【作者】 杨中纯；

【导师】 谢鼎华；

【作者基本信息】 中南大学 ， 耳鼻咽喉头颈外科， 2010， 博士

【摘要】 耳聋是引起交流障碍最常见的疾病。在常染色体隐性遗传性聋中,约50%以上耳聋是由GJB2 (CX26)基因缺陷所致,其中白种人最常见的突变是35del G,东亚人最常见的突变是235delC,该基因突变还与常染色体显性遗传性聋和遗传性综合征性聋有关,是最重要的耳聋基因。CX26(Connexin 26)是间隙连接蛋白家族中的一员。间隙连接通道(Gap Junctional Channels, GJCh)是间隙连接蛋白在相邻细胞膜上组成的一种细胞膜性通道,能允许离子(ionic coupling,离子耦联)、或诸如分子量<1000 Da的代谢产物和第二信使分子等小分子物质(biochemical coupling,生化耦联)交换通过。生物体通过这种通道进行物质和信息交换,使得细胞对内外环境的刺激作出协调一致的反应,对细胞的新陈代谢、增殖和分化、内环境稳定等生理过程起着重要的调控作用。GJCh的功能分析包括离子耦联和生化耦联两个方面的分析。目的：探讨CX26不同结构域上的各1个点突变(S19T、E47K、V84L、V95M、R165W、R32H、R143W、S199F和L214P),及导致不同长度截短蛋白的突变(35del G、235delC、572delT、465 T→A(Y155X)和631-632delGT)在体外真核细胞内的表达、定位和间隙连接功能的改变；尤其是对CX26的6个在国际上还没有功能研究报道的突变(R32H、R165W、S199F、572delT、465 T→A和631—632delGT)在体外真核细胞表达载体分析突变蛋白功能的变化。本文中的5个截短蛋白突变的共同点是CX26的羧基端(C端)缺失,5个截短蛋白突变的分析可能反应C端对CX26运输、定位和间隙连接功能的作用。本研究旨在为分析GJB2基因突变致聋的分子机制、及为防治该基因缺陷所致的耳聋奠定理论基础。方法：从CX26的9个结构域各选1个致聋点突变(错义突变),及5个导致不同长度截短蛋白的移码或无义突变,其中包括我国最常见的突变235delC,高加索人群中最常见的突变35delG,我们以前发现的一个新突变Y155X,以及572delT和631-632delGT,分别用Overlap法和长引物法快速构建CX26基因的这14个致聋突变体(p.S19T、p.R32H、p.E47K、p.V84L、p.V95M、p.R143W、p.R165W、p.S199F、p.L214P、c.35delG、c.235delC、p.Y155X、c.572delT和c.631-632delGT),将各突变体及野生型CX26分别装入pEGFP-N1质粒。脂质体转染HeLa细胞,Western印迹分析突变型和野生型CX26在HeLa细胞的表达,荧光显微镜下初步观察CX26突变蛋白和野生型蛋白在HeLa细胞的表达和定位后,进一步用共聚焦显微镜观察CX26突变蛋白和野生型蛋白的定位及在细胞膜上有无间隙连接斑样结构形成。对无间隙连接斑样结构形成的突变体,再对转染的HeLa细胞的高尔基体和内质网染色进行染色标记,共聚焦显微镜下观察突变蛋白是否定位于高尔基体或内质网,了解突变蛋白的亚细胞定位。对能形成间隙连接斑的突变体采用calcein染料转移实验分析所形成的间隙连接通道的生化耦联功能。结果：通过overlap法成功构建了p.R32H、p.E47K、p.V84L、p.V95M、p.R143W、p.R165W、p.S199F及c.35delG突变体；通过长引物法成功构建了p.S19T、p.L214P、c.235delC、p.Y155X、c.572delT及c.631-632delGT突变体。Western印迹检测结果显示,c.35delG突变体在HeLa细胞无突变蛋白的表达,其他13个突变蛋白在HeLa细胞都有表达。p.S19T、p.R32H、p.E47K、p.V84L、p.V95M、p.R143W、p.R165W、p.S199F和p.L214P突变蛋白的分子量大小与野生型CX26基本相同,c.235delC、p.Y155X、c.572delT及c.631-632delGT突变蛋白的分子量小于野生型,为截短蛋白。p.S19T、p.E47K、p.V84L、p.V95M、p.R165W突变蛋白主要定位在细胞膜上,聚集成间隙连接斑样结构。p.R32H>、p.R143W、p.S199F、p.L214P、c.235delC、p.Y155X、c.572delT、c.631-632delGT突变蛋白在细胞内呈弥散分布,主要定位于内质网,高尔基体染色部位无突变蛋白分布,在细胞膜上观察不到突变蛋白表达。Calcein染料转移实验发现p.V84L在HeLa细胞上形成的突变蛋白间隙连接和野生型CX26间隙连接具有Calcein染料转移功能,无显著性差异。而p.S19T、p.E47K、p.V95M、p.R165W突变蛋白在HeLa细胞形成的间隙连接不能进行calcein染料转移。结论：CX26的p.R32H、p.R143W、p.S199F和p.L214P错义突变体,及c.235delC、p.Y155X、c.572delT和c.631-632delGT截短蛋白突变体在体外真核细胞表达载体HeLa细胞不能运输至细胞膜上形成间隙连接,突变蛋白主要表达和定位于内质网,提示这8个突变蛋白丧失了从内质网转运到细胞膜的功能。4个不同大小的截短蛋白(c.235delC、c.465T→A、c.572delT和c.631-632delGT)均不能运输至质膜,提示其共同缺失的C端对CX26的运输可能具有重要意义。p.S19T、p.E47K、p.V84L、p.V95M和p.R165W突变体在HeLa细胞表达后能被装配和运输到细胞膜上,形成间隙连接斑样结构。其中p.V84L突变蛋白在HeLa细胞膜上形成的间隙连接有生化耦联通透功能,p.S19T、p.E47K、p.V95M和p.R165W突变体形成的间隙连接无生化耦联通透功能。CX26的c.35delG突变体不能在HeLa细胞翻译和表达。CX26的错义突变的蛋白功能改变与突变所处的结构域无关,错义突变蛋白的功能改变是由突变本身氨基酸的改变所致。更多还原

