C-reactive protein (CRP) is a major acute phase protein whose functions are not totally clear. In this study, we examined the interaction of CRP with factor H (FH), a key regulator of the alternative pathway (AP) of complement. Using the surface plasmon resonance technique and a panel of recombinantly expressed FH constructs, we observed that CRP binds to two closely located regions on short consensus repeat (SCR) domains 7 and 8–11 of FH. Also FH-like protein 1 (FHL-1), an alternatively spliced product of the FH gene, bound to CRP with its most C-terminal domain (SCR 7). The binding reactions were calcium-dependent and partially inhibited by heparin. In accordance with the finding that CRP binding sites on FH were distinct from the C3b binding sites, CRP preserved the ability of FH to promote factor I-mediated cleavage of C3b. We propose that the function of CRP is to target functionally active FH and FHL-1 to injured self tissues. Thereby, CRP could restrict excessive complement attack in tissues while allowing a temporarily enhanced AP activity against invading microbes in blood.

The human complement system (C) is comprised of at least 30 soluble or membrane-associated proteins. The classical pathway of complement (CP)3 is activated by target-bound Abs and C-reactive protein (CRP) (1), whereas the alternative pathway (AP) is activated through direct contact with foreign particles or cells. The C system has an important role in protection against microbial infections. In addition, C is also involved in the clearance of immune complexes and damaged, transformed, or apoptotic self cells (2). The AP has the capacity to enhance its own activation through the amplification loop, which can lead to massive C activation and C component depletion (3). Under normal circumstances, activation of the amplification loop is efficiently regulated by factor H (FH), a soluble plasma protein (4, 5, 6). However, during bacterial infections, a contact with a foreign surface will unleash the amplification loop. On these AP activating structures, the binding of FH to surface-associated C3b is restricted. On the other hand, FH is capable of suppressing AP activation on most cell surfaces (nonactivators) that are coated with sialic acids or glycosaminoglycans (7, 8, 9). In addition, activation of C on host cell surfaces is controlled by specific membrane-bound C inhibitors, membrane cofactor protein (CD46), decay-accelerating factor (CD55), and protectin (CD59) (10).

FH is a 150-kDa plasma protein that consists of 20 short consensus repeat (SCR) domains of ∼60 amino acid residues each (11). As a regulator of the AP, FH inhibits the binding of factor B to C3b, acts as a cofactor for the factor I-mediated cleavage of C3b to iC3b (cofactor activity), and accelerates the decay of C3bBb, the AP C3 convertase (decay-accelerating activity) (4, 5, 6). The cofactor and decay-accelerating activities of FH have been located to SCR domains 1–4 (12, 13). The binding of FH to C3b has turned out to be more complex than initially thought. It has recently been shown that FH binds to C3b through three different sites located at the N terminus, C terminus, and central part of the molecule (14, 15, 16).

FH-like protein 1 (FHL-1) is a product of alternative splicing of the FH gene. It is a 42-kDa plasma protein that consists of seven SCRs, identical to the seven N-terminal SCRs of FH, plus four unique amino acids (17, 18). FHL-1 has decay-accelerating and cofactor activities, and these characteristics suggest that FHL-1 has C regulatory activity in vivo (13, 19). Both FH and FHL-1 have been shown to bind to the M-protein of Streptococcus pyogenes (20, 21, 22). In both cases, the binding occurs through SCR 7 and is inhibited by heparin (21, 22).

The precise mechanism whereby FH can discriminate activator- and nonactivator-bound C3b is not fully understood. FH binds to heparin, sialic acids, and other polyanions through SCR domains 7, 19–20, and probably a region around SCR 13 (9, 23, 24, 25). Surfaces rich in sialic acid, like mammalian cell membranes or neisserial lipooligosaccharides, promote the binding of FH to C3b, and thereby suppress AP activation (7, 8, 26, 27, 28). Upon tissue damage, however, the structural integrity of cell membranes breaks down, and different types of structures, like certain phospholipids, cytoskeletal components and chromatin, become exposed. As the emerging structures may activate the C system, damage to self tissue creates a need for suppression of excessive AP amplification. On the other hand, clearance of nonviable structures should occur in a well-regulated and focused manner.

