A muscle-specific calpain, CAPN3, forms a homotrimer

Biochim Biophys Acta Proteins Proteom. 2020 Jul;1868(7):140411. doi: 10.1016/j.bbapap.2020.140411. Epub 2020 Mar 19.

Abstract

Calpain-3 (CAPN3), a 94-kDa member of the calpain protease family, is abundant in skeletal muscle. Mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A, indicating that CAPN3 plays important roles in muscle physiology. CAPN3 has several unique features. A crystallographic study revealed that its C-terminal penta-EF-hand domains form a homodimer, suggesting that CAPN3 functions as a homodimeric protease. To analyze complex formation of CAPN3 in a more convenient manner, we performed blue native polyacrylamide gel electrophoresis and found that the observed molecular weight of native CAPN3, as well as recombinant CAPN3, was larger than 240 kDa. Further analysis by cross-linking and sequential immunoprecipitation revealed that CAPN3 in fact forms a homotrimer. Trimer formation was abolished by the deletion of the PEF domain, but not the CAPN3-specific insertion sequences NS, IS1, and IS2. The PEF domain alone formed a homodimer, as reported, but addition of the adjacent CBSW domain to its N-terminus reinforced the trimer-forming property. Collectively, these results suggest that CAPN3 forms a homotrimer in which the PEF domain's dimer-forming ability is influenced by other domains.

Keywords: CAPN3; Calpain; Dimer; EF-hand; Protease; Skeletal muscle; Trimer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calpain / chemistry
  • Calpain / genetics
  • Calpain / metabolism*
  • Cell Line
  • EF Hand Motifs
  • Female
  • Genetic Predisposition to Disease / genetics
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Muscle Proteins / chemistry
  • Muscle Proteins / genetics
  • Muscle Proteins / metabolism*
  • Muscle, Skeletal / metabolism
  • Muscular Dystrophies, Limb-Girdle / genetics
  • Muscular Dystrophies, Limb-Girdle / metabolism*
  • Mutagenesis, Insertional
  • Mutation
  • Protein Domains

Substances

  • Muscle Proteins
  • CAPN3 protein, human
  • Calpain
  • Capn3 protein, mouse

Supplementary concepts

  • Limb-girdle muscular dystrophy type 2A