Peter Angela K, Miller Gaynor, Capote Joana, DiFranco Marino, Solares-Pérez Alhondra, Wang Emily L, Heighway Jim, Coral-Vázquez Ramón M, Vergara Julio, Crosbie-Watson Rachelle H
Department of Integrative Biology and Physiology, University of California, Los Angeles, 610 Charles E. Young Drive East, Terasaki Life Sciences Building, Los Angeles, CA, 90095, USA.
Present Address: Biofrontiers Institute, Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO, USA.
Skelet Muscle. 2017 Jun 6;7(1):11. doi: 10.1186/s13395-017-0127-9.
Sarcospan (SSPN) is a transmembrane protein that interacts with the sarcoglycans (SGs) to form a tight subcomplex within the dystrophin-glycoprotein complex that spans the sarcolemma and interacts with laminin in the extracellular matrix. Overexpression of SSPN ameliorates Duchenne muscular dystrophy in murine models.
Standard cloning approaches were used to identify nanospan, and nanospan-specific polyclonal antibodies were generated and validated. Biochemical isolation of skeletal muscle membranes and two-photon laser scanning microscopy were used to analyze nanospan localization in muscle from multiple murine models. Duchenne muscular dystrophy biopsies were analyzed by immunoblot analysis of protein lysates as well as indirect immunofluorescence analysis of muscle cryosections.
Nanospan is an alternatively spliced isoform of sarcospan. While SSPN has four transmembrane domains and is a core component of the sarcolemmal dystrophin-glycoprotein complex, nanospan is a type II transmembrane protein that does not associate with the dystrophin-glycoprotein complex. We demonstrate that nanospan is enriched in the sarcoplasmic reticulum (SR) fractions and is not present in the T-tubules. SR fractions contain membranes from three distinct structural regions: a region flanking the T-tubules (triadic SR), a SR region across the Z-line (ZSR), and a longitudinal SR region across the M-line (LSR). Analysis of isolated murine muscles reveals that nanospan is mostly associated with the ZSR and triadic SR, and only minimally with the LSR. Furthermore, nanospan is absent from the SR of δ-SG-null (Sgcd) skeletal muscle, a murine model for limb girdle muscular dystrophy 2F. Analysis of skeletal muscle biopsies from Duchenne muscular dystrophy patients reveals that nanospan is preferentially expressed in type I (slow) fibers in both control and Duchenne samples. Furthermore, nanospan is significantly reduced in Duchenne biopsies.
Alternative splicing of proteins from the SG-SSPN complex produces δ-SG3, microspan, and nanospan that localize to the ZSR and the triadic SR, where they may play a role in regulating resting calcium levels as supported by previous studies (Estrada et al., Biochem Biophys Res Commun 340:865-71, 2006). Thus, alternative splicing of SSPN mRNA generates three protein isoforms (SSPN, microspan, and nanospan) that differ in the number of transmembrane domains affecting subcellular membrane association into distinct protein complexes.
肌联蛋白（SSPN）是一种跨膜蛋白，它与肌聚糖（SGs）相互作用，在肌营养不良蛋白-糖蛋白复合物中形成紧密的亚复合物，该复合物跨越肌膜并与细胞外基质中的层粘连蛋白相互作用。在小鼠模型中，SSPN的过表达可改善杜兴氏肌营养不良症。
使用标准克隆方法鉴定纳米肌联蛋白，并制备和验证了纳米肌联蛋白特异性多克隆抗体。采用骨骼肌膜的生化分离和双光子激光扫描显微镜分析多个小鼠模型肌肉中纳米肌联蛋白的定位。通过蛋白质裂解物的免疫印迹分析以及肌肉冰冻切片的间接免疫荧光分析对杜兴氏肌营养不良症活检样本进行分析。
纳米肌联蛋白是肌联蛋白的一种可变剪接异构体。虽然SSPN有四个跨膜结构域，是肌膜肌营养不良蛋白-糖蛋白复合物的核心成分，但纳米肌联蛋白是一种II型跨膜蛋白，不与肌营养不良蛋白-糖蛋白复合物结合。我们证明纳米肌联蛋白在肌浆网（SR）组分中富集，而在T小管中不存在。SR组分包含来自三个不同结构区域的膜：T小管侧翼区域（三联体SR）、跨越Z线的SR区域（ZSR）和跨越M线的纵向SR区域（LSR）。对分离的小鼠肌肉进行分析发现，纳米肌联蛋白主要与ZSR和三联体SR相关，与LSR的相关性最小。此外，在肢体带型肌营养不良症2F的小鼠模型δ-SG基因敲除（Sgcd）骨骼肌的SR中不存在纳米肌联蛋白。对杜兴氏肌营养不良症患者的骨骼肌活检样本进行分析发现，在对照样本和杜兴氏样本中，纳米肌联蛋白均优先在I型（慢）纤维中表达。此外，在杜兴氏活检样本中纳米肌联蛋白显著减少。
来自SG-SSPN复合物的蛋白质可变剪接产生了δ-SG3、微肌联蛋白和纳米肌联蛋白，它们定位于ZSR和三联体SR，如先前研究（Estrada等人，《生物化学与生物物理研究通讯》340:865-71，2006年）所支持的，它们可能在调节静息钙水平中发挥作用。因此，SSPN mRNA的可变剪接产生了三种蛋白异构体（SSPN、微肌联蛋白和纳米肌联蛋白），它们在跨膜结构域数量上有所不同，从而影响亚细胞膜与不同蛋白复合物的结合。
