Sarcoglycan Isoforms in Skeletal Muscle*

The heterotetrameric sarcoglycan complex, composed of α-, β-, γ-, and δ-sarcoglycans, is an important component of the dystrophin-associated glycoprotein assembly in striated muscle. Mutations in any of the four genes encoding sarcoglycans cause a deficiency in all sarcoglycans in the sarcolemma and produce one of four types of limb-girdle muscular dystrophy. A fifth widely expressed sarcoglycan, ε-sarcoglycan, has been recently described. ε-Sarcoglycan is homologous to α-sarcoglycan, but whether it associates with the other sarcoglycans in muscle is not known. In this study, we use wild type and α-sarcoglycan-deficient mice to analyze the localization and association of sarcoglycans in skeletal muscle in vivo. The amounts of β-, γ-, and δ-sarcoglycans are reduced in α-sarcoglycan mutants, whereas the amount of ε-sarcoglycan is unchanged. We show here that ε-sarcoglycan is complexed with β-, γ-, and δ-sarcoglycans in both wild type and α-sarcoglycan mutant mice. We also use C2C12 myocytes to study the temporal expression and organization of sarcoglycan complexes during muscle cell differentiation in vitro. In C2C12 cells, α- and ε-sarcoglycans form separate complexes with β-, γ-, and δ-sarcoglycans. Both types of complexes are expressed at the cell surface and presumed to be functional. These results suggest that ε-sarcoglycan serves a function similar to that of α-sarcoglycan and that residual β-, γ-, and δ-sarcoglycan seen in mutant mice and α-sarcoglycan-deficient patients is due to its association with ε-sarcoglycan.

The dystrophin-associated glycoprotein complex (DAGC) 1 is a large array of membrane and cytoskeletal proteins found in striated muscle, where it is thought to function in force transmission and in protection of the muscle membrane from contraction-induced damage (1,2). The DAGC consists of many proteins including syntrophins, dystroglycan, sarcoglycans, and sarcospan that are directly or indirectly linked to dystrophin (3)(4)(5)(6)(7). The importance of these proteins is evident from the various forms of muscular dystrophy, in which primary or secondary defects in DAGC components result in muscle tissue degeneration (8 -14).
The sarcoglycan complex (SGC) is a subcomplex of the DAGC (15). In addition to playing a structural role, the SGC may have signaling functions (16,17). SGC consists of four transmembrane proteins: ␣-sarcoglycan, a type I transmembrane protein, and ␤-, ␥-, and ␦-sarcoglycans, which are type II transmembrane proteins. ␣and ␥-sarcoglycans are expressed exclusively in skeletal and cardiac muscle, whereas ␤and ␦-sarcoglycans are more widely distributed (11, 18 -21). Mutations in any one of these four sarcoglycans cause autosomal recessive limb-girdle muscular dystrophy, and mutations in the genes encoding individual sarcoglycans often lead to the concomitant loss or reduction of all sarcoglycans from the sarcolemma, suggesting that complex formation and localization of SGCs require all four subunits (9). Direct interaction between the sarcoglycans has also been demonstrated biochemically by co-immunoprecipitation (15,22). Transfection of Chinese hamster ovary cells with sarcoglycans indicates that all four sarcoglycans must be co-expressed for proper cell surface localization (23). Taken together, these data suggest that ␣-, ␤-, ␥-, and ␦-sarcoglycans are subunits of a heterotetrameric molecule (16). A fifth sarcoglycan, ⑀-sarcoglycan, was identified recently (24,25). It is highly homologous to ␣-sarcoglycan, but like ␤and ␦-sarcoglycans it is widely expressed in various tissues (24). It has been reported that ⑀-sarcoglycan associates with ␤and ␦-sarcoglycans in smooth muscle (26). Despite its homology to ␣-sarcoglycan and its presence in skeletal muscle, endogenous ⑀-sarcoglycan is unable to rescue phenotypes associated with ␣-sarcoglycan loss (27). Therefore, in skeletal muscle ⑀-sarcoglycan could exist as a monomer, form a complex with proteins distinct from those that associate with ␣-sarcoglycan, or associate with ␤-, ␥-, and ␦-sarcoglycans to form SGC that is only partially able to compensate for the loss of ␣-sarcoglycancontaining complexes.
Mice mutant in ␣-, ␤-, ␥-, and ␦-sarcoglycans have been generated experimentally by homologous recombinationdependent gene targeting (27)(28)(29)(30). A spontaneous null mutant, the ␦-sarcoglycan-deficient hamster, also exists (31). All sarcoglycan-deficient mice develop severe muscular dystrophies with varying involvement of heart muscle. The ␦-sarcoglycan null mutant hamster succumbs from cardiomyopathy with less severe involvement of skeletal muscle. The C2C12 mouse myoblast cell line, a cell line widely used for studying myogenesis and muscle differentiation, has been used as a model system to study biosynthesis and assembly of sarcoglycans (21,22,32,33).
Here we use gene targeting and C2C12 cells to demonstrate that ⑀-sarcoglycan is indeed associated with ␤-, ␥-, and ␦-sarcoglycans in skeletal muscle in vivo and in vitro. We generate ␣-sarcoglycan-deficient mice and present evidence, using immunofluorescence and protein fractionation, that SGCs containing ⑀-, ␤-, ␥-, and ␦-sarcoglycans are expressed in striated muscle and that the localization of these complexes in the muscle is similar to the ␣-sarcoglycan-containing SGC. The existence of an ⑀-sarcoglycan-containing SGC may explain why residual levels of sarcoglycan expression are seen in mutant mice and patients with ␣-sarcoglycan deficiency (27, 34 -36) and suggests that ⑀-sarcoglycan may partially compensate for the loss of ␣-sarcoglycan.

Construction of Gene Targeting Vector-
The mouse ␣-sarcoglycan gene (asg) was isolated from a 129/SvJ mouse genomic library (Stratagene, La Jolla, CA) by hybridization with a mouse ␣-sarcoglycan cDNA probe (21). The targeting vector was designed to disrupt exons 1 and 2. A 15-kb fragment containing exons 1-6 of the ␣-sarcoglycan gene was subcloned into pBluescript (Stratagene, La Jolla, CA), characterized by restriction mapping, and sequenced. A 5.5-kb genomic fragment containing sequence upstream of the ATG site in exon 1, was amplified using the Expand TM Long Template PCR System (Roche Molecular Biochemicals) with the forward primer 5Ј-GAGGGTGCGAGGGTGACAAGGAC-3Ј and the reverse primer 5Ј-GGAAGTTACTGCTGCTGCCAT-3Ј. The 5.5-kb genomic PCR product was subcloned, sequenced, and used as the 5Ј homologous region of the targeting vector. A 2-kb genomic PCR fragment containing the 3Ј portion of exon 2 through exon 6 was amplified with primers 5Ј-CTGCGCCTTC-CAGAACAC-3Ј and 5Ј-ACCAGAGACACATTGCACCAG-3Ј. The PCR product was subcloned, sequenced, and used as the 3Ј homologous region of the targeting vector. A cassette containing the LacZ with a nuclear localization signal and neo genes (nlsLacZ/neo) was inserted 21 base pairs downstream of the ATG initiation site. The diphtheria toxin A gene was inserted at the 3Ј end of the targeting vector to select against random insertion events (37). A map of the ␣-sarcoglycan gene locus and the design of the targeting vector are shown in Fig. 1.
Generation of ␣-Sarcoglycan Null Mice-SM38 ES cells, derived from 129/SvEv mice, 2 were transfected with the linearized targeting vector by electroporation and selected for growth in the presence of G418 (38). G418-resistant ES cell clones were screened by Southern blotting of BamHI-digested genomic DNA using a probe shown in Fig. 1. Two selected clones of targeted ES cells were injected separately into C57BL/6J blastocysts, and the blastocysts were transferred into pseudopregnant recipients. Chimeric male mice were identified by coat color and bred to C57BL/6J females. Genotypes were determined by Southern blot analysis of DNA from tail biopsies.
Cell Culture-The C2C12 mouse myoblast cell line (39) was obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured in dishes precoated with 50 g/ml rat tail collagen type I (Collaborative Biomedical Products, Bedford, MA) and maintained in low glucose Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 20% fetal calf serum (HyClone Laboratories, Logan, UT), 15 mM HEPES, and 20 mM glutamine at 37°C in a humidified atmosphere of 5% CO 2 . Antibiotics were not used (21,40). The culture medium was changed every 24 h. Myogenic differentiation was induced at confluence by replacing the growth medium with Dulbecco's modified Eagle's medium containing 2% horse serum (HyClone).
Indirect Immunofluorescence-Ten-m transverse cryosections of thigh muscle were prepared from 8-week-old wild type, heterozygous, and homozygous ␣-sarcoglycan mutant mice. Fluorescent staining was performed by incubating sections for 1 h at 37°C with affinity-purified polyclonal antibodies. After washing with PBS three times, sections were incubated with a fluorescein isothiocyanate-conjugated goat anti-rabbit IgG secondary antibody (ICN, Aurora, OH. 1:200) for 1 h at room temperature and then washed with PBS. Dilutions of all the antibodies were made in PBS containing 3% bovine serum albumin. Sections were mounted with Vectashield mounting medium for fluorescence (Vector, Burlingame, CA) and observed under an Axiovert 405M fluorescence microscope (Zeiss). For histological analysis, sections were stained with hematoxylin and eosin.
Biotinylation of Cell Surface Proteins and Preparation of Cell Extracts-C2C12 cells were washed three times with cold PBS and incubated on ice for 15 min with 1 mg/ml NHS-LC-biotin (Pierce). The cells were washed once with PBS, and the residual NHS-LC-biotin was inactivated by incubating the cells in 0.1 M glycine in PBS on ice for 5 min. Cells were washed three times with ice-cold PBS and solubilized in lysis buffer (50 mM Tris, pH 7.4, containing 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, and 1ϫ protease inhibitor mixture from Roche Molecular Biochemicals) on ice for 20 min and centrifuged at 12,000 ϫ g at 4°C for 10 min. The protein concentration of clarified lysates was measured with the BCA protein assay kit (Pierce) using bovine serum albumin as the standard. For isolation of biotinylated proteins, lysates were incubated overnight at 4°C with 20 l of streptavidin-Sepharose (Pierce). After washing three times with lysis buffer, bound proteins were eluted in SDS-PAGE sample buffer, separated by SDS-PAGE, and analyzed by immunoblotting as described below. In some experiments, cell extracts prepared from biotinylated cultures were immunoprecipitated with Sepharose beads coupled with either anti-␣or anti-⑀-sarcoglycan antibodies as described below.
Isolation with agitation for 30 min, and then centrifuged at 142,000 ϫ g at 4°C for 30 min. The pellet was solubilized in lysis buffer by incubation on ice for 15 min, then clarified by centrifugation at 12,000 ϫ g at 4°C for 10 min. Immunoprecipitation and Immunoblotting-One hundred g of protein from extracts of cells or microsomes were incubated with 15 l of Sepharose-bound, affinity-purified antibodies against ␣or ⑀-sarcoglycan at 4°C overnight. The Sepharose beads were washed three times with lysis buffer, and bound proteins were resolved by SDS-PAGE on 4 -20% gradient gels (Novex, San Diego, CA). Proteins were transferred from SDS-PAGE gels to nitrocellulose membranes (Bio-Rad). The Multi-Mark standard was used for markers (Novex). In immunoblotting experiments, antisera against ␣-, ␤-, and ␥-sarcoglycans were used at 1:100 dilution, and antisera against ␦-sarcoglycan at 1:10,000 dilution. Goat anti-mouse IgG conjugated with peroxidase (Bio-Rad) was used at a 1:2,000 dilution, and goat anti-rabbit IgG conjugated with peroxidase (Calbiochem, San Diego, CA) was used at a 1:5,000 dilution. For detecting cell surface localization of SGCs, streptavidin conjugated with peroxidase (Pierce) was used at a 1:1,000 dilution. Immunoreactive bands were visualized by enhanced chemiluminescence (ECLϩPLUS system; Amersham Pharmacia Biotech).

RESULTS
Generation of ␣-Sarcoglycan-deficient Mice-In order to design a targeting vector to generate ␣-sarcoglycan-null mice, we cloned a large portion of the mouse asg gene. Exons 1 and 2 were chosen for targeting to delete the signal sequence and disrupt the gene. Homologous recombination replaced the major portions of exons 1 and 2 and the entire first intron with the nlsLacZ/neo. The disruption of the asg gene in one allele of ES cells was verified by Southern blots of BamHI-digested genomic DNA with a flanking probe (Fig. 1). In properly targeted clones, this probe hybridized to two fragments: an 11-kb BamHI fragment, indicating the disrupted allele, and a 7.5-kb fragment from the wild type allele. In order to confirm a single integration of the nlsLacZ/neo gene, digested DNA from mutant clones were also hybridized to neo and LacZ gene sequences. Two ES cell clones with the correct genotype were injected into C57BL/6J blastocysts to generate chimeric mice. These mice were bred to obtain two lines of asg Ϫ/Ϫ mice.
Sarcoglycans in asg-Null Mice-Immunofluorescence analysis showed no expression of ␣-sarcoglycan in homozygous mutant mice (Fig. 2b), and absence of ␣-sarcoglycan was confirmed by immunoblotting (see below and Fig. 3A), indicating that the targeting vector used had created a null allele. Immunofluorescence analysis showed that the expression and localization of ⑀-sarcoglycan were not affected in null mutant mice (Fig. 2d). This is in agreement with previous work (27). Immunofluorescence analysis further showed that ␤-sarcoglycan was present but reduced (Fig. 2c). In agreement with other reports, ␥and ␦-sarcoglycans were also reduced in amounts as measured by immunofluorescence and/or immunoblotting (data not shown). The presence of ⑀-sarcoglycan and the residual expression of ␤-, ␥-, and ␦-sarcoglycans suggested that a complex containing ⑀-, ␤-, ␥ -, and ␦-sarcoglycans might exist in the mutant mouse muscle.
To determine whether ␤-sarcoglycan in mutant mouse muscle is associated with the other sarcoglycans, we used ␤-sarcoglycan antibody beads to isolate SGCs from wild type and mutant mouse muscle and analyzed such complexes for the presence of ␣-, ␥-, and ⑀-sarcoglycans (Fig. 3A) as well as for ␦-sarcoglycan (data not shown). All these sarcoglycans co-isolated with ␤-sarcoglycan from extracts of wild type muscle, suggesting that ␣and ⑀-sarcoglycans may be present in the same complex with ␤-sarcoglycan or form separate complexes with ␤-sarcoglycan. From asg null mutant mice, the ␤-sarcoglycan antibody co-isolated ⑀-, ␥-, and ␦-sarcoglycans, suggesting the existence of an ⑀-␤-␥-␦ complex. To determine if an ⑀-␤-␥-␦ complex is also present in normal muscle, we carried out immunoisolation with antibodies against either ␣or ⑀-sarcoglycan, followed by immunoblotting analysis to detect the Histological and immunofluorescence analysis of skeletal muscle from two-month-old mice. a, e, and i show hematoxylin and eosin staining of thigh muscle from homozygous mutant (Ϫ/Ϫ), heterozygous (ϩ/Ϫ) and wild type (ϩ/ϩ) mice. Muscle from homozygous mutants (Ϫ/Ϫ) shows dystrophic changes compared with muscle from heterozygous (ϩ/Ϫ) and wild type (ϩ/ϩ) mice. ␣-Sarcoglycan is absent from homozygous mutants (b), The intensity of ␤-sarcoglycan staining is reduced in the asg Ϫ/Ϫ mice (c), whereas the level of ␤-sarcoglycan is the same in heterozygous mice (g) as in wild type mice (k). ⑀-Sarcoglycan is not affected by the absence of ␣-sarcoglycan (d, h, l).
