Da Yuwei, Wang Wei, Liu Zhongfeng, Chen Hai, Di Li, Previch Lauren, Chen Zhiguo
a Department of Neurology , Xuan Wu Hospital, Capital Medical University , Beijing , China.
b Cell Therapy Center , Xuan Wu Hospital, Capital Medical University, and Key Laboratory of Neurodegeneration, Ministry of Education , Beijing , China.
Neurol Res. 2016 Oct;38(10):897-902. doi: 10.1080/01616412.2016.1204494. Epub 2016 Jul 1.
To determine the functional abnormalities of the Leu89Pro mutation in connexin32 (CX32), which we have previously reported is present within an X-linked dominant Charcot-Marie-Tooth disease family. In this family, male patients were moderately to severely affected.
We performed immunofluorescence to investigate whether the Leu89Pro CX32 protein was transported to the cell membrane in HeLa and Schwann cells. First, we constructed the eukaryotic express plasmids expressing CX32 (wild-type or Leu89Pro) and enhanced green fluorescent protein by the gene recombination technology. Then the recombinant plasmids were transiently transfected into communication-incompetent HeLa cells and human Schwann cells by the lipofectamine method. Later, we double-labeled cells for both CX32 and markers of the ER (calnexin) or the Golgi (58-kDa protein) at 24 h or 48 h. The images were collected using a Leica TCS SP5 II confocal microscope.
The mutant CX32 protein was localized in the endoplasmic reticulum and failed to reach the cell membrane to form gap junctions.
Our results indicated that the Leu89Pro substitution in the second transmembrane domain of CX32 disrupts the trafficking of the protein, inhibiting the assembly of CX32 gap junctions, which in turn may result in peripheral neuropathy. This functional abnormality may explain the moderate to severe phenotype seen in Leu89Pro patients, and as such represents a promising therapeutic target in the treatment of this subset of CMTX patients.
确定连接蛋白32（CX32）中Leu89Pro突变的功能异常，我们之前报道该突变存在于一个X连锁显性遗传性腓骨肌萎缩症（CMTX）家系中。在这个家系中，男性患者受到中度至重度影响。
我们进行免疫荧光检测，以研究Leu89Pro CX32蛋白是否能在HeLa细胞和施万细胞中转运至细胞膜。首先，我们通过基因重组技术构建了表达CX32（野生型或Leu89Pro）和增强型绿色荧光蛋白的真核表达质粒。然后，通过脂质体转染法将重组质粒瞬时转染至无通讯能力的HeLa细胞和人施万细胞中。之后，在24小时或48小时对细胞进行CX32与内质网标志物（钙连蛋白）或高尔基体标志物（58-kDa蛋白）的双重标记。使用徕卡TCS SP5 II共聚焦显微镜采集图像。
突变型CX32蛋白定位于内质网，无法到达细胞膜形成缝隙连接。
我们的结果表明，CX32第二个跨膜结构域中的Leu89Pro替换破坏了蛋白质的转运，抑制了CX32缝隙连接的组装，进而可能导致周围神经病变。这种功能异常可能解释了Leu89Pro患者中出现的中度至重度表型，因此代表了治疗这一亚组CMTX患者的一个有前景的治疗靶点。
