S‐nitrosylation Is Responsible for Muscle Atrophy and Weakness in GNE Myopathy

Abstract

BackgroundSialic acids are the most abundant terminal monosaccharides on glycoconjugates of eukaryotic cell surfaces and are involved in a variety of cellular functions. UDP‐N‐acetylglucosamine‐2‐epimerase/N‐acetylmannosamine 6‐kinase is a rate‐limiting enzyme for sialic acid biosynthesis, which is encoded by GNE gene. Mutations of GNE causes GNE myopathy, which is debilitating autosomal recessive myopathy, and is clinically characterized by atrophy and weakness in distal skeletal muscles. Previously, we have evidenced that sialic acid deficiency plays a key role for the pathogenesis of this myopathy with the GNE myopathy model mice, presenting muscle atrophy and weakness followed by myofiber degeneration. The phenotype was attenuated by sialic acid supplementation. And also, we found the elevation of reactive oxygen species in skeletal muscles of the model mice and demonstrated a therapeutic effect of an anti‐oxidant, N‐acetylcysteine (NAC) on muscle atrophy as well.ObjectiveWe aimed to identify protein modification(s) produced by ROS, which cause muscle atrophy and weakness in GNE myopathy.MethodsFor the analysis on S‐nitrosylation status using frozen skeletal muscles, we switched S‐nitrosocysteines to biotin‐labeled cysteins and purified the S‐nirosylated proteins by Neutravidin beads, followed by LC‐tandem mass shotgun analyses or western blotting.ResultsWe found that protein S‐nitrosylation was increased in GNE myopathy muscles. By proteome analysis, more than 60 proteins were identified in the S‐nitrosylated group, which revealed that the major proteins were involved in muscle contraction (actin and myosins) and energy production (muscle creatin kinase, enolase 3 and aldolase). We also found that sialic acid supplement significantly decreased S‐nitrosylation of these proteins as much as NAC treatment did.DiscussionOur results suggest that hyposialylation leads to failure in ROS scavenging and promotes S‐nitroso modification to deteriorate muscle contraction. Therefore, ROS cancelling would be a promising strategy to prevent muscle weakness and atrophy as well as to cure GNE myopathy.Support or Funding InformationThis study was partially supported by Intramural Research Grant (28‐6) for Neurological and Psychiatric Disorders of NCNP, by Comprehensive Research on Disability Health and Welfare (H25‐Shinkei Kin‐Ippan‐004) from Japan Agency for Medical Research and Development, AMED and by JSPS KAKENHI (23390236).