Cell-surface glycosyltransferases in gastrulating chick embryos. I. Temporally and spatially specific patterns of four endogenous glycosyltransferase activities.
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Age Factors↗
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Synthesis of plasmalemmal glycoproteins in intestinal epithelial cells. Separation of Golgi membranes from villus and crypt cell surface membranes; glycosyltransferase activity of surface membrane.
The relationship between Golgi and cell surface membranes of intestinal cells was studied. These membranes were isolated from intestinal crypt cells and villus cells. The villus cell membranes consisted of microvillus membrane, a Golgi-rich fraction, and two membrane fractions interpreted as representing lateral-basal membranes. The villus cell microvillus membrane was purified by previously published techniques while the other membranes were obtained from isolated cells by differential centrifugation and density gradient velocity sedimentation. The two membrane fractions obtained from villus cells and considered to be lateral-basal membranes were enriched for Na+,K+-ATPase activity, but one also showed enrichment in glycosyltransferase activity. The Golgi membrane fraction was enriched for glycosyltransferase activity and had low to absent Na+,K+-ATPase activity. Adenylate cyclase activity was present in all membrane fractions except the microvillus membrane but co-purified with Golgi rather than lateral-basal membranes. Electron microscopy showed that the Golgi fraction consisted of variably sized vesicles and cisternalike structures. The two lateral-basal membrane fractions showed only vesicles of smaller, more uniform size. After 125I labeling of isolated intact cells, radioactivity was found associated with the lateral-basal and microvillus membrane fractions and not with the Golgi fraction. Antibody prepared against lateral-basal membrane fractions reacted with the surface membrane of isolated villus cells. The membrane fractions from isolated crypt cells demonstrated that all had high glycosyltransferase activity. The data show that glycosyltransferase activity, in addition to its Golgi location, may be a significant property of the lateral-basal portion of the intestinal villus cell plasma membrane. Data obtained with crypt cells support earlier data and show that the crypt cell surface membrane possesses glycosyltransferase activity.
Adenosine Triphosphatases↗
Glycosyltransferases in plant and animal tissues effects of fixation and lead on enzyme activity.
As the initial step toward the cytochemical localization of glycosyl-transferases in situ, biochemical determinations of these enzyme activities from onion root tips and L1210 cells were performed before and after fixation as well as in the presence of lead ions. Glycosyltransferase activity from roots fixed in buffered formaldehyde or glutaraldehyde before homogenization decreased as the concentration of the fixative or fixation time was increased. Formaldehyde fixation was less inhibitory than glutaraldehyde; 35% of the glycosyltransferase activity was retained after 30 min fixation in 2% formaldehyde while 25% of the enzyme activity remained after a similar fixation in glutaraldehyde. Substantially higher levels of L1210 cell glycosyltransferase activity were retained after a 30 min 2% formaldehyde fixation (60% sialyltransferase; 82% galactosyltransferase), but inhibition by glutaraldehyde was similar to that observed for onion root galactosyltransferase. Glycosyltransferase from formaldehyde-fixed roots was inhbited 35% by lead nitrate, but sialytransferase from formaldehyde-fixed L1210 cells was unaffected by lead ions. These findings are encouraging for further studies aimed at the development of cytochemical technique to localize glycosyltransferase in plant and animal tissues.
Cell Line↗
[Effect of growth medium composition of glucoamylase and glycosyltransferase activity of Endomyces fibuliger].
Production of glucoamylase and glycosyltransferase by Endomyces fibuliger was found to depend on sources of carbon and nitrogen nutrition. Starch at a concentration above 0.5% in the medium stimulated biosynthesis of glycosyltransferase but inhibited production of glucoamylase by End. fibuliger 20-9. The rate of growth of the micro-organism increased by a factor of 3.3 with an increase of starch concentration from 0.5 to 6%. Synthesis of glycosyltransferase was repressed by glucose, lactose, sucrose and maltose. Synthesis of glucoamylase was repressed by lactose, sorbose and galactose. Synthesis of glycosyltransferase was stimulated by xylose, sorbose and galactose. Production of glucoamylase was stimulated by xylose and arabinose. Growth of the culture and synthesis of glucoamylase and maltase in the cultural broth were stimulated by an increase in the concentration of maize extract. Biosynthesis of glucoamylase and glycosyltransferase was stimulated by NH4H2PO4.
