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Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair.

Redpath GM, Woolger N, Piper AK, Lemckert FA, Lek A, Greer PA, North KN, Cooper ST - Mol. Biol. Cell (2014)

Bottom Line: Here we show that injury-activated cleavage of dysferlin is mediated by the ubiquitous calpains via a cleavage motif encoded by alternately spliced exon 40a.Of importance, we reveal that myoferlin and otoferlin are also cleaved enzymatically to release similar C-terminal modules, bearing two C2 domains and a transmembrane domain.Evolutionary preservation of this feature highlights its functional importance and suggests that this highly conserved C-terminal region of ferlins represents a functionally specialized vesicle fusion module.

View Article: PubMed Central - PubMed

Affiliation: Institute for Neuroscience and Muscle Research, Children's Hospital at Westmead, Sydney, NSW 2145, Australia Discipline of Paediatrics and Child Health, Faculty of Medicine, University of Sydney, Sydney, Australia.

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Cleavage of dysferlin to form mini-dysferlinC72 is conferred by exon 40a. (A) Untransfected HEK293 cells, as well as HEK293 transfected with dysferlin expression constructs with (+40a) or without exon 40a, were subjected to scrape injury 24 h posttransfection in the presence or absence of calcium. Only dysferlin expression constructs bearing exon 40a demonstrate injury-activated, calcium-dependent formation of the C-terminal mini-dysferlinC72 fragment (lane 6, Hamlet-1 and anti-Myc, black arrows). The N-terminal counterfragment can be detected with Romeo-1 (lane 6, gray arrow). Membranes were reprobed for loading controls GAPDH and β-tubulin. (B) Ubiquitous calpains specifically cleave exon 40a–containing dysferlin. MEFs were transfected by electroporation with dysferlin expression constructs with or without exon 40a and harvested 24 h posttransfection via scrape injury in the presence of calcium. Injury-activated formation of mini-dysferlinC72 requires exon 40a and is observed in wild-type MEFs (WT) but not in MEFs from CAPNS1-knockout mice (−/−) deficient for calpain-1 and -2. Retroviral rescue of CAPNS1 in knockout (−/−R) MEFs restores calpain expression (see CAPN2 immunoblot) to levels exceeding that in WT cells and increases injury-induced dysferlin cleavage. Mini-dysferlinC72 is indicated with asterisks. (C) Dysferlin bearing exon 40a is specifically cleaved by either calpain-1 or -2 in vitro, forming mini-dysferlinC72. Enhanced GFP–dysferlinFLAG was immunoprecipitated with anti-dysferlin (Romeo) and protein G–Sepharose (see Materials and Methods). Sepharose beads were incubated with the indicated dilutions of purified recombinant calpain-1 or -2 at 30°C for 3 min in the presence of 2 mM CaCl2. Digested samples were analyzed by SDS–PAGE and Western blot. Dysferlin bearing exon 40a is specifically cleaved by both calpain-1 and -2 to form mini-dysferlinC72 (black arrowhead), whereas dysferlin without exon 40a remains uncleaved.
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Figure 2: Cleavage of dysferlin to form mini-dysferlinC72 is conferred by exon 40a. (A) Untransfected HEK293 cells, as well as HEK293 transfected with dysferlin expression constructs with (+40a) or without exon 40a, were subjected to scrape injury 24 h posttransfection in the presence or absence of calcium. Only dysferlin expression constructs bearing exon 40a demonstrate injury-activated, calcium-dependent formation of the C-terminal mini-dysferlinC72 fragment (lane 6, Hamlet-1 and anti-Myc, black arrows). The N-terminal counterfragment can be detected with Romeo-1 (lane 6, gray arrow). Membranes were reprobed for loading controls GAPDH and β-tubulin. (B) Ubiquitous calpains specifically cleave exon 40a–containing dysferlin. MEFs were transfected by electroporation with dysferlin expression constructs with or without exon 40a and harvested 24 h posttransfection via scrape injury in the presence of calcium. Injury-activated formation of mini-dysferlinC72 requires exon 40a and is observed in wild-type MEFs (WT) but not in MEFs from CAPNS1-knockout mice (−/−) deficient for calpain-1 and -2. Retroviral rescue of CAPNS1 in knockout (−/−R) MEFs restores calpain expression (see CAPN2 immunoblot) to levels exceeding that in WT cells and increases injury-induced dysferlin cleavage. Mini-dysferlinC72 is indicated with asterisks. (C) Dysferlin bearing exon 40a is specifically cleaved by either calpain-1 or -2 in vitro, forming mini-dysferlinC72. Enhanced GFP–dysferlinFLAG was immunoprecipitated with anti-dysferlin (Romeo) and protein G–Sepharose (see Materials and Methods). Sepharose beads were incubated with the indicated dilutions of purified recombinant calpain-1 or -2 at 30°C for 3 min in the presence of 2 mM CaCl2. Digested samples were analyzed by SDS–PAGE and Western blot. Dysferlin bearing exon 40a is specifically cleaved by both calpain-1 and -2 to form mini-dysferlinC72 (black arrowhead), whereas dysferlin without exon 40a remains uncleaved.

