Role of a disulfide-bonded peptide loop within human complement C9 in the species-selectivity of complement inhibitor CD59.
CD59 antigen is a membrane glycoprotein that inhibits the activity of the C9 component of the C5b-9 membrane attack complex (MAC), thereby protecting human cells from lysis by human complement. The complement-inhibitory activity of CD59 is species-selective, and is most effective toward C9 derived from human or other primate plasma. The species-selective activity of CD59 was recently used to map the segment of human C9 that is recognized by this MAC inhibitor, using recombinant rabbit/human C9 chimeras that retain lytic function within the MAC [Husler, T., Lockert, D. H., Kaufman, K. M., Sodetz, J. M., & Sims, P. J. (1995) J. Biol. Chem. 270,3483-3486]. These experiments suggested that the CD59 recognition domain was contained between residues 334 and 415 in human C9. By analyzing the species-selective lytic activity of recombinant C9 with chimeric substitutions internal to this segment, we now demonstrate that the site in human C9 uniquely recognized by CD59 is centered on those residues contained between C9 Cys359/Cys384, with an additional contribution by residues C-terminal to this segment. Consistent with its role as a CD59 recognition domain, CD59 specifically bound a human C9-derived peptide corresponding to residues 359-384, and antibody (Fab) raised against this C9-derived peptide inhibited the lytic activity of human MAC. Mutant human C9 in which Ala was substituted for Cys359/384 was found to express normal lytic activity and to be fully inhibited by CD59. This suggests that the intrachain Cys359/Cys384 disulfide bond within C9 is not required to maintain the conformation of this segment of C9 for interaction with CD59.
Husler
T
T
Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee, 53233, USA.
Lockert
D H
DH
Sims
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D018957:CD59 Antigens / Q000378:metabolism* / Q000494:pharmacology; D003186:Complement C9 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D003169:Complement Inactivator Proteins / Q000378:metabolism* / Q000494:pharmacology; D015938:Complement Membrane Attack Complex; D004220:Disulfides; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013045:Species Specificity; D013329:Structure-Activity Relationship; D014162:Transfection
D018957:CD59 Antigens; D003186:Complement C9; D003169:Complement Inactivator Proteins; D015938:Complement Membrane Attack Complex; D004220:Disulfides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins