The AP C3 convertase, C3bBb(Mg 2؉ ), is subject to irreversible dissociation (decay acceleration) by three proteins: DAF, CR1, and factor H. We have begun to map the factor B (fB) sites critical to these interactions. We generated a panel of fB mutations, focusing on the type A domain because it carries divalent cation and C3b-binding elements. C3bBb complexes were assembled with the mutants and subjected to decay acceleration. Two critical fB sites were identified with a structural model. 1) Several mutations centered at adjacent alpha helices 4 and 5 (Gln-335, Tyr-338, Ser-339, Asp-382) caused substantial resistance to DAF and CR1-mediated decay acceleration but not factor H. 2) Several mutations centered at the ␣ 1 helix and adjoining loops (especially D254G) caused resistance to decay acceleration mediated by all three regulators and also increased C3b-binding affinity and C3bBb stability. In the simplest interpretation of these results, DAF and CR1 directly interact with C3bBb at ␣ 4/5; factor H likely interacts at some other location, possibly on the C3b subunit. Mutations at the C3b⅐Bb interface interfere with the normal dissociation of C3b from Bb, whether it is spontaneous or promoted by DAF, CR1, or factor H.

Decay-accelerating factor (DAF) is a complement regulator that dissociates autologous C3 convertases, which assemble on self cell surfaces. Its activity resides in the last three of its four complement control protein repeats (CCP2–4). Previous modeling on the nuclear magnetic resonance structure of CCP15–16 in the serum C3 convertase regulator factor H proposed a positively charged surface area on CCP2 extending into CCP3, and hydrophobic moieties between CCPs 2 and 3 as being primary convertase-interactive sites. To map the residues providing for the activity of DAF, we analyzed the functions of 31 primarily alanine substitution mutants based in part on this model. Replacing R69, R96, R100, and K127 in the positively charged CCP2–3 groove or hydrophobic F148 and L171 in CCP3 markedly impaired the function of DAF in both activation pathways. Significantly, mutations of K126 and F169 and of R206 and R212 in downstream CCP4 selectively reduced alternative pathway activity without affecting classical pathway activity. Rhesus macaque DAF has all the above human critical residues except for F169, which is an L, and its CCPs exhibited full activity against the human classical pathway C3 convertase. The recombinants whose function was preferentially impaired against the alternative pathway C3bBb compared with the classical pathway C4b2a were tested in classical pathway C5 convertase (C4b2a3b) assays. The effects on C4b2a and C4b2a3b were comparable, indicating that DAF functions similarly on the two enzymes. When CCP2–3 of DAF were oriented according to the crystal structure of CCP1–2 of membrane cofactor protein, the essential residues formed a contiguous region, suggesting a similar spatial relationship.

SUMMARYThe decay-accelerating factor (DAF) contains four complement control protein repeats (CCPs) with a single N-linked glycan positioned between CCPs 1 and 2. In previous studies we found that the classical pathway regulatory activity of DAF resides in CCPs 2 and 3 while its alternative pathway regulatory activity resides in CCPs 2, 3 and 4. Molecular modelling of the protein predicted that a positively charged surface area on CCPs 2 and 3 (including KKK 125±127 ) and nearby exposed hydrophobic residues (L 147 F 148 ) on CCP3 may function as ligand-binding sites. To assess the roles of the N-linked glycan and the above two sets of amino acids in the function of DAF, we mutated N 61 to Q, KKK 125±127 to TTT and L 147 F 148 to SS. Following expression of the mutated cDNAs in Chinese hamster ovary cells, the glycosylphosphatidylinositol (GPI)-anchored mutant proteins were af®nity puri®ed and their functions were assessed. In initial assays, the proteins were incorporated into sheep and rabbit erythrocytes and the effects of the mutations on regulation of classical and alternative C3 convertase activity were quanti®ed by measuring C3b deposition. Since DAF also functions on C5 convertases, comparative haemolytic assays of cells bearing each mutant protein were performed. Finally, to establish if spatial orientation between DAF and the convertases on the cell surface played any role in the observed effects,¯uid-phase C3a generation assays were performed. All three assays gave equivalent results and showed that the N-linked glycan of DAF is not involved in its regulatory function; that L 147 F 148 in a hydrophobic area of CCP3 is essential in both classical and alternative pathway C3 convertase regulation; and that KKK 125±127 in the positively charged pocket between CCPs 2 and 3 is necessary for the regulatory activity of DAF on the alternative pathway C3 convertase but plays a lesser role in its activity on the classical pathway enzyme.

