Studies on the interactions between C‐reactive protein and complement proteins

Abstract

Studies on the interactions between C-reactive protein and complement proteins IntroductionThe classical acute-phase reactant C-reactive protein (CRP) is a pentameric, disc-shaped serum protein.1 Its basic features are the control of inflammation, the stimulation of clearance of damaged cell and tissue components, and the initiation of repair functions.2 CRP shows calcium-dependent affinity for phosphate monoesters, such as phosphatidylcholine, but several other ligands of CRP have been characterized, including damaged cell membranes, small ribonucleoprotein particles, apoptotic cells and fibronectin. Native CRP can undergo subunit dissociation into individual monomeric units, for example upon association with negatively charged lipid monolayers. 4 This modified form of CRP can be produced in vitro by urea chelation, acid treatment, heating or direct immobilization of native CRP onto polystyrene.5 This modified form of CRP (mCRP) has reduced solubility and exhibits different electrophoretic characteristics as the result of a decrease of isoelectric point (pI) from 6Á4 to 5Á4. 6 The structural changes releasing CRP subunits from the pentamer are correlated with expression of a new antigenic reactivity and formation of neo-epitopes. 7 The forms of CRP expressing SummarySeveral studies have investigated the interactions between C-reactive protein (CRP) and various complement proteins but none of them took into consideration the different structural forms of CRP. The aim of our study was to investigate whether the different antigenic forms of CRP are able to bind C1q, to trigger activation of the C1 complex and to study the ability of the various CRP forms to bind complement factor H (FH) and C4b-binding protein (C4BP). Interactions between various CRP forms and complement proteins were analysed in enzyme-linked immunosorbent assay and surface plasmon resonance tests and activation of the C1 complex was followed in a reconstituted system using purified C1q, C1r and C1s in the presence of C1-INH. Native, ligand-unbound CRP activated the classical pathway weakly. After binding to phosphocholine, native CRP bound C1q and significantly activated C1. Native CRP complexed to phosphocholine did not bind the complement regulatory proteins FH and C4BP. After disruption of the pentameric structure of CRP, as achieved by urea-treatment or by site-directed mutagenesis, C1q binding and C1 activation further increased and the ability of CRP to bind complement regulatory proteins was revealed. C1q binds to CRP through its globular head domain. The binding sites on CRP for FH and C4BP seemed to be different from that of C1q. In conclusion, in parallel with the increase in the C1-activating ability of different CRP structural variants, the affinity for complement regulatory proteins also increased, providing the biological basis for limitation of excess complement activation.

Monomeric CRP contributes to complement control in fluid phase and on cellular surfaces and increases phagocytosis by recruiting factor H

“…In contrast, CRP did not bind to immobilized Factor H. 20,21 This discrepancy is explained by the structural modification of pCRP on immobilization (data not shown). 14,22 In this study, by comparing the binding of mCRP and pCRP to immobilized Factor H, we show that mCRP but not pCRP binds to immobilized Factor H (Figure 1). 23 The Factor H-mCRP interaction is detected in both settings when Factor H or mCRP is immobilized (Figure 1b and d, Figure 2b and data not shown).…”

“…23 The Factor H-mCRP interaction is detected in both settings when Factor H or mCRP is immobilized (Figure 1b and d, Figure 2b and data not shown). 22,24 mCRP binding to Factor H is calcium independent (Figure 1b) and is weakly affected by ionic strength or by heparin (Supplementary Figure 4). However, CFHR4A, an additional Factor H family protein, binds pCRP but not mCRP.…”

“…27 CRP binds several complement components and regulators such as C1q. 22 CRP-bound C1q initiates complement activation to the level of the C3 convertase, which results in C3b surface deposition and opsonization. 18,28 However, further progression of the complement cascade, for example, C5 convertase formation, C5a release, progression of the terminal pathway, membrane attack complex (MAC) assembly and membrane insertion does not occur.…”

Complement Factor H Binds at Two Independent Sites to C-reactive Protein in Acute Phase Concentrations*

“…If these findings were true, this difference would represent a molecular mechanism that leads to AMD. However recent ELISAs and analytical ultracentrifugation (AUC) analyses suggest that no FH-CRP interaction occurred and that those earlier observations of the FH-CRP interaction resulted from the use of denatured CRP (26,27).…”

Structural Characterization of PTX3 Disulfide Bond Network and Its Multimeric Status in Cumulus Matrix Organization

“…For example, native CRP inhibits platelet activation and prevents platelet capture of neutrophils, whereas monomeric CRP, resulting from loss of the pentameric symmetry of the molecule, displays potent prothrombotic activities (30,31). Moreover disruption of the pentameric structure, as achieved by urea treatment or by site-directed mutagenesis, leads to enhanced CRP binding to C1q and subsequent C1 activation as well as the ability of CRP to interact with complement-regulatory proteins factor H and C4b-binding protein (32). Neuronal pentraxin 2 (NP2), also known as neuronal activity-regulated pentraxin (Narp), and neuronal pentraxin 1 (NP1), two members of the long pentraxin subfamily selectively expressed in brain, are covalently linked by disulfide bonds into highly organized complexes (33,34).…”