A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase

C. A. Maile, J. R. Hingst, K. K. Mahalingan, A. O. O'Reilly, M. E. Cleasby, J. R. Mickelson, M. E. McCue, S. M. Anderson, Thomas Hurley, J. F P Wojtaszewski, R. J. Piercy

Research output: Contribution to journalArticle

5 Citations (Scopus)

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.

Original languageEnglish (US)
JournalBiochimica et Biophysica Acta - General Subjects
DOIs
StateAccepted/In press - Apr 19 2016

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Glycogen Storage Disease
Glycogen Synthase
Glycogen
Horses
Chemical activation
Muscle
Muscles
Phosphorylation
Glucose-6-Phosphate
Muscular Diseases
Enzymes
Uridine Diphosphate Glucose
Enzyme kinetics
Enzyme Assays
Enzyme activity
Missense Mutation
Heterozygote
Computer Simulation
Polysaccharides
Assays

Keywords

  • Glycogen
  • Glycogen storage disease
  • Glycogen synthase
  • Muscle
  • Polyglucosan
  • PSSM1

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Maile, C. A., Hingst, J. R., Mahalingan, K. K., O'Reilly, A. O., Cleasby, M. E., Mickelson, J. R., ... Piercy, R. J. (Accepted/In press). A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase. Biochimica et Biophysica Acta - General Subjects. https://doi.org/10.1016/j.bbagen.2016.08.021

A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase. / Maile, C. A.; Hingst, J. R.; Mahalingan, K. K.; O'Reilly, A. O.; Cleasby, M. E.; Mickelson, J. R.; McCue, M. E.; Anderson, S. M.; Hurley, Thomas; Wojtaszewski, J. F P; Piercy, R. J.

In: Biochimica et Biophysica Acta - General Subjects, 19.04.2016.

Research output: Contribution to journalArticle

Maile, CA, Hingst, JR, Mahalingan, KK, O'Reilly, AO, Cleasby, ME, Mickelson, JR, McCue, ME, Anderson, SM, Hurley, T, Wojtaszewski, JFP & Piercy, RJ 2016, 'A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase', Biochimica et Biophysica Acta - General Subjects. https://doi.org/10.1016/j.bbagen.2016.08.021
Maile, C. A. ; Hingst, J. R. ; Mahalingan, K. K. ; O'Reilly, A. O. ; Cleasby, M. E. ; Mickelson, J. R. ; McCue, M. E. ; Anderson, S. M. ; Hurley, Thomas ; Wojtaszewski, J. F P ; Piercy, R. J. / A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase. In: Biochimica et Biophysica Acta - General Subjects. 2016.
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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.",
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AU - Maile, C. A.

AU - Hingst, J. R.

AU - Mahalingan, K. K.

AU - O'Reilly, A. O.

AU - Cleasby, M. E.

AU - Mickelson, J. R.

AU - McCue, M. E.

AU - Anderson, S. M.

AU - Hurley, Thomas

AU - Wojtaszewski, J. F P

AU - Piercy, R. J.

PY - 2016/4/19

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N2 - 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.

AB - 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.

KW - Glycogen

KW - Glycogen storage disease

KW - Glycogen synthase

KW - Muscle

KW - Polyglucosan

KW - PSSM1

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