Sarcoglycans Are Localized to the Membrane Fraction of Differentiated Mouse Myotubes
Cell culture has been used widely as a model for studying the DGC. Localization of dystrophin to the cell membrane has been demonstrated by expression of recombinant dystrophin in COS cells, mouse C2C12 cells and mouse mdx myogenic cultures (Lee et al., 1991
; Clemens et al., 1995
; Kumar-Singh and Chamberlain, 1996
). Previous studies have shown that both endogenous dystrophin and α-sarcoglycan are localized to the sarcolemma of rat L6 myotubes (Yoshida et al., 1998
). In this report, we have used primary myoblasts established from skeletal muscle of normal mouse to study the organization of the sarcoglycan complex. Upon reaching confluency, mouse myoblasts in culture fuse to form multinucleated myotubes and begin to express skeletal muscle-specific proteins (Hauschka, 1994
). The expression of the DGC in the differentiated myotubes was confirmed by immunoblotting of the cell lysates with antibodies against components of the DGC (data not shown).
To determine if the DGC in primary mouse myotube culture is localized to the cell membrane, membrane microsomes were prepared from myotubes culture (Fig. , row A). Western analysis showed that all the components of the DGC were found in the microsomal fraction (Fig. , lane 3) but not in the soluble protein fraction (lane 2). Similar results were obtained from mouse C2C12 myotubes (data not shown). Microsomes prepared in the same way from human skeletal muscle were shown to contain all the components of the DGC as expected (Fig. , row B, lane 3).
Figure 1 Localization of the DGC. Cultured mouse myotubes (row A) and human skeletal muscle (row B) were used as starting materials to purify microsomes. Lane 1, unlysed cell pellet fraction after 1,000 g centrifugation. Lane 2, soluble protein fraction after (more ...)
When cell lysate from cultured mouse myotubes was immunoprecipitated with an anti-dystrophin antibody, 6–10, the immune complex was found to contain proteins recognized by antibodies against dystrophin, the syntrophins and α-, β-, γ-, and δ-sarcoglycan but not actin (Fig. ). Neither filamin nor troponin was coimmunoprecipitated by the 6–10 antibody (data not shown). Since the DGC is defined by copurification with dystrophin, the coimmunoprecipitation of the DGC and dystrophin which is normally at the sarcolemma implied that these components form a stable complex on the plasma membrane of mouse myotubes. The assembly of the DGC at the membrane of mouse myotubes and its similar biochemical characteristics to human skeletal muscle provides an ideal cell culture system to explore the sarcoglycan complex.
Figure 2 Coimmunoprecip-itation of the sarcoglycans with dystrophin. Cell lysate from cultured mouse myotubes was immunoprecipitated by the anti-dystrophin antibody, 6–10. The immune complex was analyzed by Western blots using antibodies (1° (more ...)
α-, β-, γ-, and δ-Sarcoglycan Exist As a Complex in Mouse Myotubes
The sarcoglycans have been shown to copurify as a subcomplex from rabbit skeletal muscle (Yoshida et al., 1994
). To determine if the sarcoglycans could also be purified as a complex from mouse myotubes, cell lysate prepared from mouse myotubes was immunoprecipitated with antibodies against different sarcoglycans followed by Western analysis. All four sarcoglycans were coprecipitated when an antibody against β-sarcoglycan (NCL–b-sarc) was used (Fig. ), thus verifying that α-, β-, γ-, and δ-sarcoglycan form a complex in the mouse myotubes.
Figure 3 Coimmunoprecipitation of the sarcoglycans using different anti-sarcoglycan antibodies. Cell lysate from cultured mouse myotubes were immunoprecipitated by an antibody directed against α-, β-, γ-, or δ-sarcoglycan. The (more ...)
When other anti-sarcoglycan antibodies were used as the primary immunoprecipitation reagents, the sarcoglycans being coimmunoprecipitated from the same cell lysate sample was different in each case (Fig. ). A polyclonal anti–δ-sarcoglycan antibody was shown to coimmunoprecipitate only β- and δ-sarcoglycan but not α- and γ-sarcoglycan from the mouse myotubes cell lysate. An anti– α-sarcoglycan antibody (NCL–a-sarc) apparently only immunoprecipitated α-sarcoglycan while an anti–γ-sarcoglycan antibody (NCL–g-sarc) only immunoprecipitated γ-sarcoglycan. This immunoprecipitation data suggests that there is a preferential association between β- and δ-sarcoglycan.
