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

Abstract

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...

Decay-Accelerating Factor Must Bind Both Components of the Complement Alternative Pathway C3 Convertase to Mediate Efficient Decay

“…DAF bound Bb via the von Willebrand factor type A (vWFA) domain rather than the serine protease domain. Functional studies with recombinant C2 and fB mutated within this domain indicated that DAF mediated its regulatory action via interactions with vWFA, and our kinetic analyses demonstrated that the Bb fragment and the vWF domain bind DAF with the same kinetic profiles and affinities (12)(13)(14). The specificity of DAF for the active convertase may be a consequence of its higher affinity for Bb compared with fB, and suggests that the regulatory function of DAF on the cell membrane is focused entirely on inhibition of activated enzymes rather than on binding to inactive proenzymes.…”

Species Selectivity in Poxviral Complement Regulators Is Dictated by the Charge Reversal in the Central Complement Control Protein Modules

“…The process of dissociation of the catalytic subunit Bb from the C3-convertase C3b,Bb is preceded by the binding of regulator to both the subunits of C3-convertase, C3b and Bb (40,41). We therefore asked whether the superior decay-acceleration activity of VCP against bovine C3-convertase is determined by its interaction with C3b or with Bb.…”

Domain Swapping Reveals Complement Control Protein Modules Critical for Imparting Cofactor and Decay-Accelerating Activities in Vaccinia Virus Complement Control Protein

“…5), suggesting a functional role for module 1 in CP DAA. Earlier examination of CP DAA by human complement regulators suggested that dissociation of the protease subunit from the convertase is a result of binding of the regulators to C4b and C2a, followed by a conformational change in the von Willebrand factor type A (vWFA) domain (45,47,63,64). More recently, it has been suggested that dissociation of the convertases could also be a result of displacement of the protease subunit by the regulator owing to a competition posed by the regulator for the protease interaction site on the noncatalytic subunit of the convertase (51).…”

“…DAA involves binding of the viral complement regulator to C3b or C4b and to Bb or C2a (44,45), whereas CFA requires interaction of the viral complement regulator with C3b or C4b, followed by interaction of factor I with C3b or C4b and the complement regulator (46). To determine whether loss or gain in the CFA and DAA of the domain swap mutants was owing to difference in their binding to C3b and C4b, we measured binding of VCP and the mutants to C3b and C4b using SPR assay described earlier (41,47).…”