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Peter J. Roach - One of the best experts on this subject based on the ideXlab platform.

  • Glycogenin is dispensable for glycogen synthesis in human muscle and Glycogenin deficiency causes polyglucosan storage
    The Journal of Clinical Endocrinology and Metabolism, 2020
    Co-Authors: Kittichate Visuttijai, Carola Hedbergoldfors, Christer Thomsen, Emma Glamuzina, C Kornblum, Giorgio Tasca, Aurelio Hernandezlain, Joakim Sandstedt, Goran Dellgren, Peter J. Roach
    Abstract:

    CONTEXT Glycogenin is considered to be an essential primer for glycogen biosynthesis. Nevertheless, patients with Glycogenin-1 deficiency due to biallelic GYG1 (NM_004130.3) mutations can store glycogen in muscle. Glycogenin-2 has been suggested as an alternative primer for glycogen synthesis in patients with Glycogenin-1 deficiency. OBJECTIVE The objective of this article is to investigate the importance of Glycogenin-1 and Glycogenin-2 for glycogen synthesis in skeletal and cardiac muscle. DESIGN, SETTING, AND PATIENTS Glycogenin-1 and Glycogenin-2 expression was analyzed by Western blot, mass spectrometry, and immunohistochemistry in liver, heart, and skeletal muscle from controls and in skeletal and cardiac muscle from patients with Glycogenin-1 deficiency. RESULTS Glycogenin-1 and Glycogenin-2 both were found to be expressed in the liver, but only Glycogenin-1 was identified in heart and skeletal muscle from controls. In patients with truncating GYG1 mutations, neither Glycogenin-1 nor Glycogenin-2 was expressed in skeletal muscle. However, nonfunctional Glycogenin-1 but not Glycogenin-2 was identified in cardiac muscle from patients with cardiomyopathy due to GYG1 missense mutations. By immunohistochemistry, the mutated Glycogenin-1 colocalized with the storage of glycogen and polyglucosan in cardiomyocytes. CONCLUSIONS Glycogen can be synthesized in the absence of Glycogenin, and Glycogenin-1 deficiency is not compensated for by upregulation of functional Glycogenin-2. Absence of Glycogenin-1 leads to the focal accumulation of glycogen and polyglucosan in skeletal muscle fibers. Expression of mutated Glycogenin-1 in the heart is deleterious, and it leads to storage of abnormal glycogen and cardiomyopathy.

  • INTERACTION BETWEEN Glycogenin AND GLYCOGEN SYNTHASE
    Archives of Biochemistry and Biophysics, 2006
    Co-Authors: Alexander V. Skurat, Amy D. Dietrich, Peter J. Roach
    Abstract:

    Glycogen synthase plays a key role in regulating glycogen metabolism. In a search for regulators of glycogen synthase, a yeast two-hybrid study was performed. Two glycogen synthase-interacting proteins were identified in human skeletal muscle, Glycogenin-1, and nebulin. The interaction with Glycogenin was found to be mediated by the region of Glycogenin which contains the 33 COOH-terminal amino acid residues. The regions in glycogen synthase containing both NH 2 - and COOH-terminal phosphorylation sites are not involved in the interaction. The core segment of glycogen synthase from Glu 21 to Gly 503 does not bind COOH-terminal fragment of Glycogenin. However, this region of glycogen synthase binds full-length Glycogenin indicating that Glycogenin contains at least one additional interacting site for glycogen synthase besides the COOH-terminus. We demonstrate that the COOH-terminal fragment of Glycogenin can be used as an effective high affinity reagent for the purification of glycogen synthase from skeletal muscle and liver.

