Sakagami Hiroyuki, Katsumata Osamu, Hara Yoshinobu, Sasaoka Toshikuni, Fukaya Masahiro
Department of Anatomy, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan.
Department of Comparative and Experimental Medicine, Brain Research Institute, Niigata University, Niigata, Japan.
Invest Ophthalmol Vis Sci. 2017 Jul 1;58(9):3795-3803. doi: 10.1167/iovs.17-21746.
Mutations in genes encoding the dystrophin-associated glycoprotein complex (DGC) can cause muscular dystrophy and disturb synaptic transmission in the photoreceptor ribbon synapse. However, the molecular composition and specific functions of the photoreceptor DGC remain unknown. Brefeldin A-resistant Arf-GEF 2 (BRAG2), also known as IQSEC1, is a guanine nucleotide exchange factor for ADP-ribosylation factor 6 (Arf6), a critical GTPase that regulates endosomal trafficking and actin cytoskeleton remodeling. In the present study, we characterized the expression of BRAG2a, an alternative splicing isoform of BRAG2, in the adult mouse photoreceptor.
Immunofluorescence and immunoelectron microscopic analyses of adult mouse retinas were performed using a novel anti-BRAG2a antibody. Pull-down, immunoprecipitation, and in situ proximity ligation assays were performed to examine the interaction between BRAG2a and the DGC in vivo.
Immunofluorescence demonstrated punctate colocalization of BRAG2a with β-dystroglycan in the outer plexiform layer. Immunoelectron microscopy revealed the localization of BRAG2a at the plasma membrane of lateral walls and processes of photoreceptor terminals within the synaptic cavity. Pull-down and immunoprecipitation assays using retinal lysates demonstrated the protein complex formation between BRAG2a with the DGC. In situ proximity ligation assays further detected a close spatial relationship between BRAG2a and β-dystroglycan in the outer plexiform layer.
The present study provided evidence that BRAG2a is a novel component of the photoreceptor DGC, suggesting functional involvement of the BRAG2a-Arf6 pathway downstream of the DGC.
编码抗肌萎缩蛋白相关糖蛋白复合体（DGC）的基因突变可导致肌肉萎缩，并扰乱光感受器带状突触中的突触传递。然而，光感受器DGC的分子组成和特定功能仍不清楚。抗布雷菲德菌素A的Arf-GEF 2（BRAG2），也称为IQSEC1，是ADP-核糖基化因子6（Arf6）的鸟嘌呤核苷酸交换因子，Arf6是一种关键的GTP酶，可调节内体运输和肌动蛋白细胞骨架重塑。在本研究中，我们对成年小鼠光感受器中BRAG2的一种可变剪接异构体BRAG2a的表达进行了表征。
使用新型抗BRAG2a抗体对成年小鼠视网膜进行免疫荧光和免疫电子显微镜分析。进行下拉、免疫沉淀和原位邻近连接分析以检查BRAG2a与DGC在体内的相互作用。
免疫荧光显示BRAG2a与β-肌营养不良聚糖在外部丛状层呈点状共定位。免疫电子显微镜显示BRAG2a定位于突触腔内光感受器终末侧壁和突起的质膜上。使用视网膜裂解物进行的下拉和免疫沉淀分析证明BRAG2a与DGC之间形成了蛋白质复合物。原位邻近连接分析进一步检测到外部丛状层中BRAG2a与β-肌营养不良聚糖之间存在紧密的空间关系。
本研究提供了证据表明BRAG2a是光感受器DGC的一种新成分，提示DGC下游的BRAG2a-Arf6途径具有功能作用。
