Publications

• Molecular characterization of nucleocytosolic O-GlcNAc transferases of Giardia lamblia and Cryptosporidium parvum.

  Banerjee S., Robbins P.W., Samuelson J.

  O-Linked N-acetylglucosaminyltransferase (OGT) catalyzes the transfer of a single GlcNAc to the Ser or Thr of nucleocytoplasmic proteins. OGT activity, which may compete with that of kinases, is involved in signaling in animals and plants, and abnormalities in OGT activities have been associated w ... >> More

  O-Linked N-acetylglucosaminyltransferase (OGT) catalyzes the transfer of a single GlcNAc to the Ser or Thr of nucleocytoplasmic proteins. OGT activity, which may compete with that of kinases, is involved in signaling in animals and plants, and abnormalities in OGT activities have been associated with type 2 diabetes. Here, we show that ogt genes that predict enzymes with characteristic tetratricopeptide repeats and a spindly domain are present in some protists (Giardia, Cryptosporidium, Toxoplasma, and Dictyostelium) but are absent from the majority of protists examined (e.g., Plasmodium, Trypanosoma, Entamoeba, and Trichomonas). Similarly, ogt genes are present in some fungi but are absent from numerous other fungi, suggesting that secondary loss is an important contributor to the evolution of ogt genes. Nucleocytosolic extracts of Giardia and Cryptosporidium show OGT activity, and recombinant Giardia and Cryptosporidium OGTs are active in yeast and bacteria, respectively. These results suggest the possibility that O-GlcNAc modification of nucleocytosolic proteins also has function(s) in simple eukaryotes. << Less

  Glycobiology 19:331-336(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• O-Linked-N-acetylglucosamine on extracellular protein domains mediates epithelial cell-matrix interactions.

  Sakaidani Y., Nomura T., Matsuura A., Ito M., Suzuki E., Murakami K., Nadano D., Matsuda T., Furukawa K., Okajima T.

  The O-linked-N-acetylglucosamine (O-GlcNAc) modification of cytoplasmic and nuclear proteins regulates basic cellular functions and is involved in the aetiology of diabetes and neurodegeneration. This intracellular O-GlcNAcylation is catalyzed by a single O-GlcNAc transferase, OGT. Here we report ... >> More

  The O-linked-N-acetylglucosamine (O-GlcNAc) modification of cytoplasmic and nuclear proteins regulates basic cellular functions and is involved in the aetiology of diabetes and neurodegeneration. This intracellular O-GlcNAcylation is catalyzed by a single O-GlcNAc transferase, OGT. Here we report a novel OGT, EOGT, responsible for extracellular O-GlcNAcylation. Although both OGT and EOGT are regulated by hexosamine flux, EOGT localizes to the lumen of the endoplasmic reticulum and transfers GlcNAc to epidermal growth factor-like domains in an OGT-independent manner. Loss of Eogt gives phenotypes similar to those caused by defects in the apical extracellular matrix. Dumpy (Dp), a membrane-anchored extracellular protein, is O-GlcNAcylated, and EOGT is required for Dp-dependent epithelial cell-matrix interactions. Thus, O-GlcNAcylation of secreted and membrane glycoproteins is a novel mediator of cell-cell or cell-matrix interactions at the cell surface. << Less

  Nat. Commun. 2:583-583(2011) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structural insights into the mechanism and inhibition of eukaryotic O-GlcNAc hydrolysis.

  Rao F.V., Dorfmueller H.C., Villa F., Allwood M., Eggleston I.M., van Aalten D.M.

  O-linked N-acetylglucosamine (O-GlcNAc) modification of specific serines/threonines on intracellular proteins in higher eukaryotes has been shown to directly regulate important processes such as the cell cycle, insulin sensitivity and transcription. The structure, molecular mechanisms of catalysis ... >> More

  O-linked N-acetylglucosamine (O-GlcNAc) modification of specific serines/threonines on intracellular proteins in higher eukaryotes has been shown to directly regulate important processes such as the cell cycle, insulin sensitivity and transcription. The structure, molecular mechanisms of catalysis, protein substrate recognition/specificity of the eukaryotic O-GlcNAc transferase and hydrolase are largely unknown. Here we describe the crystal structure, enzymology and in vitro activity on human substrates of Clostridium perfringens NagJ, a close homologue of human O-GlcNAcase (OGA), representing the first family 84 glycoside hydrolase structure. The structure reveals a deep active site pocket highly conserved with the human enzyme, compatible with binding of O-GlcNAcylated peptides. Together with mutagenesis data, the structure supports a variant of the substrate-assisted catalytic mechanism, involving two aspartic acids and an unusually positioned tyrosine. Insights into recognition of substrate come from a complex with the transition state mimic O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (Ki=5.4 nM). Strikingly, the enzyme is inhibited by the pseudosubstrate peptide Ala-Cys(-S-GlcNAc)-Ala, and has OGA activity against O-GlcNAcylated human proteins, suggesting that the enzyme is a suitable model for further studies into the function of human OGA. << Less

  EMBO J. 25:1569-1578(2006) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Structure of human O-GlcNAc transferase and its complex with a peptide substrate.

