Sarcospan protects against LGMD R5 via remodeling of the sarcoglycan complex composition in dystrophic mice

Abstract

The dystrophin-glycoprotein complex (DGC) is composed of peripheral and integral membrane proteins at the muscle cell membrane that link the extracellular matrix with the intracellular cytoskeleton. While it is well established that genetic mutations that disrupt the structural integrity of the DGC result in numerous muscular dystrophies, the 3D structure of the complex has remained elusive. Two recent elegant cryoEM structures of the DGC illuminate its molecular architecture and reveal the unique structural placement of sarcospan (SSPN) within the complex. SSPN, a 25 kDa tetraspanin-like protein, anchors β-dystroglycan to the β-, γ- and δ-sarcoglycan trimer, supporting the conclusions of biochemical studies that SSPN is a core element for DGC assembly and stabilization. Here, we advance these studies by revealing that SSPN provides scaffolding in δ-sarcoglycanopathies, enabling substitution of δ-sarcoglycan by its homolog, ζ-sarcoglycan, leading to the structural integrity of the DGC and prevention of limb-girdle muscular dystrophy R5. Three-dimensional modeling reveals that ζ-sarcoglycan preserves protein-protein interactions with the sarcospan, sarcoglycans, dystroglycan, and dystrophin. The structural integrity of the complex maintains myofiber attachment to the extracellular matrix and protects the cell membrane from contraction-induced damage. These findings demonstrate that sarcospan prevents limb-girdle muscular dystrophy R5 by remodeling of the sarcoglycan complex composition.

Keywords: Cell biology; Muscle biology; Skeletal muscle.

Figure 1. SSPN reduces fiber crosssectional area and fiber size variability.

Crosssectional area analysis of quadriceps muscle fibers from g-sarcoglycan deficient (A and D), α-sarcoglycan–deficient (B and E) and β-sarcoglycan-deficient mice and (C and F) SSPN-transgenic littermates. Overexpression of SSPN reduced crosssectional area and fiber size variability across all three lines, most notable in γ-sarcoglycan–deficient muscle overexpressing SSPN. Data in (D–F) shows muscle fiber crosssectional area distributions (total 1,500 individual fibers), median (solid black line), mean (solid black circle), and first and third quartiles (dashed black line). Muscle fiber crosssectional area distributions were compared using pairwise Kolmogorov–Smirnov tests with Bonferroni correction. n = 3 mice per genotype, 500 fibers were measured per mouse.

Figure 2. SSPN ameliorates central nucleation and reduces dystrophic pathology in γ-sarcoglycanopathy muscles.

(A–F) Quantification of centrally located nuclei (CN, central nucleation) from H&E-stained quadriceps cryosections from (A and B) Sgcg, (C and D) Sgca, and (E and F) Sgcb mice and SSPN-transgenic littermates (n = 3–5 mice per genotype). The percentage of myofibers with centrally located nuclei was significantly increased in all 3 sarcoglycan-deficient lines. Overexpression of SSPN reduced central nucleation in Sgcg mice to WT levels, but not in Sgca or Sgcb mice. (G–L) Diaphragms from Sgcg^TG mice exhibit reduced dystrophic pathology. Low-magnification view of diaphragm muscle from (G) Sgcg, (I) Sgca, and (K) Sgcb mice and SSPN-transgenic littermates. The magnification is identical in all images. SSPN overexpression reduced diaphragm thickness in (H) Sgcg mice, but not in (J) Sgca or (L) Sgcb mice (n = 4–6 mice per genotype). Statistical analysis by 1-way ANOVA and Tukey’s test. Scale bars: 50 μm (A, C, and E); 200 μm (G, I, and K).

Figure 3. Fibrosis is prevented in Sgcg^TG muscle.

Transverse cryosections and quantification of collagen area from (A and B) quadriceps and (C and D) diaphragm stained with Picrosirius Red. Overexpression of SSPN reduced collagen deposition throughout the diaphragm and quadriceps compared with Sgcg. n = 3–4 mice per genotype. Statistical analysis by 1-way ANOVA and Tukey’s test. Scale bars: 50 μm.

Figure 4. SSPN reduces membrane damage and improves muscle physiology in Sgcg mice.

(A) Plasma creatine kinase (CK) levels of WT, Sgcg, and Sgcg^TG mice. Plasma CK in Sgcg mice was elevated approximately 4× that of WT mice. Overexpression of SSPN reduced CK back to WT levels. n = 12–18 per genotype (12–20 weeks of age). Statistical analysis by 1-way ANOVA and Tukey’s test. (B) Representative images of transverse sections of quadriceps muscles from 12-week-old Sgcg and Sgcg^TG mice stained with anti-mouse IgG antibody (green) and laminin (red). (C) IgG⁺ fibers counted from whole quadriceps images, n = 3–7. (D) eMHC (green) positive fibers were imaged as a measure of regeneration. Laminin (red) was used to outline fibers. (E) eMHC⁺ fibers were quantified and normalized to whole quadriceps image area. Both IgG⁺ and eMHC⁺ stains exhibited high variability across Sgcg mice, while Sgcg^TG muscle had very few positively stained fibers in both cases. Statistical analysis for IgG⁺ and eMHC⁺ fibers conducted by Kruskal-Wallis test and subsequent Conover-Iman test with Bonferroni correction. (F–H) Transgenic SSPN expression improved muscle physiology in Sgcg mice. (F) Representative traces of mouse ambulation in an open field during a 6-minute recording time. (G and H) Quantification of after-exercise activity distances from 30-week-old mice. Sgcg^TG mice traveled significantly farther distances compared with Sgcg littermates. n = 3–5 per genotype. Statistical analysis by 1-way ANOVA and Tukey’s test. Circles, male mice; triangles, female mice. Scale bars: 50 μm.