  • direct detection of Glycogenin reaction products during glycogen initiation
    Biochemical and Biophysical Research Communications, 2006
    Co-Authors: Thomas D Hurley, Peter J. Roach, Chad D Walls, John R Bennett, Mu Wang
    Abstract:

    Glycogenin initiates glycogen synthesis in an autocatalytic reaction in which individual glucose residues are covalently linked to Tyrosine 194 in order to form a short priming chain of glucose residues that is a substrate for glycogen synthase which, combined with the branching enzyme, catalyzes the bulk synthesis of glycogen. We sought to develop a new enzymatic assay to better characterize both the chemical and enzymatic characteristics of this unusual reaction. By directly detecting the reaction products using electrospray mass spectrometry this procedure permits both the visualization of the intact individual reaction species produced as a function of time and quantitation of the levels of each of species. The quantitation of the reaction agrees well with previous measurements of both catalytic rate and the change in rate as a function of average glucosylation. The results from this assay provide new insight into the mechanism by which Glycogenin catalyzes the initiation reaction.

  • requirements for catalysis in mammalian Glycogenin
    Journal of Biological Chemistry, 2005
    Co-Authors: Thomas D Hurley, Stephanie L Stout, Emily Miner, Jing Zhou, Peter J. Roach
    Abstract:

    Abstract Glycogenin is a glycosyltransferase that functions as the autocatalytic initiator for the synthesis of glycogen in eukaryotic organisms. Prior structural work identified the determinants responsible for the recognition and binding of UDP-glucose and the catalytic manganese ion and implicated two aspartic acid residues in the reaction mechanism for self-glucosylation. We examined the effects of substituting asparagine and serine for the aspartic acid residues at positions 159 and 162. We also examined whether the truncation of the protein at residue 270 (Δ270) was compatible with its structural integrity and its functional role as the initiator for glycogen synthesis. The truncated form of the enzyme was indistinguishable from the wild-type enzyme by all measures of activity and could support glycogen accumulation in a Glycogenin-deficient yeast strain. Substitution of aspartate 159 by either serine or asparagine eliminated self-glucosylation and reduced trans-glucosylation activity by at least 260-fold but only reduced UDP-glucose hydrolytic activity by 4-14-fold. Substitution of aspartate 162 by either serine or asparagine eliminated self-glucosylation activity and reduced UDP-glucose hydrolytic activity by at least 190-fold. The trans-glucosylation of maltose was reduced to undetectable levels in the asparagine 162 mutant, whereas the serine 162 enzyme showed only an 18-30-fold reduction in its ability to trans-glucosylate maltose. These data support a role for aspartate 162 in the chemical step for the glucosyltransferase reaction and a role for aspartate 159 in binding and activating the acceptor molecule.

  • biochemical characterization of neurospora crassa Glycogenin gnn the self glucosylating initiator of glycogen synthesis
    FEBS Letters, 2005
    Co-Authors: Renato De Paula, Peter J. Roach, Wayne A Wilson, Hector Francisco Terenzi, Maria Celia Bertolini
    Abstract:

    Abstract Glycogenin acts in the initiation step of glycogen biosynthesis by catalyzing a self-glucosylation reaction. In a previous work [de Paula et al., Arch. Biochem. Biophys. 435 (2005) 112–124], we described the isolation of the cDNA gnn, which encodes the protein Glycogenin (GNN) in Neurospora crassa. This work presents a set of biochemical and functional studies confirming the GNN role in glycogen biosynthesis. Kinetic experiments showed a very low GNN Km (4.41 μM) for the substrate UDP-glucose. Recombinant GNN was produced in Escherichia coli and analysis by mass spectroscopy identified a peptide containing an oligosaccharide chain attached to Tyr196 residue. Site-directed mutagenesis and functional complementation of a Saccharomyces cerevisiae mutant strain confirmed the participation of this residue in the GNN self-glucosylation and indicated the Tyr198 residue as an additional, although less active, glucosylation site. The physical interaction between GNN and glycogen synthase (GSN) was analyzed by the two-hybrid assay. While the entire GSN was required for full interaction, the C-terminus in GNN was more important. Furthermore, mutation in the GNN glucosylation sites did not impair the interaction with GSN.

Juan A. Curtino - One of the best experts on this subject based on the ideXlab platform.

