Dysferlin is a member of a mammalian gene family sharing homology with the Caenorhabditis elegans spermatogenesis factor fer-1 gene, which mediates vesicle fusion to the plasma membrane in spermatids [1].

The human dysferlin gene is located on chromosome 2p13 [2]. Mutations in dysferlin are linked to two clinically autosomal recessive forms of muscle disorders, limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM) [3,4]. Although the onset of these two diseases is generally in the late teens, they are clinically distinct because they differ in the muscle groups that initially show the onset of the disease. LGMD2B is a predominantly proximal muscular dystrophy. By contrast, MM is a predominantly distal muscular dystrophy. Both diseases progress slowly, and high levels of creatine kinase are detected in the serum of the patients [5,6].

The muscle fibres of dysferlin-null mice maintain a functional dystrophin-glycoprotein complex and a stable structure of the plasma membrane [7,8]. However, these mice develop a progressive muscular dystrophy caused by inefficient resealing of muscle cell plasma membrane [7]. These results suggest that dysferlin has a role in the process of membrane repair in muscle fibres and highlight the importance of this basic cellular function.

Dysferlin is the first identified member of the membrane repair machinery in skeletal muscle. It is highly probable that the protein-binding partners of dysferlin are also members of

this machinery. These proteins could serve as potential candidate for other muscular dystrophies and muscle-diseases with unknown aetiology. Therefore, we attempted to identify the interacting partners of dysferlin and used the tandem affinity purification (TAP) method for this purpose.

To allow purification of proteins associated with dysferlin, we constructed an alphaviral TAP vector. As shown in Fig. (1), the TAP cassette, which contains two copies of the IgG binding domains of ProtA of Staphylococcus aureus, the TEV protease cleavage site, and a CBP tag, was fused to the C-terminus of murine dysferlin and was inserted into the pSFV vector.

Fig. (1) Schematic representation of dysferlin with the C-terminal TAP tag. The IgG binding domain of Staphylococcus aureus protein A (ProtA) is separated by a tobacco etch virus protease cleavage site (TEV) from the calmodulin binding peptide (CBP).

To asses whether these tandem affinity tags are in a correct fusion format and to determine the activity of the virus, mouse myoblast cell line was maintained in 6-well plates and transfected with increasing amounts of SFV (10 μl to 500 μl). The expression of the tagged dysferlin in the cell lysates was checked by Western blot. For the detection, we used a complex of PAP which binds to ProtA and allows us to distinguish between endogenous and tagged dysferlin. A specific band of about 230 kDa was visualized in the Western blot (Fig. 2). The use of higher amount of the virus results at first in an increase and then in a decrease of the expression yield of the tagged dysferlin. This can be explained due to the cytotoxicity of SFV for the host cell. Therefore, the amount in lane e was defined as optimum and was up-scaled for transfection in 15 cm plates.

Fig. (2) Western blot analysis of dysferlin-TAP expression using a complex of PAP. Equal amounts of total cell lysates were loaded in each lane. Endogenous dysferlin is not recognized by PAP (lane a), and only the recombinant dysferlin-TAP tag was detected (lane b-i). Increasing amounts of the virus have been used for the transfection of the cells in lane b to i: b: 10 µl, c: 25 µl, d: 50 µl, e: 100 µl, f: 200 µl, g: 300 µl, h: 400 µl, i: 500 µl.

ProtA binds tightly to IgG-coated beads, which requires the use of TEV protease to elute materials under native conditions. For further purification, the eluate is incubated with calmodulin-coated beads in the presence of calcium. Finally, the complex of dysferlin and its associated proteins is released with EGTA. Fig. (3) shows a Coomassie-stained gel with proteins which were recovered from the purification of TAP-tagged dysferlin.

Fig. (3) TAP purification of dysferlin-TAP tag and its associated proteins. After both purification steps, the proteins were separated using a 10% SDS-PAGE gel and visualized by Coomassie staining.

Bands shown in Fig. (3) were excised and processed for identification with MS. Table 1 shows the list of the identified proteins with their accession numbers of the UniProt database. Annexin A2, which was previously described as an interacting partner of dysferlin [12], can be found on this list.

Table 1.

List of Proteins Identified with TAP Tag Technique with the Corresponding Accession Number of the UniProt Database.

