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

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          Abstract

          O-GlcNAc transferase (OGT) is an essential mammalian enzyme that couples metabolic status to the regulation of a wide variety of cellular signaling pathways by acting as a nutrient sensor 1. OGT catalyzes the transfer of N-acetyl-glucosamine from UDP-GlcNAc to serines and threonines of cytoplasmic, nuclear and mitochondrial proteins 2, 3, including numerous transcription factors 4, tumor suppressors, kinases 5, phosphatases 1, and histone-modifying proteins 6. Aberrant O-GlcNAcylation by OGT has been linked to insulin resistance 7, diabetic complications 8, cancer 9 and neurodegenerative diseases including Alzheimer’s 10. 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 A) and a ternary complex with UDP and a peptide substrate (1.95 A). 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 and the design of inhibitors for use as cellular probes and to assess its potential as a therapeutic target.

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          Most cited references 21

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          The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny.

          Nuclear and cytoplasmic protein glycosylation is a widespread and reversible posttranslational modification in eukaryotic cells. Intracellular glycosylation by the addition of N-acetylglucosamine (GlcNAc) to serine and threonine is catalyzed by the O-GlcNAc transferase (OGT). This "O-GlcNAcylation" of intracellular proteins can occur on phosphorylation sites, and has been implicated in controlling gene transcription, neurofilament assembly, and the emergence of diabetes and neurologic disease. To study OGT function in vivo, we have used gene-targeting approaches in male embryonic stem cells. We find that OGT mutagenesis requires a strategy that retains an intact OGT gene as accomplished by using Cre-loxP recombination, because a deletion in the OGT gene results in loss of embryonic stem cell viability. A single copy of the OGT gene is present in the male genome and resides on the X chromosome near the centromere in region D in the mouse spanning markers DxMit41 and DxMit95, and in humans at Xq13, a region associated with neurologic disease. OGT RNA expression in mice is comparably high among most cell types, with lower levels in the pancreas. Segregation of OGT alleles in the mouse germ line with ZP3-Cre recombination in oocytes reveals that intact OGT alleles are required for completion of embryogenesis. These studies illustrate the necessity of conditional gene-targeting approaches in the mutagenesis and study of essential sex-linked genes, and indicate that OGT participation in intracellular glycosylation is essential for embryonic stem cell viability and for mouse ontogeny.
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            Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc.

             C Torres,  Adam Hart (1984)
            Bovine milk galactosyltransferase has been used, in conjunction with UDP-[3H]galactose, as an impermeant probe for accessible GlcNAc residues on the surfaces of lymphocytes. Galactosylation of living thymic lymphocytes is dependent upon cell number, enzyme concentration, UDP-galactose concentration, and Mn2+ concentration. Kinetics of labeling are biphasic, leveling off at approximately 30 min. The data strongly indicate vectorial surface labeling and covalent attachment of galactose. Thymocytes, T-lymphocytes, and B-lymphocytes have approximately 10(6), 3 X 10(6), and 5 X 10(6) galactosylatable sites on their cell surfaces, respectively. Numerous proteins are exogalactosylated that differ quantitatively among the major functional subsets of lymphocytes. Negligible radioactivity is found in lipid. In thymocytes, 49% of the exogalactosylated oligosaccharides are alkali labile, whereas 80 and 90% of that derived from T-lymphocytes and B-lymphocytes can be beta-eliminated, respectively. Sensitivity of the intact proteins or tryptic peptides to the peptide: N-glycosidase also confirms the relative amounts of cell surface, N-linked and O-linked oligosaccharides which are exogalactosylated. Composition, size, and high performance liquid chromatography on two types of high resolution columns establish that the bulk of the exogalactosylated, beta-eliminated oligosaccharides are Gal beta 1-4GlcNAcitol. These data suggest the presence of O-glycosidically linked GlcNAc monosaccharide on many lymphocyte cell-surface proteins. However, additional experiments indicate that the majority of these moieties appear to be cryptic or inside the cell. Thus, these studies not only describe dramatic differences in the amounts and distribution of terminal GlcNAc residues on phenotypically different lymphocyte populations, but they also describe the presence of a novel protein-saccharide linkage, which is present on numerous lymphocyte proteins.
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              O-GlcNAcylation regulates phosphorylation of tau: a mechanism involved in Alzheimer's disease.

              Microtubule-associated protein tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles in brains of individuals with Alzheimer's disease (AD) and other tauopathies. Tau pathology is critical to pathogenesis and correlates to the severity of dementia. However, the mechanisms leading to abnormal hyperphosphorylation are unknown. Here, we demonstrate that human brain tau was modified by O-GlcNAcylation, a type of protein O-glycosylation by which the monosaccharide beta-N-acetylglucosamine (GlcNAc) attaches to serine/threonine residues via an O-linked glycosidic bond. O-GlcNAcylation regulated phosphorylation of tau in a site-specific manner both in vitro and in vivo. At most of the phosphorylation sites, O-GlcNAcylation negatively regulated tau phosphorylation. In an animal model of starved mice, low glucose uptake/metabolism that mimicked those observed in AD brain produced a decrease in O-GlcNAcylation and consequent hyperphosphorylation of tau at the majority of the phosphorylation sites. The O-GlcNAcylation level in AD brain extracts was decreased as compared to that in controls. These results reveal a mechanism of regulation of tau phosphorylation and suggest that abnormal hyperphosphorylation of tau could result from decreased tau O-GlcNAcylation, which probably is induced by deficient brain glucose uptake/metabolism in AD and other tauopathies.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                0028-0836
                1476-4687
                16 November 2010
                16 January 2011
                27 January 2011
                27 July 2011
                : 469
                : 7331
                : 564-567
                Affiliations
                [1 ] Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA
                [2 ] Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
                [3 ] Laboratory of Molecular Medicine, Children’s Hospital, Boston, Massachusetts 02115, USA
                [4 ] Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA
                Author notes
                Correspondence and requests for materials should be addressed to: S.W. ( suzanne_walker@ 123456hms.harvard.edu ) or P.S. ( piotr_sliz@ 123456hms.harvard.edu )
                [*]

                These authors contributed equally to this work

                Article
                nihpa250431
                10.1038/nature09638
                3064491
                21240259

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                Funding
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM078477-04 ||GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM078477-03 ||GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM076710-04 ||GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM076710-03 ||GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM076710-02 ||GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM076710-01A1 ||GM
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