An investigation of the interaction between human complement factor H and C3b.

The complement system in human blood is a major effector system in humoraVinnate immunity (1). Factor H is a 155kD glycoprotein which is involved in the regulation of the complement alternative pathway (2). Factor H regulates C3 turnover by 2 activities: decay acceleration activity describes the ability of factor H to bind to C3b thus displacing Bb from the active C3bBb enzyme (C3 convertase) of the alternative pathway (3). Once factor H is bound to C3b, factor I, a serine protease, cleaves the 111 kD alpha’ chain of C3b to 68 and 43 kD fragments, thus abrogating the action of C3b (4). The latter activity of factor H is termed “Factor I-cofactor activity”. Interaction of factor H with C3b is mainly ionic, and of low affinity, involving several (probably 3-4) weak interaction sites (5). Structurally, factor H is composed of 20 complement control protein modules (CCPs), each about 60 aminoacids long and possessing 4 conserved cysteine residues. 2D proton NMR has revealed that each CCP has a hydrophobic core, with 3 protruding loops of variable sequence, which are likely to form the binding sites for ligands of proteins which contain CCPs(6). Other complement control proteins, DAF, MCP, C4bp and CR1, which have similar activities to factor H, are also composed of CCPs. In CR1, up to 4 contiguous CCPs form a C3b binding site (7). In factor H, previous studies have shown that C3b-binding and cofactor activities are localised to a 38kD fragment containing CCPs 1-5 and part of CCP 6 (8). In this study a further investigation of the regions within factor H responsible for C3b binding and factor Icofactor activity was carried out. Assays for factor I-cofactor activity were done as previously described (4). Factor H, factor I and C3b, isolated and prepared from human plasma were used (9, 10). Chemical modifications of factor H we carried out to identify residue types important for factor I cofactor activity (11). Histidine and carboxyl modifications (by diethyl pyrocarbonate and EDAC, respectively) were found to inhibit completely the cofactor activity. Lysine modifications (by cyanate, acetic anhydride or a succinimide) produced a substantial inhibition whilst arginine modifications (phenylglyoxal) resulted in only minor reduction in the factor I cofactor activity of factor H. A number of monoclonal antibodies (mabs) against factor H were assessed for their effect on cofactor activity. Several inhibitory mabs, e.g MAH1, MAH3, 82216, MH9 and MH5 could totally inhibit the cofactor activity, suggesting that they bind within the C3b binding site. Mabs OX23 and OX24 have very minor effects on cofactor activity, suggesting that they do not bind within the C3b binding site. Proteolytic digestions of factor H were carried out to localise the epitopes of these mabs by SDS-PAGE and Western blotting. OX23 binds to a 12kD fragment produced by the action of pepsin on factor H. This corresponds to the first 1.6 CCPs of factor H. As this MAb has only a small effect on factor H activity, CCP 1 and part of CCP 2 may not be required for cofactor activity and C3b binding. OX24 appears to bind to the junction of CCPs 5 and 6 , again suggesting that this region may not be required for the cofactor activity. The inhibitory antibodies MAHl and MAH3 bind to a 32kD pepsin fragment, encompassing half of CCP 3, plus CCPs 4-7, and also, consistent with previous findings(8) to the 38kD tryptic fragment composed of CCPs