A subset of the interacting partners was additionally assessed by co-IP and Western blots. Cell lysates were prepared from our mouse myoblast cell line to determine the interaction of endogenous dysferlin with the following three proteins from the list in Table 1: striatin, adaptin alpha and utrophin. These three proteins were chosen because they are involved either in vesicular trafficking (striatin, adaptin alpha) or muscle regeneration (utrophin). Two different co-IPs were performed for confirming the TAP tag result. First, we used an anti-dysferlin antibody for co-immunoprecipitating the interacting partners of dysferlin. Second, the set-up for co-IP was reversed: we utilised an anti-striatin, -adaptin alpha or -utrophin antibody for co-immunoprecipitating dysferlin. We confirmed by Western blot that either the interacting partners of dysferlin (set-up one, Fig. 4) or dysferlin (set-up two, Fig. 5) were immunoprecipitated. As controls the co-IP procedure was performed in both set-ups without antibodies. The analysis of the co-IPs confirmed that proteins involved in vesicular trafficking or muscle regeneration do indeed interact with dysferlin.

Fig. (4) Immunoprecipitation of dysferlin-interacting partners with anti-dysferlin. The levels of the indicated proteins in the lysates before immunoprecipitation were detected by Western blot analysis (lane 1). To confirm interactions detected by the TAP tag technique, immunoprecipitations were conducted with a dysferlinspecific antibody (lane 2). As control, no dysferlin-specific antibody was added to the lysates (lane 3).

Fig. (5) Confirmation of the immunoprecipitation of dysferlininteracting partners. Lane 1 shows the level of dysferlin in the lysate before immunoprecipitation. As controls no lysate were added to the beads (lane 2) or the lysate was not pre-incubated with any antibody (lane 3). For the immunoprecipitation antibodies against the following proteins were used: dysferlin (lane 4), adaptin alpha (lane 5), striatin (lane 6) and utrophin (lane 7).

Disruption of the plasma membrane is a common event in various normal cells. Plasma membrane repair is a basic cellular process required to reseal membrane disruptions [13-15]. The membrane repair machinery allows the cell to overcome physical injuries to the plasma membrane and is essential to prevent disruption-induced cell death. As the primary function of skeletal muscle is to generate physical force, muscle fibres are often exposed to varying degrees of mechanical stress. Therefore, muscle cells are often susceptible to damages of the plasma membrane and require efficient membrane repair machinery.

Dysferlin is a predicted transmembrane protein [8,16]. Mutations in the dysferlin gene cause proximal (LGMD2B) and distal (MM) forms of recessively inherited muscular dystrophies [3,4]. Recent work showed that dysferlin-null mice, like LGMD2B and MM patients, develop a slowly progressive muscular dystrophy [7]. The results of this work suggest a direct role of dysferlin in the repair process of plasma membrane in muscle fibres.

Nearly every major cellular process is carried out by assemblies of large complexes of proteins [17]. It can be assumed that this also pertains for the process of plasma membrane repair. Dysferlin is the first molecule to be discovered that is possibly involved in the membrane repair machinery in skeletal muscle. It is very probable that proteins which interact with dysferlin are associated with this machinery. Furthermore, these proteins are potential candidate genes for other muscular dystrophies. In this work we used the TAP tag purification approach for their identification.

The TAP tag technique allows a spectrometric analysis of dysferlin associated proteins. As usual with such procedures, the interactions must be confirmed by other techniques. This was done by co-IP for the following three proteins: striatin, adaptin alpha and utrophin.

Striatin is an intracellular protein and is involved in ve-sicular trafficking [18]. In the light of recent finding, a dys-ferlin-mediated membrane repair model was already postu-lated [19]. The model states that vesicles are targeted to dis-ruption sites, where they fuse with each other and the plasma membrane. This causes an addition of membrane to the plasma membrane, thereby patching and resealing the dis-rupted membrane. Dysferlin is thought to play a role at the fusion step of this repair process by facilitating the docking and fusion of the vesicles with the plasma membrane through interactions with other proteins.

Adaptins are subunits of adaptor protein (AP) complexes. Four basic AP complexes have been described: AP-1, AP-2, AP-3 and AP-4. Each of these complexes is composed of two large adaptins, one medium adaptin and one small adaptin [20-22]. Adaptin alpha belongs to the group of large adaptins and is part of AP-2. Like striatin, AP complexes are also involved in vesicular trafficking [23].

Early time point of muscle regeneration is characterized by the activation of satellite cells: upon muscle damage quiescent satellite cells start proliferating, migrate to the injury site, and fuse together to repair the damaged fibres [24]. A second type of muscle membrane fusion relates to reseal membrane disruptions which occur under physiological conditions [19]. In both types of membrane fusion, it is thought that vesicles fuse with the plasma membrane to facilitate fusion of apposed membranes [25]. The induction of utrophin during muscle regeneration suggests an important role of it during this process [26-28]. Our TAP data show that dysferlin interacts with utrophin. This result was also confirmed by IP. Therefore, we propose that there is a link between muscle regeneration and the repair of muscle membrane and that dysferlin plays an important role during both processes. Inefficient regeneration of muscle fibres contribute to reduced muscle mass seen with age. Thus, dysferlin and its associated proteins could not only be important for disease states, but also for muscle degeneration associated with aging.