Role of a Disulfide-Bonded Peptide Loop within Human Complement C9 in the Species-Selectivity of Complement Inhibitor CD59

Abstract

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.

Identity of the Residues Responsible for the Species-restricted Complement Inhibitory Function of Human CD59

“…Whereas definitive identification of the residues forming the active site of CD59 awaits a direct demonstration of which side chains of the protein actually contact the C8 and C9 components of MAC, it is of interest to note the following. (i) In the case of both human C8␣ and C9 polypeptides, the peptide segment within each protein that has been directly shown to provide the specific binding site for attachment of human CD59 to these complement components represents a distinct region within each polypeptide that is most notable for marked divergence in aligned sequence when human C8␣ and C9 are each compared with its corresponding polypeptide of rabbit and other species (7)(8)(9)26). This suggests that the motif within each of these complement proteins to which CD59 attaches has itself undergone considerable evolutionary drift relative to other portions of the polypeptide, implying a similar evolutionary drift of the complementary binding site in CD59 as would be required to maintain interaction between the proteins.…”

“…This implies that the CD59 homologue expressed on the surface of rabbit cells is also selective for homologous complement and cannot effectively interact with the human C8 and C9 components to inhibit MAC. We have previously taken advantage of the species selectivity inherent to the interaction of CD59 with C8 and C9 to map the peptide-binding sites within the human C8 and C9 proteins that are recognized by human CD59 (7,8,26). In those studies, the capacity of human CD59 to inhibit MAC assembly was measured using recombinant human/rabbit chimeras of C8 and C9 in order to identify the residues within the C8␣ and C9 polypeptides that are specifically required to confer recognition of these complement components by human CD59.…”

Application of a novel inhibitor of human CD59 for the enhancement of complement-dependent cytolysis on cancer cells

“…17,18,[25][26][27][28][29] Recently, we developed a novel, potent, non-toxic and specific antihCD59 inhibitor: the domain 4 of intermedilysin (ILY). This inhibitor is defined as rILYd4.…”

The good and evil of complement activation in HIV-1 infection

“…142,143 (ii) Peptides derived from C8 and C9: extensive studies of hCD59 structure, as well as of its interaction with C8 and C9, revealed the region of hCD59 critical for inhibiting MAC formation through preventing binding of C8 and C9. [144][145][146][147][148] Small peptides derived from C8 and C9 that bind to this region are effective against hCD59 function at concentrations greater than 300 mmol/l of IC 50 , limiting their therapeutic usefulness. 147,148 Recently, we reported the development of a recombinant form of the fourth domain of the bacterial toxin intermedilysin (rILYd4), a 114 amino-acid protein that inhibits human CD59 function with high affinity and specificity.…”