Differential Expression of Sarcoglycans in Vitro-First, we used RT-PCR to determine transcript levels of individual sarcoglycans during myogenic differentiation. RNA was isolated from C2C12 cells at different time points. By varying the number of cycles in RT-PCR, we found that transcripts for ␣and ␥-sarcoglycans, the striated muscle-specific sarcoglycans, increased during differentiation. In contrast, no difference in transcript levels of ␤-, ␦-, and ⑀-sarcoglycans was detected regardless of the number of PCR cycles used (Fig. 4A). Next, we determined protein levels by immunoblotting using specific antibodies against each sarcoglycan. ␣and ␥-sarcoglycans were barely detectable in C2C12 myoblasts (Fig. 4B, lane 1 day) but were present at high levels in cultures of differentiated cells (Fig. 4B, lanes 2-5 days). The protein levels for ␤-sarcoglycan also increased slightly, while ␦and ⑀-sarcoglycans were present at constant levels throughout the culture period (Fig. 4B). Hence, the expression of ␣and ␥-sarcoglycans, which are striated muscle-specific, appeared to correlate with myogenic differentiation at both transcriptional and translational levels, while ␤-, ␦-, and ⑀-sarcoglycans were expressed constitutively.
␣and ⑀-Sarcoglycans Characterize Separate Complexes in Muscle Cells-The ␣-, ␤-, ␥-, and ␦-sarcoglycans have been shown to exist in a stable complex, as they can be co-isolated from rabbit skeletal muscle (15,43) and mouse C2C12 myocytes (22). To test whether ⑀-sarcoglycan also exists in such a complex with other sarcoglycans in C2C12 cells, lysates prepared from C2C12 cells were used for immunoisolation with antibodies against either ␣or ⑀-sarcoglycan, followed by immunoblotting analysis of bound proteins using antibodies specific for each sarcoglycan. As shown in Fig. 5A, the anti-␣sarcoglycan antibody co-isolated ␤-, ␥-, and ␦-sarcoglycans but not ⑀-sarcoglycan. The amount of the complex, as judged from the intensity of each sarcoglycan band, increased dramatically around day 3 after induction of differentiation (data not shown). The ␤-, ␥-, and ␦-sarcoglycans were also co-isolated by the anti-⑀-sarcoglycan antibody (Fig. 5A), but ␣-sarcoglycan was not detected in such preparations even after prolonged exposure of the immunoblot. These results confirm in cultured muscle cells that ␤-, ␥-, and ␦-sarcoglycans associate with either ␣or ⑀-sarcoglycan, forming distinct complexes.
