The tick-over theory revisited: Formation and regulation of the soluble alternative complement C3 convertase (C3(H2O)Bb)

Abstract

The molecular interactions between the components of the C3 convertase of the alternative pathway (AP) of complement and its regulators, in both surface-bound and fluid-phase form, are still incompletely understood. The fact that the AP convertase is labile makes studies difficult to perform. According to the so called tick-over theory, hydrolyzed C3, called C3(H(2)O), forms the initial convertase in fluid phase together with factor B. In the present study, we have applied western blot analysis and ELISA together with fluorescence resonance energy transfer (FRET) to study the formation of the fluid-phase AP convertases C3(H(2)O)Bb and C3bBb and their regulation by factor H and factor I at specific time points and, with FRET, in real time. In our hands, factor B showed a higher affinity for C3(H(2)O) than for C3b, although in both cases it was readily activated to Bb. However, the convertase activity of C3bBb was approximately twice that of C3(H(2)O)Bb, as monitored by the generation of C3a. But in contrast, the C3(H(2)O)Bb convertase was more resistant to inactivation by factor H and factor I than was the C3bBb convertase. Under conditions that totally inactivated C3bBb, C3(H(2)O)Bb still retained approximately 25% of its initial activity.

Use of time‐resolved FRET to validate crystal structure of complement regulatory complex between C3b and factor H (N terminus)

“…While FRET based on specifically positioned donor and acceptor fluorophores has been widely employed for protein-structure determination, its use to investigate interactions between the difficult-to-study multiple-domain and disulfide-rich proteins of the complement system has received only limited attention. 38,39 By using FRET in the current work to validate the physiological relevance of previous crystallography and EMbased studies we have not only enhanced confidence in these results but also explored the potential of FRET for looking at further complexes, for which high-resolution structural information is lacking. Incorporation of free cysteine residues into proteins that naturally contain multiple disulfides carries the risk of disulfide shuffling and misfolding.…”

Candidate inhibitors of porcine complement

“…The LP is activated by mannose-binding lectin and ficolins binding to in particular carbohydrate structures (58,59). The AP pathway is continuously activated by hydrolysis of C3 and triggered by a range of different compounds like lipids, carbohydrates and proteins (60). In the 50's properdin was proposed as an initiator of this pathway and recent evidence supports these early findings (61,62).…”

Neutrophils Induce Astroglial Differentiation and Migration of Human Neural Stem Cells via C1q and C3a Synthesis

“…This process can be triggered spontaneously, via generation of C3(H20) in the fluid phase. Critically, although C3bBb is more active than C3(H20)Bb, generating 2-fold the amount of C3a, factor B has a greater affinity for C3(H20), and the resistance of C3(H20)Bb to inactivation results in prolonged activity (55). PMN also synthesize and secrete properdin (Fig.…”