential, we have generated a number of clonal cell lines over-expressing the alpha2,3(N) sialyltransferase enzyme (ST3N). In general, there was a decrease in total sialyltransferase (ST) enzyme activity in the clones transfected with the ST3N cDNA, with this decrease being inversely proportional to the quantity of the mRNA coding for the enzyme. The ST3N enzyme was, however, functional and there was an increase in both MAA lectin staining and the expression of polysialic acid, which is attached to the NCAM protein backbone primarily via an alpha2,3 linkage. These results suggest that the overexpression of a sialyltransferase may upset the sialylation potential of the cell.
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PMID:Overexpression of alpha2,3 sialyltransferase in neuroblastoma cells results in an upset in the glycosylation process. 1097 43

Glycopolypeptide (1) carrying the beta-D-Gal-(1-->3)-alpha-D-GalNAc unit as a kind model of asialo-type mucin was synthesized through three steps: enzymatic synthesis of p-nitrophenyl disaccharide glycoside, reduction of the p-nitrophenyl group, and coupling of the amino group with the carboxyl group of poly(L-glutamic acid)s (PGA). In a similar manner, glycopolypeptides (2-7) carrying beta-D-Gal-(1-->3)-beta-D-GalNAc, beta-D-Gal-(1-->3)-beta-D-GlcNAc, beta-D-Gal-(1-->6)-alpha-D-GalNAc, beta-D-Gal-(1-->6)-beta-D-GalNAc, alpha-D-GalNAc, and beta-D-GalNAc, respectively, were synthesized as analogous polymers of polymer 1. Glycopolypeptides 8 and 9 as a mimic of sialo-type mucin were further prepared from polymers 1 and 2 as the acceptor of CMP-Neu5Ac by alpha2,3-(O)-sialyltransferase, respectively. Interactions of these glycopolypeptides with lectins were investigated with the double-diffusion test and the hemagglutination-inhibition assay and in terms of an optical biosensor based on surface plasmon resonance. Polymers 1 and 2 reacted strongly with peanut (Arachis hypogaea) agglutinin (PNA) and Agaricus bisporus agglutinin (ABA). On the other hand, polymers 8 and 9 through sialylation from polymers 1 and 2 reacted with ABA, but did not with PNA. Other polymers 3-7 did not show any reactivity for both the lectins. These results show that PNA acts precisely in an exo manner on the beta-D-Gal-(1-->3)-D-GalNAc sequence, while ABA acts in an endo manner. Polymers 6 and 7 substituted with GalNAc reacted strongly with soybean (Glycine max) agglutinin and Vicia villosa agglutinin B4, regardless of the configuration of the glycosidic linkage. The interaction of all polymers with Bauhinia purpurea agglutinin was much stronger than that of the corresponding sugars. Polymers 8 and 9 reacted with wheat germ (Triticum vulgaris) agglutinin (WGA), to which Neu5Ac residues are needed for binding, but polymers 1 and 2 did not. These sugar-substituted glycopolypeptides interacted specifically with the corresponding lectins. Furthermore, polymers 4-7 reacted with WGA, but the corresponding sugars did not. It suggests that the N-acetyl group along the PGA backbone has a cluster effect for WGA. The artificial glycopolypeptides were shown to be useful as tools and probes of carbohydrate recognition and modeling in the analysis of glycoprotein-lectin interactions.
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PMID:Chemoenzymatic synthesis of glycopolypeptides carrying alpha-Neu5Ac-(2-->3)-beta-D-Gal-(1-->3)-alpha-D-GalNAc, beta-D-Gal-(1-->3)-alpha-D-GalNAc, and related compounds and analysis of their specific interactions with lectins. 1109 73

Sialyltransferase activity has traditionally been studied by determining the rate at which the enzyme transfers a labeled donor sugar to an acceptor substrate. These types of assays can be difficult to quantitate, and the separation of untransfered donor sugar from the sialylated acceptor is time-consuming. The biosensor-based method described here is both rapid and semi-automated. The NeuAc-alpha2-6Gal-R-specific lectin Sambucus nigra agglutinin (SNA) immobilized to the carboxymethyl dextran surface of a BIAcore sensor chip was used to detect and measure the formation of the NeuAc-alpha2-6Gal-R moieties. The sialyltransferase assays were carried out using modified protocols based on the method described in Rearick, J.I., Sadler, J.E., Paulson, J.C., and Hill, R.L. (1979) Enzymatic characterization of betaD-galactoside alpha2-3 sialyltransferase from porcine submaxillary gland. J. Biol. Chem., 254, 4444-4451. The complete assay mixture was simply diluted before injection into the instrument. All injections were performed automatically using the robotics of the BIAcore instrument. Using this technique it is possible to detect product from 0.4 microU of commercial Galbeta1-4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) (ST6Gal I). One unit of sialyltransferase is defined as the quantity that will transfer 1 micromol of N-acetylneuraminic acid from cytidine monophosphate (CMP)-N-acetylneuraminic acid to asialofetuin per min at pH 6.5 and 37 degrees C. The method described here requires as little as 10 microl total assay volume, thus reducing the consumption of reagents. In addition, the sample is completely recoverable from the sensor chip surface, which allows for downstream analysis of the reaction product if desired. This method eliminates the need for labeled donor and acceptor molecules and does not require the separation of the substrates from the product before analysis. Although some kinetic properties of the enzyme can be estimated using this method, further development and validation is required. The method is most useful in determining qualitative estimates of ST6Gal I activity in tissue extracts and in characterizing the production of enzymes in cultured cell systems. The use of a microtiter plate assay format enables the rapid screening of multiple fractions for sialyltransferase activity.
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PMID:A rapid, semi-automated method for detection of Galbeta1-4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) activity using the lectin Sambucus nigra agglutinin. 1144 35

The potential of surface glycoengineering for biomaterials and biosensors originates from the importance of carbohydrate-protein interactions in biological systems. The strategy employed here utilises carbene generated by illumination of diazirine to achieve covalent bonding of carbohydrates. Here, we describe the synthesis of an aryl diazirine containing a disaccharide (lactose). Surface analysis techniques [X-ray photoelectron spectroscopy (XPS) and time of flight secondary ion mass spectroscopy (ToF-SIMS)] demonstrate its successful surface immobilisation on polystyrene (PS). Results are compared to those previously obtained with an aryl diazirine containing a monosaccharide (galactose). The biological activity of galactose- or lactose-modified PS samples is studied using rat hepatocytes, Allo A lectin and solid-phase semi-synthesis with alpha-2,6-sialyltransferase. Allo A shows some binding to galactose-modified PS but none to lactose-modified surfaces. Similar results are obtained with rat hepatocytes. In contrast, sialylation of lactose-modified PS is achieved but not with galactose-modified surfaces. The different responses indicate that the biological activity depends not only on the carbohydrate per se but also on the structure and length of the spacer.
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PMID:Immobilisation on polystyrene of diazirine derivatives of mono- and disaccharides: biological activities of modified surfaces. 1159 76


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