Limits...
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.

Show MeSH

Related in: MedlinePlus

Exon 40a–containing dysferlin is ubiquitously expressed, and mini-dysferlinC72 can be generated in multiple tissues. (A) Exon 40a is widely expressed in human tissues (∼40–60% transcripts), with lower relative levels in skeletal muscle, heart, and brain (∼10–15% transcripts). Dysferlin alternately spliced exons 5a, 17, and 40a were PCR amplified from a human tissue cDNA panel (Clontech) using primers flanking each of the exons. PCR amplification was performed for 30, 35, and 40 cycles to derive a simple standard curve and control for saturation. Ctrl; plasmid control. (B) Endogenous dysferlin from multiple tissues is cleaved by calpains in vitro, releasing mini-dysferlinC72. Mouse tissues were sectioned and lysed in RIPA, and endogenous dysferlin was immunoprecipitated with Romeo and protein G–Sepharose. Dysferlin-bound Sepharose beads were incubated with 0.2 active unit (A.U.) of purified recombinant calpain-1 at 30°C for 10 s in the presence of 2 mM CaCl2. Dysferlin was detected by Western analysis with the C-terminal antibody Hamlet-1. Mini-dysferlinC72­ is indicated with a black arrow. (C) An anti–exon 40a antibody (α-40a) is specific to exon 40a-containing dysferlin in transfected HEK293 cells. Membranes were probed with anti–exon 40a and then reprobed with Hamlet-1 to reveal total dysferlin expression. GAPDH indicates even loading. (D) Anti–exon 40a antibody recognizes full-length dysferlin-exon 40a and cleaved mini-dysferlinC72 but not the N-terminal counterfragment. Dysferlin was immunopurified from transfected HEK293 cells and subject to in vitro calpain cleavage. R1 (Romeo) reveals the N-terminal counterfragment, H1 reveals mini-dysferlinC72, and α-40a shows reactivity to full-length dysferlin and mini-dysferlinC72. (E) Dysferlin exon 40a is expressed at similar levels in human muscle and heart. Total dysferlin was immunoprecipitated with Hamlet-1 from three control human muscles (1–3, ages 5, 18, and 37 yr, respectively, from young adults subject to testing for malignant hypothermia and shown to be normal) and two human hearts (1 and 2, donor hearts from young adults). Dysferlin-exon 40a was identified by Western blot with pAb α-40a. Membranes were reprobed with Romeo to reveal total immunoprecipitated dysferlin.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230592&req=5

Figure 3: Exon 40a–containing dysferlin is ubiquitously expressed, and mini-dysferlinC72 can be generated in multiple tissues. (A) Exon 40a is widely expressed in human tissues (∼40–60% transcripts), with lower relative levels in skeletal muscle, heart, and brain (∼10–15% transcripts). Dysferlin alternately spliced exons 5a, 17, and 40a were PCR amplified from a human tissue cDNA panel (Clontech) using primers flanking each of the exons. PCR amplification was performed for 30, 35, and 40 cycles to derive a simple standard curve and control for saturation. Ctrl; plasmid control. (B) Endogenous dysferlin from multiple tissues is cleaved by calpains in vitro, releasing mini-dysferlinC72. Mouse tissues were sectioned and lysed in RIPA, and endogenous dysferlin was immunoprecipitated with Romeo and protein G–Sepharose. Dysferlin-bound Sepharose beads were incubated with 0.2 active unit (A.U.) of purified recombinant calpain-1 at 30°C for 10 s in the presence of 2 mM CaCl2. Dysferlin was detected by Western analysis with the C-terminal antibody Hamlet-1. Mini-dysferlinC72­ is indicated with a black arrow. (C) An anti–exon 40a antibody (α-40a) is specific to exon 40a-containing dysferlin in transfected HEK293 cells. Membranes were probed with anti–exon 40a and then reprobed with Hamlet-1 to reveal total dysferlin expression. GAPDH indicates even loading. (D) Anti–exon 40a antibody recognizes full-length dysferlin-exon 40a and cleaved mini-dysferlinC72 but not the N-terminal counterfragment. Dysferlin was immunopurified from transfected HEK293 cells and subject to in vitro calpain cleavage. R1 (Romeo) reveals the N-terminal counterfragment, H1 reveals mini-dysferlinC72, and α-40a shows reactivity to full-length dysferlin and mini-dysferlinC72. (E) Dysferlin exon 40a is expressed at similar levels in human muscle and heart. Total dysferlin was immunoprecipitated with Hamlet-1 from three control human muscles (1–3, ages 5, 18, and 37 yr, respectively, from young adults subject to testing for malignant hypothermia and shown to be normal) and two human hearts (1 and 2, donor hearts from young adults). Dysferlin-exon 40a was identified by Western blot with pAb α-40a. Membranes were reprobed with Romeo to reveal total immunoprecipitated dysferlin.

