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

Abstract

Several 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.

Figure 1

Structural modification of C-reactive protein upon immobilization. Polystyrene ELISA plates were coated either directly (at 2 μg/ml, black bars) or indirectly [through anti-CRP polyclonal antibodies (1 : 1000) at 2 μg/ml, open bars] with different C-reactive protein preparations: (a) native CRP; (b) recombinant, modified CRP. A set of anti-CRP monoclonal antibodies (1DH, 8D8, 2C10, 3H12 at 0·1 μg/ml; 3G12, 9C9 at 0·2 μg/ml; 12D7 at 0·9 μg/ml; and 8C10 at 1·6 μg/ml) was used to detect structural modifications. Each value is the mean ± SD of two parallel experiments. (c) Binding of 1D6 and CRP8 anti-CRP antibodies to immobilized native CRP was performed in 0·01 m HEPES, 0·15 m NaCl, 0·67 mm CaCl₂, 0·005% v/v surfactant P20, pH 7·4 at a flow rate of 20 μl/min.

Figure 2

Analysis by surface plasmon resonance spectroscopy of the interaction of native CRP and modified CRP with immobilized C1q. Sensorchips were coated with C1q (17 400 RU) as described in the Materials and methods. (a) Sixty microlitres of nCRP or Cr-mCRP (100 nm) was injected in the running buffer (PBS containing 0·005% surfactant P20) at a flow rate of 20 μl/min. Resonance units are indicated as a function of time. (b) Dose–response analysis of the binding of Cr-mCRP to immobilized C1q. Representative sensorgrams (after background subtraction) illustrating the binding of Cr-mCRP at varying concentrations (bottom to top curves: 2, 5, 7·5, 10, 20 nm) to immobilized C1q.

Figure 3

(a) Binding of different CRP preparations to immobilized C1q in ELISA. C1q (2 μg/ml) was coated onto ELISA plates and incubated with different CRP preparations at varying concentrations. The extent of CRP binding to immobilized C1q was measured by reaction with polyclonal anti-CRP antibodies (1 : 1000) and is expressed as OD₄₉₀ values. (b) Binding of C1q to ligand-complexed nCRP. ELISA plates were coated with PC-KLH and native CRP was added at increasing concentrations. In the next step varying C1q concentrations were added and the amount of bound C1q was measured by specific antibodies. PC-KLH and uncoated wells were used as controls. The data shown represent the means ± SD of two experiments.

Figure 4

Competitive inhibition of the binding of Cr-mCRP to immobilized C1q by soluble C1q (a) or its globular regions (GR) (b) C1q (2 μg/ml) was coated onto ELISA plates and incubated with Cr-mCRP at varying concentrations. Before addition to the plate, Cr-mCRP was incubated with C1q (a) or C1q GR (b) at varying concentrations. The extent of Cr-mCRP binding to immobilized C1q was measured by polyclonal anti-CRP antibodies (1 : 1000) and is indicated as OD values. The data shown represent the means ± SD of three experiments.

Figure 5

Binding of various CRP forms to human complement FH. (a) ELISA plates were coated directly with Cr-mCRP or urea-mCRP, or indirectly (through PC-KLH) with native CRP at 2 μg/ml concentration. Uncoated wells and wells coated with PC-KLH only were used as controls. Increasing amounts of purified human FH were added and detected by anti-FH antibodies. (b) ELISA plates were coated with human complement FH (2 μg/ml) and incubated with either nCRP or Cr-mCRP (10 μg/ml). Monoclonal antibodies specific for the native (1D6) or modified (9C9 and CRP-8) CRP conformation were used to determine the amounts of CRP bound to the plate. The data shown represent the means ± SD of two experiments.

Figure 6

Binding of various CRP forms to human CD binding protein (C4BP). (a) ELISA plates were coated directly with nCRP, Cr-mCRP, urea-mCRP or with nCRP complexed to PC-KLH at 2 μg/ml concentration. PC-KLH-coated wells and uncoated wells were used as controls. Increasing amounts of purified human C4BP were added and detected by anti-C4BP antibodies. (b) ELISA plates were coated with human C4BP (2 μg/ml) and incubated with either nCRP or Cr-mCRP (10 μg/ml). Monoclonal antibodies specific of the native (1D6) or modified (9C9 and CRP-8) CRP conformation were used to determine the amounts of CRP bound to the plate. The data shown represent the means ± SD of two experiments.

Figure 7

Analysis by surface plasmon resonance spectroscopy of the interaction between native CRP or modified CRP and immobilized C4BP. Sensor chips were coated with C4BP (5230 RU) as described in the Materials and methods. Sixty microlitres of nCRP (100 nm), or Cr-mCRP (25–200 nm) were injected in the running buffer (0·01 m HEPES, 0·15 m NaCl, 0·67 mm CaCl₂, 0·005% v/v surfactant P20, pH 7·4) at a flow rate of 20 μl/min. Resonance units are indicated as a function of time.

Figure 8

Mapping of the binding sites for C1q, FH and C4BP on CRP by competitive ELISA experiments. ELISA plates coated with Cr-mCRP (2 μg/ml) were incubated with C1q and then allowed to interact with complement regulatory proteins FH and C4BP. Binding was detected by specific antibodies and secondary antibodies as described in the Materials and methods. The observed differences were not statistically significant. The data shown represent the means ± SD of three experiments.