CRP is a 120-kDa acute phase protein that belongs to the family of pentraxins. It consists of five identical nonglycosylated subunits and binds to various ligands, including phosphocholine, chromatin complexes, and pneumococcal C-polysaccharide (29, 30, 31, 32). The serum level of CRP is usually <1 μg/ml, but during inflammation or extensive tissue damage it may rise up to 500 μg/ml within 24 h due to increased synthesis of CRP in liver. CRP expression is stimulated by IL-1 and IL-6 and quickly subsides once the triggering factor has been eliminated (33).

Although CRP has a putative role in clearance of pneumococci, its main physiological function is uncertain. Judging from its preservation and prevalence among mammals, CRP probably serves an essential and beneficial role during the acute phase reaction. The level of plasma CRP rises rapidly in bacterial infections and also following noninfectious tissue injury, e.g., surgical operations. CRP binds to C1q and is able to activate the CP of complement in vitro and in vivo (1, 34, 35). CRP has been suggested to inhibit activation of the AP (36). We have shown earlier that CRP binds directly to FH (37), a finding recently demonstrated also by Mold et al. (38). On the basis of these results, we have postulated that CRP may regulate complement activation induced by damaged tissue.

The aim of the present study was to explore a potential physiological function for CRP by examining the interactions between FH and CRP. By using the surface plasmon resonance analysis and a series of FH mutants, we mapped the binding sites of CRP to SCR domain 7 of FH and FHL-1 and domains 8–11 of FH. Our data showed that CRP-bound FH remained functionally active. These findings lead us to suggest that CRP acts to restrict inflammation induced by apoptotic or damaged self cells by targeting the regulatory activities of FH and FHL-1 to areas affected by injury. While removing FH away from the fluid phase, CRP may simultaneously allow a temporarily enhanced AP amplification and stronger AP activity against invading microorganisms.

FH, factor I, and C3 were purified from human plasma, and C3b was generated with factors B and D in the presence of Mg2+ ions, as described previously (39, 40). FH constructs SCR1–5, 1–6, 1–7 (FHL-1), 8–11, and 8–20 were cloned and produced in the baculovirus expression system as described previously (13, 16, 41, 42). C1q was purchased from Quidel (San Diego, CA). CRP was obtained from Sigma (St. Louis, MO).

Surface plasmon resonance measurements were performed using the Biacore 2000 instrument and analyzed with the BIAevaluation 3.0 software (Biacore AB, Uppsala, Sweden). CRP, FH, C1q, and C3b were immobilized on carboxylated dextran CM5 chips (Biacore AB) by using the amine-coupling procedure, according to the protocol of the manufacturer. Binding analyses were performed using 1/3 veronal buffered saline (VBS; 49 mM NaCl, 0.6 mM sodium barbital, 1.1 mM barbituric acid (pH 7.4)) at a flow rate of 5 μl/min. The CRP-coupled chip was used for determining the FH domains for CRP binding. Before injecting into the Biacore flowcell, FH and recombinant constructs of FH were dialyzed against 1/3 VBS and mixed with Ca2+ (CaCl2, final concentration 0.67 mM) or EDTA (final concentration 6.7 mM). The protein concentrations of the reagents were measured using the BCA Protein Assay (Pierce, Rockford, IL). The final concentrations of proteins in the CRP binding assay were: FH, 250 μg/ml; SCR 1–5, 27 μg/ml; SCR 1–6, 105 μg/ml; SCR 1–7, 50 μg/ml; SCR 8–20, 45 μg/ml; and SCR 15–20, 45 μg/ml. The reverse binding of CRP to FH was examined by injecting CRP (37 μg/ml) to a FH-coupled chip. Binding of CRP to C3b was tested by injecting CRP (37 μg/ml) to a C3b-coated chip. As a positive control, we examined the binding of CRP (37 μg/ml) to a C1q-coated chip. All assays were performed in the presence of 0.67 mM Ca2+ or 6.7 mM EDTA. As controls, all binding tests were also performed using a blank chip (activated and deactivated without any coupled proteins). After each binding experiment, the surface was regenerated by 30 μl of 3 M NaCl in acetate buffer (pH 4.6) (regeneration buffer).

The possible inhibitory effect of heparin on the interactions of CRP with FH was tested in the Biacore 2000 system. The CM5 chip was coupled with CRP as above. FH, FHL-1, and SCR 8–11 (at concentrations 250, 50, and 50 μg/ml, respectively) were mixed with 0.67 mM Ca2+ and varying amounts of unfractionated heparin (Heparin 5000 IU/ml; Lövens Kemiske Fabrik, Ballerup, Denmark) or low-m.w. heparin (dalteparine, Fragmin; Pharmacia & Upjohn, Kalamazoo, MI) in 1/3 VBS buffer. The heparin and dalteparine concentrations are expressed as IU and Xa-units per ml, respectively. Binding analyses were performed using the standard conditions described above.

