C A Maile1, J R Hingst2, K K Mahalingan3, A O O'Reilly4, M E Cleasby5, J R Mickelson6, M E McCue7, S M Anderson7, T D Hurley3, J F P Wojtaszewski2, R J Piercy8. 1. Comparative Neuromuscular Diseases Laboratory, Department of Clinical Sciences and Services, Royal Veterinary College, London, UK. 2. Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, Denmark. 3. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, USA. 4. School of Natural Sciences and Psychology, Liverpool John Moores University, Liverpool, UK. 5. Department of Comparative Biomedical Sciences, Royal Veterinary College, London, UK. 6. Veterinary Biomedical Sciences Department, University of Minnesota, St. Paul, MN, USA. 7. Veterinary Population Medicine Department, University of Minnesota, St. Paul, MN, USA. 8. Comparative Neuromuscular Diseases Laboratory, Department of Clinical Sciences and Services, Royal Veterinary College, London, UK. Electronic address: rpiercy@rvc.ac.uk.
Abstract
BACKGROUND: Equine type 1 polysaccharide storage myopathy (PSSM1) is associated with a missense mutation (R309H) in the glycogen synthase (GYS1) gene, enhanced glycogen synthase (GS) activity and excessive glycogen and amylopectate inclusions in muscle. METHODS: Equine muscle biochemical and recombinant enzyme kinetic assays in vitro and homology modelling in silico, were used to investigate the hypothesis that higher GS activity in affected horse muscle is caused by higher GS expression, dysregulation, or constitutive activation via a conformational change. RESULTS: PSSM1-affected horse muscle had significantly higher glycogen content than control horse muscle despite no difference in GS expression. GS activity was significantly higher in muscle from homozygous mutants than from heterozygote and control horses, in the absence and presence of the allosteric regulator, glucose 6 phosphate (G6P). Muscle from homozygous mutant horses also had significantly increased GS phosphorylation at sites 2+2a and significantly higher AMPKα1 (an upstream kinase) expression than controls, likely reflecting a physiological attempt to reduce GS enzyme activity. Recombinant mutant GS was highly active with a considerably lower Km for UDP-glucose, in the presence and absence of G6P, when compared to wild type GS, and despite its phosphorylation. CONCLUSIONS: Elevated activity of the mutant enzyme is associated with ineffective regulation via phosphorylation rendering it constitutively active. Modelling suggested that the mutation disrupts a salt bridge that normally stabilises the basal state, shifting the equilibrium to the enzyme's active state. GENERAL SIGNIFICANCE: This study explains the gain of function pathogenesis in this highly prevalent polyglucosan myopathy. Copyright Â
BACKGROUND:Equine type 1 polysaccharide storage myopathy (PSSM1) is associated with a missense mutation (R309H) in the glycogen synthase (GYS1) gene, enhanced glycogen synthase (GS) activity and excessive glycogen and amylopectate inclusions in muscle. METHODS:Equine muscle biochemical and recombinant enzyme kinetic assays in vitro and homology modelling in silico, were used to investigate the hypothesis that higher GS activity in affected horse muscle is caused by higher GS expression, dysregulation, or constitutive activation via a conformational change. RESULTS: PSSM1-affected horse muscle had significantly higher glycogen content than control horse muscle despite no difference in GS expression. GS activity was significantly higher in muscle from homozygous mutants than from heterozygote and control horses, in the absence and presence of the allosteric regulator, glucose 6 phosphate (G6P). Muscle from homozygous mutant horses also had significantly increased GS phosphorylation at sites 2+2a and significantly higher AMPKα1 (an upstream kinase) expression than controls, likely reflecting a physiological attempt to reduce GS enzyme activity. Recombinant mutant GS was highly active with a considerably lower Km for UDP-glucose, in the presence and absence of G6P, when compared to wild type GS, and despite its phosphorylation. CONCLUSIONS: Elevated activity of the mutant enzyme is associated with ineffective regulation via phosphorylation rendering it constitutively active. Modelling suggested that the mutation disrupts a salt bridge that normally stabilises the basal state, shifting the equilibrium to the enzyme's active state. GENERAL SIGNIFICANCE: This study explains the gain of function pathogenesis in this highly prevalent polyglucosan myopathy. Copyright Â
Authors: Leigh Willard; Anuj Ranjan; Haiyan Zhang; Hassan Monzavi; Robert F Boyko; Brian D Sykes; David S Wishart Journal: Nucleic Acids Res Date: 2003-07-01 Impact factor: 16.971
Authors: Clara Prats; Joan A Cadefau; Roser Cussó; Klaus Qvortrup; Jakob N Nielsen; Jørgen F P Wojtaszewski; Jørgen F P Wojtaszewki; D Grahame Hardie; Greg Stewart; Bo F Hansen; Thorkil Ploug Journal: J Biol Chem Date: 2005-04-19 Impact factor: 5.157
Authors: S J Valberg; T L Ward; B Rush; H Kinde; H Hiraragi; D Nahey; J Fyfe; J R Mickelson Journal: J Vet Intern Med Date: 2001 Nov-Dec Impact factor: 3.333
Authors: Wayne A Wilson; Peter J Roach; Manuel Montero; Edurne Baroja-Fernández; Francisco José Muñoz; Gustavo Eydallin; Alejandro M Viale; Javier Pozueta-Romero Journal: FEMS Microbiol Rev Date: 2010-11 Impact factor: 16.408
Authors: Alejandro Buschiazzo; Juan E Ugalde; Marcelo E Guerin; William Shepard; Rodolfo A Ugalde; Pedro M Alzari Journal: EMBO J Date: 2004-07-22 Impact factor: 11.598
Authors: Kurt Højlund; Peter Staehr; Bo Falck Hansen; Kevin A Green; D Grahame Hardie; Erik A Richter; Henning Beck-Nielsen; Jørgen F P Wojtaszewski Journal: Diabetes Date: 2003-06 Impact factor: 9.461