【Abstract】 Hearing impairment is the most common sensory disorder causing communication disturbance.Nearly 50% of autosomal recessive non-syndromic hearing loss is associated with mutations in GJB2. The 35delG mutation showed a high prevalence among Caucasian populations while the 235delC mutation was found predominantly within individuals of Asian decent. Mutations in the connexin26 gene (GJB2) are also associated with autosomal dominant non-syndromic hearing loss and inherited syndromic hearing loss. It’s the most common cause of genetic deafness. CX26 (Connexin 26) is a gap junctional protein that is encoded by GJB2. Gap junction channels,formed at the appositional plasma membranes by a family of related proteins named connexins,allow the diffusion of ions and small molecules (molecular weight<1000 Da,such as metabolites and the second messengers) between adjacent cells and provide a mechanism of synchronizing response of groups of cells to environment stimuli. Gap junction intercellular communication (GJIC) plays a key role in cell metabolism,cell proliferation and cell differentiation and homeostasis,Gap junction intercellular communication includes ionic coupling and biochemical coupling.Objective Our objective is to study the effect of various point mutations (S19T,E47K,V84L,V95M,R165W,R32H,R143W,S199F and L214P) in different domains of CX26 as well as mutations (35delG,235delC, 572delT,465 T→A and 631-632delGT) causing truncated proteins of various lenghth on assembly, localization and function of CX26. Among these mutations, R32H, R165W, S199F,465T→A,572delT and 631-632delGT have not been reported to be studied in exogenous expression system.All of the 5 deletion mutations have no carboxyl terminus.The possible effects of the absence of carboxyl terminus on trafficking,localization and function of mutant proteins will be discussed.Our study will improve the knowledge on the mechanism of how GJB2 mutations cause hearing loss and the development of therapeutic methods.Method We constructed expression plasmids of 9 missense mutations each of which is located in a domain of CX26 and 5 mutations causing truncated proteins including c.235delC and c.35delG which are most common in Asia and Caucasian population respectively,c.465 T→A that we discovered previously,c.572del and c.631-632delGT.We used overlap extension PCR and "long-primer PCR" to introduce mutations in CDS of CX26.The CDS with mutations as well as the CDS of wild type CX26 were directionally subcloned into pEGFP-Nl respectively. After successfully constructed,these plasmids were transfected into Hela cells using lipofectamine 2000.The expression of mutants and wild type CX26 was analyzed by Western-blot and the localization were observed under fluorescence microscopy with immunofluorescence technics.The subcellular localization of the mutants which couldn’t form gap junctions were identified by analyzing the colocalization of these mutants with Endoplasmic Reticulum (ER) marker or Golgi marker. Biochemical coupling of the mutants which can form gap junctions was tested by calceinAM dye transfer experiments.Result The mutations p.R32H,p.E47K,p.V84L,p.V95M,p.R143W, p.R165W,p.S199F and c.35delG,were introduced by overlap PCR,and the mutations p.S19T,p.L214P,c.235delC,c.465T→A,c.572delT and c.631-632delGT were introduced by long-primer PCR.The expression of all mutant proteins except c.35delG was visible as the results of western blot showed.Mutants p.R32H,p.E47K,p.V84L,p.V95M,p.R143W, p.R165W,p.S199F,p.S19T and p.L214P encoded full-length products while the molecular weights of the mutant proteins c.235delC,c.465T→A,c.572delT and c.631-632delGT were lower than that of wild type CX26. The mutant proteins p.S19T, p.E47K, p.V84L, p.V95M, p.R165W meanly located on cell membrane and could form gap junction plaques.The mutant proteins p.R32H,p.R143W, p.S199F, p.L214P,c.35delG,c.235delC,c.465 T→A, c.572delT,c.631-632delGT displayed cytoplasmic accumulation and couldn’t be transported to plasma membrane.Further study showed that,these mutant proteins were colocalized with ER marker but not Golgi maker,indicating that they were accumulated in ER.The dye transfer rate of p.V84L showed no significant difference with wild type CX26,but p.S19T,p.E47K,p.V95M, p.R165W couldn’t mediate dye transfer.Conclusion Mutant proteins p.R32H,p.R143W,p.S199F,p.L214P, c.235delC,p.Y155X,c.572delT and c.631-632delGT could not form gap junction and accumulated in ER after synthesis probably demonstrating that these 8 mutant proteins got defect in ER-to-plasma membrane trafficking.4 truncated proteins c.235delC,c.465T→A,c.572delT and c.631-632delGT, also accumulated in ER and couldn’t be transport to plasma membrane,indicating that the C-terminal of CX26 contains sequence important for trafficking.p.V84L can form functional gap junction with biochemical coupling while p.S19T, p.E47K, p.V95M, p.R165W couldn’t,although they could form gap junction plaques.The mutant c.35delG couldn’t express in HeLa cells.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 mutant protein.更多还原