Cell Surface Localization of Two Sarcoglycan Complexes-The SGC containing ␣-sarcoglycan is present in the membrane fraction of muscle tissue and differentiated mouse myotubes (22,32,43). Furthermore, transfection of Chinese hamster ovary cells has demonstrated that cell surface localization of ␣-, ␤-, ␥-, and ␦-sarcoglycans requires co-expression of all four sarcoglycans (23). To determine if the ⑀-sarcoglycan-containing SGC, like the ␣-SGC, is localized to the cell surface in C2C12 cells, cell surface proteins were labeled with biotin before lysis of the cells. SGCs were then isolated with either anti-␣-or anti-⑀-sarcoglycan antibodies, and labeled polypeptides visualized with streptavidin-conjugated peroxidase following separation on SDS-PAGE. A strong band at 35 kDa, which corresponds to the migration position of ␥and ␦-sarcoglycans, and a weaker band at 43 kDa, corresponding to ␤-sarcoglycan, were observed in anti-␣-sarcoglycan isolates (Fig. 5B). We did not detect a band corresponding to ␣-sarcoglycan (ϳ50 kDa). There are five lysine residues within the extracellular portion of mouse ␣-sarcoglycan (21), but ␣-sarcoglycan may be poorly labeled by biotin. Anti-⑀-sarcoglycan isolates contained the same 35-and 43-kDa bands as were present in anti-␣-sarcoglycan isolates, and in addition a band at ϳ47 kDa corresponding to the expected M r of ⑀-sarcoglycan (Fig. 5B). ⑀-Sarcoglycan has 12 lysine residues, which may potentially be labeled with biotin (25). The reverse experiment, using streptavidin-Sepharose as the primary reagent for isolation, followed by immuno- FIG. 3. A, co-isolation of sarcoglycans in asg Ϫ/Ϫ mice and wild type littermates using anti-␤-sarcoglycan antibodies. Heavy microsomes were isolated from wild type and mutant mouse muscle, extracted, and incubated with ␤-sarcoglycan antibodies. The isolates from 80 g of extract were analyzed by SDS-PAGE, and immunoblotting. ␣-sarcoglycan is absent in the isolates from mutant mice, whereas ⑀-sarcoglycan is associated with ␤-sarcoglycan in both wild type and mutant mice. B, co-isolation of sarcoglycans in normal muscle using anti-␣-or anti-⑀sarcoglycan antibodies. One hundred g of protein from extracts of microsomes was immunoprecipitated with either anti-␣-or anti-⑀-sarcoglycan antibodies. The immune complexes were analyzed by SDS-PAGE and immunoblotted with antibodies against ␣-, ␤-, ␥-, ␦-, and ⑀-sarcoglycans. ⑀-Sarcoglycan was undetectable in the preparation isolated with anti-␣-sarcoglycan antibodies and vice versa.

FIG. 4. Expression of sarcoglycans during myogenic differentiation of mouse C2C12 cells.
A, mRNA expression of sarcoglycans. Total RNA was purified from C2C12 cells on day 0, 1, and 5 of differentiation and subjected to 30 cycles of PCR. Amplified PCR products were resolved on 1% agarose gels and stained with ethidium bromide. mRNA levels of ␣and ␥-sarcoglycans increased during differentiation, while no differences were detected in mRNA levels of ␤-, ␦-, and ⑀-sarcoglycans. B, analysis of protein expression levels in differentiating C2C12 cultures. Total protein (30 g/well) prepared from C2C12 cell extracts on day 1, 2, 3, and 5 of differentiation was separated by SDS-PAGE under reducing condition, transferred to nitrocellulose membranes, and blotted with specific antibodies against each sarcoglycan. ␣and ␥-sarcoglycans increased from day 2 of differentiation, ␤-sarcoglycan increased slightly, while no change was detected in levels of ␦and ⑀-sarcoglycans.
blotting with antibodies against ␣-, ␤-, ␥-, ␦-, and ⑀-sarcoglycans, confirmed the expression of ⑀-sarcoglycan at the cell surface (data not shown). Taken together, these results indicate that both ␣and ⑀-SGCs are expressed at the cell surface. DISCUSSION The importance of sarcoglycans in skeletal muscle is evident from the various forms of muscular dystrophy that result from their absence. In order to analyze the molecular events in the pathogenesis of ␣-sarcoglycanopathy, we generated an ␣-sarcoglycan-deficient mouse by homologous recombination in ES cells. The homozygous mutant mice generated from the mutant ES cells expressed no detectable ␣-sarcoglycan mRNA or protein. Although not described in detail in this report, the mutant mice we generated are similar in phenotype to the ones previously reported (27). They are outwardly normal, similar to dystrophin-deficient mdx mice but different from mice with laminin ␣2-deficiency (38) that have a much more severe phenotype affecting their behavior and viability at an early age. However, the ␣-sarcoglycan-deficient mice do show the hallmarks of muscular dystrophy, including early stage muscle hypertrophy and regeneration and fibrosis at later stages.
