A muscle-specific calpain, CAPN3, forms a homotrimer

https://doi.org/10.1016/j.bbapap.2020.140411Get rights and content
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Highlights

  • Mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A (LGMD2A).

  • The molecular entity of CAPN3 has not been so far definitely identified.

  • CAPN3 functions as a homotrimer that represents a novel functional entity of calpain.

  • Activated CAPN3 also forms a trimeric structure without dissociation.

  • The characterization of CAPN3 homotrimer will lead to better understanding of LGMD2A.

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
Protease
Dimer
Trimer
EF-hand
Skeletal muscle

Abbreviations

CysPc
calpain-type Cys protease conserved domain
PC
protease core domain
PEF
penta-EF-hand domain
NS
N-terminal sequence
IS
internal sequence
BN-PAGE
Blue-Native PAGE
FL
full-length
Ab
antibody
GA
Glutaraldehyde
IP
immunoprecipitation
iMOC
intermolecular complementation
KO
knock-out
KI
knock-in
aa
amino acid residues.

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