Ascomycota↗
Different effects of concanavalin A and E-phytohemagglutinin upon lymphocyte glycosyltransferase activities.
We have studied glycosyltransferase activities in human lymphocytes stimulated with the plant mitogens E-phytohemagglutinin or concanavalin A (Con A) and have compared the results with activities found in resting lymphocytes. Compared to resting lymphocytes, Con A-stimulated lymphocytes possess an enhanced capacity to transfer the sugars, sialic acid, galactose, and N-acetyl-glucosamine from their respective nucleotide donors to both endogenous cellular acceptors and added exogenous glycoprotein acceptors. The enhanced glycosyltransferase activity induced by Con A is not inhibited by puromycin despite effective inhibition of de novo protein synthesis, indicating that synthesis of new glycosyltransferase enzymes is not necessary for the observed increases in glycosylation activity. By contrast to the findings in Con A-stimulated cells, the corresponding glycosyltransferase activities of E-phytohemagglutinin-stimulated lymphocytes do not differ from those of unstimulated lymphocytes. These data indicate that individual plant lectins have different effects on the biosynthesis of complex saccharides by cultured human lymphocytes.
Cell Membrane↗
Tandem Duplication-Driven Neofunctionalization of UDP-Glycosyltransferases Shapes the Diversification of Triterpenoid Saponins in the Cucurbitaceae.
Tandem duplication of tailoring enzymes allows evolutionary innovation that diversifies plant specialized metabolism. Here, we present an interesting example of how tandem duplicated UDP-glycosyltransferases undergo neofunctionalization and shape the chemical diversity of triterpenoid saponins in the Cucurbitaceae family. A chromosome-level genome of Siraitia grosvenorii was assembled and aligned with multiple cucurbit genomes, revealing a specific UGT73AM tandem duplication responsible for regio-selective glycosylation (e.g. the rare 1,4-linked disaccharide) of diverse saponins such as mogrosides, ginsenosides, and momordicines. Comparative genomics depicted the evolutionary trajectory of a universal saponin-biosynthesizing UGT73 tandem arrays syntenously preserved across core eudicots, where lineage-specific UGT copies contribute to distinct metabolic phenotypes. A crystal structure of SgUGT73AM30 (mogrol 25-O-glycosyltransferase) in complex with UDP and mogrol was obtained to elucidate the molecular basis of the regio-specific decoration on vicinal diol of the substrates. Altogether, these findings provide insights into tandem duplication-driven diversification of glycosyltransferases and lay the foundation for engineered glycosylation of valuable triterpenoid saponins.
Saponins↗
Cell surface glycosyltransferases--do they exist?
The presence of glycosyltransferases on surfaces of mammalian cells has been reported by many investigators and a biological role for these enzymes in cell adhesion and cell recognition has been postulated. Critical analysis, however, showed 2 major complications regarding the assay for cell surface glycosyltransferases: 1) hydrolysis of the nucleotide sugar by cell surface enzymes and subsequent intracellular use of the free sugar and 2) loss of cell integrity if trypsinized or EDTA-treated cells were used in suspension assays. We have assayed intact, viable cells in monolayer for cell surface glycosyltransferases using conditions under which intracellular utilization of free sugars generated by hydrolysis of the nucleotide sugar was prevented. Our data demonstrate that the presence of galactosyltransferases on the surface of a variety of cells, including established (normal and virally transformed) as well as nonestablished cells, is unlikely. No evidence for the existence of cell surface fucosyl- and sialytransferases could be obtained, but our data do not exclude the possibility that low levels of these enzymes are present.
Cell Line↗
Regional and cellular localization of glycosyltransferases in rat small intestine. Changes in enzymes with differentiation of intestinal epithelial cells.
