N端八肽重复区数目对人重组PrP与金属离子结合后的蛋白酶抗性及与tau蛋白结合能力的影响  

Influence of the Numbers of Octapeptide Repeats within N-terminus of Recombinant Human PrP Proteins on the Protease Resistance after Interacting with Metal Ions and the Binding Ability with Tau Protein

作　　者：高建梅[1] 万言珍[1] 韩俊[1] 王小凡[1] 陈岚[1] 聂凯[1] 周伟[1] 董小平[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052

出　　处：《病毒学报》2005年第5期376-383,共8页Chinese Journal of Virology

基　　金：国家自然科学基金委项目(30070038);国家自然科学基金委重点项目(30130070);国家863计划项目(2001AA215391);欧盟项目(QLRT200001441);国家科技攻关计划项目(2003BA712A04-02)

摘　　要：人类朊病毒病中约10%～15%具有家族遗传特性,其中插入或缺失突变多发生于PrP蛋白N末端的八肽重复区域.运用PCR成功地构建并表达了含不同八肽重复数目的PrP蛋白,并观察八肽重复数目的增加对PrP与Cu2+等二价离子以及tau蛋白的相互作用的影响.实验结果显示:各种纯化后的PrP蛋白对常规浓度PK消化是敏感的,而与Cu2+共同孵育可使PrP蛋白的PK抗性增强;八肽重复序列的数目及Cu2+的浓度决定了PK抗性的出现和强弱.另外,Mn2+可诱导产生与Cu2+相似的结果,但其诱导效应似乎低于Cu2+,而Zn2+对PrP蛋白的PK抗性无影响.GST-tau包被的ELISA检测证实,重组的PrP呈现出明显的tau蛋白结合能力,并且与八肽重复序列的数量相关,重复序列数量越多,结合能力越强.这些结果提示,Cu2+诱导产生的PrP蛋白PK抗性是通过八肽重复序列区域产生的,并且直接与重复序列的数量相关.另外,PrP蛋白八肽重复序列的存在和数量直接影响PrP与tau蛋白的结合效应.除了八肽区域外,PrP蛋白其它区域似乎也具有一定的tau蛋白结合能力.About 10 % - 15 % human prion diseases are associated with inheritance, including familial CJD, GSS and FFI with insertion or deletion mutations within octapeptide repeats region of PrP gene （ PRNP ）. To observe whether the number of octapeptide repeats in rHuPrPs had effects on the interactions of PrP with bivalent metal such as copper, magnesium, or GST-tau, we constructed PRNP mutants with different number of octapeptide repeats with PCR technique and various PrP proteins were expressed successfully in E. coli . The results demonstrated that all purified rHuPrPs with different octapeptide repeats were sensitive to PK digestion. However, after incubated with copper, the rHuPrPs acquired PK resistance. The number of octapeptide and the concentration of Cu^2＋ determined the acquisition and the intensity of PK resistance. In addition, PrP（PG9） also showed the similar PK resistance when incubated with various concentrations of Mn^2＋ but not Zn^2＋ as that induced by Cu^2＋ , but the intensity of induction by Mn^2＋ was slightly lower than that by Cu^2＋ . The tau-coated ELISA demonstrated that the recombinant PrP appeared significant ability of binding with GST-tau in a dosedependent manner, moreover, it was associated with the number of octapeptide, the more the octapeptides the much stronger binding ability with GST-tau. These results suggested that the PK resistance of rHuPrPs induced by Cu^2＋ was mediated through octapeptide repeat domains and the intensity of the effect was related with the number of the repeats. Moreover, the efficiency of molecular interaction between PrP and tau relied on the existence and the number of octapeptide repeats. Besides the octapeptide repeat domain, other regions within PrP seems to be also responsible for binding with GST-tau protein.

关 键 词：重组人PrP N端八肽重复区突变 生化特性 

分 类 号：R373[医药卫生—病原生物学]