title: Amplified gene expression in CD59-transfected Chinese hamster ovary cells confers protection against the membrane attack complex of human complement.
abstract: Protection against the pore-forming activity of the human C5b-9 proteins was conferred on a nonprimate cell by transfection with cDNA encoding the human complement regulatory protein CD59. CD59 was stably expressed in Chinese hamster ovary cells using the pFRSV mammalian expression vector. After cloning and selection, the transfected cells were maintained in media containing various concentrations of methotrexate, which induced surface expression of up to 4.2 x 10(6) molecules of CD59/cell. Phosphatidylinositol-specific phospholipase C removed greater than 95% of surface-expressed CD59 antigen, confirming that recombinant CD59 was tethered to the Chinese hamster ovary plasma membrane by a lipid anchor. The recombinant protein exhibited an apparent molecular mass of 21-24 kDa (versus 18-21 kDa for human erythrocyte CD59). After N-glycanase digestion, recombinant and erythrocyte CD59 comigrated with apparent molecular masses of 12-14 kDa, suggesting altered structure of asparagine-linked carbohydrate in recombinant versus erythrocyte CD59. The function of the recombinant protein was evaluated by changes in the sensitivity of the CD59 transfectants to the pore-forming activity of human C5b-9. Induction of cell-surface expression of CD59 antigen inhibited C5b-9 pore formation in a dose-dependent fashion. CD59 transfectants expressing greater than or equal to 1.2 x 10(6) molecules of CD59/cell were completely resistant to human serum complement. By contrast, CD59 transfectants remained sensitive to the pore-forming activity of guinea pig C8 and C9 (bound to human C5b67). Functionally blocking antibody against erythrocyte CD59 abolished the human complement resistance observed for the CD59-transfected Chinese hamster ovary cells. These results confirm that the C5b-9 inhibitory function of the human erythrocyte membrane is provided by CD59 and suggest that the gene for this protein can be expressed in xenotypic cells to confer protection against human serum complement.
authors.affiliation: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
authors.forename: J
authors.initials: J
authors.lastname: Zhao
authors.forename: S A
authors.initials: SA
authors.lastname: Rollins
authors.forename: S E
authors.initials: SE
authors.lastname: Maher
authors.forename: A L
authors.initials: AL
authors.lastname: Bothwell
authors.forename: P J
authors.initials: PJ
authors.lastname: Sims
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000502:physiology*; D000943:Antigens, Differentiation / Q000235:genetics / Q000502:physiology*; D018957:CD59 Antigens; D002460:Cell Line; D015938:Complement Membrane Attack Complex / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D010053:Ovary; D011994:Recombinant Proteins / Q000276:immunology; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000943:Antigens, Differentiation; D018957:CD59 Antigens; D015938:Complement Membrane Attack Complex; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D010738:Type C Phospholipases