P.3.12 Characterization of CAPN3-dependent proteolysis of C-terminal titin

The titinopathies tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J) are caused by mutations in the C-terminus of titin, residing in the sarcomeric M-band. Mutations identified so far affect the last Ig domain M10 or the preceding is7 region. The FINmaj mutation, underlying TMD/LGMD2J in patients of Finnish origin, causes the exchange of four amino acids in M10 and presumably leads to domain unfolding. The other known mutations cause missense changes of single amino acids or truncation of the ultimate C-terminus. Loss of C-terminal titin epitopes in IF microscopy and reduced amount of C-terminal titin fragments in western blotting suggest that increased or abnormal proteolytic turnover of mutant titin may contribute to the pathomechanism. The muscle-specific protease calpain 3 (CAPN3) binds M-band titin at the is7 region; the interaction is thought to regulate the autolytic activation of CAPN3. LGMD2J patients and FINmaj knock-in mice show secondary CAPN3 deficiency, reflecting loss of the binding site and consequent dysregulation of CAPN3 activity. To elucidate the proteolytic events involved in the pathogenesis of M-band titinopathies, we investigated the cleavage of C-terminal titin by CAPN3. Cleavage fragments generated by CAPN3 were identified by coexpressing various wild-type and mutant titin constructs with active or inactive CAPN3 in cell culture, and comparing the resulting fragment patterns. Active CAPN3 produced several titin fragments, which were characterized by western blotting, protein sequencing and mass spectrometry for identification of cleavage sites. Furthermore, targeted mutagenesis of predicted CAPN3 recognition sites was utilized for understanding the sequence determinants of CAPN3 cleavage and studying the order of cleavage events.