title: CAPN3-mediated processing of C-terminal titin replaced by pathological cleavage in titinopathy.
abstract: 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.
authors: Généthon, CNRS UMR 8115, Evry, France.
authors: Karine
authors: K
authors: Charton
authors: Department of Medical Genetics, Folkhälsan Institute of Genetics, University of Helsinki, Helsinki, Finland.
authors: Jaakko
authors: J
authors: Sarparanta
authors: Department of Medical Genetics, Folkhälsan Institute of Genetics, University of Helsinki, Helsinki, Finland.
authors: Anna
authors: A
authors: Vihola
authors: Généthon, CNRS UMR 8115, Evry, France.
authors: Astrid
authors: A
authors: Milic
authors: Department of Medical Genetics, Folkhälsan Institute of Genetics, University of Helsinki, Helsinki, Finland.
authors: Per Harald
authors: PH
authors: Jonson
authors: Généthon, CNRS UMR 8115, Evry, France.
authors: Laurence
authors: L
authors: Suel
authors: Department of Medical Genetics, Folkhälsan Institute of Genetics, University of Helsinki, Helsinki, Finland.
authors: Helena
authors: H
authors: Luque
authors: Généthon, CNRS UMR 8115, Evry, France.
authors: Imène
authors: I
authors: Boumela
authors: Généthon, CNRS UMR 8115, Evry, France, richard@genethon.fr.
authors: Isabelle
authors: I
authors: Richard
authors: Department of Medical Genetics, Folkhälsan Institute of Genetics, University of Helsinki, Helsinki, Finland, Neuromuscular Research Center, Tampere University Hospital and University of Tampere, Tampere, Finland and Department of Neurology, Vaasa Central Hospital, Vaasa, Finland.
authors: Bjarne
authors: B
authors: Udd
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D064211:Connectin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D049310:Distal Myopathies / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000201:enzymology / Q000378:metabolism; D049288:Muscular Dystrophies, Limb-Girdle / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D059748:Proteolysis
chemical_list: D064211:Connectin; D009124:Muscle Proteins; C577285:TTN protein, human; D011494:Protein Kinases; C579730:titin protein, mouse; C105884:CAPN3 protein, human; D002154:Calpain; C491373:Capn3 protein, mouse