PDF(Pubmed)
Abstract：
Sarcospan (SSPN) is a 25-kDa transmembrane protein that is broadly expressed at the cell surface of many tissues, including, but not limited to, the myofibers from skeletal and smooth muscles, cardiomyocytes, adipocytes, kidney epithelial cells, and neurons. SSPN is a core component of the dystrophin-glycoprotein complex (DGC) that links the intracellular actin cytoskeleton with the extracellular matrix. It is also associated with integrin α7β1, the predominant integrin expressed in skeletal muscle. As a tetraspanin-like protein with four transmembrane spanning domains, SSPN functions as a scaffold to facilitate protein-protein interactions at the cell membrane. Duchenne muscular dystrophy, Becker muscular dystrophy, and X-linked dilated cardiomyopathy are caused by the loss of dystrophin at the muscle cell surface and a concomitant loss of the entire DGC, including SSPN. SSPN overexpression ameliorates Duchenne muscular dystrophy in the mdx murine model, which supports SSPN being a viable therapeutic target. Other rescue studies support SSPN as a biomarker for the proper assembly and membrane expression of the DGC. Highly specific and robust antibodies to SSPN are needed for basic research on the molecular mechanisms of SSPN rescue, pre-clinical studies, and biomarker evaluations in human samples. The development of SSPN antibodies is challenged by the presence of its four transmembrane domains and limited antigenic epitopes. To address the significant barrier presented by limited commercially available antibodies, we aimed to generate a panel of robust SSPN-specific antibodies that can serve as a resource for the research community. We created antibodies to three SSPN protein epitopes, including the intracellular N- and C-termini as well as the large extracellular loop (LEL) between transmembrane domains 3 and 4. We developed a panel of rabbit antibodies (poly- and monoclonal) against an N-terminal peptide fragment of SSPN. We used several assays to show that the rabbit antibodies recognize mouse SSPN with a high functional affinity and specificity. We developed mouse monoclonal antibodies against the C-terminal peptide and the large extracellular loop of human SSPN. These antibodies are superior to commercially available antibodies and outperform them in various applications, including immunoblotting, indirect immunofluorescence analysis, immunoprecipitation, and an ELISA. These newly developed antibodies will significantly improve the quality and ease of SSPN detection for basic and translational research.
摘要：
Sarcospan（SSPN）是一种25kDa的跨膜蛋白，在许多组织的细胞表面广泛表达，包括,但不限于,骨骼肌和平滑肌的肌纤维，心肌细胞，脂肪细胞,肾上皮细胞，和神经元。SSPN是连接细胞内肌动蛋白细胞骨架与细胞外基质的肌营养不良蛋白-糖蛋白复合物（DGC）的核心成分。它还与整合素α7β1(骨骼肌中表达的主要整合素)相关。作为具有四个跨膜域的四跨膜蛋白样蛋白，SSPN充当支架以促进细胞膜上的蛋白质-蛋白质相互作用。杜氏肌营养不良症,Becker肌营养不良症,和X连锁扩张型心肌病是由肌细胞表面的肌营养不良蛋白的丢失以及伴随的整个DGC的丢失引起的，包括SSPN。SSPN过表达改善mdx小鼠模型中的Duchenne肌营养不良,这支持SSPN成为可行的治疗靶标。其他拯救研究支持SSPN作为DGC的正确组装和膜表达的生物标志物。SSPN拯救分子机制的基础研究需要针对SSPN的高度特异性和强大的抗体，临床前研究,和人类样本中的生物标志物评估。SSPN抗体的开发受到其四个跨膜结构域和有限的抗原表位的存在的挑战。为了解决有限的市售抗体带来的重大障碍，我们的目标是产生一组强大的SSPN特异性抗体，可以作为研究界的资源。我们创建了针对三个SSPN蛋白表位的抗体，包括细胞内N-和C-末端以及跨膜结构域3和4之间的大细胞外环(LEL)。我们开发了一组针对SSPNN末端肽片段的兔抗体（多抗体和单克隆抗体）。我们使用了几种测定法来显示兔抗体以高功能亲和力和特异性识别小鼠SSPN。我们开发了针对人SSPN的C末端肽和大细胞外环的小鼠单克隆抗体。这些抗体优于市售抗体，并在各种应用中胜过它们。包括免疫印迹,间接免疫荧光分析，免疫沉淀,和ELISA。这些新开发的抗体将显着提高SSPN检测的质量和易用性，用于基础和翻译研究。
