Identity of a peptide domain of human C9 that is bound by the cell-surface complement inhibitor, CD59.
The CD59 antigen is a plasma membrane glycoprotein that serves as an inhibitor of the C5b-9 complex of complement. This inhibitory activity appears related to the capacity of CD59 to bind with high affinity to sites that are nascently exposed in the alpha-chain subunit of human C8, as well as within the C9b domain (amino acid residues 245-538) of human C9, during assembly of the C5b-9 complex on the target membrane (Ninomiya, H., and Sims, P. J. (1992) J. Biol. Chem. 267, 13675-13680). The CD59 binding site in C9 was first investigated by N-terminal sequencing of CD59-binding peptides generated by limited digest of the isolated C9b domain. These experiments revealed a 17-kDa fragment (starting at C9 residue Thr-320) that retained affinity for CD59, suggesting the possibility for localizing the CD59 binding site by mapping with small C9-derived peptides. Peptides spanning the entire C9b sequence were expressed in Escherichia coli and then probed with CD59. CD59 bound specifically to all peptides starting N-terminal to C9 residue 359 with C termini extending beyond residue 411. Little to no CD59 binding was observed for various C9-derived peptides that started C-terminal to residue 359 or that were truncated N-terminal to residue 411. Affinity-purified antibody against C9 residues 320-411 inhibited CD59 binding to C9 by > 50% and completely inhibited its binding to the isolated C9b domain. Little to no specific binding of CD59 was detected for peptides restricted to the putative hinge domain within C9b (residues 245-271). These results indicate that a CD59 binding site is located between residues 320 and 411 of the C9 polypeptide and suggest that the affinity of this site is principally determined by residues 359-411.
Chang
C P
CP
Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee 53233.
Hüsler
T
T
Zhao
J
J
Wiedmer
T
T
Sims
P J
PJ
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000378:metabolism*; D001665:Binding Sites; D018957:CD59 Antigens; D003186:Complement C9 / Q000378:metabolism*; D015938:Complement Membrane Attack Complex / Q000037:antagonists & inhibitors; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*
D015703:Antigens, CD; D018957:CD59 Antigens; D003186:Complement C9; D015938:Complement Membrane Attack Complex; D008562:Membrane Glycoproteins; D010446:Peptide Fragments