  • from the seminal discovery of proteoglycogen and Glycogenin to emerging knowledge and research on glycogen biology
    Biochemical Journal, 2019
    Co-Authors: Juan A. Curtino, Miguel A Aon
    Abstract:

    Although the discovery of glycogen in the liver, attributed to Claude Bernard, happened more than 160 years ago, the mechanism involved in the initiation of glucose polymerization remained unknown. The discovery of Glycogenin at the core of glycogen's structure and the initiation of its glucopolymerization is among one of the most exciting and relatively recent findings in Biochemistry. This review focuses on the initial steps leading to the seminal discoveries of proteoglycogen and Glycogenin at the beginning of the 1980s, which paved the way for subsequent foundational breakthroughs that propelled forward this new research field. We also explore the current, as well as potential, impact this research field is having on human health and disease from the perspective of glycogen storage diseases. Important new questions arising from recent studies, their links to basic mechanisms involved in the de novo glycogen biogenesis, and the pervading presence of Glycogenin across the evolutionary scale, fueled by high throughput -omics technologies, are also addressed.

  • structural and biochemical insight into Glycogenin inactivation by the glycogenosis causing t82m mutation
    FEBS Letters, 2012
    Co-Authors: M E Carrizo, Federico M Issoglio, Jorge M Romero, Juan A. Curtino
    Abstract:

    Abstract The X-ray structure of rabbit Glycogenin containing the T82M (T83M according to previous authors amino acid numbering [1] ) mutation causing glycogenosis showed the loss of Thr82 hydrogen bond to Asp162, the residue involved in the activation step of the glucose transfer reaction mechanism. Autoglucosylation, maltoside transglucosylation and UDP-glucose hydrolyzing activities were abolished even though affinity and interactions with UDP-glucose and positioning of Tyr194 acceptor were conserved. Substitution of Thr82 for serine but not for valine restored the maximum extent of autoglucosylation as well as transglucosylation and UDP-glucose hydrolysis rate. Results provided evidence sustaining the essential role of the lost single hydrogen bond for UDP-glucose activation leading to Glycogenin-bound glycogen primer synthesis.

  • mechanisms of monomeric and dimeric Glycogenin autoglucosylation
    Journal of Biological Chemistry, 2012
    Co-Authors: Federico M Issoglio, Jorge M Romero, Mara A E Carrizo, Juan A. Curtino
    Abstract:

    Initiation of glucose polymerization by Glycogenin autoglucosylation at Tyr-194 is required to prime de novo biosynthesis of glycogen. It has been proposed that the synthesis of the primer proceeds by intersubunit glucosylation of dimeric Glycogenin, even though it has not been demonstrated that this mechanism is responsible for the described polymerization extent of 12 glucoses produced by the dimer. We reported previously the intramonomer glucosylation capability of Glycogenin without determining the extent of autoglucopolymerization. Here, we show that the maximum specific autoglucosylation extent (MSAE) produced by the non-glucosylated Glycogenin monomer is 13.3 ± 1.9 glucose units, similar to the 12.5 ± 1.4 glucose units measured for the dimer. The mechanism and capacity of the dimeric enzyme to carry out full glucopolymerization were also evaluated by construction of heterodimers able to glucosylate exclusively by intrasubunit or intersubunit reaction mechanisms. The MSAE of non-glucosylated Glycogenin produced by dimer intrasubunit glucosylation was 16% of that produced by the monomer. However, partially glucosylated Glycogenin was able to almost complete its autoglucosylation by the dimer intrasubunit mechanism. The MSAE produced by heterodimer intersubunit glucosylation was 60% of that produced by the wild-type dimer. We conclude that both intrasubunit and intersubunit reaction mechanisms are necessary for the dimeric enzyme to acquire maximum autoglucosylation. The full glucopolymerization capacity of monomeric Glycogenin indicates that the enzyme is able to synthesize the glycogen primer without the need for prior dimerization.