Optimal assay conditions were determined for four glycosyltransferases in rat small intestinal mucosal homogenates and the regional distribution and cellular localization of these enzymes was studied. For each glycosyltransferase, similar levels of activity were found in duodenal, proximal jejunal and distal ileal segments; activities of the galactosyltransferases were lower in the distal jejunal-proximal ileal segment. Planar section studies indicated that the undifferentiated crypt cells had significantly higher levels of sialyltransferase activities in the jejunum and ileum than the mature villus cells. A similar crypt to villus gradient was found for a galactosyltransferase in the ileum. These data suggest that glycoprotein synthesis may be active in the undifferentiated crypt cells and that certain glycosyltransferases may serve as marker enzymes for cellular differentiation in the intestine.
Animals↗
Resolution of the glycosyltransferase activities from two strains of Streptococcus mutans by polyacrylamide gel electrophoresis in the presence of Tween 80.
The glycosyltransferase complex from Streptococcus mutans can be resolved by polyacrylamide gel electrophoresis following treatment with Tween 80. The enzyme complex was treated with Tween 80 and electrophoresis was performed in the presence of Tween 80. Discrete bands of glycosyltransferase activity were observed when the gels were incubated in buffered sucrose. The enzyme aggregation that occurs with the exocellular glycosyltransferase may involve lipophilic interactions that are disrupted by the Tween 80.
Electrophoresis, Polyacrylamide Gel↗
Developmental changes in collagen glycosyltransferase activities in chick embryos.
The developmental pattern of collagen galactosyltransferase and collagen glucosyltransferase activities was determined in chick embryos between the 4th and 21st day of growth. Both enzyme activities increased up to the 16th day and decreased thereafter in whole chick embryos and in most tissues studied. The highest collagen glycosyltransferase activities were found in the leg tendons of the 16-day-old embryos, and the activities found in cartilage were higher than those noted in either skin or skull, indicating that the the activities of the collagen glycosyltransferases may play a part in the regulation of the carbohydrate content of the collagen synthesized by a given tissue. The changes observed in the collagen glycosyltransferase activities agree with previous data on the development of prolyl and lysyl hydroxylase activities and also with findings on collagen turnover in the developing chick embryo.
Animals↗
Chemical and enzymic degradations of nucleoside mono- and diphosphate sugars. I. Determination of the degradation rate during the glycosyltransferase assays.
In incorporation experiments used for the determination of glycosyltransferase activities, we demonstrated that the nucleoside diphosphate sugars are decomposed in three different ways: 1, transfer of the monosaccharide to acceptor molecule, catalyzed by glycosyltransferases; 2, degradation of the glycosyl nucleotides by nucleotide pyrophosphatase into monosaccharide 1-phosphates which are further hydrolyzed into free monosaccharides by phosphatases; 3, chemical decomposition of UDP-D-[14C]Gal; UDP-D-[14C]Glc and UDP-D-[14C]GlcUA into 1,2-cyclic phosphate derivatives of the corresponding monosaccharide. All the breakdown products of the nucleoside mono- and diphosphate sugars which are obtained during the incorporation experiments may be separated by paper chromatography and their amounts may be determined. Galactosyltransferase assays on human and rat serum have shown that the three different ways of decomposition of the nucleoside diphosphate sugars are dependent mostly on the concentration of divalent cations (Mn2+, Mg2+). Inhibition of the nucleotide pyrophosphatase activity is obtained with low concentrations of UMP, but increasing concentrations of UMP inhibit also the galactosyltransferase activity and consequently enhance the formation of galactose 1,2-monophosphate. A partial elimination of the nucleotide pyrophosphatase activity was achieved by the addition of increasing concentrations of UDP-D-Gal. These results demonstrate that the determination of glycosyltransferase activities in tissues and in biological fluids is not possible without a concomitant determination of the nucleotide pyrophosphatase activity present in the assay.
Adult↗
Genetic and biochemical screens identify MGAT1 as a druggable glycosyltransferase target in STK11-mutant lung cancer.