To test the effect of CRP on FH-C3b interactions, a chip was coupled with C3b. Samples were prepared by mixing FH (65 μg/ml), Ca2+ (0.67 mM), and varying amounts of CRP in 1/3 VBS. A total of 10 μl of the samples was injected into the C3b-coated or blank flowcell. The buffers and test conditions were as described above.

To analyze the effect of heparin on the binding of FH to CRP, an ELISA assay was also used. Microtiter plates (Greiner, Frickenhausen, Germany) were first coated with CRP or BSA (Sigma), both at 10 μg/ml in VBS, during an overnight incubation at room temperature. Nonspecific binding sites on the wells were blocked by treating the plates for 20 min with VBS containing 0.1% gelatin and 0.1% BSA and washing three times with 0.1% Tween 20/VBS. The samples were prepared by mixing FH (final concentration 1 μg/ml) with Ca2+ (final concentration 1 mM) and 0–1000 IU/ml of heparin in VBS. A total of 100 μl of the samples was pipetted to the wells in duplicate and incubated for 2 h at +37°C. After washing the plate, 100 μl of goat anti-human FH Ab (Incstar, Stillwater, MN), diluted 1:2000 in 0.1%Tween/VBS, was added to the wells and incubated for 2 h at +37°C. After washing, HRP-conjugated rabbit anti-goat IgG Ab (Jackson ImmunoResearch, West Grove, PA) (100 μl/well) was added and incubated for 2 h at +37°C. The wells were washed, the substrate was added, and the optical density determined at 492 nm using a Multiskan 340 MCC spectrophotometer (Labsystems, Helsinki, Finland).

The effect of CRP on the fluid phase cofactor activity of FH in factor I-mediated cleavage of C3b was tested essentially as described previously (43). Purified C3b was radiolabeled to an initial specific activity of 106 cpm/μg using the Iodogen method (Pierce). Factor I (30 μg/ml), FH (20 μg/ml) 125I-C3b (80,000 cpm), 1 mM Ca2+, and varying amounts of CRP (0–100 μg/ml) were mixed in VBS buffer and incubated for 1 h at +37°C. Experiments conducted in the absence of factor I or FH were taken as negative controls. After incubation, the samples were heated to 95°C in a reducing buffer (containing 2.5% β-mercaptoethanol) and run in a 10% SDS-PAGE gel. The gels were fixed with 5% acetic acid for 30 min, dried, and autoradiographed with the Fujifilm BAS 2500 instrument (Fuji Photo Film, Tokyo, Japan).

The binding of FH to CRP was examined by surface plasmon resonance analysis (Biacore 2000). Binding of FH to the CRP-coated flowcell could be clearly seen when 0.67 mM Ca2+ was present (Fig. 1,A), while EDTA (6.7 mM) completely inhibited the binding (Fig. 1,B). Ca2+ by itself gave only a weak signal (Fig. 1,B). The reverse binding of fluid-phase CRP to surface-coupled FH was also observed in the presence of Ca2+ (Fig. 1,C), and EDTA inhibited this binding (Fig. 1,D). For comparison, and as a positive control, we examined the binding of CRP to C1q that was coupled to the Biacore 2000 chip. CRP bound to C1q in the presence of 0.67 mM Ca2+ and, again, 6.7 mM EDTA totally inhibited the binding (Fig. 1, E and F).

FIGURE 1.

Analysis of CRP binding to FH and C1q by surface plasmon resonance. Biacore chips were coated with CRP (A, B), FH (C, D), C1q (E, F) or no protein (controls). The indicated proteins in 1/3 VBS with 0.67 mM Ca2+ or 6.7 mM EDTA were injected into CRP-, FH- or C1q-coupled flowcell and into a control flowcell (blank). Resonance units are indicated as a function of time. Only bulk effects caused by ligands in solution are seen in the controls. The bulk effects vary according to the inherent properties of the proteins and constructs. A, FH binding to CRP in the presence of Ca2+. B, Inhibition of FH binding to CRP by EDTA and a control containing 0.67 mM Ca2+ in 1/3 VBS but no FH. C, The binding of CRP to surface-bound FH in the presence of Ca2+ and inhibition by EDTA (D). E, The binding of CRP to C1q in the presence of Ca2+. F, In the presence of EDTA, no binding of CRP to C1q occurs.

