A Study Of Relationship Between CD59 Mutagenisis And Protein Glycation

Objective To construct two mutant CD59 eukaryotic expression system and investigate whether glycation could inhibit the activity of mutant CD59.Methods Site-directed mutagenesis to replace residue 37or 38 with histidine (H), was performed by recombinant PCR .The mutagenic primers were designed according to two mutant CD59s. Successful mutagenesis was confirmed by sequence analysis. mutant CD59 DNAs were cloned into the mammalian expression vector pALTER-MAX and transfected into CHO together with the selection marker pcDNA3, which confers resistance to G418. Expression of mutant CD59s in the G418-resistant clones were tested by Western blot, immunohistochernistry and FACS,a functional dye release assay is used to measure protection role of CD59s against human complement.Results Recombinant plasmids of pALTER-MAX-HMCD59 have been sucssesfully constructed according to sequence and enzyme digestion analysis. The mutant gene is about 500 bp. Stable transfectants were selected by the addition of G418. stable populations of CHO cells of expressing relatively high levels of recombinant protein were sorted by immunolabled technique . In Western blot assay, the recombinant proteins expressed on CHO is about 20 KD. dye release assays confirm both mutants still preserved CD59 activity of anticomplement .glycation of CD59 in CHO increases their sensitivity to MAC-mediated lysis. The mutant clones were propagated in a great deal for more research of them.Conclusion Recombinant plasmids of pALTER-MAX-HMCD59 have been sucssesfully constructed , Stable transfectants were selected and stable populations of CHO cells of expressing relatively high levels of recombinant protein were sorted . Residues by Site-directed mutagenesis to replace residue 37or 38 with histidine (H) still form a preferential glycation motif in human CD59. mutant CD59s can be glycated in vitro and glycated , mutant CD59 loses its most MAC-inhibitory function.The presence of this glycation motif in human mutant CD59, may help explain the distinct propensity of humans to develop vascular proliferative complications of diabetes.