Christopher J Contreras

Indiana University-Purdue University Indianapolis, Indianapolis, Indiana, United States

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Publications (4)35.76 Total impact

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    ABSTRACT: Glycogen is a branched polymer of glucose that acts as an energy reserve in many cell types. Glycogen contains trace amounts of covalent phosphate, in the range of one phosphate per 500-2000 glucose residues, depending on the source. The function, if any, is unknown but in at least one genetic disease, the progressive myooclonic epilepsy Lafora disease, excessive phosphorylation of glycogen has been implicated in the pathology by disturbing glycogen structure. Some 90% of Lafora cases are attributed to mutations of the EPM2A or EPM2B genes and mice with either gene disrupted accumulate hyperphosphorylated glycogen. It is therefore of importance to understand the chemistry of glycogen phosphorylation. Rabbit skeletal muscle glycogen contained covalent phosphate as monoesters of C2, C3 and C6 carbons of glucose residues based on analyses of phospho-oligosaccharides by NMR. Furthermore, using a sensitive assay for glucose-6-P in hydrolysates of glycogen coupled with measurement of total phosphate, we determined the proportion of C6 phosphorylation in rabbit muscle glycogen to be ~20%. C6 phosphorylation also accounted for ~20% of the covalent phosphate in wild type mouse muscle glycogen. Glycogen phosphorylation in Epm2a-/- and Epm2b-/- mice was increased eight- and four-fold compared to wild type mice but the proportion of C6 phosphorylation remained unchanged at ~20%. Therefore, our results suggest that C2, C3 and/or C6 phosphate could all contribute to abnormal glycogen structure or to Lafora disease. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 11/2014; 290(2). DOI:10.1074/jbc.M114.607796 · 4.60 Impact Factor
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    ABSTRACT: Glycogen is a glucose polymer that contains minor amounts of covalently attached phosphate. Hyperphosphorylation is deleterious to glycogen structure and can lead to Lafora disease. Recently, it was demonstrated that glycogen synthase catalyzes glucose-phosphate transfer in addition to its characteristic glucose transfer reaction. Glucose-1,2-cyclic-phosphate (GCP) was proposed to be formed from UDP-Glc breakdown and subsequently transferred, thus providing a source of phosphate found in glycogen. To gain further insight into the molecular basis for glucose-phosphate transfer, two structures of yeast glycogen synthase were determined; a 3.0-Å resolution structure of the complex with UMP/GCP and a 2.8-Å resolution structure of the complex with UDP/glucose. Structural superposition of the complexes revealed that the bound ligands and most active site residues are positioned similarly, consistent with the use of a common transfer mechanism for both reactions. The N-terminal domain of the UDP⋅glucose complex was found to be 13.3° more closed compared with a UDP complex. However, the UMP⋅GCP complex was 4.8° less closed than the glucose complex, which may explain the low efficiency of GCP transfer. Modeling of either α- or β-glucose or a mixture of both anomers can account for the observed electron density of the UDP⋅glucose complex. NMR studies of UDP-Glc hydrolysis by yeast glycogen synthase were used to verify the stereochemistry of the product, and they also showed synchronous GCP accumulation. The similarities in the active sites of glycogen synthase and glycogen phosphorylase support the idea of a common catalytic mechanism in GT-B enzymes independent of the specific reaction catalyzed.
    Proceedings of the National Academy of Sciences 12/2013; DOI:10.1073/pnas.1310106111 · 9.81 Impact Factor
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    ABSTRACT: Glycogen synthase is a rate-limiting enzyme in the biosynthesis of glycogen and has an essential role in glucose homeostasis. The three-dimensional structures of yeast glycogen synthase (Gsy2p) complexed with maltooctaose identified four conserved maltodextrin-binding sites distributed across the surface of the enzyme. Site-1 is positioned on the N-terminal domain, site-2 and site-3 are present on the C-terminal domain, and site-4 is located in an interdomain cleft adjacent to the active site. Mutation of these surface sites decreased glycogen binding and catalytic efficiency toward glycogen. Mutations within site-1 and site-2 reduced the V(max)/S(0.5) for glycogen by 40- and 70-fold, respectively. Combined mutation of site-1 and site-2 decreased the V(max)/S(0.5) for glycogen by >3000-fold. Consistent with the in vitro data, glycogen accumulation in glycogen synthase-deficient yeast cells (Δgsy1-gsy2) transformed with the site-1, site-2, combined site-1/site-2, or site-4 mutant form of Gsy2p was decreased by up to 40-fold. In contrast to the glycogen results, the ability to utilize maltooctaose as an in vitro substrate was unaffected in the site-2 mutant, moderately affected in the site-1 mutant, and almost completely abolished in the site-4 mutant. These data show that the ability to utilize maltooctaose as a substrate can be independent of the ability to utilize glycogen. Our data support the hypothesis that site-1 and site-2 provide a "toehold mechanism," keeping glycogen synthase tightly associated with the glycogen particle, whereas site-4 is more closely associated with positioning of the nonreducing end during catalysis.
    Journal of Biological Chemistry 08/2011; 286(39):33999-4006. DOI:10.1074/jbc.M111.264531 · 4.60 Impact Factor
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    ABSTRACT: Glycogen is a branched polymer of glucose that serves as an energy store. Phosphate, a trace constituent of glycogen, has profound effects on glycogen structure, and phosphate hyperaccumulation is linked to Lafora disease, a fatal progressive myoclonus epilepsy that can be caused by mutations of laforin, a glycogen phosphatase. However, little is known about the metabolism of glycogen phosphate. We demonstrate here that the biosynthetic enzyme glycogen synthase, which normally adds glucose residues to glycogen, is capable of incorporating the β-phosphate of its substrate UDP-glucose at a rate of one phosphate per approximately 10,000 glucoses, in what may be considered a catalytic error. We show that the phosphate in glycogen is present as C2 and C3 phosphomonoesters. Since hyperphosphorylation of glycogen causes Lafora disease, phosphate removal by laforin may thus be considered a repair or damage control mechanism.
    Cell metabolism 03/2011; 13(3):274-82. DOI:10.1016/j.cmet.2011.01.017 · 16.75 Impact Factor