Decay Accelerating Activity of Complement Receptor Type 1 (CD35)

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151

Membrane cofactor protein (MCP; CD46), a widely distributed regulator of complement activation, is a cofactor for the factor I-mediated degradation of C3b and C4b deposited on host cells. MCP possesses four extracellular, contiguous complement control protein modules (CCPs) important for this inhibitory activity. The goal of the present study was to delineate functional sites within these modules. We employed multiple approaches including mutagenesis, epitope mapping, and comparisons to primate MCP to make the following observations. First, functional sites were located to each of the four CCPs. Second, some residues were important for both C3b and C4b interactions while others were specific for one or the other. Third, while a reduction in ligand binding was invariably accompanied by a parallel reduction in cofactor activity (CA), other mutants lost or had reduced CA but retained ligand binding. Fourth, two C4b-regulatory domains overlapped measles virus interactive regions, indicating that the hemagglutinin docks to a site important for complement inhibition. Fifth, several MCP regulatory areas corresponded to functionally critical, homologous positions in other CCP-bearing C3b/C4b-binding proteins. Based on these data and the recently derived crystal structure of repeats one and two, computer modeling was employed to predict MCP structure and examine active sites.

“…3, there are two highly homologous regions to MCP in C4BP and CR1. These regions are ones likely to be involved in C4b regulation as they are in MCP (52). 27)).…”

Section: Discussionmentioning

confidence: 99%

Dissecting Sites Important for Complement Regulatory Activity in Membrane Cofactor Protein (MCP; CD46)

Liszewski¹,

Leung²,

Cui³

et al. 2000

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151

“…Armed with this information on DAF, we then analyzed the extent of conservation of residues in DAF and in other C3 convertase regulators determined by mutagenesis to be important, either for cofactor activity or for DAA (12,18,21,22,(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). Whereas we found an absence of strict conservation of residues which distinguish the two functions, by focusing alignment on the sequence flanking the CCP2 and -3 junction where DAF function resides, we identified differences between those regulators that 1) mediate DAA and 2) mediate cofactor activity.…”

Section: Discussionmentioning

confidence: 99%

Structure-based Mapping of DAF Active Site Residues That Accelerate the Decay of C3 Convertases

Kuttner-Kondo

Hourcade

Anderson

et al. 2007

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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...

“…The interaction of C4b2a with DAF may afford the simplest model for decay acceleration because as indicated (see the Introduction) only two DAF CCPs, CCP2 and CCP3 (9,10), are involved, whereas decay acceleration by all other RCA proteins appears to involve at least three CCPs (12,13,30). Moreover, unlike the other regulators, DAF does not have cofactor activity for the factor I-mediated cleavage of C4b or C3b.…”

Section: Decay Acceleration Sites Of the Cp C3 Convertasementioning

confidence: 99%

“…In both cases about 10 amino acids, located in DAF CCPs 2 and 3 and the inter-CCP segment, appear critical (11). For CR1, CCPs 1-3 (of a total of 30 CCPs) are required for decay acceleration of both C3bBb and C4b2a (12), with a critical site in CCPs 1 and 2 structurally similar to that in DAF CCP2 and 3. For C4BP, CCPs 1-3 (of 8 CCPs) are required for decay acceleration of C4b2a (13).…”

mentioning

confidence: 99%

A Corresponding Tyrosine Residue in the C2/Factor B Type A Domain Is a Hot Spot in the Decay Acceleration of the Complement C3 Convertases

Kuttner-Kondo

Dybvig

Mitchell

et al. 2003

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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...