AHNAK a novel component of the dysferlin protein complex, redistributes to the cytoplasm with dysferlin during skeletal muscle regeneration

Abstract

Mutations in dysferlin cause limb girdle muscular dystrophy 2B, Miyoshi myopathy and distal anterior compartment myopathy. Dysferlin is proposed to play a role in muscle membrane repair. To gain functional insight into the molecular mechanisms of dysferlin, we have searched for dysferlin-interacting proteins in skeletal muscle. By coimmunoprecipitation coupled with mass spectrometry, we demonstrate that AHNAK interacts with dysferlin. We defined the binding sites in dysferlin and AHNAK as the C2A domain in dysferlin and the carboxyterminal domain of AHNAK by glutathione S-transferase (GST)-pull down assays. As expected, the N-terminal domain of myoferlin also interacts with the carboxyterminal domain of AHNAK. In normal skeletal muscle, dysferlin and AHNAK colocalize at the sarcolemmal membrane and T-tubules. In dysferlinopathies, reduction or absence of dysferlin correlates with a secondary muscle-specific loss of AHNAK. Moreover, in regenerating rat muscle, dysferlin and AHNAK showed a marked increase and cytoplasmic localization, consistent with the direct interaction between them. Our data suggest that dysferlin participates in the recruitment and stabilization of AHNAK to the sarcolemma and that AHNAK plays a role in dysferlin membrane repair process. It may also have significant implications for understanding the biology of AHNAK-containing exocytotic vesicles, "enlargosomes," in plasma membrane remodeling and repair.

Genetic Manipulation of Dysferlin Expression in Skeletal Muscle

“…Dysferlin interacts with a number of other proteins that could affect the health of the membrane such as annexins, AHNAK, and MG53, which have also been implicated in membrane trafficking and resealing. [17][18][19][20] Dysferlin also interacts with affixin, a focal adhesion protein, the L-type calcium channel, caveolin 3, and calpain 3, the later two of which themselves can cause a limb-girdle MD when deficient. [21][22][23] Our results clearly show that replacement of dysferlin by transgenesis only in skeletal muscle of A/J mice completely rescued muscle pathology and fully restored functional recovery from injury caused by large strain lengthening contractions.…”

Membrane damage-induced vesicle–vesicle fusion of dysferlin-containing vesicles in muscle cells requires microtubules and kinesin

“…Dysferlin localizes to the plasma membrane and intracellular vesicles in developing myotubes, and interacts with numerous proteins involved in membrane transport, including caveolin-3 (22,23), annexin-4 (5), annexin-6 (24), enlargeosomal marker AHNAK (25) and tubulin (26), but the exact contribution of dysferlincontaining vesicles to resealing following wounding remains elusive, as few studies have examined the behavior of dysferlincontaining vesicles in live cells following cellular wounding. Therefore, we sought to investigate the behavior of dysferlincontaining vesicles in live-muscle cells prior to and following wounding, and examine the role of kinesin and microtubules in dysferlin vesicle biology.…”

Structure of a C-terminal AHNAK peptide in a 1:2:2 complex with S100A10 and an acetylated N-terminal peptide of annexin A2

“…AHNAK, a large 629 kDa protein, has been implicated in membrane repair, and the annexin A2-S100A10 heterotetramer [(p11) 2 (AnxA2) 2 ] has high affinity for several regions of the long (1002-amino-acid) C-terminal domain of AHNAK (Shtivelman & Bishop, 1993;Benaud et al, 2004;Huang et al, 2007;Lennon et al, 2003;De Seranno et al, 2006;Rezvanpour et al, 2011).…”

“…Depletion of AnxA2 by siRNA causes release of AHNAK into the cytoplasm, suggesting that (p11) 2 (AnxA2) 2 may recruit AHNAK to the plasma membrane and act as a scaffold, locating it nearby for cell membrane-repair activities and possibly regulation of membrane cyto-architecture (Benaud et al, 2004). Both AHNAK and annexins A1 and A2 have been shown to interact with dysferlin, a 230 kDa muscle membrane protein with roles in skeletal muscle regeneration and wound healing (Huang et al, 2007;Lennon et al, 2003;Cacciottolo et al, 2011). Furthermore, AHNAK-deficient mice were highly susceptible to Leishmania major infection owing to the proposed role of AHNAK in T-cell Ca 2+ signaling mediated by Ca v 1 channels based on loss-of-function experiments, the expression characteristics of AHNAK in T cells and the requirement of AHNAK for the expression of L-type calcium channels (Matza et al, 2008).…”