Mapping the Regions of the Complement Inhibitor CD59 Responsible for Its Species Selective Activity

Abstract

CD59 is a widely distributed membrane-bound glycoprotein that inhibits the formation of the cytolytic membrane attack complex (MAC) of complement on host cells. CD59 from different species varies in its capacity to inhibit heterologous complement, and this species selective function of CD59 contributes to the phenomenon of homologous restriction. Here, we demonstrate that human CD59 is not an effective inhibitor of rat complement, although rat CD59 inhibits rat and human complement equally well. By constructing human-rat CD59 chimeric proteins, we have mapped the residues important in conferring human CD59 species selectivity to two regions; 40-47 and 47-66 in the primary structure. Analysis of a model of the molecular surface of human CD59 revealed that residues 40-66 mapped to a region in the three-dimensional structure that surrounds residues previously identified as important for CD59 function.

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

“…residues 40 -66) might be responsible for regulating interaction of CD59 with the C8 and C9 components of MAC. Data implicating various residues of the CD59 polypeptide as responsible for its MAC inhibitory function have also been reported from studies of an apparent MAC inhibitory activity observed for CD59-derived peptides and by analysis of site-specific mutations in CD59 resulting in loss of its MAC inhibitory function (20,21,29,(33)(34)(35). Unfortunately, these data do not provide a consistent mapping of the potential active-site residues of the protein, and the actual amino acid side chains of the CD59 polypeptide that participate in its attachment to the C8 and C9 components of MAC remain to be identified (see below).…”

“…These data imply that the active-site residues in CD59 are contained in the central portion of the polypeptide (between residues 17 and 56) without direct contribution of the flanking sequences. Additional insight into potential active-site residues was provided by a subsequent study of the species selectivity of human/rat CD59 chimeras (21). Whereas human CD59 is restricted in its activity to inhibit human MAC and does not inhibit rat MAC, rat CD59 shows activity toward both rat and human MACs.…”

“…The complement inhibitory function of CD59 exhibits marked selectivity for MAC assembled from C8 and C9 of human or primate origin, and CD59 shows little inhibitory function toward these same complement components of rabbit and several other non-primate species (8,10,13,(21)(22)(23). Conversely, whereas rabbit erythrocytes are well protected from lysis by rabbit C5b-9, they are exquisitely sensitive to the lytic action of C5b-9 assembled from human C8 or C9 (24,25).…”

“…Our choice of constructs reflected (i) identity of amino acid residues exposed on the surface of human CD59 that were not conserved in the aligned polypeptide sequence of rabbit CD59 (selection was based on the reported solution structure of the glycosylated protein, utilizing surface residues considered not to be occluded by the N-linked carbohydrate (1, 14, 15)); (ii) an attempt to group these various nonconserved amino acid side chains into contiguously clustered spatial arrays; and (iii) a consideration of prior data relating to potential identity of the active-site residues in human CD59 as deduced from peptide studies, site-directed mutagenesis in the protein, or analysis of other CD59 chimeras (see "Discussion") (20,21,29,(33)(34)(35). Based upon these considerations, we chose to analyze six human/rabbit CD59 chimeras (and the six complementary rabbit/human CD59 chimeras) constructed so as to replace the surface-exposed side chains contributed by (i) residues 8, 10, 12, and 14 (chimeras Ch1 and Ch1R; Fig.…”

Complement receptor 2–mediated targeting of complement inhibitors to sites of complement activation

“…Human CR2 binds human and mouse C3 ligands with similar affinities (54), and the biodistribution and microscopy studies establish that a CR2 fusion protein retains targeting function in vivo. However, human CD59 and soluble human DAF are poor inhibitors of rodent complement relative to their activity against human complement (with the exception of human DAF activity against the rat alternative pathway of activation) (43,55,56), and so the complement-inhibitory…”