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The human complement system: assembly of the classical pathway C3 convertase.

ABSTRACT: The assembly of the classical pathway C3 convertase in the fluid phase has been studied. The enzyme is assembled from C2 and C4 on cleavage of these proteins by C1s. Once assembled, the enzyme activity decays rapidly. Kinetic evidence has been obtained that this decay is even more rapid than previously suggested (kdecay is 2.0 min-1 at 37 degrees C). As a result, optimal C3 convertase activity is only observed with high C1s levels, which result in rapid rates of cleavage of C2 and increased rates of formation of the C3 convertase. Using high concentrations of C1s at lower temperatures (22 degrees C) in the presence of excess substrate we have demonstrated kinetically that the enzyme comprises an equimolar complex of C4b and cleaved C2. We have obtained direct evidence from gel-filtration experiments for the role of C2a as the catalytic subunit of the enzyme. C2b appears to mediate the interaction between C4 (or C4b) and C2 at pH 8.5 and at low ionic strength where the interactions can easily be detected. It may therefore be important in the assembly of the enzyme, though it is not involved in the catalytic activity. The decay of the C3 convertase reflects the release of C2a from the C4b x (C2b) x C2a complex, and the stabilizing effect of iodine on the C3 convertase is therefore apparently one of stabilizing the C4b-C2z interaction, which is otherwise weak. C1s is not a part of the C3 convertase enzyme.

SUBMITTER: Kerr MA

PROVIDER: S-EPMC1161929 | biostudies-other | 1980 Jul

REPOSITORIES: biostudies-other

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Project description:Complement is essential for the protection against infections; however, dysregulation of complement activation can cause onset and progression of numerous inflammatory diseases. Convertase enzymes play a central role in complement activation and produce the key mediators of complement: C3 convertases cleave C3 to generate chemoattractant C3a and label target cells with C3b, which promotes phagocytosis; C5 convertases cleave C5 into chemoattractant C5a, and C5b, which drives formation of the membrane attack complex. Since convertases mediate nearly all complement effector functions, they are ideal targets for therapeutic complement inhibition. A unique feature of convertases is their covalent attachment to target cells, which effectively confines complement activation to the cell surface. However, surface localization precludes detailed analysis of convertase activation and inhibition. In our previous work, we developed a model system to form purified alternative pathway (AP) C5 convertases on C3b-coated beads and quantify C5 conversion via functional analysis of released C5a. Here, we developed a C3aR cell reporter system that enables functional discrimination between C3 and C5 convertases. By regulating the C3b density on the bead surface, we observe that high C3b densities are important for conversion of C5, but not C3, by AP convertases. Screening of well-characterized complement-binding molecules revealed that differential inhibition of AP C3 convertases (C3bBb) and C5 convertases [C3bBb(C3b)n] is possible. Although both convertases contain C3b, the C3b-binding molecules Efb-C/Ecb and FHR5 specifically inhibit C5 conversion. Furthermore, using a new classical pathway convertase model, we show that these C3b-binding proteins not only block AP C3/C5 convertases but also inhibit formation of a functional classical pathway C5 convertase under well-defined conditions. Our models enable functional characterization of purified convertase enzymes and provide a platform for the identification and development of specific convertase inhibitors for treatment of complement-mediated disorders.

Project description:The complement network is increasingly recognized as an important triage system that is able to differentiate between healthy host cells, microbial intruders, cellular debris and immune complexes, and tailor its actions accordingly. At the center of this triage mechanism is the alternative pathway C3 convertase (C3bBb), a potent enzymatic protein complex capable of rapidly converting the inert yet abundant component C3 into powerful effector fragments (C3a and C3b), thereby amplifying the initial response on unprotected surfaces and inducing a variety of effector functions. A fascinating molecular mechanism of convertase assembly and intrinsic regulation, as well as the interplay with a panel of cell surface-bound and soluble inhibitors are essential for directing complement attack to intruders and protecting healthy host cells. While efficiently keeping immune surveillance and homeostasis on track, the reliance on an intricate cascade of interaction and conversion steps also renders the C3 convertase vulnerable to derail. On the one hand, tissue damage, accumulation of debris, or polymorphisms in complement genes may unfavorably shift the balance between activation and regulation, thereby contributing to a variety of clinical conditions. On the other hand, pathogens developed powerful evasion strategies to avoid complement attack by targeting the convertase. Finally, we increasingly challenge our bodies with foreign materials such as biomaterial implants or drug delivery vehicles that may induce adverse effects that are at least partially caused by complement activation and amplification via the alternative pathway. The involvement of the C3 convertase in a range of pathological conditions put this complex into the spotlight of complement-targeted drug discovery efforts. Fortunately, the physiological regulation and microbial evasion approaches provide a rich source of inspiration for the development of powerful treatment options. This review provides insight into the current knowledge about the molecular mechanisms that drive C3 convertase activity, reveals common and divergent strategies of convertase inhibition employed by host and pathogens, and how this inhibitory arsenal can be tapped for developing therapeutic options to treat complement-related diseases.

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The human complement system: assembly of the classical pathway C3 convertase.

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