  Lazarus M.B., Nam Y., Jiang J., Sliz P., Walker S.

  The essential mammalian enzyme O-linked β-N-acetylglucosamine transferase (O-GlcNAc transferase, here OGT) couples metabolic status to the regulation of a wide variety of cellular signalling pathways by acting as a nutrient sensor. OGT catalyses the transfer of N-acetylglucosamine from UDP-N-acety ... >> More

  The essential mammalian enzyme O-linked β-N-acetylglucosamine transferase (O-GlcNAc transferase, here OGT) couples metabolic status to the regulation of a wide variety of cellular signalling pathways by acting as a nutrient sensor. OGT catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine (UDP-GlcNAc) to serines and threonines of cytoplasmic, nuclear and mitochondrial proteins, including numerous transcription factors, tumour suppressors, kinases, phosphatases and histone-modifying proteins. Aberrant glycosylation by OGT has been linked to insulin resistance, diabetic complications, cancer and neurodegenerative diseases including Alzheimer's. Despite the importance of OGT, the details of how it recognizes and glycosylates its protein substrates are largely unknown. We report here two crystal structures of human OGT, as a binary complex with UDP (2.8 Å resolution) and as a ternary complex with UDP and a peptide substrate (1.95 Å). The structures provide clues to the enzyme mechanism, show how OGT recognizes target peptide sequences, and reveal the fold of the unique domain between the two halves of the catalytic region. This information will accelerate the rational design of biological experiments to investigate OGT's functions; it will also help the design of inhibitors for use as cellular probes and help to assess its potential as a therapeutic target. << Less

  Nature 469:564-567(2011) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• O-linked GlcNAc transferase is a conserved nucleocytoplasmic protein containing tetratricopeptide repeats.

  Lubas W.A., Frank D.W., Krause M., Hanover J.A.

  O-Linked GlcNAc addition and phosphorylation may compete for sites on nuclear pore proteins and transcription factors. We sequenced O-linked GlcNAc transferase from rabbit blood and identified the homologous Caenorhabditis elegans transferase gene on chromosome III. We then isolated C. elegans and ... >> More

  O-Linked GlcNAc addition and phosphorylation may compete for sites on nuclear pore proteins and transcription factors. We sequenced O-linked GlcNAc transferase from rabbit blood and identified the homologous Caenorhabditis elegans transferase gene on chromosome III. We then isolated C. elegans and human cDNAs encoding the transferase. The enzymes from the two species appear to be highly conserved; both contain multiple tetratricopeptide repeats and nuclear localization sequences. The C. elegans transferase accumulated in the nucleus and in perinuclear aggregates in overexpressing transgenic lines. O-Linked GlcNAc transferase activity was also elevated in HeLa cells transfected with the human cDNA. At least four human transcripts were observed in the tissues examined ranging in size from 4.4 to 9.3 kilobase pairs. The two largest transcripts (7.9 and 9.3 kilobase pairs) were enriched at least 12-fold in the pancreas. Based on its substrate specificity and molecular features, we propose that O-linked GlcNAc transferase is part of a glucose-responsive pathway previously implicated in the pathogenesis of diabetes mellitus. << Less

  J. Biol. Chem. 272:9316-9324(1997) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• O-linked-N-acetylglucosamine modification of mammalian Notch receptors by an atypical O-GlcNAc transferase Eogt1.

  Sakaidani Y., Ichiyanagi N., Saito C., Nomura T., Ito M., Nishio Y., Nadano D., Matsuda T., Furukawa K., Okajima T.

