CAPN3-mediated processing of C-terminal titin replaced by pathological cleavage in titinopathy

Hum Mol Genet. 2015 Jul 1;24(13):3718-31. doi: 10.1093/hmg/ddv116. Epub 2015 Apr 15.


Mutations in the extreme C-terminus of titin (TTN), situated in the sarcomeric M-band, cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J). The mutations ultimately cause a loss of C-terminal titin, including a binding site for the protease calpain 3 (CAPN3), and lead to a secondary CAPN3 deficiency in LGMD2J muscle. CAPN3 has been previously shown to bind C-terminal titin and to use it as a substrate in vitro. Interestingly, mutations in CAPN3 underlie limb-girdle muscular dystrophy 2A (LGMD2A). Here, we aimed to clarify the relationship of CAPN3 and M-band titin in normal and pathological muscle. In vitro analyses identified several CAPN3 cleavage sites in C-terminal titin that were defined by protein sequencing. Furthermore, cleavage products were detected in normal muscle extracts by western blotting and in situ by immunofluorescence microscopy. The TMD/LGMD2J mutation FINmaj proved to alter this processing in vitro, while binding of CAPN3 to mutant titin was preserved. Unexpectedly, the pathological loss of M-band titin due to TMD/LGMD2J mutations was found to be independent of CAPN3, whereas the involvement of ubiquitous calpains is likely. We conclude that proteolytic processing of C-terminal titin by CAPN3 may have an important role in normal muscle, and that this process is disrupted in LGMD2A and in TMD/LGMD2J due to CAPN3 deficiency and to the loss of C-terminal titin, respectively.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Animals
  • Calpain / genetics
  • Calpain / metabolism*
  • Connectin / chemistry*
  • Connectin / genetics
  • Connectin / metabolism*
  • Distal Myopathies / genetics
  • Distal Myopathies / metabolism*
  • Female
  • Humans
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Muscle Proteins / genetics
  • Muscle Proteins / metabolism*
  • Muscle, Skeletal / enzymology
  • Muscle, Skeletal / metabolism
  • Muscular Dystrophies, Limb-Girdle / enzymology*
  • Muscular Dystrophies, Limb-Girdle / genetics
  • Protein Binding
  • Protein Kinases / chemistry*
  • Protein Kinases / genetics
  • Protein Kinases / metabolism*
  • Protein Processing, Post-Translational
  • Proteolysis


  • Connectin
  • Muscle Proteins
  • TTN protein, human
  • Protein Kinases
  • titin protein, mouse
  • CAPN3 protein, human
  • Calpain
  • Capn3 protein, mouse