Mentions: To determine whether there is one dominant exon 40a-containing dysferlin transcript, we derived primer sets flanking each of the alternately spliced exons 5a, 17, and 40a and analyzed their relative abundance among dysferlin transcripts derived from a panel of human tissues (Clontech Multiple Tissue cDNA panels I and II; Figure 3A). Exon 40a-containing dysferlin transcripts are abundantly expressed in most human tissues (40–60% of transcripts in liver, kidney, lung, placenta, and pancreas; Figure 3A). Skeletal muscle expresses the lowest relative levels of exon 40a–containing transcripts (∼10–15%), with heart and brain expressing intermediate levels (∼15–25%). Exon 5a–containing transcripts dominate in liver, kidney, lung, placenta, and pancreas (80–90% of transcripts). Exon 17 shows variable tissue-specific expression, comprising the dominant mRNA species in liver, heart, brain, and muscle but only a minor mRNA species in kidney, lung, placenta, and pancreas. Our results therefore suggest that in human tissues exon 40a is regularly copresent with exon 5a and sometimes copresent with exon 17; this is consistent with previous results by Pramono et al. (2009), who compared splice isoforms of dysferlin in skeletal muscle and peripheral blood monocytes.


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)

Exon 40a–containing dysferlin is ubiquitously expressed, and mini-dysferlinC72 can be generated in multiple tissues. (A) Exon 40a is widely expressed in human tissues (∼40–60% transcripts), with lower relative levels in skeletal muscle, heart, and brain (∼10–15% transcripts). Dysferlin alternately spliced exons 5a, 17, and 40a were PCR amplified from a human tissue cDNA panel (Clontech) using primers flanking each of the exons. PCR amplification was performed for 30, 35, and 40 cycles to derive a simple standard curve and control for saturation. Ctrl; plasmid control. (B) Endogenous dysferlin from multiple tissues is cleaved by calpains in vitro, releasing mini-dysferlinC72. Mouse tissues were sectioned and lysed in RIPA, and endogenous dysferlin was immunoprecipitated with Romeo and protein G–Sepharose. Dysferlin-bound Sepharose beads were incubated with 0.2 active unit (A.U.) of purified recombinant calpain-1 at 30°C for 10 s in the presence of 2 mM CaCl2. Dysferlin was detected by Western analysis with the C-terminal antibody Hamlet-1. Mini-dysferlinC72­ is indicated with a black arrow. (C) An anti–exon 40a antibody (α-40a) is specific to exon 40a-containing dysferlin in transfected HEK293 cells. Membranes were probed with anti–exon 40a and then reprobed with Hamlet-1 to reveal total dysferlin expression. GAPDH indicates even loading. (D) Anti–exon 40a antibody recognizes full-length dysferlin-exon 40a and cleaved mini-dysferlinC72 but not the N-terminal counterfragment. Dysferlin was immunopurified from transfected HEK293 cells and subject to in vitro calpain cleavage. R1 (Romeo) reveals the N-terminal counterfragment, H1 reveals mini-dysferlinC72, and α-40a shows reactivity to full-length dysferlin and mini-dysferlinC72. (E) Dysferlin exon 40a is expressed at similar levels in human muscle and heart. Total dysferlin was immunoprecipitated with Hamlet-1 from three control human muscles (1–3, ages 5, 18, and 37 yr, respectively, from young adults subject to testing for malignant hypothermia and shown to be normal) and two human hearts (1 and 2, donor hearts from young adults). Dysferlin-exon 40a was identified by Western blot with pAb α-40a. Membranes were reprobed with Romeo to reveal total immunoprecipitated dysferlin.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230592&req=5

