Publications

• Glycogenin is the priming glucosyltransferase required for the initiation of glycogen biogenesis in rabbit skeletal muscle.

  Pitcher J., Smythe C., Cohen P.

  Purified preparations of glycogen synthase are a complex of two proteins, the catalytic subunit of glycogen synthase and glycogenin, present in a 1:1 molar ratio [J. Pitcher, C. Smythe, D. G. Campbell & P. Cohen (1987) Eur. J. Biochem. 169, 497-502]. This complex has now been found to contain a fu ... >> More

  Purified preparations of glycogen synthase are a complex of two proteins, the catalytic subunit of glycogen synthase and glycogenin, present in a 1:1 molar ratio [J. Pitcher, C. Smythe, D. G. Campbell & P. Cohen (1987) Eur. J. Biochem. 169, 497-502]. This complex has now been found to contain a further glucosyltransferase activity that catalyses the transfer of glucose residues from UDP-Glc to glucosylated-glycogenin. The glucosyltransferase, which is of critical importance in forming the primer required for de novo glycogen biosynthesis, is distinct from glycogen synthase in several ways. It has an absolute requirement for divalent cations, a 1000-fold lower Km for UDP-Glc and its activity is unaffected by incubation with UDP-pyridoxal or exposure to 2 M LiBr, which inactivate glycogen synthase by 95% and 100%, respectively. The priming glucosyltransferase and glycogen synthase activities coelute on Superose 6, and the rate of glycosylation of glycogenin is independent of enzyme concentration, suggesting that the reaction is catalysed intramolecularly by a subunit of the glycogen synthase complex. This component has been identified as glycogenin, following dissociation of the subunits in 2 M LiBr and their separation on Superose 12. The glycosylation of isolated glycogenin reaches a plateau when five additional glucose residues have been added to the protein, and digestion with alpha-amylase indicates that all the glycogenin molecules contain at least one glucosyl residue prior to autoglucosylation. The priming glucosyltransferase activity of glycogenin is unaffected by either glucose 6-phosphate or by phosphorylation of the catalytic subunit of glycogen synthase. The mechanism of primer formation is discussed in the light of the finding that glycogenin is an enzyme that catalyses its own autoglucosylation. << Less

  Eur J Biochem 176:391-395(1988) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Catalytic activities of glycogenin additional to autocatalytic self-glucosylation.

  Alonso M.D., Lomako J., Lomako W.M., Whelan W.J.

  Glycogenin is the autocatalytic, self-glucosylating protein that initiates glycogen synthesis in muscle and other tissues. We have sequenced the cDNA for rabbit muscle glycogenin and expressed and purified the protein in high yield as well as two mutant proteins in which Phe or Thr replaces Tyr-19 ... >> More

  Glycogenin is the autocatalytic, self-glucosylating protein that initiates glycogen synthesis in muscle and other tissues. We have sequenced the cDNA for rabbit muscle glycogenin and expressed and purified the protein in high yield as well as two mutant proteins in which Phe or Thr replaces Tyr-194, the site of glucosylation. While the wild-type protein can self-glucosylate, the mutants cannot, but all three utilize alternative acceptors by intermolecular glucose transfer for which the mutants have altered specificity. Tyr-194 is therefore not essential for the catalytic activity of glycogenin. All three proteins also hydrolyze UDP-glucose to glucose at rates comparable with the rate of self-glucosylation. The hydrolysis is competitive with glucose transfer to p-nitrophenyl alpha-maltoside. Self-glucosylation, glucosylation of other acceptors, and hydrolysis all appear to be catalyzed by the same active center. In the absence of peptidase inhibitors, the homogenous recombinant proteins of M(r) 37,000 break down to equally active species having M(r) 32,000. The kinetics of self-glucosylation catalyzed by the wild-type enzyme suggest that the reaction could be intermolecular rather than, as previously reported, intramolecular. The wild-type recombinant enzyme and native muscle glycogenin, which is phosphorylated, are inhibited quite differently by ATP at physiological concentration. << Less

  J Biol Chem 270:15315-15319(1995) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Crystal structure of the autocatalytic initiator of glycogen biosynthesis, glycogenin.

  Gibbons B.J., Roach P.J., Hurley T.D.

