Mutations in the muscle-specific, non-lysosomal cysteine protease calpain 3 cause LGMD2A. The function of calpain 3 and its role in LGMD2A pathogenesis are largely unknown but recent evidence points towards an important function in cytoskeleton remodeling and cytoskeleton–membrane interactions (7
). Our previous studies demonstrated that calpain 3 and AHNAK are in complex with dysferlin (14
). Moreover, calpain 3 showed the ability to cleave several cytoskeletal components and actin binding proteins (5
). As AHNAK also associates with actin (19
) and is proposed to be cleaved by yet unidentified proteases (20
), we hypothesized that AHNAK is a substrate for calpain 3.
The first step to test our hypothesis was to investigate a potential interaction between calpain 3 and AHNAK by GST pull-down assays. Using different recombinant AHNAK domains we showed a clear interaction between the C-terminus of AHNAK and full-length calpain 3.
In previous studies using the C-terminal Tail-antibody (not CQL), western blots of total protein lysates from rat heart and skeletal muscle revealed the presence of protein bands of 700, 500, 340, 170 kDa in addition to low molecular mass immunoreactive fragments (19
). Moreover, a band of ~120 kDa was also detected by the CQL antibody in lung and kidney tissues (20
). It was suggested that this band, corresponding to a C-terminal AHNAK proteolytic product, was cleaved by ubiquitous calpains, possibly μ-calpain or m-calpain, but to date the identity of the proteases that cleave AHNAK is unknown. We analyzed COS-1 cells transiently transfected with active and inactive calpain 3 using the CQL antibody and revealed a 120 kDa fragment by western blot only in cells expressing active calpain 3. Thus, these results indicate that calpain 3 can cleave endogenous AHNAK in a cell culture system.
The CQL antiserum that identified the AHNAK cleavage fragment was raised against the COOH-terminus of AHNAK. It is therefore not a suitable tool to investigate cleavage if calpain 3 cleavage of AHNAK would occur at multiple sites. The possible interaction between calpain 3 and several T7-AHNAK fusion protein fragments in our GST pull-down assay seems to suggest that calpain 3 can bind AHNAK also at locations other than the C2-domain. To investigate this, four domains of AHNAK were fused to an N-terminal VSV and C-terminal HA tag and analyzed in COS-1 cells in the presence of active or inactive calpain 3. N-, C1- and C2-AHNAK fusion proteins were clearly cleaved in cells expressing calpain 3. In contrast, specific cleavage products of AHNAK were not observed in cells transfected with a mutated, inactive calpain 3 or GFP. No cleavage was observed for the repeat domain of AHNAK (M-AHNAK).
This apparent discrepancy for N-AHNAK for which we found evidence of cleavage without observing a direct interaction may be explained by the different experimental design. Due to its autocatalytic activity calpain 3 is very unstable and only inactive calpain 3 can be heterologously expressed for interaction assays. Therefore, all GST pull-down assays were performed with inactive calpain 3 C129S. As the conversion from inactive to active calpain 3 involves a major conformational change including excision of insertion sequences (22
), it is possible that only active, but not inactive calpain 3 C129S binds the N-terminus of AHNAK. Alternatively, the interaction between the N-terminus of AHNAK and calpain 3 may be more transient compared with the C-terminus, similar to what has been shown for the Annexin 2 binding sequence (23
The influence of calpain 3 on the expression of endogenous AHNAK was also investigated in cell culture by immunostaining. This showed that AHNAK was absent in cells expressing active calpain 3 while normal or even increased signals were observed in cells expressing inactive calpain 3 C129S. In agreement with this observation, AHNAK immunoreactivity was increased along the sarcolemma of muscle fibers of calpainopathy patients in addition to an intense signal in blood vessels and extracellular matrix compared with normal control muscle and muscle of an unrelated MD patient. Dystrophin showed constant immunoreactivity at sarcolemma in all muscle biopsies. Moreover, semiquantitative western blot analysis confirmed the increase of AHNAK in skeletal muscle of patients with a calpainopathy. Taken together, these findings suggest that in skeletal muscle AHNAK protein turnover is regulated by calpain 3 activity and that this turnover is perturbed in patients with a calpainopathy.
Although the exact biological function of AHNAK is unknown, based on its interaction with other proteins, AHNAK has been implicated in several essential biological functions. First, in resting neuronal PC12–27 cells, AHNAK is localized within the lumen of specific vesicles called ‘enlargeosomes’, and is redistributed to the external surface of the plasma membrane in response to large increases in Ca2+
). These enlargeosomes have been proposed to participate in cell membrane enlargement, differentiation and repair. Second, in cardiomyocytes, AHNAK interacts specifically with the β2
subunit of cardiac L-type calcium channels at the plasma membrane (18
) where it modulates channel function (25
), and is suggested to play a role in the protein kinase A (PKA)-mediated signal transduction pathway (26
). Third, in vitro
, the C-terminal AHNAK region (amino acids 5262–5643) binds to G-actin and cosediments with F-actin (18
). Finally, a C-terminal 72 kDa AHNAK fragment was found in heart tissue to stabilize muscle contraction (19
). These observations suggest a role for AHNAK in the maintenance of the structural and functional organization of the subsarcolemmal cytoarchitecture. The finding that calpain 3 regulates AHNAK, combined with the observation that both proteins are found in the dysferlin protein complex raises the interesting possibility that they may function in muscle membrane repair. For m and µ calpain it has been shown that they are essential for Ca2+
induced cell membrane repair (27
). Moreover, stabilization of m calpain by fetuin A (OMIM# 38680) increases the membrane resealing potential of fibroblasts (28
). Fetuin A can also bind calpain 3 (28
). It is therefore an interesting possibility that calpain 3 may exert the same function in muscle by regulating AHNAK levels. Based on the proteolysis of AHNAK by calpain 3, we suggest that in healthy muscle calpain 3 works as a regulatory enzyme modulating AHNAK function. Such a regulatory role would be similar to the well-documented calpain 3-dependent accumulation of IκBα in the cytoplasm and nucleus (29
) and the calpain 3-mediated interaction between FLNC and γ- and δ-sarcoglycan (6
The loss of calpain 3 activity as the cause of LGMD2A indicates that calpain 3-dependent proteolysis is required for the normal biological function and homeostasis of skeletal muscle (29
). Recently, increased ubiquitination, correlated with an increase of calpain 3 expression, was observed during muscle reloading in wild-type mice, which was absent in Capn3
−/− mice (30
). It was suggested that calpain 3 acts upstream of the ubiquitination–proteasome pathway to release myofibrillar proteins and provide them for proteasomal breakdown. In the present study, we provide evidence that calpain 3 activity promotes turnover of AHNAK in cell culture and in skeletal muscle. Thus, in the future, it is essential to establish whether AHNAK is ubiquitinated after calpain 3 cleavage in skeletal muscle.
To date, three major pathological mechanisms have been suggested for MD. Membrane disruption, caused by mutations in proteins of the dystrophin–glycoprotein complex, is a well established pathogenic mechanism in a large group of MD (31
). Besides this membrane fragility, defects in membrane repair caused by mutations in dysferlin have been identified as second pathological mechanism (11
). Recently, a new pathogenic mechanism, deregulation of sarcomere remodeling due to the mutations in calpain 3, was suggested as the cause of LGMD2A (7
). Thus, the regulatory role of calpain 3 in the dysferlin protein complex may implicate an intimate relationship between muscle membrane repair and remodeling of sarcomere and subsarcolemmal cytoskeleton architecture, which thus far have been considered independent pathological mechanisms for LGMDs.