Single Chain Variable Fragment against Nicastrin Inhibits the γ-Secretase Activity
Single Chain Variable Fragment against Nicastrin Inhibits the γ-Secretase Activity
复制标题
DOI：
10.1074/jbc.m109.055061
复制
发表时间：
2009-10-09
影响因子：
4.8
通讯作者：
Iwatsubo, Takeshi
中科院分区：
文献类型：
--
作者：
Hayashi, Ikuo;Takatori, Sho;Iwatsubo, Takeshi
γ-分泌酶是一种膜蛋白复合物，催化多种底物的膜内蛋白水解，包括阿尔茨海默病的淀粉样β前体蛋白。Nicastrin（NCT）是一种具有较大胞外结构域的单通道膜糖蛋白，是γ-分泌酶复合物的重要组成部分。在这里，我们报告了针对NCT的单链可变片段（scFv）作为胞内抗体的过表达抑制了γ-分泌酶活性。生物化学分析表明，scFv破坏了内源性NCT的适当折叠和适当的糖基成熟，这分别是γ-分泌酶复合物的稳定性和内在的蛋白水解活性所需的，暗示了NCT在γ-分泌酶复合物中的双重作用。我们的研究结果还强调了钙连接蛋白循环在γ-分泌酶复合物功能成熟中的重要性。工程化的胞内抗体可以作为合理设计的分子靶向工具，用于发现膜蛋白的新作用。
gamma-Secretase is a membrane protein complex that catalyzes intramembrane proteolysis of a variety of substrates including the amyloid beta precursor protein of Alzheimer disease. Nicastrin (NCT), a single-pass membrane glycoprotein that harbors a large extracellular domain, is an essential component of the gamma-secretase complex. Here we report that overexpression of a single chain variable fragment (scFv) against NCT as an intrabody suppressed the gamma-secretase activity. Biochemical analyses revealed that the scFv disrupted the proper folding and the appropriate glycosyl maturation of the endogenous NCT, which are required for the stability of the gamma-secretase complex and the intrinsic proteolytic activity, respectively, implicating the dual role of NCT in the gamma-secretase complex. Our results also highlight the importance of the calnexin cycle in the functional maturation of the gamma-secretase complex. The engineered intrabodies may serve as rationally designed, molecular targeting tools for the discovery of novel actions of the membrane proteins.