Hayashi Ikuo, Takatori Sho, Urano Yasuomi, Iwanari Hiroko, Isoo Noriko, Osawa Satoko, Fukuda Maiko A, Kodama Tatsuhiko, Hamakubo Takao, Li Tong, Wong Philip C, Tomita Taisuke, Iwatsubo Takeshi
Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
J Biol Chem. 2009 Oct 9;284(41):27838-27847. doi: 10.1074/jbc.M109.055061. Epub 2009 Aug 14.
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.
γ-分泌酶是一种膜蛋白复合物，可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前体蛋白。尼卡斯特林（NCT）是一种具有大细胞外结构域的单次跨膜糖蛋白，是γ-分泌酶复合物的重要组成部分。在此我们报告，作为胞内抗体的抗NCT单链可变片段（scFv）的过表达抑制了γ-分泌酶活性。生化分析表明，scFv破坏了内源性NCT的正确折叠和适当的糖基成熟，这分别是γ-分泌酶复合物稳定性和内在蛋白水解活性所必需的，这暗示了NCT在γ-分泌酶复合物中的双重作用。我们的结果还突出了钙连蛋白循环在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作为合理设计的分子靶向工具，用于发现膜蛋白的新作用。
