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