Aberrant trafficking of a Leu89Pro connexin32 mutant associated with X-linked dominant Charcot–Marie–Tooth disease

<b>Objective:</b> 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. <b>Methods:</b> 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. <b>Results:</b> The mutant CX32 protein was localized in the endoplasmic reticulum and failed to reach the cell membrane to form gap junctions. <b>Conclusion:</b> 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.

**研究目的**：明确本团队此前已报道的、存在于某X连锁显性夏科-马里-图思病（X-linked dominant Charcot–Marie–Tooth disease, CMTX）家系中的连接蛋白32（connexin32, CX32）Leu89Pro突变的功能异常情况。该家系内男性患者呈现中度至重度的患病表型。
**研究方法**：本研究采用免疫荧光（immunofluorescence）实验，探究Leu89Pro突变型CX32蛋白能否在海拉细胞（HeLa cells）及人源雪旺细胞（Schwann cells）中成功转运至细胞膜。首先，借助基因重组技术（gene recombination technology）构建分别表达野生型或Leu89Pro突变型CX32，以及增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）的真核表达质粒。随后，通过脂质体转染法（lipofectamine method）将重组质粒瞬时转染至无细胞通讯能力的海拉细胞与人源雪旺细胞中。转染后24小时与48小时，分别对细胞进行CX32与内质网标志物钙联蛋白（calnexin）或高尔基体标志物58kDa蛋白的双重免疫荧光标记。最终使用徕卡TCS SP5 II共聚焦显微镜（Leica TCS SP5 II confocal microscope）采集成像数据。
**研究结果**：突变型CX32蛋白定位于内质网内，无法抵达细胞膜以形成缝隙连接（gap junctions）。
**研究结论**：本研究结果表明，CX32第二个跨膜结构域（transmembrane domain）中的Leu89Pro氨基酸替换会破坏蛋白的转运过程，抑制CX32缝隙连接的组装，进而可能引发周围神经病（peripheral neuropathy）。这一功能异常可解释Leu89Pro突变患者所呈现的中度至重度患病表型，同时也为该亚型CMTX患者的治疗提供了极具潜力的靶向治疗靶点。