Immunofluorescent analysis of sarcoglycans in the muscle of the asg Ϫ/Ϫ mice showed a reduction in staining intensity for the ␤-, ␥-, and ␦-sarcoglycans relative to wild type and heterozygous mice. In contrast, the staining intensity for ⑀-sarcoglycan was similar in mutant and wild type mice. A reduction in the staining intensity for ␤-, ␥-, and ␦-sarcoglycans in ␣-sarcoglycan deficiency has been reported for numerous patients (34 -36). We show here that the residual amount of ␤-, ␥-, and ␦-sarcoglycans is due to their presence in a complex with ⑀-sarcoglycan. Although ⑀-SGC do not seem to be more abundant in our ␣-sarcoglycan mutant mice than in wild type mice, it is likely that the ⑀-SGC, even at low levels, play some compensatory role in skeletal muscle of both mutant mice and human patients with a primary ␣-sarcoglycan defect. Varying levels of ⑀-sarcoglycan expression, and consequently of ⑀-SGC, in the skeletal muscle of these patients could be a determinant of disease severity, i.e. low levels of ⑀-sarcoglycan could mean more severe disease. In fact, great variability in disease severity has been noted in patients (36). That ␣and ⑀-sarcoglycans form similar complexes and presumably substitute for each other to some extent in skeletal muscle is in agreement with their high homology. At this point, we do not know if ⑀-sarcoglycan also exists as a monomer or in association with unidentified proteins.
⑀-Sarcoglycan is expressed at higher levels in blood vessels and nerves than in skeletal muscle fibers (24). However, the sarcoglycans co-isolated with ⑀-sarcoglycan from muscle extracts in our experiments must have originated, at least in part, from muscle cells, because ␥-sarcoglycan, which was present in the isolates, is specific for striated muscle (11). To confirm the existence of an ⑀-SGC of muscle origin, we also isolated such a SGC from clonal C2C12 cells, which originate from skeletal muscle. SGCs from these cells also included complexes composed of ⑀-␤-␥-␦ SGC in addition to the well known ␣-␤-␥-␦ SGC. We also show in C2C12 cells that SGCs containing ⑀-sarcoglycan in place of ␣-sarcoglycan are present at the cell surface, a location that suggests that they are functional.
We found that ⑀-, ␤-, and ␦-sarcoglycans are expressed in undifferentiated C2C12 myoblasts, and that only the expression of the two striated muscle-specific sarcoglycans, ␣and ␥-sarcoglycans, is induced during differentiation of the cells into myotubes. This suggests that some of the sarcoglycans play a role in the undifferentiated cells as well as in the differentiated cells, and that skeletal myoblasts contain a SGC consisting of ⑀-, ␤-, and ␦-sarcoglycans. An ⑀-␤-␦ SGC has been described in smooth muscle cells (26). Interestingly, as most studies indicate the necessity for a heterotetrameric SGC as a functional unit, the skeletal myoblast and the smooth muscle SGCs may either contain an additional copy of one of the sarcoglycans, or they may contain a homologue of the missing ␥-sarcoglycan.