Checkpoint inhibitors are standard-of-care therapies for non-small cell lung cancer (NSCLC), but their efficacy is limited in tumors with STK11 mutations, highlighting the need for new therapeutic strategies. Here, we performed complementary in vivo and in vitro CRISPR-Cas9 functional genomic screens to identify genes whose loss restores sensitivity to anti-PD-1 therapy. We found that loss of MGAT1, a Golgi glycosyltransferase critical for the maturation of high-mannose N-glycans into hybrid and complex glycan structures, reversed resistance to anti-PD-1 treatment in syngeneic mouse tumor models harboring STK11 mutations. Parallel co-culture screens with antigen-matched CD8+ T cells further showed that disruption of N-glycosylation strongly sensitized tumor cells to T cell-mediated killing. Genetic rescue studies demonstrated that this immune-evasion phenotype depends on MGAT1 catalytic activity, supporting direct biochemical interrogation of the enzyme. Using purified human MGAT1 and a UDP-Glo™ glycosyltransferase assay, we established a tractable screening platform and performed a 500,000-compound biochemical high-throughput screen, identifying an initial hit (compound 1; IC50 = 197 μM). Subsequent medicinal chemistry optimization delivered progressively more potent analogs, including TNG-9333 (0.814 μM) and TNG-2673 (0.043 μM) and represented a >1000-fold improvement in biochemical potency from the starting hit. Crystal structures of human MGAT1 in apo, UDP-bound, UDP-GlcNAc-bound, and inhibitor-bound states, together with SPR and DSF analyses, revealed that this chemical series engages a previously unrecognized allosteric pocket and inhibits MGAT1 through a UDP-noncompetitive mechanism. Collectively, our work implicates N-glycosylation as a key mediator of immune evasion and establishes MGAT1 as a ligandable, structurally tractable target for small-molecule drug discovery.
CRISPR/Cas9 target discovery↗
The use of liposomes as acceptors for the assay of lipid glycosyltransferases from rat brain.
Preparation and characterization of sonicated vesicles of various lipid composition containing hydroxy and normal fatty acid ceramides are reported. Such vesicles have been successfully used for the first time as acceptors for the assays of lipid glycosyltransferases, UDP-galactose:ceramide galactosyltransferase and UDPglucose: ceramide glucosyltransferase. Stability of the vesicles and the optimal enzyme activities were the criteria used to select the final composition of the vesicles. The activities of the glycosyltransferases were dependent not only on the appropriate assay conditions but also on the type and source of the phospholipids used to form the liposomes. Ceramides containing normal fatty acids were incorporated into phosphatidylcholine vesicles in a molar ratio of 1 : 3.4 and used as the acceptor for the assay of UDPglucose:ceramide glucostyltransferase. For the assay UDP-galactose:ceramide galactosyltransferase, vesicles were prepared by sonication of bovine brain ethanolamine phospholipids, phosphatidylcholine and ceramide containing alpha-hydroxy fatty acids, in a molar ratio of 6 : 0.57 : 1. The size of the vesicles as determined by electron microscopic measurement ranged mostly between 200--500 A. The results obtained by selective labelling of the outer surface amino groups with the membrane-impermeable reagent, 2,4,6-trinitrobenzenesulfonic acid, indicated that the ethanolamine phospholipid-containing liposomes consisted of closed vesicles. After incubation with the appropriate cofactors and labelled sugar nucleotides, the radioactive reaction products were shown to cochromatograph with the authentic standards by thin-layer chromatography and autoradiography.
Animals↗
Putative rhamnogalacturonan-II glycosyltransferase identified through callus gene editing which bypasses embryo lethality.
