Activity after site-directed mutagenesis of CD59 on complement-mediated cytolysis.
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 CHO 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.
Zhu
Xinhong
X
Department of Immunology, Medical College of Qingdao University, Qingdao, Shandong, China.
Gao
Meihua
M
Ren
Shurong
S
Wang
Qiubo
Q
Lin
Cunzhi
C
D000818:Animals; D018957:CD59 Antigens / Q000235:genetics* / Q000378:metabolism*; D016466:CHO Cells; D003165:Complement System Proteins / Q000276:immunology*; D006224:Cricetinae; D003412:Cricetulus; D003602:Cytotoxicity, Immunologic / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D017403:In Situ Hybridization; D016297:Mutagenesis, Site-Directed; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection
D018957:CD59 Antigens; D012333:RNA, Messenger; D003165:Complement System Proteins