EXPRESSION OF RECOMBINANT CD59 WITH AN N-TERMINAL PEPTIDE EPITOPE FACILITATES ANALYSIS OF RESIDUES CONTRIBUTING TO ITS COMPLEMENT-INHIBITORY FUNCTION

CD59 is a plasma membrane-anchored glycoprotein that serves to protect human cells from lysis by the C5b-9 complex of complement. The immuno dominant epitopes of CD59 are known to be sensitive to disruption of n ative tertiary structure, complicating immunological measurement of ex pressed mutant constructs for structure-function analysis. In order to quantify cell-surface expression of wild-type and mutant forms of thi s complement inhibitor, independent of CD59 antigen, an 11-residue pep tide (TAG) recognized by monoclonal antibody (mAb) 9E10 was inserted b efore the N-terminal codon (L1) of mature CD59, in a pcDNA3 expression plasmid. SV-T2 cells were transfected with this plasmid, yielding cel l lines expressing 0 to > 10(5) CD59/cell. The TAG-CD59 fusion protein was confirmed to be GPI-anchored, N-glycosylated and showed identical complement-inhibitory function to wild-type CD59, lacking the TAG pep tide sequence. Using this construct, the contribution of each of four surface-localized aromatic residues (4Y, 47F, 61Y, and 62Y) to CD59's complement-inhibitory function was examined. These assays revealed nor mal surface expression with complete loss of complement-inhibitory fun ction in the 4Y --> S, 47F --> G and 61Y --> S mutants. By contrast, 6 2Y --> S mutants retained approximately 40% of function of wild-type C D59. These studies confirmed the utility of the TAG-CD59 construct for quantifying CD59 surface expression and activity, and implicate surfa ce aromatic residues 4Y, 47F, 61Y and 62Y as essential to maintenance of CD59's normal complement-regulatory function. (C) 1997 Elsevier Sci ence Ltd.