title: Expression of recombinant CD59 with an N-terminal peptide epitope facilitates analysis of residues contributing to its complement-inhibitory function.
abstract: CD59 is a plasma membrane-anchored glycoprotein that serves to protect human cells from lysis by the C5b-9 complex of complement. The immunodominant epitopes of CD59 are known to be sensitive to disruption of native tertiary structure, complicating immunological measurement of expressed mutant constructs for structure function analysis. In order to quantify cell-surface expression of wild-type and mutant forms of this complement inhibitor, independent of CD59 antigen, an 11-residue peptide (TAG) recognized by monoclonal antibody (mAb) 9E10 was inserted before the N-terminal codon (L1) of mature CD59, in a pcDNA3 expression plasmid. SV-T2 cells were transfected with this plasmid, yielding cell 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 peptide 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 normal surface expression with complete loss of complement-inhibitory function in the 4Y --> S, 47F --> G and 61Y --> S mutants. By contrast, 62Y --> S mutants retained approximately 40% of function of wild-type CD59. These studies confirmed the utility of the TAG-CD59 construct for quantifying CD59 surface expression and activity, and implicate surface aromatic residues 4Y, 47F, 61Y and 62Y as essential to maintenance of CD59's normal complement-regulatory function.
authors.affiliation: The Blood Research Institute of The Blood Center of Southeastern Wisconsin, Milwaukee 53201, USA.
authors.forename: Q
authors.initials: Q
authors.lastname: Zhou
authors.forename: J
authors.initials: J
authors.lastname: Zhao
authors.forename: T
authors.initials: T
authors.lastname: Hüsler
authors.forename: P J
authors.initials: PJ
authors.lastname: Sims
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D018957:CD59 Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D003169:Complement Inactivator Proteins / Q000032:analysis*; D000939:Epitopes / Q000096:biosynthesis* / Q000276:immunology; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000276:immunology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D000911:Antibodies, Monoclonal; D018957:CD59 Antigens; D003169:Complement Inactivator Proteins; D000939:Epitopes; D010446:Peptide Fragments; D011994:Recombinant Proteins