Mentions: To examine whether cleavage of dysferlin is conferred by exon 40a, a region flanking exon 40a was PCR amplified from human skeletal muscle and subcloned into our dysferlin expression construct lacking exon 40a. Human embryonic kidney 293 (HEK293) cells, which do not express significant levels of endogenous dysferlin, were transfected with dysferlin expression constructs with and without exon 40a and then subjected to membrane injury via cell scraping in the presence or absence of extracellular calcium. Cell injury induced the calcium-dependent cleavage of dysferlin to release C-terminal mini-dysferlinC72 (Figure 2A, left) and the N-terminal counterfragment (Figure 2A, middle) only in cells transfected with the dysferlin expression construct bearing exon 40a. HEK293 cells expressing the canonical skeletal muscle isoform of dysferlin (without exon 5a, with exon 17, and without exon 40a) did not show injury-activated, calcium-dependent cleavage of dysferlin. We also established that the cleaved mini-dysferlinC72 product bears the extreme luminal/extracellular domain by probing a triplicate membrane with anti-Myc (Figure 2A, right).


Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair.

Redpath GM, Woolger N, Piper AK, Lemckert FA, Lek A, Greer PA, North KN, Cooper ST - Mol. Biol. Cell (2014)

Cleavage of dysferlin to form mini-dysferlinC72 is conferred by exon 40a. (A) Untransfected HEK293 cells, as well as HEK293 transfected with dysferlin expression constructs with (+40a) or without exon 40a, were subjected to scrape injury 24 h posttransfection in the presence or absence of calcium. Only dysferlin expression constructs bearing exon 40a demonstrate injury-activated, calcium-dependent formation of the C-terminal mini-dysferlinC72 fragment (lane 6, Hamlet-1 and anti-Myc, black arrows). The N-terminal counterfragment can be detected with Romeo-1 (lane 6, gray arrow). Membranes were reprobed for loading controls GAPDH and β-tubulin. (B) Ubiquitous calpains specifically cleave exon 40a–containing dysferlin. MEFs were transfected by electroporation with dysferlin expression constructs with or without exon 40a and harvested 24 h posttransfection via scrape injury in the presence of calcium. Injury-activated formation of mini-dysferlinC72 requires exon 40a and is observed in wild-type MEFs (WT) but not in MEFs from CAPNS1-knockout mice (−/−) deficient for calpain-1 and -2. Retroviral rescue of CAPNS1 in knockout (−/−R) MEFs restores calpain expression (see CAPN2 immunoblot) to levels exceeding that in WT cells and increases injury-induced dysferlin cleavage. Mini-dysferlinC72 is indicated with asterisks. (C) Dysferlin bearing exon 40a is specifically cleaved by either calpain-1 or -2 in vitro, forming mini-dysferlinC72. Enhanced GFP–dysferlinFLAG was immunoprecipitated with anti-dysferlin (Romeo) and protein G–Sepharose (see Materials and Methods). Sepharose beads were incubated with the indicated dilutions of purified recombinant calpain-1 or -2 at 30°C for 3 min in the presence of 2 mM CaCl2. Digested samples were analyzed by SDS–PAGE and Western blot. Dysferlin bearing exon 40a is specifically cleaved by both calpain-1 and -2 to form mini-dysferlinC72 (black arrowhead), whereas dysferlin without exon 40a remains uncleaved.