  • evidence for Glycogenin autoglucosylation cessation by inaccessibility of the acquired maltosaccharide
    Biochemical and Biophysical Research Communications, 2008
    Co-Authors: Jorge M Romero, Federico M Issoglio, Maria Eugenia Carrizo, Juan A. Curtino
    Abstract:

    Abstract Glycogenin initiates the biosynthesis of proteoglycogen, the mammalian Glycogenin-bound glycogen, by intramolecular autoglucosylation. The incubation of Glycogenin with UDP-glucose results in formation of a tyrosine-bound maltosaccharide, reaching maximum polymerization degree of 13 glucose units at cessation of the reaction. No exhaustion of the substrate donor occurred at the autoglucosylation end and the full autoglucosylated enzyme continued catalytically active for transglucosylation of the alternative substrate dodecyl-maltose. Even the autoglucosylation cessation once Glycogenin acquired a mature maltosaccharide moiety, proteoglycogen and Glycogenin species ranging rM 47–200 kDa, derived from proteoglycogen, showed to be autoglucosylable. The results describe for the first time the ability of polysaccharide-bound Glycogenin for intramolecular autoglucosylation, providing evidence for cessation of the glucose polymerization initiated into the tyrosine residue, by inaccessibility of the acquired maltosaccharide moiety to further autoglucosylation.

  • the intramolecular autoglucosylation of monomeric Glycogenin
    Biochemical and Biophysical Research Communications, 2008
    Co-Authors: Soledad Bazan, Federico M Issoglio, Maria Eugenia Carrizo, Juan A. Curtino
    Abstract:

    Abstract The ability of monomeric Glycogenin to autoglucosylate by an intramolecular mechanism of reaction is described using non-glucosylated and partially glucosylated recombinant Glycogenin. We determined that monomer Glycogenin exists in solution at concentration below 0.60–0.85 μM. The specific autoglucosylation rate of non-glucosylated and glucosylated monomeric Glycogenin represented 50 and 70% of the specific rate of the corresponding dimeric Glycogenin species. The incorporation of a unique sugar unit into the tyrosine hydroxyl group of non-glucosylated Glycogenin, analyzed by autoxylosylation, occurred at a lower rate than the incorporation into the glucose hydroxyl group of the glucosylated enzyme. The intramonomer autoglucosylation mechanism here described for the first time, confers to a just synthesized Glycogenin molecule the capacity to produce maltosaccharide primer for glycogen synthase, without the need to reach the concentration required for association into the more efficient autoglucosylating dimer. The monomeric and dimeric interconversion determining the different autoglucosylation rate, might serve as a modulation mechanism for the de novo biosynthesis of glycogen at the initial glucose polymerization step.

Alexander V. Skurat - One of the best experts on this subject based on the ideXlab platform.

  • INTERACTION BETWEEN Glycogenin AND GLYCOGEN SYNTHASE
    Archives of Biochemistry and Biophysics, 2006
    Co-Authors: Alexander V. Skurat, Amy D. Dietrich, Peter J. Roach
    Abstract:

    Glycogen synthase plays a key role in regulating glycogen metabolism. In a search for regulators of glycogen synthase, a yeast two-hybrid study was performed. Two glycogen synthase-interacting proteins were identified in human skeletal muscle, Glycogenin-1, and nebulin. The interaction with Glycogenin was found to be mediated by the region of Glycogenin which contains the 33 COOH-terminal amino acid residues. The regions in glycogen synthase containing both NH 2 - and COOH-terminal phosphorylation sites are not involved in the interaction. The core segment of glycogen synthase from Glu 21 to Gly 503 does not bind COOH-terminal fragment of Glycogenin. However, this region of glycogen synthase binds full-length Glycogenin indicating that Glycogenin contains at least one additional interacting site for glycogen synthase besides the COOH-terminus. We demonstrate that the COOH-terminal fragment of Glycogenin can be used as an effective high affinity reagent for the purification of glycogen synthase from skeletal muscle and liver.

