Mapping of O-linked beta-N-acetylglucosamine modification sites in key contractile proteins of rat skeletal muscle

Proteomics. 2009 Apr;9(8):2139-48. doi: 10.1002/pmic.200800617.

Abstract

O-linked beta-N-acetylglucosamine (O-GlcNAc) is a widespread modification of serine/threonine residues of nucleocytoplasmic proteins. Recently, several key contractile proteins in rat skeletal muscle (i.e., myosin heavy and light chains and actin) were identified as O-GlcNAc modified. Moreover, it was demonstrated that O-GlcNAc moieties involved in contractile protein interactions could modulate Ca(2+) activation parameters of contraction. In order to better understand how O-GlcNAc can modulate the contractile activity of muscle fibers, we decided to identify the sites of O-GlcNAc modification in purified contractile protein homogenates. Using an MS-based method that relies on mild beta-elimination followed by Michael addition of DTT (BEMAD), we determined the localization of one O-GlcNAc site in the subdomain four of actin and four O-GlcNAc sites in the light meromyosin region of myosin heavy chains (MHC). According to previous reports concerning the role of these regions, our data suggest that O-GlcNAc sites might modulate the actin-tropomyosin interaction, and be involved in MHC polymerization or interactions between MHC and other contractile proteins. Thus, the results suggest that this PTM might be involved in protein-protein interactions but could also modulate the contractile properties of skeletal muscle.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / metabolism*
  • Actins / isolation & purification
  • Actins / metabolism
  • Animals
  • Glycosylation
  • Muscle Proteins / chemistry*
  • Muscle Proteins / isolation & purification
  • Muscle Proteins / metabolism*
  • Muscle, Skeletal / metabolism*
  • Myosin Heavy Chains / isolation & purification
  • Myosin Heavy Chains / metabolism
  • Myosin Light Chains / isolation & purification
  • Myosin Light Chains / metabolism
  • Peptide Mapping
  • Protein Processing, Post-Translational*
  • Rats
  • Serine / metabolism
  • Tandem Mass Spectrometry

Substances

  • Actins
  • Muscle Proteins
  • Myosin Light Chains
  • Serine
  • Myosin Heavy Chains
  • Acetylglucosamine