Rhamnogalacturonan II (RG-II) is a structurally complex and conserved domain of the pectin present in the primary cell walls of vascular plants. Borate cross-linking of RG-II is required for plants to grow and develop normally. Mutations that alter RG-II structure also affect cross-linking and are lethal or severely impair growth. Thus, few genes involved in RG-II synthesis have been identified. Here, we developed a method to generate viable loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in callus tissue via CRISPR/Cas9-mediated gene editing. We combined this with a candidate gene approach to characterize the male gametophyte defective 2 (MGP2) gene that encodes a putative family GT29 glycosyltransferase. Plants homozygous for this mutation do not survive. We showed that in the callus mutant cell walls, RG-II does not cross-link normally because it lacks 3-deoxy-D-manno-octulosonic acid (Kdo) and thus cannot form the α-L-Rhap-(1→5)-α-D-kdop-(1→sidechain). We suggest that MGP2 encodes an inverting RG-II CMP-β-Kdo transferase (RCKT1). Our discovery provides further insight into the role of sidechains in RG-II dimerization. Our method also provides a viable strategy for further identifying proteins involved in the biosynthesis of RG-II.
Arabidopsis↗
Production of Schardinger beta-dextrin by soluble and immobilized cyclodextrin glycosyltransferase of an alkalophilic Bacillus sp.
Succinylated cyclodextrin glycosyltransferase (EC 3.2.1.19) of an alkalophilic Bacillus sp. was adsorbed on a vinylpyridine copolymer. The enzyme had about 25% of the activity of soluble enzyme added. No increase of pH or thermal stability of the enzyme was observed by the adsorption, whereas optimum temperature for the enzyme action was shifted from 50 to 55 degrees C. The enzyme converted starch to cyclodextrine without significant loss of activity under the conditions of 4 times reusing of 6 hr conversion by the batch system or 2 weeks continuous reaction by the column system at 55 degrees C and pH 8.0. About 46% of the potato starch solution [15% (w/v)] was converted to cyclodextrins by the enzyme, and 52% was converted by the simultaneous action of the enzyme and alkaline pullulanase of alkalophilic Bacillus sp. (No. 202-1). These values were almost the same as those obtained by the soluble enzyme or enzymes system.
Bacillus↗
Steric requirements for the stimulation of glycosyltransferase activity by lysophosphatidylcholine.
1-Palmitoyl-sn-glycerol-3-phosphocholine and 3-palmitoyl-sn-glycerol-1-phosphocholine have been found to be equipotent in the stimulation of membrane-bound glycosyltransferases in microsomes of rat intestinal villus cells. This indicates that the stimulatory effect of lysophosphatidylcholine is not stereospecific, but that it may be related to a specific detergent property dependent upon the peculiar balance of hydrophilic and hydrophobic components in the molecule.
Animals↗
Evidence for glycosyltransferases in trout liver microsomes (Salmo gairdneri).
1. Trout (Salmo gairdneri) serum is rich in glycoproteins which are synthetized in liver. 2. An attempt to localize glycosyltransferases in hepatocytes is described, using cellular fractionation and marker enzyme determination. 3. Galactosyltransferase, mannosyltransferase, N-acetyl-glucosaminyl transferase, glucosyltransferase, sialyltransferase (on exogenous acceptor) are found in a microsomal fraction obtained by centrifugation at 117 X 10(5) g min of the post-mitochondrial supernatant. 4. Mannose is transferred to endogenous lipids and proteins.
Animals↗
Purification and characterization of a glycosyltransferase complex from the culture broth of Streptococcus mutans FA1.
The extracellular glycosyltransferases from Streptococcus mutans FA1 were purified by using the following procedures: ammonium sulfate precipitation, poly-(acrylamide) gel filtration, DEAE-cellulose chromatography, and agarose-gel filtration. The dextransucrase and levansucrase activities were purified 350- and 500-fold, repsectively, and the ratio of the two activities remained almost constant throughout the purification. Both enzymes have a pH optimum of 6.0, a Km for sucrose of 55mM, and isoelectric points of 3.7 and 4.6. The enzymes are inactivated by repeated freezing and thawing, but retain partial activity even after heating at 100 degrees. The enzyme preparation contains a carbohydrate moiety which does not appear to be either bound levan or dextran.
Chromatography, DEAE-Cellulose↗