  Glycogen is an important storage reserve of glucose present in many organisms, from bacteria to humans. Its biosynthesis is initiated by a specialized protein, glycogenin, which has the unusual property of transferring glucose from UDP-glucose to form an oligosaccharide covalently attached to itse ... >> More

  Glycogen is an important storage reserve of glucose present in many organisms, from bacteria to humans. Its biosynthesis is initiated by a specialized protein, glycogenin, which has the unusual property of transferring glucose from UDP-glucose to form an oligosaccharide covalently attached to itself at Tyr194. Glycogen synthase and the branching enzyme complete the synthesis of the polysaccharide. The structure of glycogenin was solved in two different crystal forms. Tetragonal crystals contained a pentamer of dimers in the asymmetric unit arranged in an improper non-crystallographic 10-fold relationship, and orthorhombic crystals contained a monomer in the asymmetric unit that is arranged about a 2-fold crystallographic axis to form a dimer. The structure was first solved to 3.4 A using the tetragonal crystal form and a three-wavelength Se-Met multi-wavelength anomalous diffraction (MAD) experiment. Subsequently, an apo-enzyme structure and a complex between glycogenin and UDP-glucose/Mn2+ were solved by molecular replacement to 1.9 A using the orthorhombic crystal form. Glycogenin contains a conserved DxD motif and an N-terminal beta-alpha-beta Rossmann-like fold that are common to the nucleotide-binding domains of most glycosyltransferases. Although sequence identity amongst glycosyltransferases is minimal, the overall folds are similar. In all of these enzymes, the DxD motif is essential for coordination of the catalytic divalent cation, most commonly Mn2+. We propose a mechanism in which the Mn2+ that associates with the UDP-glucose molecule functions as a Lewis acid to stabilize the leaving group UDP and to facilitate the transfer of the glucose moiety to an intermediate nucleophilic acceptor in the enzyme active site, most likely Asp162. Following transient transfer to Asp162, the glucose moiety is then delivered to the final acceptor, either directly to Tyr194 or to glucose residues already attached to Tyr194. The positioning of the bound UDP-glucose far from Tyr194 in the glycogenin structure raises questions as to the mechanism for the attachment of the first glucose residues. Possibly the initial glucosylation is via inter-dimeric catalysis with an intra-molecular mechanism employed later in oligosaccharide synthesis. << Less

  J. Mol. Biol. 319:463-477(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of human glycogenin-2, a self-glucosylating initiator of liver glycogen metabolism.

  Mu J., Roach P.J.

  Glycogenin-2 is a recently described self-glucosylating protein potentially involved in the initiation of glycogen biosynthesis (Mu, J., Skurat, A. V., and Roach, P. J. (1997) J. Biol. Chem. 272, 27589-27597). In human liver extracts, most of the glycogenin-2 was only detectable after treatment wi ... >> More

  Glycogenin-2 is a recently described self-glucosylating protein potentially involved in the initiation of glycogen biosynthesis (Mu, J., Skurat, A. V., and Roach, P. J. (1997) J. Biol. Chem. 272, 27589-27597). In human liver extracts, most of the glycogenin-2 was only detectable after treatment with alpha-amylase. Similarly, purifed high Mr glycogen was only detected after release by alpha-amylase treatment. Based on analysis by polymerase chain reaction, the predominant isoform in liver was glycogenin-2beta. Glycogenin-2 was found in Ewing's sarcoma RD-ES cells where, however, it was not associated with high Mr carbohydrate. Both human liver and human RD-ES cell extracts also contained glycogenin-1. Glycogenin-1 and glycogenin-2 interact with one another, based on in vitro interactions and co-immunoprecipitation from liver and cell extracts. Mutation of Tyr-196 in glycogenin-2 to a Phe residue abolished the ability of glycogenin-2 to self-glucosylate but not to interact with glycogenin-1. Stable overexpression of glycogenin-2alpha in Rat-1 fibroblast cells resulted in a 5-fold increase in the level of glycogen present in the low speed pellet but little change in the low speed supernatant. This result is important since it indicates that the level of glycogenin-2 can determine glycogen accumulation and hence has the potential to control glycogen synthesis. << Less

  J. Biol. Chem. 273:34850-34856(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• A self-glucosylating protein is the primer for rabbit muscle glycogen biosynthesis.

  Lomako J., Lomako W.M., Whelan W.J.