Focused complement activation on foreign targets depends on regulatory proteins that decay the bimolecular C3 convertases. Although this process is central to complement control, how the convertases engage and disassemble is not established. The second and third complement control protein (CCP) modules of the cell surface regulator, decay-accelerating factor (DAF, CD55), comprise the simplest structure mediating this activity. Positioning the functional effects of 31 substitution mutants of DAF CCP2 to -4 on partial structures was previously reported. In light of the high resolution crystal structure of the DAF four-CCP functional region, we now reexamine the effects of these and 40 additional mutations. Moreover, we map six monoclonal antibody epitopes and overlap their effects with those of the amino acid substitutions. The data indicate that the interaction of DAF with the convertases is mediated predominantly by two patches ϳ13 Å apart, one centered around Arg 69 and Arg 96 on CCP2 and the other around Phe 148 and Leu 171 on CCP3. These patches on the same face of the adjacent modules bracket an intermodular linker of critical length (16 Å ). Although the key DAF residues in these patches are present or there are conservative substitutions in all other C3 convertase regulators that mediate decay acceleration and/or provide factor I-cofactor activity, the linker region is highly conserved only in the former. Intra-CCP regions also differ. Linker region comparisons suggest that the active CCPs of the decay accelerators are extended, whereas those of the cofactors are tilted. Intra-CCP comparisons suggest that the two classes of regulators bind different regions on their respective ligands.The C3 convertases of the classical and alternative pathways are the central enzymes of the complement cascade (1). These bimolecular complexes, C4b2a and C3bBb, produce anaphylatoxin C3a, locally amplify C3b deposition, and serve as sites for the assembly of the C5 convertases, C4b2a3b and C3bBb3b. These trimeric convertases, in turn, generate anaphylatoxin C5a and initiate the terminal pathway, leading to formation of lytic C5b-9 membrane attack complexes. The relative rates of assembly and disassembly of the C3 convertases lie at the heart of complement regulation, and their physiologic modulation ensures that complement acts in a proportionate and targeted fashion.To focus complement activation on foreign targets, prevent nearby activation in the fluid phase, and simultaneously protect self tissues from complement-mediated injury, the C3 convertases are controlled by both serum-and cell-associated regulatory proteins (2, 3). Because the convertases assemble on C4b and C3b fragments that condense indiscriminately with free hydroxyl and amino groups on foreign targets and these same acceptor groups are present on all biological membranes, their formation on self cells at the single enzyme level cannot be avoided. To prevent further propagation of the cascade, which would induce self cell injury, self cells possess decay-acc...

A model of the regulatory region of human decay accelerating factor (DAF) was built based on the known coordinates of a fragment of the structurally and functionally homologous serum protein, factor H. According to this model, the four short consensus repeats (SCRs) in DAF are arranged in a helical fashion. A positively charged surface area on SCRs 2 and 3, two of the three repeating units essential for function, is postulated to be the primary recognition site for the C3 convertases C4b2a and C3bBb. This area encompasses a cavity on SCR 2, as well as part of the groove on the SCR 2-SCR 3 interface. Two additional surface depressions are centered around the C-terminal disulfide bridges of SCRs 3 and 4. These are likely to provide additional ligand binding sites. Based on this model in conjunction with sequence homology to the Ba fragment of factor B, a mechanism of DAF's accelerated convertase decay action is postulated.

The cleavage of C3 by the C3 convertases (C3bBb and C4b2a) determines whether complement activation proceeds. Dissociation (decay acceleration) of these central enzymes by the regulators decay-accelerating factor (DAF), complement receptor 1 (CR1), factor H, and C4-binding protein (C4BP) controls their function. In a previous investigation, we obtained evidence implicating the ␣4/5 region of the type A domain of Bb (especially Tyr 338 ) in decay acceleration of C3bBb and proposed this site as a potential interaction point with DAF and long homologous repeat A of CR1. Because portions of only two DAF complement control protein domains (CCPs), CCP2 and CCP3, are necessary to mediate its decay of the CP C3 convertase (as opposed to portions of at least three CCPs in all other cases, e.g. CCPs 1-3 of CR1), DAF/C4b2a provides the simplest structural model for this reaction. Therefore, we examined the importance of the C2 ␣4/5 site on decay acceleration of C4b2a. Functional C4b2a complexes made with the C2 Y327A mutant, the C2 homolog to factor B Y338A, were highly resistant to DAF, C4BP, and long homologous repeat A of CR1, whereas C2 substitutions in two nearby residues (N324A and L328A) resulted in partial resistance. Our new findings indicate that the ␣4/5 region of C2a is critical to decay acceleration mediated by DAF, C4BP, and CR1 and suggest that decay acceleration of C4b2a and C3bBb requires interaction of the convertase ␣4/5 region with a CCP2/CCP3 site of DAF or structurally homologous sites of CR1 and C4BP.The complement system consists of about 30 proteins that play critical roles in both innate and adaptive immunity. Three different activation pathways initiate the complement response (1): the classical pathway (CP), 1 the alternative pathway (AP), and the lectin pathway. Although each responds to different activators, all three pathways converge at the assembly of the C3 convertases. These two-component serine proteases (C4b2a and C3bBb) cleave the serum protein C3 at a single site, forming C3b and C3a activation fragments. Nearly all of the biological consequences associated with complement depend on this enzymatic cleavage. C3b binds covalently to and opsonizes activating targets, which marks them for lysis and/or immune clearance (1) and primes them for the production of the high affinity antibody (2-4). C3a is an anaphylactic agent that focuses inflammatory reactions around foreign substances by inducing local vasodilation, the influx of leukocytes, the upregulation of surface receptors, and the release of inflammatory mediators (5).There are two structurally different C3 convertases (1): 1) The classical and lectin pathway convertase, C4b2a, traditionally termed the CP convertase and 2) the AP C3 convertase, C3bBb. Each is formed first through the association of C4b or C3b with a zymogen (C2 or factor B) in the presence of Mg 2ϩ . C2 and factor B are homologous proteins composed of three amino-terminal globular domains (complement control protein domains (CCPs)) followed by a type A domain and a se...

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