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

Seline A Zwarthoff,Evelien T M Berends,Mihály Józsi,Piet C Aerts,Suzan H M Rooijakkers,Carla J C De Haas,Maartje Ruyken,Ronald D Gorham,Sanne Mol

doi:10.3389/fimmu.2018.01691

Seline A Zwarthoff, Evelien T M Berends + Show 7 more

Open Access

https://doi.org/10.3389/fimmu.2018.01691

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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.

Highlights

The human complement system comprises a family of proteins that are essential to the human immune response against infections [1]
These biotinylated C3b molecules were subsequently loaded onto small magnetic streptavidin (SA) beads (2.8 μm diameter) and incubated with factor B (FB) and factor D (FD) to form surface-bound convertases
C3b-coated beads were incubated with FB, FD and C3 and release of C3a was determined in supernatants via calcium mobilization in U937C3aR (Figure 2B)

Summary

Introduction

The human complement system comprises a family of proteins that are essential to the human immune response against infections [1]. C3 convertases cleave C3 into C3a, a chemoattractant molecule, and C3b, which covalently binds to target surfaces and triggers phagocytosis. Upon binding to the antibody platforms [6], C1q-associated protease C1s converts C4 and C2 to generate a C3 convertase enzyme (C4b2a) on the cell surface (Figure 1A). The lectin pathway forms C4b2a via activation of mannose-binding lectin-associated serine proteases. The proconvertase C3bB is cleaved by factor D (FD) to form an active C3 convertase complex that consists of C3b and the protease fragment Bb (C3bBb) (Figure 1B). When the density of C3b molecules on the cell surface becomes sufficiently high, the existing C3 convertases (C4b2a and C3bBb) gain the ability to cleave C5, leading to formation of C5a and C5b (Figures 1A,B) [7, 8]

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