An important question is whether a SGC contains four proteins, or whether the complex is a protein composed of four subunits? Implicit in the latter view are two assumptions: (i) that the sarcoglycan functional unit is the tetramer rather than any single sarcoglycan, and (ii) that the presence of one sarcoglycan in a cell signals the presence of a tetrameric sarcoglycan assembly. The subunit hypothesis suggests that cell types expressing fewer than four of the known sarcoglycans must contain unidentified subunits. The subunit hypothesis is supported by the many observations showing that a deficiency of one sarcoglycan reduces the levels of other sarcoglycans (27, 34 -36); sarcoglycans unable to form a complex due to lack of a partner subunit may be unstable. Our experiments support the subunit hypothesis in that four sarcoglycans remained assembled with each other throughout all extraction and fractionation procedures, and that ␣and ⑀-sarcoglycans were never found in the same assembly. In contrast, in immunoprecipitation experiments with an antibody raised to the extracellular domain of ␣-sarcoglycan, Chan et al. (22) detected only free ␣-sarcoglycan and were unable to co-immunoprecipitate other known sarcoglycans. Previous studies have shown that sarcoglycans interact with each other primarily via their extracellular domains (44). Since the antibody used by Chan et al. binds to a site in ␣-sarcoglycan involved in interaction with ␤-, ␥-, and ␦-sarcoglycans, this antibody may have dissociated SGCs after their extraction (22). In our study, the antibodies used for immunoisolation were raised against the intracellular domain of sarcoglycans and are less likely to interfere with proteinprotein interactions between individual sarcoglycans.
Although the functions of sarcoglycans are largely unknown, the current focus is on their association with the DAGC, and on the muscular dystrophy that develops in the absence of sarcoglycans. The dystroglycan and associated DAGC components are proposed to link the cytoskeleton with the extracellular basement membrane, thereby stabilizing muscle and protect- FIG. 5. A, sarcoglycan complexes in differentiated C2C12 cells analyzed by co-immunoprecipitation using anti-␣-or anti-⑀-sarcoglycan antibodies. One hundred g of cellular protein, prepared from cultured C2C12 cells on day 5 of differentiation, were immunoprecipitated with either anti-␣-or anti-⑀-sarcoglycan antibodies. The immunocomplexes were analyzed by SDS-PAGE and immunoblotted with antibodies against ␣-, ␤-, ␥-, ␦-, and ⑀-sarcoglycans. ⑀-Sarcoglycan was undetectable in anti-␣-sarcoglycan isolates and vice versa. B, cell surface expression of sarcoglycan subcomplexes. Cultured C2C12 cells were labeled with NHS-LC-biotin. One hundred g of protein from cell lysates were immunoprecipitated with antibodies against either ␣or ⑀sarcoglycan, and precipitated proteins were analyzed by SDS-PAGE. After proteins in the gel were transferred to nitrocellulose, biotin-labeled proteins were detected by streptavidin conjugated with peroxidase.
ing it from contraction-induced damage. However, dystroglycan apparently plays several non-structural roles; it is essential for formation of the basement membrane in the early embryo (45) and for a laminin-containing extracellular matrix in vitro (46) and mediates a laminin-dependent assembly of a cytoskeletal network in cells (47). There is no firm evidence that sarcoglycans play a structural role in muscle. In fact, the recent report by Hack et al. (16) shows that there is no apparent structural damage to the skeletal muscle of ␥-sarcoglycandeficient mice, even with excessive exercise. Increased uptake of a dye into sarcoglycan-deficient muscle indicates that the muscle cell membrane is leaky, as it is in muscle deficient in dystrophin (48,49). The leaky membranes observed in sarcoglycan deficiencies may result from loss of functional integrity of the cell membrane (16). In this regard, a recent report that ␣-sarcoglycan has ATPase activity is noteworthy (17).
Only muscle-associated defects have been observed in sarcoglycan deficiencies; however, sarcoglycans are likely to play important roles in other tissues. Our preliminary data indicate that individual sarcoglycans are also expressed in endothelial cells, Schwann cells, and macrophages. 3 These observations suggest the presence of novel SGCs in these cells. Both the Drosophila melanogaster and Caenorhabditis elegans genomes contain single ␣/⑀-like, ␤-like, and ␥/␦-like sarcoglycans. The observation that vertebrates contain up to four genes for each gene present in Drosophila or C. elegans (50) suggests that additional sarcoglycans await discovery. In summary, we show here that ⑀-sarcoglycan is functionally similar to ␣-sarcoglycan in skeletal muscle, and suggest that novel sarcoglycans may be responsible for sarcoglycan function in non-muscle as well as muscle cells.