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.
Xinhong ZhuMeihua GaoShurong RenQiubo WangCunzhi Lin
CD59,belonging to membrane complement regulatory proteins(mCRPs) ,inhibits the cytolytic activity of complement and is over-expressed in solid cancers,including ovary cancer. The aim of the present study was to construct recombinant retrovirus encoding shRNA targeted human CD59 and infect A2780 cells in order to investigate the relationship between decreased CD59 expression and tumorigenesis of ovary cancer. siCD59 and siCD59-C were successfully constructed and identified by PCR,restriction endonuclease analyses and DNA sequencing,respectively. The siCD59 was able to efficiently infect A2780 cells,which was confirmed by Western blotting. When incubated with fresh normal human serum(8%,v/v) for 1 h at 37°C,the cell viability was decreased and cell damage was increased in siCD59 infected A2780 cells compared to siCD59-C infected cells. This led to the activation of caspase-3. The apoptosis in siCD59 infected cells was shown with hypercondensed nuclei using Hoechst staining. Meanwhile,the weight of ovary tumor graft in nude mice was significantly decreased in siCD59 group compared to that of siCD59-C group. And the expression of CD59 protein in tumor tissue in siCD59 group was significantly decreased. These results suggested that CD59 silencing in ovary cancer cells via retrovirusmediated RNAi can enhance complement-mediated cell damage,inhibiting growth of ovary cancer. CD59 might be a potential target for gene therapy in ovary cancer.