Figure 3: Exon 40a–containing dysferlin is ubiquitously expressed, and mini-dysferlinC72 can be generated in multiple tissues. (A) Exon 40a is widely expressed in human tissues (∼40–60% transcripts), with lower relative levels in skeletal muscle, heart, and brain (∼10–15% transcripts). Dysferlin alternately spliced exons 5a, 17, and 40a were PCR amplified from a human tissue cDNA panel (Clontech) using primers flanking each of the exons. PCR amplification was performed for 30, 35, and 40 cycles to derive a simple standard curve and control for saturation. Ctrl; plasmid control. (B) Endogenous dysferlin from multiple tissues is cleaved by calpains in vitro, releasing mini-dysferlinC72. Mouse tissues were sectioned and lysed in RIPA, and endogenous dysferlin was immunoprecipitated with Romeo and protein G–Sepharose. Dysferlin-bound Sepharose beads were incubated with 0.2 active unit (A.U.) of purified recombinant calpain-1 at 30°C for 10 s in the presence of 2 mM CaCl2. Dysferlin was detected by Western analysis with the C-terminal antibody Hamlet-1. Mini-dysferlinC72­ is indicated with a black arrow. (C) An anti–exon 40a antibody (α-40a) is specific to exon 40a-containing dysferlin in transfected HEK293 cells. Membranes were probed with anti–exon 40a and then reprobed with Hamlet-1 to reveal total dysferlin expression. GAPDH indicates even loading. (D) Anti–exon 40a antibody recognizes full-length dysferlin-exon 40a and cleaved mini-dysferlinC72 but not the N-terminal counterfragment. Dysferlin was immunopurified from transfected HEK293 cells and subject to in vitro calpain cleavage. R1 (Romeo) reveals the N-terminal counterfragment, H1 reveals mini-dysferlinC72, and α-40a shows reactivity to full-length dysferlin and mini-dysferlinC72. (E) Dysferlin exon 40a is expressed at similar levels in human muscle and heart. Total dysferlin was immunoprecipitated with Hamlet-1 from three control human muscles (1–3, ages 5, 18, and 37 yr, respectively, from young adults subject to testing for malignant hypothermia and shown to be normal) and two human hearts (1 and 2, donor hearts from young adults). Dysferlin-exon 40a was identified by Western blot with pAb α-40a. Membranes were reprobed with Romeo to reveal total immunoprecipitated dysferlin.
Mentions: To determine whether there is one dominant exon 40a-containing dysferlin transcript, we derived primer sets flanking each of the alternately spliced exons 5a, 17, and 40a and analyzed their relative abundance among dysferlin transcripts derived from a panel of human tissues (Clontech Multiple Tissue cDNA panels I and II; Figure 3A). Exon 40a-containing dysferlin transcripts are abundantly expressed in most human tissues (40–60% of transcripts in liver, kidney, lung, placenta, and pancreas; Figure 3A). Skeletal muscle expresses the lowest relative levels of exon 40a–containing transcripts (∼10–15%), with heart and brain expressing intermediate levels (∼15–25%). Exon 5a–containing transcripts dominate in liver, kidney, lung, placenta, and pancreas (80–90% of transcripts). Exon 17 shows variable tissue-specific expression, comprising the dominant mRNA species in liver, heart, brain, and muscle but only a minor mRNA species in kidney, lung, placenta, and pancreas. Our results therefore suggest that in human tissues exon 40a is regularly copresent with exon 5a and sometimes copresent with exon 17; this is consistent with previous results by Pramono et al. (2009), who compared splice isoforms of dysferlin in skeletal muscle and peripheral blood monocytes.

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