p.G38R和p.D40G突变通过改变ANXA11蛋白亚细胞定位引发肌萎缩侧索硬化症

Mutations p.G38R and p.D40G induce amyotrophic lateral sclerosis by changing the subcellular localization of Annexin A11 protein

目的本研究旨在研究Annexin A11蛋白(ANXA11)N端突变p.G38R和p.D40G对该蛋白亚细胞定位的影响,探索其引起肌萎缩侧索硬化症(ALS)的机制。方法构建表达ANXA11野生型蛋白(wt)及p.G38R和p.D40G突变蛋白的真核表达载体,采用激光共聚焦显微镜检测3种蛋白在HEK293细胞中的亚定位。结果 ANXA11wt、p.G38R及p.D40 G蛋白均可在HEK 293细胞中形成囊泡状结构,其中p.G38 R组囊泡面积大于wt组(P=0.002)和p.D40 G组(P=0.006),后两组间无明显差异(P=0.791)。在单个细胞中,ANXA11 wt形成的囊泡数目大于p.G38 R组(P=0.002)和p.D40 G组(P<0.001),而p.G38 R组和p.D40 G组差异不明显(P=0.516)。与ANXA11 wt相比,p.G38 R蛋白(P<0.001)和p.D40 G蛋白(P<0.001)在细胞核内的分布显著减少,后两者间无明显差异(P=0.519)。结论 p.G38 R和p.D40 G突变减少了ANXA11蛋白相关囊泡状结构的数目和ANXA11蛋白在细胞核的分布,这可能是引起ALS发病的机制之一。

Objective To investigate the influence of the N-terminal mutations of Annexin A11 （ANXA11 ） protein, p. G38R and p. D40G, on the subcellular localization of this protein and their mechanism of action in inducing amyotrophic lateral sclerosis （ALS）. Methods Eukaryotic expression vectors which expressed ANXA11 wild-type protein, ANXA11 p. G38R protein, or ANXA11 p. D40G protein were constructed, and a laser scanning confocal microscope was used to investigate the subeellular localization of these three proteins in HEK293 ceils. Results ANXAll wild-type protein, ANXAll p. G38R protein, and ANXAll p. D40G protein formed a vesicular structure in HEK293 cells, and the p. G38R group had significantly larger vesicles than the ANXA11 wild-type group （P = 0.002） and the p. D40G group （P =0.006）, while there was no significant difference between the latter two groups （P =0.791 ）. In a single cell, the ANXA11 wild-type group had significantly more vesicles than the p. G38R group （P = 0.002） and the p. D40G group （ P 〈 0.001 ）, and there was no significant difference between the p. G38R group and the p. D10G group （ P = 0.516）. Compared with ANXAll wild-type protein, p. G38R protein and p. D40G protein had significantly reduced distribution in the nucleus （both P 〈 0. 001 ）, and there was no significant difference between p. G38R protein and p. D40G protein （P = 0. 519）. Conclusions The p.G38R and p. D40G mutations reduce the number of vesicles of ANXA11 protein and the distribution of ANXA11 protein in the nucleus, which may be one of the mechanism of ALS.