TY - JOUR

T1 - A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase

AU - Maile, C A

AU - Hingst, Janne Rasmuss

AU - Mahalingan, K K

AU - O'Reilly, A O

AU - Cleasby, Mark E

AU - Mickelson, James R

AU - McCue, M E

AU - Anderson, S M

AU - Hurley, T D

AU - Wojtaszewski, Jørgen

AU - Piercy, R J

N1 - CURIS 2017 NEXS 013

PY - 2017

Y1 - 2017

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 - Faculty of Science

KW - PSSM1

KW - Polyglucosan

KW - Glycogen synthase

KW - Glycogen

KW - Muscle

KW - Glycogen storage disease

U2 - 10.1016/j.bbagen.2016.08.021

DO - 10.1016/j.bbagen.2016.08.021

M3 - Journal article

C2 - 27592162

VL - 1861

SP - 3388

EP - 3398

JO - B B A - General Subjects

JF - B B A - General Subjects

SN - 0304-4165

IS - 1, Part A

ER -