Activity after Site-Directed Mutagenesis of CD59 on Complement-Mediated Cytolysis

作     者：Xinhong Zhu Meihua Gao Shurong Ren Qiubo Wang Cunzhi Lin 

作者机构：Department of Immunology Medical College of Qingdao University Qingdao Shandong China Clinical Immune Institution Qingdao Chest Hospital 896 Chongqing Road Qingdao 266043 China 

出 版 物：《Cellular & Molecular Immunology》 (中国免疫学杂志（英文版）)

年 卷 期：2008年第5卷第2期

页      面：141-146页

核心收录：

学科分类：1001[医学-基础医学(可授医学、理学学位)] 100101[医学-人体解剖与组织胚胎学] 10[医学] 

基　　金：National Natural Science Foundation of China (No.30671936) 

主　　题：CD59 complement active site site-directed mutagenesis overlap.extension PCR 

摘      要：CD59 may inhibit the cytolytic activity of complement by binding to C8/C9 and protect host cell membranes against homologous membrane attack complex （MAC）. However, CD59 is widely overexpressed on tumor cells, which has been implicated in tumorigenesis. The active site of CD59 relative to MAC is still confused. As reported the MAC binding site is located in the vicinity of a hydrophobic groove on the membrane distal face of the protein centered around residue W40. Here two site-directed mutagenesis were performed by overlapping extension PCR to delete residue W40 site （Mutant 1, M1） or to change C39W40K41 to W39W40W41 （Mutant 2, M2）. Then we constructed mutant CD59 eukaryotic expression system and investigated their biological function on CI-IO cells compared with wild-type CD59. Stable populations of CHO cells expressing recombinant proteins were screened by immunotechnique. After 30 passages culturing, proteins could be tested. Dye release assays suggest that M1CD59 loses the activity against complement, while M2CD59 increases the anti-complement activity slightly. Results indicate that W40 of human CD59 is important to its activity, and prohibition of this site may be a potential way to increase complement activity and to treat tumors. Cellular ＆ Molecular Immunology.