  In this paper we elucidate part of the mechanism of the early stages of the biosynthesis of glycogen. This macromolecule is constructed by covalent apposition of glucose units to a protein, glycogenin, which remains covalently attached to the mature glycogen molecule. We have now isolated, in a 35 ... >> More

  In this paper we elucidate part of the mechanism of the early stages of the biosynthesis of glycogen. This macromolecule is constructed by covalent apposition of glucose units to a protein, glycogenin, which remains covalently attached to the mature glycogen molecule. We have now isolated, in a 3500-fold purification, a protein from rabbit muscle that has the same Mr as glycogenin, is immunologically similar, and proves to be a self-glucosylating protein (SGP). When incubated with UDP-[14C]glucose, an average of one molecular proportion of glucose is incorporated into the protein, which we conclude is the same as glycogenin isolated from native glycogen. The native SGP appears to exist as a high-molecular-weight species that contains many identical subunits. Because the glucose that is self-incorporated can be released almost completely from the acceptor by glycogenolytic enzymes, the indication is that it was added to a preformed chain or chains of 1,4-linked alpha-glucose residues. This implies that SGP already carries an existing maltosaccharide chain or chains to which the glucose is added, rather than glucose being added directly to protein. The putative role of SGP in glycogen synthesis is confirmed by the fact that glucosylated SGP acts as a primer for glycogen synthase and branching enzyme to form high-molecular-weight material. SGP itself is completely free from glycogen synthase. The quantity of SGP in muscle is calculated to be about one-half the amount of glycogenin bound in glycogen. << Less

  FASEB J 2:3097-3103(1988) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• A novel glycosyl-amino acid linkage: rabbit-muscle glycogen is covalently linked to a protein via tyrosine.

  Rodriguez I.R., Whelan W.J.

  A recent review summarizes our identification in rabbit-muscle glycogen of a protein that resists all attempts at removal by means that should displace non-covalently bound protein [Kennedy et al. (1985) In Membranes and Muscle (Berman, M.C., Gevers, W. and Opie, L.H. eds.) pp. 65-84, ICSU Press/I ... >> More

  A recent review summarizes our identification in rabbit-muscle glycogen of a protein that resists all attempts at removal by means that should displace non-covalently bound protein [Kennedy et al. (1985) In Membranes and Muscle (Berman, M.C., Gevers, W. and Opie, L.H. eds.) pp. 65-84, ICSU Press/IRL Press, Oxford]. Here we confirm that the glycogen is covalently bonded to the protein and report that the attachment is via a novel glycosidic linkage involving the hydroxyl group of tyrosine. << Less

  Biochem Biophys Res Commun 132:829-836(1985) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Identification of the 38-kDa subunit of rabbit skeletal muscle glycogen synthase as glycogenin.

  Pitcher J., Smythe C., Campbell D.G., Cohen P.

  Glycogen synthase from rabbit skeletal muscle has been shown to be a complex of two types of subunit which have apparent molecular masses of 86 kDa and 38 kDa and are present in a 1:1 molar ratio. The 38-kDa component was separated from the 86-kDa catalytic subunit by gel filtration in the presenc ... >> More

  Glycogen synthase from rabbit skeletal muscle has been shown to be a complex of two types of subunit which have apparent molecular masses of 86 kDa and 38 kDa and are present in a 1:1 molar ratio. The 38-kDa component was separated from the 86-kDa catalytic subunit by gel filtration in the presence of 2 M LiBr, and a number of chymotryptic peptides were sequenced. This demonstrated that the 38-kDa subunit was glycogenin, the protein that is bound covalently to glycogen and believed to be the 'primer' involved in the initiation of de novo glycogen synthesis. << Less

  Eur J Biochem 169:497-502(1987) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• A new look at the biogenesis of glycogen.

  Alonso M.D., Lomako J., Lomako W.M., Whelan W.J.