  • structure function analysis of gnip the Glycogenin interacting protein
    Archives of Biochemistry and Biophysics, 2004
    Co-Authors: Lanmin Zhai, Alexander V. Skurat, Amy D. Dietrich, Peter J. Roach
    Abstract:

    Glycogenin is a self-glucosylating protein that initiates glycogen biosynthesis. We recently identified a family of proteins, GNIPs, that interact with Glycogenin and stimulate its self-glucosylating activity [J. Biol. Chem. 277 (2002) 19331]. The GNIP gene (also called TRIM7) encodes at least four distinct isoforms of GNIP, three of which (GNIP1, GNIP2, and GNIP3) have in common a COOH-terminal B30.2 domain and predicted coiled-coil regions. Based on Western blot analysis, the GNIP1 protein is widely distributed in tissues. From analysis of a series of deletion mutants of GNIP2 using the yeast two-hybrid system, the B30.2 domain was found to be responsible for the interaction with Glycogenin. A truncated form of recombinant GNIP2, lacking the NH2-terminal coiled-coil region, was cross-linked to Glycogenin by glutaraldehyde treatment, supporting the idea that the B30.2 domain was sufficient for the interaction. In the course of this study, GNIP2 was also found to interact with itself, via the coiled-coil domain. Heterologous interactions between GNIP1 and GNIP2 were also detected. Since Glycogenin is also a dimer, higher order multimeric complexes between Glycogenin and GNIPs would be possible.

  • gnip a novel protein that binds and activates Glycogenin the self glucosylating initiator of glycogen biosynthesis
    Journal of Biological Chemistry, 2002
    Co-Authors: Alexander V. Skurat, Amy D. Dietrich, Lanmin Zhai, Peter J. Roach
    Abstract:

    Glycogenin is a self-glucosylating protein involved in the initiation of glycogen biosynthesis. Self-glucosylation leads to the formation of an oligosaccharide chain, which, when long enough, supports the action of glycogen synthase to elongate it and form a mature glycogen molecule. To identify possible regulators of Glycogenin, the yeast two-hybrid strategy was employed. By using rabbit skeletal muscle Glycogenin as a bait, cDNAs encoding three different proteins were isolated from the human skeletal muscle cDNA library. Two of the cDNAs encoded Glycogenin and glycogen synthase, respectively, proteins known to be interactors. The third cDNA encoded a polypeptide of unknown function and was designated GNIP (Glycogenin interacting protein). Northern blot analysis revealed that GNIP mRNA is highly expressed in skeletal muscle. The gene for GNIP generates at least four isoforms by alternative splicing. The largest isoform GNIP1 contains, from NH(2)- to COOH-terminal, a RING finger, a B box, a putative coiled-coil region, and a B30.2-like motif. The previously identified protein TRIM7 (tripartite motif containing protein 7) is also derived from the GNIP gene and is composed of the RING finger, B box, and coiled-coil regions. The GNIP2 and GNIP3 isoforms consist of the coiled-coil region and B30.2-like domain. Physical interaction between GNIP2 and Glycogenin was confirmed by co-immunoprecipitation, and in addition GNIP2 was shown to stimulate Glycogenin self-glucosylation 3-4-fold. GNIPs may represent a novel participant in the initiation of glycogen synthesis.

  • do rodents have a gene encoding Glycogenin 2 the liver isoform of the self glucosylating initiator of glycogen synthesis
    Iubmb Life, 2001
    Co-Authors: Lanmin Zhai, Alexander V. Skurat, Jill M Schroeder, Peter J. Roach
    Abstract:

    The discovery of a second human gene, GYG2, encoding a liver-specific isoform of Glycogenin, the self-glucosylating initiator of glycogen biosynthesis, raised the possibility for differential controls of this protein in liver and muscle. The new protein, Glycogenin-2, had several properties similar biochemically to the muscle isoform, Glycogenin-1, but unlike Glycogenin-1, stable expression in fibroblasts led to a significant overaccumulation of glycogen. Ensuing attempts to generate reagents suitable for use with rodents, to examine the physiological regulation of Glycogenin-2 by nutritional and hormonal factors, have been unsuccessful. Proof of a negative is difficult but the weight of the evidence is beginning to mitigate against the existence of a second Glycogenin gene in rodents leading us to hypothesize that the presence of the GYG2 gene is limited to primates.