β- and δ-Sarcoglycan Are Preferentially Associated with One Another
To examine the interaction between β- and δ-sarcoglycan more closely, the sarcoglycan complex was disrupted in the presence of different concentrations of SDS. Proteins immunoprecipitated by the anti–β-sarcoglycan antibody were immobilized on protein G–Sepharose beads and washed with buffers containing different concentrations of SDS. The final composition of the post-SDS–washed immune complex was then determined by Western blot using different anti-sarcoglycan antibodies. All four sarcoglycans were present in the immunoprecipitated complex after being washed with buffer containing no SDS (Fig. a, lane 2) or 0.2% SDS (lane 3). However, increasing the SDS concentration in the washing buffer to 0.3% or above caused the complete dissociation of α-sarcoglycan from the complex (Fig. a, lane 4 and 5, respectively). A slight reduction in the amount of γ-sarcoglycan in the complex was also observed at 0.3% SDS washing conditions (Fig. a, lane 4). Further dissociation of γ-sarcoglycan was seen when the immune complex was washed with 0.4% SDS (lane 5). β- and δ-Sarcoglycan remained relatively well associated even after the immune complex was washed with 0.4% SDS (Fig. a, lane 5).
Figure 4 Coimmunoprecip-itation of the sarcoglycans under different stringencies. (A) Cell lysate from cultured mouse myotubes was immunoprecipitated by the anti– β-sarcoglycan antibody (NCL– b-sarc). The immune complex was washed (more ...)
To increase the stringency of the disruption of the sarcoglycan complex, mouse myotubes were lysed in different concentrations of SDS for 1 h before immunoprecipitation was carried out. Under these conditions, the sarcoglycan complex was likely to undergo further dissociation due to the prolonged exposure to high concentrations of SDS. Similar to the results shown above, all four sarcoglycans were detected in the immunoprecipitated complex when mouse myotubes were lysed under relatively mild conditions with no SDS, 0.1% or 0.2% SDS (Fig. b, lanes 2–4). α-Sarcoglycan was completely separated from the complex when myotubes were lysed at either 0.3 or 0.4% SDS (Fig. b, lanes 5 and 6). However, in contrast to the previous observation that washing the immune complex with 0.3% SDS only caused a mild dissociation of γ-sarcoglycan from the complex (Fig. a, lane 4), a drastic reduction of γ-sarcoglycan in the complex was observed when myotubes were lysed in 0.3% SDS (Fig. b, lane 5). The amount of β- and δ-sarcoglycan found in the immunoprecipitated complex was also reduced to a greater extent in the more stringent conditions but nevertheless, a substantial amount of both sarcoglycans were still associated with one another (Fig. b, lane 5). When the concentration of SDS in the lysis buffer was increased to 0.4%, all evidence of the immunoprecipitation was lost probably because such stringent conditions abolished the binding of the antibody to its antigen or the binding of the immune complex to the protein G–Sepharose beads.
The same pattern of sarcoglycan dissociation was observed when immunoprecipitation was performed on cell lysates prepared from other cell lines, such as murine C2C12 cells or a normal mouse cell line transformed with v-myc (data not shown). In addition, the experiment was repeated on cultured human myotubes using a human δ-sarcoglycan–specific antibody (NCL–d-sarc). As with the anti–β-sarcoglycan antibody, the complex immunoprecipitated by this antibody under low stringency also contained all four sarcoglycans. When the SDS concentration in the lysis buffer was increased above 0.2%, both α- and γ-sarcoglycan were found to dissociate from the complex but β- and δ-sarcoglycan remained bound together (data not shown).
Sarcoglycans in Mouse Myotubes Can Be Cross-linked In Vivo
Proteins that are tightly associated or in close proximity with one another can frequently be cross-linked with chemical reagents. To explore the spatial relationship of the sarcoglycans in the DGC, cultured mouse myotubes were cross-linked with 1 mM DTSSP (Jung and Moroi, 1983
). Both uncross-linked and DTSSP cross-linked mouse myotubes were lysed in 0.3% SDS and then immunoprecipitated with the anti–β-sarcoglycan antibody. Cross-linked proteins immunoprecipitated from the DTSSP-treated myotubes were cleaved with 10 mM DTT before loading on SDS-PAGE gels. Western blots of the immunoprecipitated complex from uncross-linked myotubes showed that only β- and δ-sarcoglycan were present in the complex as one would expect under this SDS concentration (Fig. a
, lane 2
). On the other hand, mouse myotubes cross-linked with DTSSP resulted in co-immunoprecipitation of all four sarcoglycans (Fig. a
, lane 3
), indicating that they were in close enough proximity to be cross-linked and were located at the plasma membrane because the cross-linking reagent used in the experiments is membrane impermeable and can therefore only cross-link proteins on the extracellular surface of the cells. However, not all sarcoglycans were cross-linked to the same extent in the experiments. Comparison of the amount of sarcoglycans being immunoprecipitated (Fig. b
, lane 3
) to the original input materials (lane 1
) suggests that α-sarcoglycan was less efficiently cross-linked to the complex relative to other sarcoglycans.
