title: A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase.
abstract: 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.
  
  
  
  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.
  
  
  
  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 Kmfor UDP-glucose, in the presence and absence of G6P, when compared to wild type GS, and despite its phosphorylation.
  
  
  
  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.
  
  
  
  This study explains the gain of function pathogenesis in this highly prevalent polyglucosan myopathy.
authors: Comparative Neuromuscular Diseases Laboratory, Department of Clinical Sciences and Services, Royal Veterinary College, London, UK.
authors: C A
authors: CA
authors: Maile
authors: Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, Denmark.
authors: J R
authors: JR
authors: Hingst
authors: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, USA.
authors: K K
authors: KK
authors: Mahalingan
authors: School of Natural Sciences and Psychology, Liverpool John Moores University, Liverpool, UK.
authors: A O
authors: AO
authors: O'Reilly
authors: Department of Comparative Biomedical Sciences, Royal Veterinary College, London, UK.
authors: M E
authors: ME
authors: Cleasby
authors: Veterinary Biomedical Sciences Department, University of Minnesota, St. Paul, MN, USA.
authors: J R
authors: JR
authors: Mickelson
authors: Veterinary Population Medicine Department, University of Minnesota, St. Paul, MN, USA.
authors: M E
authors: ME
authors: McCue
authors: Veterinary Population Medicine Department, University of Minnesota, St. Paul, MN, USA.
authors: S M
authors: SM
authors: Anderson
authors: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, USA.
authors: T D
authors: TD
authors: Hurley
authors: Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, Denmark.
authors: J F P
authors: JFP
authors: Wojtaszewski
authors: Comparative Neuromuscular Diseases Laboratory, Department of Clinical Sciences and Services, Royal Veterinary College, London, UK. Electronic address: rpiercy@rvc.ac.uk.
authors: R J
authors: RJ
authors: Piercy
mesh_terms: D000263:Adenylate Kinase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001947:Breeding; D004789:Enzyme Activation; D051275:Glucose Transporter Type 4 / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism; D006008:Glycogen Storage Disease / Q000201:enzymology* / Q000453:epidemiology*; D006006:Glycogen Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta / Q000378:metabolism; D006736:Horses / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000201:enzymology; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D010766:Phosphorylation; D015995:Prevalence; D021122:Protein Subunits / Q000378:metabolism; D040681:Structural Homology, Protein; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
chemical_list: D051275:Glucose Transporter Type 4; D050505:Mutant Proteins; D021122:Protein Subunits; D019298:Glucose-6-Phosphate; D006003:Glycogen; D006006:Glycogen Synthase; D000071679:Glycogen Synthase Kinase 3 beta; D000263:Adenylate Kinase; D014532:Uridine Diphosphate Glucose
keywords: Glycogen; Glycogen storage disease; Glycogen synthase; Muscle; PSSM1; Polyglucosan