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.
Bibliographical noteFunding Information:
This work was partly funded by The Petplan Charitable Trust (Award S12-25 ), The Morris Animal Foundation (Award D14EQ-404 ) awarded to RJ Piercy and C Maile and by the National Institutes of Health , National Institute of Arthritis, Musculoskeletal and Skin Diseases award 1K08AR055713-01A2 to ME McCue. TD Hurley and KK Mahalingan were supported by NIH grant R01-79887 . JFP Wojtaszewski and JR Hingst were supported by the Danish Council for Independent Research and the Ministry of Culture. This manuscript was approved by The Royal Veterinary College's Research Office (reference number: CSS_00938).
- Glycogen storage disease
- Glycogen synthase