  • Glycogenin 2 a novel self glucosylating protein involved in liver glycogen biosynthesis
    The FASEB Journal, 1997
    Co-Authors: Alexander V. Skurat, Peter J. Roach
    Abstract:

    Glycogenin is a self-glucosylating protein involved in the initiation phase of glycogen biosynthesis. A single mammalian gene had been reported to account for glycogen biogenesis in liver and muscle, the two major repositories of glycogen. We describe the characterization of novel forms of Glycogenin, designated Glycogenin-2 (GN-2), encoded by a second gene that is expressed preferentially in certain tissues, including liver, heart, and pancreas. Cloning of cDNAs encoding Glycogenin-2 indicated the existence of multiple species, including three liver forms (GN-2α, GN-2β, and GN-2γ) generated in part by alternative splicing. Overall, GN-2 has 40–45% identity to muscle Glycogenin but is 72% identical over a 200-residue segment thought to contain the catalytic domain. GN-2 expressed in Escherichia coli or COS cells is active in self-glucosylation assays, and self-glucosylated GN-2 can be elongated by skeletal muscle glycogen synthase. Antibodies raised against GN-2 produced in E. coli recognized proteins of M r ∼66,000 present in extracts of rat liver and in cultured H4IIEC3 hepatoma cells. In H4IIEC3 cells, most of the GN-2 was present as a free protein but some was covalently associated with glycogen fractions and was only released by treatment with α-amylase. H4IIEC3 cells also expressed the muscle form of Glycogenin (Glycogenin-1), which was attached to a chromatographically separable glycogen fraction.

William J Whelan - One of the best experts on this subject based on the ideXlab platform.

  • why the linkage of glycogen to Glycogenin was so difficult to determine
    Biochemistry and Molecular Biology Education, 2007
    Co-Authors: William J Whelan
    Abstract:

    Glycogenin is the self-glucosylating enzyme that primes mammalian and yeast glycogen synthesis. It proved to be the long-suspected, covalently bound protein component of glycogen. One of the most difficult aspects in elucidating the role of Glycogenin was to learn the nature of its covalent bond to glycogen.

  • Glycogenin the primer for mammalian and yeast glycogen synthesis
    Biochimica et Biophysica Acta, 2004
    Co-Authors: Joseph Lomako, Wieslawa M Lomako, William J Whelan
    Abstract:

    Glycogen synthesis, whether in mammalian tissue, yeast, or Agrobacterium tumefaciens or other bacteria, is initiated by autoglucosylation of a protein. Initiation in muscle, by a self-glucosylating protein, Glycogenin-1, is the most thoroughly studied system, as is described here. These relatively recent findings have prompted a rekindling of interest in the intermediates lying between the primer and mature mammalian glycogen.

  • the human intron containing gene for Glycogenin maps to chromosome 3 band q24
    Genomics, 1996
    Co-Authors: Joseph Lomako, Wieslawa M Lomako, William J Whelan, Miriam D. Alonso, K Mazuruk, Ignacio R Rodriguez
    Abstract:

    Glycogenin is the autocatalytic, self-glucosylating primer for glycogen synthesis, providing the anchor on which the macromolecule is constructed. We have sequenced the cDNA coding for human muscle Glycogenin and have deduced the corresponding amino acid sequence. By means of the polymerase chain reaction and fluorescence in situ hybridization, we have found the chromosomal location of the gene coding for Glycogenin. This is localized to human chromosome 3, band q24.

  • glycogen metabolism in quail embryo muscle the role of the Glycogenin primer and the intermediate proglycogen
    FEBS Journal, 1995
    Co-Authors: Joseph Lomako, Wieslawa M Lomako, William J Whelan
    Abstract:

    Cultured quail embryo muscle has proven to be an excellent model system for studying the synthesis of macromolecular glycogen from, and its degradation to, Glycogenin, the autocatalytic, self-glucosylating primer for glycogen synthesis. We recently demonstrated that proglycogen, a low-Mr form of glycogen, is an intermediate in the synthesis. Here we show that proglycogen also functions as an intermediate in macroglycogen degradation and, in one set of circumstances, represents an arrest point in glycogen breakdown, which does not continue to Glycogenin. We suggest that in the nutritionally dependent turnover of glycogen in tissues, the molecules cycle between proglycogen and macromolecular glycogen and are not normally degraded to Glycogenin. Nevertheless, when this does happen, the released Glycogenin is active, capable of re-initiating glycogen synthesis. Under culture conditions where the conversion of proglycogen into Glycogenin does take place, the intermediates lying between form a discrete rather than a continuous series, suggestive of a cluster structure for proglycogen and indicating that breakdown is stepwise. Evidence of post-translational modification of Glycogenin was obtained by the finding that, in glycogen from cultured muscle, Glycogenin is phosphorylated.

  • Catalytic activities of Glycogenin additional to autocatalytic self-glucosylation.
    Journal of Biological Chemistry, 1995
    Co-Authors: Miriam D. Alonso, Joseph Lomako, Wieslawa M Lomako, William J Whelan
    Abstract:

    Abstract 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 α-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 Mr 37,000 break down to equally active species having Mr 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.

Anders Oldfors - One of the best experts on this subject based on the ideXlab platform.

  • functional characterization of gyg1 variants in two patients with myopathy and Glycogenin 1 deficiency
    Neuromuscular Disorders, 2019
    Co-Authors: Carola Hedbergoldfors, Kittichate Visuttijai, Willem De Ridder, Ognian Kalev, Klaus Bock, Georg Caravias, Ana Topf, Volker Straub, Jonathan Baets, Anders Oldfors
    Abstract:

    Glycogen storage disease XV is caused by variants in the Glycogenin-1 gene, GYG1, and presents as a predominant skeletal myopathy or cardiomyopathy. We describe two patients with late-onset myopathy and biallelic GYG1 variants. In patient 1, the novel c.144-2A>G splice acceptor variant and the novel frameshift variant c.631delG (p.Val211Cysfs*30) were identified, and in patient 2, the previously described c.304G>C (p.Asp102His) and c.487delG (p.Asp163Thrfs*5) variants were found. Protein analysis showed total absence of Glycogenin-1 expression in patient 1, whereas in patient 2 there was reduced expression of Glycogenin-1, with the residual protein being non-functional. Both patients showed glycogen and polyglucosan storage in their muscle fibers, as revealed by PAS staining and electron microscopy. Age at onset of the myopathy phenotype was 53 years and 70 years respectively, with the selective pattern of muscle involvement on MRI corroborating the pattern of weakness. Cardiac evaluation of patient 1 and 2 did not show any specific abnormalities linked to the Glycogenin-1 deficiency. In patient 2, who was shown to express the p.Asp102His mutated Glycogenin-1, cardiac evaluation was still normal at age 77 years. This contrasts with the association of the p.Asp102His variant in homozygosity with a severe cardiomyopathy in several cases with an onset age between 30 and 50 years. This finding might indicate that the level of p.Asp102His mutated Glycogenin-1 determines if a patient will develop a cardiomyopathy.

  • is Glycogenin essential for glycogen synthesis
    Cell Metabolism, 2017
    Co-Authors: Anders Oldfors
    Abstract:

    Glycogen synthesis requires a priming oligosaccharide, formed by autoglucosylation of Glycogenin, a core protein in glycogen particles. In this edition of Cell Metabolism, Testoni et al. (2017) challenge this generally accepted concept by demonstrating that Glycogenin inactivation in mice results in an increased amount of glycogen and not glycogen depletion.

  • muscle pathology and whole body mri in a polyglucosan myopathy associated with a novel Glycogenin 1 mutation
    Neuromuscular Disorders, 2015
    Co-Authors: Sushan Luo, Anders Oldfors, Carola Hedbergoldfors, Wenhua Zhu, Dongyue Yue, Jie Lin, Yin Wang, Zhen Zhu, Wenjuan Qiu, Chongbo Zhao
    Abstract:

    We report a 46-year-old female with late-onset skeletal myopathy affecting proximal limb muscles. Muscle biopsy revealed a polyglucosan myopathy with PAS-positive inclusions predominantly in glycogen-depleted fibers, which were demonstrated as type I fibers by ATPase staining. Whole-body magnetic imaging disclosed that the paravertebral, scapular, and pelvic girdle muscles, the anterior compartment of the arms, and the posterior compartment of the thighs were preferentially involved. Genetic analysis revealed a homozygous novel mutation in exon 6 of the Glycogenin-1 gene (GYG1) (c.634C>T, p.His212Tyr). Protein analysis revealed normal levels of Glycogenin-1 even before alpha-amylase digestion indicating preserved protein expression but impaired glucosylation. In vitro functional assay demonstrated that this variant impaired the autoglucosylating ability resulting in a non-functional protein. We report a Glycogenin-1 related myopathy with a distinct histopathology and unique muscle imaging pattern.

  • LC-MS/MS characterization of combined Glycogenin-1 and Glycogenin-2 enzymatic activities reveals their self-glucosylation preferences.
    Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2014
    Co-Authors: Johanna Nilsson, Adnan Halim, Erik Larsson, Ali-reza Moslemi, Anders Oldfors, Göran Larson, Jonas Nilsson
    Abstract:

    Glycogen synthesis is initiated by self-glucosylation of the glycosyltransferases Glycogenin-1 and -2 that, in the presence of UDP-glucose, form both the first glucose-O-tyrosine linkage, and then stepwise add a series of α1,4-linked glucoses to a growing chain of variable length. Glycogen-1 and -2 coexist in liver glycogen preparations where the proteins are known to form homodimers, and they also have been shown to interact with each other. In order to study how Glycogenin-1 and -2 interactions may influence each other's glucosylations we setup a cell-free expression system for in vitro production and glucosylation of Glycogenin-1 and -2 in various combinations, and used a mass spectrometry based workflow for the characterization and quantitation of tryptic glycopeptides originating from Glycogenin-1 and -2. The analysis revealed that the self-glucosylation endpoint was the incorporation of 4-8 glucose units on Tyr 195 of Glycogenin-1, but only 0-4 glucose units on Tyr-228 of Glycogenin-2. The glucosylation of Glycogenin-2 was enhanced to 2-4 glucose units by the co-presence of enzymatically active Glycogenin-1. Glycogenin-2 was, however, unable to glucosylate inactive Glycogenin-1, at least not an enzymatically inactivated Thr83Met Glycogenin-1 mutant, recently identified in a patient with severe glycogen depletion.

  • molecular pathogenesis of a new glycogenosis caused by a Glycogenin 1 mutation
    Biochimica et Biophysica Acta, 2012
    Co-Authors: Johanna Nilsson, Adnan Halim, Ali-reza Moslemi, Göran Larson, Jonas Nilsson, Anders Pedersen, Anders Oldfors
    Abstract:

    Glycogenin-1 initiates the glycogen synthesis in skeletal muscle by the autocatalytic formation of a short oligosaccharide at tyrosine 195. Glycogenin-1 catalyzes both the glucose-O-tyrosine linkage and the α1,4 glucosidic bonds linking the glucose molecules in the oligosaccharide. We recently described a patient with glycogen depletion in skeletal muscle as a result of a non-functional Glycogenin-1. The patient carried a Thr83Met substitution in Glycogenin-1. In this study we have investigated the importance of threonine 83 for the catalytic activity of Glycogenin-1. Non-glucosylated Glycogenin-1 constructs, with various amino acid substitutions in position 83 and 195, were expressed in a cell-free expression system and autoglucosylated in vitro. The autoglucosylation was analyzed by gel-shift on western blot, incorporation of radiolabeled UDP-(14)C-glucose and nano-liquid chromatography with tandem mass spectrometry (LC/MS/MS). We demonstrate that Glycogenin-1 with the Thr83Met substitution is unable to form the glucose-O-tyrosine linkage at tyrosine 195 unless co-expressed with the catalytically active Tyr195Phe Glycogenin-1. Our results explain the glycogen depletion in the patient expressing only Thr83Met Glycogenin-1 and why heterozygous carriers without clinical symptoms show a small proportion of unglucosylated Glycogenin-1.