Abstract
BackgroundComplement is a large protein network in plasma that is crucial for human immune defenses and a major cause of aberrant inflammatory reactions. The C5 convertase is a multi-molecular protease complex that catalyses the cleavage of native C5 into its biologically important products. So far, it has been difficult to study the exact molecular arrangement of C5 convertases, because their non-catalytic subunits (C3b) are covalently linked to biological surfaces through a reactive thioester. Through development of a highly purified model system for C5 convertases, we here aim to provide insights into the surface-specific nature of these important protease complexes.ResultsAlternative pathway (AP) C5 convertases were generated on small streptavidin beads that were coated with purified C3b molecules. Site-specific biotinylation of C3b via the thioester allowed binding of C3b in the natural orientation on the surface. In the presence of factor B and factor D, these C3b beads could effectively convert C5. Conversion rates of surface-bound C3b were more than 100-fold higher than fluid-phase C3b, confirming the requirement of a surface. We determine that high surface densities of C3b, and its attachment via the thioester, are essential for C5 convertase formation. Combining our results with molecular modeling explains how high C3b densities may facilitate intermolecular interactions that only occur on target surfaces. Finally, we define two interfaces on C5 important for its recognition by surface-bound C5 convertases.ConclusionsWe establish a highly purified model that mimics the natural arrangement of C5 convertases on a surface. The developed model and molecular insights are essential to understand the molecular basis of deregulated complement activity in human disease and will facilitate future design of therapeutic interventions against these critical enzymes in inflammation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0203-8) contains supplementary material, which is available to authorized users.
Highlights
Complement is a large protein network in plasma that is crucial for human immune defenses and a major cause of aberrant inflammatory reactions
To assay C5 convertase activity, C3b-loaded beads were incubated with purified factor B (FB), factor D (FD) and C5 after which C5 conversion was quantified by analyzing C5a release into the supernatant using a calcium mobilization assay with C5a receptor (C5aR) transfected cells [17, 18]
We showed that C3b beads incubated with C5 alone, C5 + FB, or C5 + FD, did not convert C5 (Fig. 1c)
Summary
Complement is a large protein network in plasma that is crucial for human immune defenses and a major cause of aberrant inflammatory reactions. All initiation pathways converge in the formation of short-lived C3 convertase enzymes on the target surface These C3 convertases cleave the major complement protein C3 into the large, reactive C3b molecule that can covalently attach to target surfaces to label them for rapid opsonization and phagocytosis [7]. Molecular studies highlight two important interfaces for the recognition of substrate C5 by surface-bound convertase enzymes. These increased molecular insights into C5 convertase enzymes are essential to understand the molecular basis for deregulated and excessive convertase activity in human disease and will be critical for future design of therapeutic interventions against the undesired activation of complement during systemic infections and acute inflammatory processes
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