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 105,000 g centrifugation. Lane 3, microsome fraction. The composition of each fraction was determined by Western blots using antibodies (1° Ab) against the components of the DGC. Note that some α-dystroglycan from human skeletal muscle was found in the soluble protein fraction which is probably due to the presence of an extensive extracellular matrix network in human tissue but not in cell culture. Also note that actin can exist as both unpolymerized monomers in the cytoplasm (soluble protein fraction) and filaments associated with the membrane (microsome fraction).

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° Ab) against the sarcoglycans, dystrophin, the syntrophins, and the actin. Lane 1, cell lysate from mouse myotubes (11% input). Lane 2, immunoprecipitated products. Note that α-sarcoglycan was the least efficiently precipitated component of the DGC.

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 immune complex was analyzed by Western blots using antibodies (1° Ab) against different sarcoglycans. Lane 1, cell lysate from mouse myotubes (13–16% input). Lane 2, immunoprecipitated products. Note that different sarcoglycans were precipitated by different anti-sarcoglycan antibodies.

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 in 1% NP-40 buffer containing no SDS (lane 2), 0.2% SDS (lane 3), 0.3% SDS (lane 4), and 0.4% SDS (lane 5). Lane 1, cell lysate from mouse myotubes (33% input). (B) Immunoprecipitation was carried out after cultured mouse myotubes were lysed in 1% NP-40 buffer containing no SDS (lane 2), 0.1% SDS (lane 3), 0.2% SDS (lane 4), 0.3% SDS (lane 5), and 0.4% SDS (lane 6). Lane 1, cell lysate from mouse myotubes (16% input). The final composition of the immune complex was determined by Western blots using antibodies (1° Ab) against different sarcoglycans. Note that α-sarcoglycan was the most sensitive to increasing SDS concentration, resulting in dissociation from the complex in both cases.

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 1, cell lysate from mouse myotubes (26–34% input). Lanes 2 and 4, immunoprecipitated products from uncross-linked myotubes. Lanes 3 and 5, immunoprecipitated products from equal amount of DTSSP cross-linked myotubes. The composition of the immune complex was determined by Western blots using antibodies (1° Ab) against different sarcoglycans and dystroglycans.

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 proteins; two circles joined by a line, cross-linked product. Shown in the examples are Western blots using antibodies against α-sarcoglycan, β- and α-dystroglycan (B), against β- and γ-sarcoglycan (C), and against β- and δ-sarcoglycan (D). Cross-linked proteins that appear below the diagonal line are represented by Arabic numerals: 1, α-dystroglycan; 2, β-dystroglycan; 4a and 4b, β-sarcoglycan; 5a, γ-sarcoglycan; and 6a, 6b, and 7, δ-sarcoglycan. The estimated molecular weight of the three cross-linked products X, X₁, and X₂ identified in this experiment are ∼200, 120, and 80 kD, respectively. Note that not all sarcoglycans and dystroglycans were cross-linked in the experiments. Uncross-linked proteins were found on the diagonal line and represented in Roman numerals: I, α-dystroglycan; II, β-dystroglycan; III, α-sarcoglycan; IV, β-sarcoglycan; V, γ-sarcoglycan; and VI, δ-sarcoglycan.

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, Oxford, UK). Right, numbers correspond to the last amino acid residue in the protein; asterisk, stop codon; underline, four conserved cysteine residues. The cysteine 283 in γ-sarcoglycan (italics) is changed to tyrosine in patients with a severe form of early onset autosomal recessive muscular dystrophy (Piccolo et al., 1996). According to the consensus EGF-like repeat motif, the fifth cysteine is linked to the sixth cysteine by a disulfide bond (Abe et al., 1998). This corresponds to the two middle conserved cysteine residues in the sarcoglycans. (B) Cell lysates from cultured mouse myotubes were electrophoresed in SDS-PAGE gel under nonreducing conditions (lane 1) and reducing conditions (lane 2). After transferred to nitrocellulose membranes, blots were examined by Western using antibodies (1° Ab) against different sarcoglycans. The size of the sarcoglycans is indicated by a single arrow (nonreduced form) or a double arrow (reduced form). Note that the extra bands appearing on the nonreducing lane on the Western blot using the anti–β-sarcoglycan antibody were determined as nonspecific products unrelated to the DGC by 2-D diagonal gel electrophoresis (data not shown).

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 is the Western blot using antibodies against α-, β-, and δ-sarcoglycan. Note that no extra spot was observed below the diagonal line (dashed line).

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 (γ-sar), and δ-sarcoglycan (δ-sar). Note that patients AL and CR showed different patterns of immunostaining for each of the sarcoglycans.

Structural model of the sarcoglycan complex and the dystroglycan complex. The four sarcoglycans (left) are represented by α, β, γ, and δ. α-DG and β-DG denote α- and β-dystroglycan (right), respectively. Branch structure corresponds to N-glycoside sugar chain. SH, disulfide linkage. Double-headed arrow, potential interaction between δ-sarcoglycan and the dystroglycan complex. In the model, β-sarcoglycan is tightly associated with δ-sarcoglycan. α-Sarcoglycan is placed apart from other sarcoglycans and is viewed as a separate subunit within the sarcoglycan complex.