Functional Characterization of Alternative and Classical Pathway C3/C5 Convertase Activity and Inhibition Using Purified Models

Abstract

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.

An inhibitor of complement C5 provides structural insights into activation

“…The C3 convertases C4b2a (classic/lectin pathways) and C3bBb (alternative pathway) generate surface-associated C3b (hereafter termed C3b′), which subsequently associates with additional factor Bb, and thus provides a potent amplification loop. Following the deposition of critically high concentrations of C3b′ on a surface, a shift in convertase activity is created, permitting C5 as a substrate (21)(22)(23)(24)(25)(26). Current models of C5 activation include docking of C5 to C3b, complexed with either of the activating proteases C2a or Bb (11,26).…”

A novel inhibitor of complement C5 provides structural insights into activation

“…The C3 convertases C4b2a (classical/lectin pathways) and C3bBb (alternative pathway) generate novel surface-associated C3b (hereafter termed C3b'), which subsequently associates with additional factor Bb, and thus provides a potent amplification loop. Following the deposition of critically high concentrations of C3b' on a surface, a shift in convertase activity is created, permitting C5 as a substrate [18][19][20][21][22] . Current models of C5 activation include docking of C5 to C3b, complexed with either of the activating proteases C2a or Bb 9,22 .…”

“…Following the deposition of critically high concentrations of C3b' on a surface, a shift in convertase activity is created, permitting C5 as a substrate [18][19][20][21][22] . Current models of C5 activation include docking of C5 to C3b, complexed with either of the activating proteases C2a or Bb 9,22 . Previous work utilizing the C3b homolog cobra venom factor (CVF) has provided structural information of how this docking may occur 14 .…”

“…Our structural data thus support the CVF-generated model of C5convertase docking, and provides further evidence for this being a crucial part of the mechanism of C5-activation in humans ( Figure 6B and C). C5-convertase activity is dependent on a high density of surface-associated C3b 22,23 . Combined with structural knowledge of C5inhibitors such as OmCI and RaCI, a novel hypothesis suggesting two distinct binding interfaces between C5 and C3b has emerged.…”

“…Combined with structural knowledge of C5inhibitors such as OmCI and RaCI, a novel hypothesis suggesting two distinct binding interfaces between C5 and C3b has emerged. 9,22,23 . The data presented here indicate, that the binding-interface utilizing C5_MG4 is essential for sufficient binding of C5 to C3b.…”