Figure 5 In vivo cross-linking of the sarcoglycans. Mouse myotubes were chemically cross-linked with 1 mM DTSSP and immunoprecipitated by anti–β-sarcoglycan antibody (A) or anti–α- or β-dystroglycan antibody (B). Lane (more ...)
In Vivo Cross-linked Sarcoglycans Consist of Two Major Subcomplexes
The status of the DTSSP cross-linked myotubes was examined in more detail by two-dimensional (2-D) diagonal gel electrophoresis (Traut et al., 1989
). The first dimension of electrophoresis was performed under nonreducing conditions and the second dimension of electrophoresis was performed under reducing conditions. Proteins that are not cross-linked will have the same electrophoretic mobility in both dimensions and will be detected on a diagonal line in the gel. On the other hand, cross-linked proteins will dissociate into their monomeric species in the second dimension under reduction and will therefore appear below the diagonal line (Fig. a
). By Western blot analysis, both α- and β-dystroglycan were found to cross-link to form a product of ~200 kD in the first dimension (Fig. b
) and upon reduction with DTT, they separated from one another according to their molecular weights in the second dimension (spot 1
). This is consistent with previous studies that α-dystroglycan was intimately associated with β-dystroglycan (Ibraghimov-Beskrovnaya et al., 1992
; Yoshida et al., 1994
) and thus validated the use of 2-D diagonal gel to detect cross-linked proteins.
Figure 6 Analysis of the cross-linked sarcoglycans. Cell lysates from DTSSP cross-linked myotubes were examined by 2-D diagonal gel using antibodies against different sarcoglycans. (A) Schematic diagram of the principle of 2-D diagonal gel. Single circle, uncross-linked (more ...)
When a replicate blot was immunoblotted with the anti–β- and anti–γ-sarcoglycan antibodies, three spots were detected below the diagonal line (Fig. c). Two of the spots were recognized by antibodies against β-sarcoglycan (spot 4a) and γ-sarcoglycan (spot 5a) and together they were cross-linked to form the product X1 of ~120 kD. The third spot was also recognized by the anti–β-sarcoglycan antibody (spot 4b) but was cross-linked to form a smaller product X2 of ~80 kD. When the sum of the molecular weight of the sarcoglycans identified in the cross-linked species X1 was obtained (43 kD + 35 kD = 78 kD), there was a missing mass of ~40 kD in the product. Similarly, when we added up the sum of the molecular weights of the sarcoglycans identified in the cross-linked species X2 (43 kD = 43 kD), there was also a missing mass of ~40 kD. Since δ-sarcoglycan has a molecular weight of 35 kD, it is possible that δ-sarcoglycan could be part of the cross-linked 120- and 80-kD complex. Western analysis of a replicate blot using anti–β- and anti–δ-sarcoglycan antibodies revealed at least four spots below the diagonal line (Fig. d, spots 4a, 4b, 6a, and 6b). Two of the spots (4a and 6a) were recognized by the anti–β- and anti–δ-sarcoglycan antibody, respectively, and were shown to cross-link to form the same 120-kD product (Fig. d, X1) identified in Fig. c. The other two spots (4b and 6b) were also recognized by the anti–β- and anti–δ-sarcoglycan antibodies, respectively, and were cross-linked to form the same 80-kD product (Fig. d, X2) identified in Fig. c. Intriguingly, α-sarcoglycan was found to migrate as a single spot on the diagonal line and did not seem to cross-link to any proteins in these experiments (Fig. b, spot III). Similar results were obtained when mouse myotubes were treated with another cross-linker, sulfo-EGS which has a longer spacer arm length (data not shown). Collectively, we have demonstrated that β- and δ-sarcoglycan could be cross-linked to form two major subcomplexes (X1 and X2) in which the 120-kD product X1 also consisted of γ-sarcoglycan. Our results suggest that both β- and δ-sarcoglycan are in close physical proximity and are consistent with our earlier observations that they are tightly associated with one another.