  O-linked-β-N-acetylglucosamine (O-GlcNAc) modification is a unique cytoplasmic and nuclear protein modification that is common in nearly all eukaryotes, including filamentous fungi, plants, and animals. We had recently reported that epidermal growth factor (EGF) repeats of Notch and Dumpy are O-Gl ... >> More

  O-linked-β-N-acetylglucosamine (O-GlcNAc) modification is a unique cytoplasmic and nuclear protein modification that is common in nearly all eukaryotes, including filamentous fungi, plants, and animals. We had recently reported that epidermal growth factor (EGF) repeats of Notch and Dumpy are O-GlcNAcylated by an atypical O-GlcNAc transferase, EOGT, in Drosophila. However, no study has yet shown whether O-GlcNAcylation of extracellular proteins is limited to insects such as Drosophila or whether it occurs in other organisms, including mammals. Here, we report the characterization of A130022J15Rik, a mouse gene homolog of Drosophila Eogt (Eogt 1). Enzymatic analysis revealed that Eogt1 has a substrate specificity similar to that of Drosophila EOGT, wherein the Thr residue located between the fifth and sixth conserved cysteines of the folded EGF-like domains is modified. This observation is supported by the fact that the expression of Eogt1 in Drosophila rescued the cell-adhesion defect caused by Eogt downregulation. In HEK293T cells, Eogt1 expression promoted modification of Notch1 EGF repeats by O-GlcNAc, which was further modified, at least in part, by galactose to generate a novel O-linked-N-acetyllactosamine structure. These results suggest that Eogt1 encodes EGF domain O-GlcNAc transferase and that O-GlcNAcylation reaction in the secretory pathway is a fundamental biochemical process conserved through evolution. << Less

  Biochem. Biophys. Res. Commun. 419:14-19(2012) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Glycosylation of nuclear and cytoplasmic proteins. Purification and characterization of a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase.

  Haltiwanger R.S., Blomberg M.A., Hart G.W.

  Using a combination of conventional and affinity chromatographic techniques, we have purified a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase (O-GlcNAc transferase) over 30,000-fold from rat liver cytosol. The transferase is soluble and very large, migratin ... >> More

  Using a combination of conventional and affinity chromatographic techniques, we have purified a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase (O-GlcNAc transferase) over 30,000-fold from rat liver cytosol. The transferase is soluble and very large, migrating with an apparent molecular weight of 340,000 on molecular sieve chromatography. Analysis of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals two protein species migrating at 110 (alpha subunit) and 78 (beta subunit) kDa in approximately a two-to-one ratio. Thus, the enzyme likely exists as a heterotrimer complex with two subunits of 110 kDa and one of 78 kDa (alpha 2 beta). The alpha subunit appears to contain the enzyme's active site since it is selectively radiolabeled by a specific photoaffinity probe (4-[beta-32P]thiouridine diphosphate). Photoinactivation and photolabeling of the enzyme are dependent on time and long wavelength ultraviolet light. Photolabeling of the alpha subunit is specifically blocked by UDP. The enzyme has an extremely high affinity for UDP-GlcNAc (Km = 545 nM). This unusually high affinity for the sugar nucleotide donor probably provides the enzyme an advantage over the nucleotide transporters in the endoplasmic reticulum and Golgi apparatus which compete for available cytoplasmic UDP-GlcNAc. The multimeric state and large size of the O-GlcNAc transferase imply that its activity may be highly regulated within the cell. << Less

  J. Biol. Chem. 267:9005-9013(1992) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structural insights into mechanism and specificity of O-GlcNAc transferase.

  Clarke A.J., Hurtado-Guerrero R., Pathak S., Schuttelkopf A.W., Borodkin V., Shepherd S.M., Ibrahim A.F., van Aalten D.M.

  Post-translational modification of protein serines/threonines with N-acetylglucosamine (O-GlcNAc) is dynamic, inducible and abundant, regulating many cellular processes by interfering with protein phosphorylation. O-GlcNAcylation is regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase, both enc ... >> More

  Post-translational modification of protein serines/threonines with N-acetylglucosamine (O-GlcNAc) is dynamic, inducible and abundant, regulating many cellular processes by interfering with protein phosphorylation. O-GlcNAcylation is regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase, both encoded by single, essential, genes in metazoan genomes. It is not understood how OGT recognises its sugar nucleotide donor and performs O-GlcNAc transfer onto proteins/peptides, and how the enzyme recognises specific cellular protein substrates. Here, we show, by X-ray crystallography and mutagenesis, that OGT adopts the (metal-independent) GT-B fold and binds a UDP-GlcNAc analogue at the bottom of a highly conserved putative peptide-binding groove, covered by a mobile loop. Strikingly, the tetratricopeptide repeats (TPRs) tightly interact with the active site to form a continuous 120 A putative interaction surface, whereas the previously predicted phosphatidylinositide-binding site locates to the opposite end of the catalytic domain. On the basis of the structure, we identify truncation/point mutants of the TPRs that have differential effects on activity towards proteins/peptides, giving first insights into how OGT may recognise its substrates. << Less

  EMBO J 27:2780-2788(2008) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.