Figure 5. The basement membrane is expanded in Sgcg and Sgcg^TG muscle.

Proteomics analysis was conducted using quantitative concatemers (QconCATs) to determine concentrations of basement membrane proteins. (A) Schematic overview of proteomics protocol designed to accurately capture difficult-to-extract ECM protein. The figure was partly generated using Servier Medical Art, provided by Servier and OpenStax, both licensed under Creative Commons Attribution 4.0 International. (B) Percentages of basement membrane proteins in WT, Sgcg, and Sgcg^TG muscle. (C) Comparison of total basement membrane protein content across the 3 genotypes. Basement membrane proteins were more abundant in both Sgcg and Sgcg^TG muscle compared with WT muscle. (D–F) Absolute quantity of LAMA2, LAMB1, and LAMC1 (protein components of laminin-211) from quantitative proteomics analysis. The laminin-211 proteins were elevated above WT in Sgcg and Sgcg^TG muscle. n = 4–7. Statistical analysis by 1-way ANOVA and Tukey’s test. Circles, male mice; triangles, female mice.

Figure 6. Distinct changes in basement membrane proteins in Sgcg and Sgcg^TG muscle.

Quantitative proteomics plots of basement membrane proteins classified as (A) glycoproteins, (B) collagens, (C) proteoglycans, and (D) matrikines. The matrikines canstatin and endorepellin are peptide sequences found in the C-terminus of COL4A2 and HSPG2, respectively. NID1/2 represents the peptide sequence GNLYWTDWNR found in both NID1 and NID2. COL4A1/5 represents the peptide sequence SAPFIECHGR found in both COL4A1 and COL4A5. Type IV collagen was increased in Sgcg^TG samples, while the expanded basement membrane in Sgcg muscle was largely driven by regeneration-associated proteins HSPG2, endorepellin, NID1, and NID1/2. n = 4–7. COL4A5 and LAMB3 values were compared using Kruskal-Wallis test followed by Dunn’s test. LAMA4 values were compared using Welch’s ANOVA and Dunnett test. All other protein comparisons were conducted by 1-way ANOVA and Tukey’s test. Circles, male mice; triangles, female mice.

Figure 7. SSPN does not affect the presence of ECM receptors at the membrane or laminin binding capacity in Sgcg muscle.

(A) Immunofluorescence assay quadriceps cryosections probed for laminin and dystroglycan (DAG) with (B) quantification. Scale bar: 50 mm; n = 3–6 mice per genotype, 49 individual myofiber measurements per mouse (total 147–294). Data are presented as FC relative to WT (mean ± SD) for individual mice within a genotype. The mean ± SD for each genotype is provided with black solid bars (dashed line represents mean of WT samples). Statistical analysis by fitting a generalized linear model. (C) Immunoblotting of eluate fractions of lectin (sWGA) purifications using the indicated antibodies (n = 2–3). IIH6 detects the laminin binding glycoepitope of α-DAG. (D) Proteomics analysis quadriceps muscle, n = 4–7. Data presented as log₂(FC) relative to WT. Statistical analysis by unpaired t test and Benjamini-Hochberg procedure. (E) Dag1 expression in myonuclei from single nuclei RNA-seq of quadriceps muscles. (F) Laminin overlays of sWGA-enriched eluates. (G) Laminin binding capacity of sWGA-enriched muscle lysates by solid phase laminin binding assay, n = 2–3. Circles, male mice; triangles, female mice.

Figure 8. SSPN restores membrane expression protein interactions of α-, β- and δ-sarcoglycans.

(A) Indirect immunofluorescence assays of transverse WT, Sgcg, and Sgcg^TG quadriceps cryosections using the indicated antibodies. Scale bar: 50 μm. (B–D) Quantification of sarcoglycan abundance at the sarcolemma, n = 4–6 mice per genotype, 49 individual myofiber measurements per mouse (total 196–294 myofibers per genotype). Data are presented as fold change (FC) relative to WT (mean ± SD) for individual mice within a genotype. The mean ± SD for each genotype is provided with black solid bars (dashed line represents mean of WT samples). Statistical analysis was performed by fitting a generalized linear model. (E–J) Immunoblotting of the eluate fractions of lectin (sWGA) purifications from all 3 genotypes using the indicated antibodies (n = 2–3 per genotype). Proteins that interact in a complex can be detected in eluate fractions.

Figure 9. SSPN stabilizes DGC complexes containing ζ-sarcoglycan.

(A) Indirect immunofluorescence assays of transverse WT, Sgcg, and Sgcg^TG quadriceps cryosections using the indicated antibodies. Scale bar: 50 μm. (B) Quantification of ε- and ζ-sarcoglycan abundance at the sarcolemma, n = 3–5 mice per genotype, 49 individual myofiber measurements per mouse (total 147–245). Data are presented as FC relative to WT (mean ± SD) for individual mice within a genotype. The mean ± SD for each genotype is provided with black solid bars (dashed line represents mean of WT samples). Statistical analysis was performed by fitting a generalized linear model. (C) Immunoblotting of the eluate fractions of lectin (sWGA) purifications from all 3 genotypes using the indicated antibodies (n = 2–3 per genotype). Proteins that interact in a complex can be detected in eluate fractions.

Figure 10. SSPN restores dystrophin-dystroglycan association.

(A) Immunofluorescence staining and (B) sarcolemmal intensity quantification of dystrophin and utrophin, n = 4–5 mice per genotype, 49 individual myofiber measurements per mouse (total 196–245); data presented as fold change (FC) relative to WT, mean ± SD within each mouse and ± SD of all measurements per genotype. Statistical analysis by fitting a generalized linear model described in the methods section. (C and D) Relative protein levels from proteomics show dystrophin was reduced in the Sgcg and Sgcg^TG, despite similar membrane intensity values, n = 4–7. Adjusted P values (Benjamini–Hochberg procedure) from differential protein expression analysis shown. (E and F) Dmd and Utrn mRNA expression in myonuclei from single nuclei RNA-seq. Dmd expression was slightly reduced in both Sgcg (average log₂ FC = –0.48) and Sgcg^TG (average log₂ FC = –0.35) compared with WT, with Sgcg^TG slightly higher than Sgcg (average log₂ FC = 0.14). Utrn expression was elevated in Sgcg myonuclei (average log₂ FC = 3.23) above WT. (G and H) sWGA enrichment of muscle lysates and immunoblotting for dystrophin and utrophin, n = 2–3. Dystrophin-dystroglycan association was reduced in Sgcg but restored in Sgcg^TG samples while utrophin-dystroglycan association was similarly increased in Sgcg^TG and Sgcg compared with WT.

Figure 11. ζ-sarcoglycan replaces γ-sarcoglycan in predicted SSPN-SG structure.

AlphaFold 3 predicted structures of (A) SSPN with α-, β-, δ-, γ-SG complex. (B) An inset (orange box in A) showing hydrophobic interactions consisting of Leu145 of SSPN in a hydrophobic pocket created by Phe88 of γ-SG and Tyr125 of β-SG. (C) An inset (grey box in A) showing a salt bridge between Arg57 of SSPN and Asp64 of δ-SG. (D) SSPN with α-, β-, δ-, ζ-SG complex demonstrated similarity to the canonical complex shown in A. (E) An inset (orange box in D) showing conserved hydrophobic interactions consisting of Leu145 of SSPN in a hydrophobic pocket created by Leu101 of ζ-SG and Tyr125 of β-SG. (F) An inset (grey box in D) showing a conserved salt bridge formed Arg57 of SSPN and Asp64 of δ-SG. Dotted yellow lines, hydrogen bonds involved in a salt bridge; yellow arcs, hydrophobic interactions.

Figure 12. Coevolutionary analysis of protein pairs in the SG-SSPN complex and control proteins.

Coevolution of residues from SSPN, α-SG, β-SG, δ-SG, γ-SG, ε-SG, ζ-SG, dystroglycan, and 2 control proteins GAPDH and ATP1A1 was analyzed. (A) Workflow schematic of the mutual information-based coevolutionary analysis. (B) Dot plot integrating mutual information scores and coevolution significance for each protein pair. Dot size represents the proportion of significantly coevolving residue pairs (q-value < 0.05) and color (yellow to red) indicates the fraction of residue pairs with nonzero mutual information scores. The more intense yellow color between SSPN and γ-SG indicates a higher fraction of nonzero mutual information scores for this protein pair. This analysis revealed a strong coevolutionary relationship between SSPN and γ-SG.

Figure 13. Rebuilding, rewiring and stabilization — how sarcospan prevents limb-girdle muscular dystrophy R5.

The SSPN-sargoclycan complex interacts with and stabilizes the DGC and maintains dystrophin-dystroglycan association. Though expressed at lower levels in skeletal muscle, ε- and ζ-sarcoglycan form alternative sarcoglycan complexes (see discussion section for details and references). Our model suggests that SSPN overexpression restores membrane stability in γ-sarcoglycan–deficient muscle by supporting the stabilization of a lower abundance sarcoglycan complex (α-, β-, δ-, ζ-sarcoglycan). This supports restoration of dystrophin-dystroglycan binding, thereby protecting against muscle injury.