δ-Sarcoglycan Can Be Cross-linked to the Dystroglycan Complex
In the previous 2-D diagonal gel electrophoresis of DTSSP cross-linked mouse myotubes, a third spot recognized by the anti–δ-sarcoglycan antibody was sometimes observed below the diagonal line (Fig. d, spot 7). This spot was cross-linked to form a product with an estimated molecular weight between 200–220 kD which is similar in size to the cross-linked α-/β-dystroglycan complex identified in Fig. b. To determine if δ-sarcoglycan could be cross-linked to the dystroglycan complex, cell lysates from both DTSSP cross-linked and uncross-linked mouse myotubes were immunoprecipitated by antibodies against β-dystroglycan (Fig. b, lanes 2 and 3) or α-dystroglycan (lane 4 and 5). Western analysis of the immune complex showed α-dystroglycan was coprecipitated with β-dystroglycan from both uncross-linked (lanes 2 and 4) and DTSSP cross-linked myotube samples (lanes 3 and 5). No sarcoglycan was found to be associated with the dystroglycans without cross-linking (lanes 2 and 4), whereas both antibodies immunoprecipitated δ-sarcoglycan from DTSSP cross-linked myotube cell lysate (lanes 3 and 5). There was no evidence that the immunoprecipitated materials contained the other three sarcoglycans as revealed by Western blot using antibodies against α-, β-, or γ-sarcoglycan (lanes 2–5 and data not shown).
Sarcoglycans in Mouse Myotubes Form Intramolecular Disulfide Bonds
Sequence analysis of the sarcoglycans revealed that there are clusters of conserved cysteine residues in their extracellular domains which have the potential to form intra- and/or intermolecular disulfide bonds (Fig. a
). To determine whether or not there are disulfide bonds in the sarcoglycans, their electrophoretic mobility was compared under reducing and nonreducing conditions using cell lysates prepared from mouse myotubes. Western analysis showed that β-, γ-, δ-sarcoglycan, and β-dystroglycan all had a faster electrophoretic mobility under nonreducing conditions (Fig. b
, lane 1
, single arrowhead
). This apparent shift of mobility indicated the presence of intramolecular disulfide bonds because proteins that contain such disulfide bonds will often adapt a more compact conformation and are expected to migrate faster in SDS-PAGE gel under nonreducing conditions. This approach has been used by others to demonstrate the presence of intramolecular disulfide bonds in β-dystroglycan (Deyst et al., 1995
). No discernible difference in the electrophoretic mobility was observed for α-sarcoglycan or the actin negative control.
Figure 7 Detection of intramolecular disulfide bond in the sarcoglycans. (A) Alignment of the carboxyl termini of β-, γ-, δ-, and C. elegans sarcoglycan with the consensus EGF-like repeat by MacVector Program (Oxford Molecular Group, (more ...)
The possibility of intermolecular disulfide bonds formation in sarcoglycans was examined by 2-D diagonal gel method. The expectation would be that molecules linked by disulfide bonds will migrate in the first dimension as a large disulfide-linked complex and will dissociate into monomers upon reduction in the second dimension. All four sarcoglycans from mouse myotubes were detected on the diagonal line (Fig. ). Since no extra spot was detected below the diagonal line, the sarcoglycans are not likely to form disulfide bridges with other proteins in mouse myotubes.
Figure 8 Absence of intermolecular disulfide bonds between the sarcoglycans. Cell lysate from cultured mouse myotubes was electrophoresed in 2-D diagonal gel and examined by Western blots using antibodies against different sarcoglycans. Shown in the example (more ...)
β- and γ-Sarcoglycan Mutations Have Different Effects on the Sarcoglycan Complex
Although mutations in one sarcoglycan can disrupt the sarcoglycan complex and lead to the loss or reduction of other sarcoglycans, the degree of reduction of each sarcoglycan observed in patients is often not the same. To assess the effect of different sarcoglycan mutations on the complex, we compared the immunostaining pattern of sarcoglycans in two autosomal recessive muscular dystrophy patients with homozygous mutations in different sarcoglycan genes. Both patients have a normal dystrophin pattern at the muscle membrane as revealed by immunohistochemistry (Fig. ). Patient AL has a homozygous nonsense mutation that changes a tyrosine residue to a premature stop codon at codon 178 in the β-sarcoglycan (Bönnemann et al., 1998
). This mutation is predicted to produce a truncated protein ~25 kD. Immunofluorescence studies showed that all four sarcoglycans were completely absent from the sarcolemma (Fig. ). Patient CR, on the other hand, carries a homozygous Δ521-T deletion in the γ-sarcoglycan gene. This patient has been previously described in another report (Bönnemann et al., 1998
). The mutation has also been seen in other patients (McNally et al., 1996
) and is predicted to generate a truncated γ-sarcoglycan of ~23 kD which is similar in size to the truncated β-sarcoglycan. This patient showed a complete absence of γ-sarcoglycan staining in the muscle biopsy by immunofluorescence but the staining of the other three sarcoglycans was preserved at a significant level (Fig. ).
Figure 9 Immunofluorescence of muscle biopsies from patients with autosomal recessive muscular dystrophy. Muscle section was stained with antibodies against dystrophin (dys), α-sarcoglycan (α-sar), β-sarcoglycan (β-sar), γ-sarcoglycan (more ...)