摘要
目的探讨CX26蛋白质的9个结构域上的不同错义突变体在细胞内的表达及功能改变的异同,初步研究CX26不同错义突变体的致聋机理。方法在CX26的9个结构域中各选择1个致聋的错义突变(p.S19T、p.R32H、p.E47K、p.V84L、p.V95M、p.R143W、p.R165W、p.S199F、p.L214P),应用重叠区扩增基因拼接法和长引物快速法,构建成与增强型绿色荧光蛋白(EGFP)融合表达的载体,野生型CX26-EGFP作对照,脂质体转染HeLa细胞,Westernblot分析蛋白的表达,共聚焦显微镜下观察各突变体在细胞膜上有无间隙连接斑形成。结果 CX26的9个结构域上的各1个错义突变体在HeLa细胞中均有表达,其中p.S19T、p.E47K、p.V84L、p.V95M和p.vR165W突变体能在细胞膜上表达并形成间隙连接斑,p.R32H、p.R143W、p.S199F和p.L214P突变体在细胞浆中表达,在细胞膜无表达,无间隙连接斑形成。结论 CX26的p.S19T、p.E47K、p.V84L、p.V95M和p.R165W突变体在细胞内能被转运到细胞膜并形成间隙连接,而p.R32H、p.R143W、p.S199F和p.L214P突变体失去被转运到细胞膜的功能,不能形成间隙连接。CX26的不同错义突变的致聋机制可能不同,与突变点所在的结构域可能没有相关性。
Objective To explore the deafness--causing underlying mechanisms of CX26 gene recessive mutations through functional analyzing nine missense mutations （p. S19T, p. R32H, p. E47K, p. V84L, p. V95M, p. R143W, p. R165W, p. S199F, p. L214P） in exogenous expression system Hela cells. Methods The nine recessive missense mutations of CX26, which are in the different domains of CX26 protein, and the wild type CX26 were subcloned into pEGFP--NI vector directively, following to transfect into HeLa cells by the liposome complex method. The expressions of the mutated proteins were analyzed using western blot method. The localizations of the mutated proteins and whether there were gap junction--plaques formation were observed under confocal microscopy with immunofluorescence technique. Results The nine constructs were all expressed in HeLa cells. In which, the mutated proteins of p. S19T, p. E47K, p. V84L, p. V95M and p. R165W localized at the cytoplasmic membrane of HeLa cells and formed gap junction-plaques at contact points between two cells, and the mutated proteins of p. R32H, p. R143W, p. S199F and p. L214P accumulated and localized only intracellularly and did not form gap junction-- plaques on cell membrenes. Conclusion The mutations of p. S19T, p. E47K, p. V84L, p. V95M and p. R165W do not interfere the mutated connxins trafficking and inserting into the plasma membrane. The mutations of p. R32 H, p. R143W, p. S199F and p. L214P impared the proteins trafficking to the cell surface. The deafness--causing mechanisms of different missense mutations might not be identical and no correlation could be observed between the mutation and the topological domain of the mutated protein.
出处
《听力学及言语疾病杂志》
CAS
CSCD
北大核心
2010年第3期249-253,共5页
Journal of Audiology and Speech Pathology
基金
国家自然科学基金(30000094
30572021)
中南大学博士后基金(56890)资助
作者简介
杨中纯，女，湖南人，医师，在读博士研究生，主要从事耳科学基础研究。
肖自安（Email：xiaozian@sina.com）通信作者
谢鼎华（Email：huaxie@2118.cn）通信作者