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Figure 2: Cleavage of dysferlin to form mini-dysferlinC72 is conferred by exon 40a. (A) Untransfected HEK293 cells, as well as HEK293 transfected with dysferlin expression constructs with (+40a) or without exon 40a, were subjected to scrape injury 24 h posttransfection in the presence or absence of calcium. Only dysferlin expression constructs bearing exon 40a demonstrate injury-activated, calcium-dependent formation of the C-terminal mini-dysferlinC72 fragment (lane 6, Hamlet-1 and anti-Myc, black arrows). The N-terminal counterfragment can be detected with Romeo-1 (lane 6, gray arrow). Membranes were reprobed for loading controls GAPDH and β-tubulin. (B) Ubiquitous calpains specifically cleave exon 40a–containing dysferlin. MEFs were transfected by electroporation with dysferlin expression constructs with or without exon 40a and harvested 24 h posttransfection via scrape injury in the presence of calcium. Injury-activated formation of mini-dysferlinC72 requires exon 40a and is observed in wild-type MEFs (WT) but not in MEFs from CAPNS1-knockout mice (−/−) deficient for calpain-1 and -2. Retroviral rescue of CAPNS1 in knockout (−/−R) MEFs restores calpain expression (see CAPN2 immunoblot) to levels exceeding that in WT cells and increases injury-induced dysferlin cleavage. Mini-dysferlinC72 is indicated with asterisks. (C) Dysferlin bearing exon 40a is specifically cleaved by either calpain-1 or -2 in vitro, forming mini-dysferlinC72. Enhanced GFP–dysferlinFLAG was immunoprecipitated with anti-dysferlin (Romeo) and protein G–Sepharose (see Materials and Methods). Sepharose beads were incubated with the indicated dilutions of purified recombinant calpain-1 or -2 at 30°C for 3 min in the presence of 2 mM CaCl2. Digested samples were analyzed by SDS–PAGE and Western blot. Dysferlin bearing exon 40a is specifically cleaved by both calpain-1 and -2 to form mini-dysferlinC72 (black arrowhead), whereas dysferlin without exon 40a remains uncleaved.
Mentions: To examine whether cleavage of dysferlin is conferred by exon 40a, a region flanking exon 40a was PCR amplified from human skeletal muscle and subcloned into our dysferlin expression construct lacking exon 40a. Human embryonic kidney 293 (HEK293) cells, which do not express significant levels of endogenous dysferlin, were transfected with dysferlin expression constructs with and without exon 40a and then subjected to membrane injury via cell scraping in the presence or absence of extracellular calcium. Cell injury induced the calcium-dependent cleavage of dysferlin to release C-terminal mini-dysferlinC72 (Figure 2A, left) and the N-terminal counterfragment (Figure 2A, middle) only in cells transfected with the dysferlin expression construct bearing exon 40a. HEK293 cells expressing the canonical skeletal muscle isoform of dysferlin (without exon 5a, with exon 17, and without exon 40a) did not show injury-activated, calcium-dependent cleavage of dysferlin. We also established that the cleaved mini-dysferlinC72 product bears the extreme luminal/extracellular domain by probing a triplicate membrane with anti-Myc (Figure 2A, right).

Bottom Line: Here we show that injury-activated cleavage of dysferlin is mediated by the ubiquitous calpains via a cleavage motif encoded by alternately spliced exon 40a.Of importance, we reveal that myoferlin and otoferlin are also cleaved enzymatically to release similar C-terminal modules, bearing two C2 domains and a transmembrane domain.Evolutionary preservation of this feature highlights its functional importance and suggests that this highly conserved C-terminal region of ferlins represents a functionally specialized vesicle fusion module.

View Article: PubMed Central - PubMed

Affiliation: Institute for Neuroscience and Muscle Research, Children's Hospital at Westmead, Sydney, NSW 2145, Australia Discipline of Paediatrics and Child Health, Faculty of Medicine, University of Sydney, Sydney, Australia.

Show MeSH
Related in: MedlinePlus