  The discovery of glycogenin as a self-glucosylating protein that primes glycogen synthesis has significantly increased our understanding of the structure and metabolism of this storage polysaccharide. The amount of glycogenin will influence how much glycogen the cell can store. Therefore, the prod ... >> More

  The discovery of glycogenin as a self-glucosylating protein that primes glycogen synthesis has significantly increased our understanding of the structure and metabolism of this storage polysaccharide. The amount of glycogenin will influence how much glycogen the cell can store. Therefore, the production of active glycogenin primer in the cell has the potential to be the overall rate-limiting process in glycogen formation, capable of overriding the better understood hormonally controlled mechanisms of protein phosphorylation/dephosphorylation that regulate the activities of glycogen synthase and phosphorylase. There are indications that a similar covalent modification control is also being exerted on glycogenin. Glycogenin has the ability to glucosylate molecules other than itself and to hydrolyze UDPglucose. These are independent of self-glucosylation, so that glycogenin, even when it has completed its priming role and become part of the glycogen molecule, retains its catalytic potential. Another new component of glycogen metabolism has been discovered that may have even greater influence on total glycogen stores than does glycogenin. This is proglycogen, a low molecular mass (approximately 400 kDa) form of glycogen that serves as a stable intermediate on the pathways to and from depot glycogen (macroglycogen, mass 10(7) Da, in muscle). It is suggested that glycogen oscillates, according to glucose supply and energy demand, between the macroglycogen and proglycogen, but not usually the glycogenin, forms. The proportion of proglycogen to macroglycogen varies widely between liver, skeletal muscle, and heart, from 3 to 15% to 50% by weight, respectively. On a molar basis, proglycogen is greatly in excess over macroglycogen in muscle and heart, meaning that if the proglycogen in these tissues could be converted into macroglycogen, they could store much more total glycogen. Discovering the factors that regulate the balance between glycogenin, proglycogen, and macroglycogen may have important implications for the understanding and management of noninsulin-dependent diabetes and for exercise physiology. << Less

  FASEB J 9:1126-1137(1995) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structural basis for the recruitment of glycogen synthase by glycogenin.

  Zeqiraj E., Tang X., Hunter R.W., Garcia-Rocha M., Judd A., Deak M., von Wilamowitz-Moellendorff A., Kurinov I., Guinovart J.J., Tyers M., Sakamoto K., Sicheri F.

  Glycogen is a primary form of energy storage in eukaryotes that is essential for glucose homeostasis. The glycogen polymer is synthesized from glucose through the cooperative action of glycogen synthase (GS), glycogenin (GN), and glycogen branching enzyme and forms particles that range in size fro ... >> More

  Glycogen is a primary form of energy storage in eukaryotes that is essential for glucose homeostasis. The glycogen polymer is synthesized from glucose through the cooperative action of glycogen synthase (GS), glycogenin (GN), and glycogen branching enzyme and forms particles that range in size from 10 to 290 nm. GS is regulated by allosteric activation upon glucose-6-phosphate binding and inactivation by phosphorylation on its N- and C-terminal regulatory tails. GS alone is incapable of starting synthesis of a glycogen particle de novo, but instead it extends preexisting chains initiated by glycogenin. The molecular determinants by which GS recognizes self-glucosylated GN, the first step in glycogenesis, are unknown. We describe the crystal structure of Caenorhabditis elegans GS in complex with a minimal GS targeting sequence in GN and show that a 34-residue region of GN binds to a conserved surface on GS that is distinct from previously characterized allosteric and binding surfaces on the enzyme. The interaction identified in the GS-GN costructure is required for GS-GN interaction and for glycogen synthesis in a cell-free system and in intact cells. The interaction of full-length GS-GN proteins is enhanced by an avidity effect imparted by a dimeric state of GN and a tetrameric state of GS. Finally, the structure of the N- and C-terminal regulatory tails of GS provide a basis for understanding phosphoregulation of glycogen synthesis. These results uncover a central molecular mechanism that governs glycogen metabolism. << Less

  Proc. Natl. Acad. Sci. U.S.A. 111:E2831-2840(2014) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• A precursor of glycogen biosynthesis: alpha-1,4-glucan-protein.

  Krisman C.R., Barengo R.

  The mechanism of glycogen biosynthesis in the absence of added primers takes place at least in two different steps. The first step could be initiated by a new enzyme named glycogen intiator synthase that catalyzes the transfer of glucose from UDP-glucose to an acceptor protein. This step takes pla ... >> More

  The mechanism of glycogen biosynthesis in the absence of added primers takes place at least in two different steps. The first step could be initiated by a new enzyme named glycogen intiator synthase that catalyzes the transfer of glucose from UDP-glucose to an acceptor protein. This step takes place in vitro only in the presence of some salts at high concentration. The complex product from this reaction has been isolated and demonstrated to act as a precursor for the synthesis of glycogen which takes place in the second step and is catalyzed by the already known glycogen synthase. << Less

  Eur J Biochem 52:117-123(1975) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.