Sarcospan protects against LGMD R5 via remodeling of the sarcoglycan complex composition in dystrophic mice
Ekaterina I. Mokhonova, Daniel Helzer, Ravinder Malik, Hafsa Mamsa, Jackson Walker, Mark Maslanka, Tess S. Fleser, Mohammad H. Afsharinia, Shiheng Liu, Johan Holmberg, Z. Hong Zhou, Eric J. Deeds, Kirk C. Hansen, Elizabeth M. McNally, Rachelle H. Crosbie
Ekaterina I. Mokhonova, Daniel Helzer, Ravinder Malik, Hafsa Mamsa, Jackson Walker, Mark Maslanka, Tess S. Fleser, Mohammad H. Afsharinia, Shiheng Liu, Johan Holmberg, Z. Hong Zhou, Eric J. Deeds, Kirk C. Hansen, Elizabeth M. McNally, Rachelle H. Crosbie
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Research Article Cell biology Muscle biology

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.

Authors

Ekaterina I. Mokhonova, Daniel Helzer, Ravinder Malik, Hafsa Mamsa, Jackson Walker, Mark Maslanka, Tess S. Fleser, Mohammad H. Afsharinia, Shiheng Liu, Johan Holmberg, Z. Hong Zhou, Eric J. Deeds, Kirk C. Hansen, Elizabeth M. McNally, Rachelle H. Crosbie

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Figure 4

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

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SSPN reduces membrane damage and improves muscle physiology in Sgcg mice...
(A) Plasma creatine kinase (CK) levels of WT, Sgcg, and SgcgTG 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 SgcgTG 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 SgcgTG 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. SgcgTG 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.