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.
Highlights
Protection against the pore-forming activity of the The cytolytic activity of human serum resides in a complex human C5b-9 proteins was conferreodn a nonprimate composed of complement proteins C5b, C6, C7, C8, and C9 cell by transfection with cDNA encoding the human [1, 2]
After N-glycanase digestion, recombinantand andthe leukocyte antigen CD59, a glycoprotein with an erythrocyte CD59 comigrated with apparentmolecu- apparent molecular mass of 18-21 kDa [7,8,9,10,11,12]
The function of the recombinant protein was evaluated by changes in the sensitivity of the CD59 transfectants to the pore-forming proteins exhibit quitesimilar properties, including the following. 1)Both HRF and CD59 are tethered to the cell surface by a glycolipid anchor, and these proteins are deleted from activity of human C5b-9
Summary
Experiments confirmed that this concentration of antibody was saturating (data not shown). Data for CD59 transfectants was expressed as to incrementally amplify the DNA flanking the dihydrofolate increase in surface antigen relative to nontransfected CHO cell controls. After incubation for 1 h at 37 "C, 25 pl of each cell suspension was added to tubes containing 25 p1 of monoclonal antibody 1F1 (final concentration, pg/ml in HBSS). Monoclonal antibody against this antigen, cell-surface CD59 was increased from 0 (in nontransfected CHO controls)to. Recombinant CD59 exanti-mouse IgG was added (final concentration, 67 pg/ml), and cells pressed on the surface of these cells was susceptible to removal were incubated for an additional 15 min a t 23 "C. Western Blotting-Purified human erythrocyte CD59 (1 pg) and this antigen from CD59-transfected CHO cells were denatured (3 by phosphatidylinositol-specific phospholipase C digestion, consistent with its attachment to themembrane via a glycolipid anchor (Fig. 2).
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