FIGURE 1.

Analysis of CRP binding to FH and C1q by surface plasmon resonance. Biacore chips were coated with CRP (A, B), FH (C, D), C1q (E, F) or no protein (controls). The indicated proteins in 1/3 VBS with 0.67 mM Ca2+ or 6.7 mM EDTA were injected into CRP-, FH- or C1q-coupled flowcell and into a control flowcell (blank). Resonance units are indicated as a function of time. Only bulk effects caused by ligands in solution are seen in the controls. The bulk effects vary according to the inherent properties of the proteins and constructs. A, FH binding to CRP in the presence of Ca2+. B, Inhibition of FH binding to CRP by EDTA and a control containing 0.67 mM Ca2+ in 1/3 VBS but no FH. C, The binding of CRP to surface-bound FH in the presence of Ca2+ and inhibition by EDTA (D). E, The binding of CRP to C1q in the presence of Ca2+. F, In the presence of EDTA, no binding of CRP to C1q occurs.

Close modal

The binding site of CRP on FH was located by using the surface plasmon resonance assay and different recombinant constructs of FH that spanned through the whole molecule. The binding site was first roughly determined by using a recombinant construct of FHL-1, an alternatively spliced product of the FH gene representing SCRs 1–7 and a FH fragment containing SCRs 8–20. Both of these constructs bound to CRP in the presence of 0.67 mM Ca2+ (Fig. 2, A–D). Binding was strictly Ca2+-dependent because in both cases EDTA inhibited the binding. This finding suggested at least two binding sites for CRP on the FH molecule. To further define locations of the CRP binding sites, smaller constructs of FH were used. Neither SCR 1–5 (data not shown) nor SCR 1–6 (Fig. 2,E) showed any reaction with CRP. Therefore, the first binding site for CRP on both FH and FHL-1 was located to SCR 7. In this approach, however, the possibility that both SCR 6 and 7 constitute the binding site cannot be excluded. Next, to map the second site, constructs containing FH SCRs 8–11 and 15–20 were tested. As shown in Fig. 2, F and G, SCR 8–11 bound to CRP in a Ca2+-dependent fashion, but SCR 15–20 showed no binding. The shape of the curve for SCR 15–20 does not indicate binding, as the association did not increase with time but was rather an effect caused by the buffer conditions. Thus, a second binding site was located to a region within SCR domains 8–11 of FH.

FIGURE 2.

Mapping of the CRP binding sites on FH by using recombinant fragments of FH and surface plasmon resonance analysis. The binding site was first located by using SCR constructs 1–7 (FHL-1) and 8–20. A, FHL-1 bound in the presence of 0.67 mM Ca2+ to the CRP-coated flowcell but not to the control flowcell. B, Binding of FHL-1 was totally inhibited by 6.7 mM EDTA. C, Also, SCR 8–20 bound to CRP in a Ca2+-dependent manner but did not bind when EDTA was present (D). The binding sites were further mapped by using smaller constructs. E, SCR 1–6 showed no binding to the CRP-coupled flowcell or the control flowcell. F, SCR 8–11 bound to CRP in the presence of Ca2+. EDTA totally abolished the binding (data not shown). SCR 15–20 (G) did not bind to CRP, as the association does not increase with time but decreases.

FIGURE 2.

Mapping of the CRP binding sites on FH by using recombinant fragments of FH and surface plasmon resonance analysis. The binding site was first located by using SCR constructs 1–7 (FHL-1) and 8–20. A, FHL-1 bound in the presence of 0.67 mM Ca2+ to the CRP-coated flowcell but not to the control flowcell. B, Binding of FHL-1 was totally inhibited by 6.7 mM EDTA. C, Also, SCR 8–20 bound to CRP in a Ca2+-dependent manner but did not bind when EDTA was present (D). The binding sites were further mapped by using smaller constructs. E, SCR 1–6 showed no binding to the CRP-coupled flowcell or the control flowcell. F, SCR 8–11 bound to CRP in the presence of Ca2+. EDTA totally abolished the binding (data not shown). SCR 15–20 (G) did not bind to CRP, as the association does not increase with time but decreases.

Close modal

As heparin has previously been shown to bind to SCR 7 in FH and FHL-1 (24), we wanted to see whether heparin inhibited the binding of CRP to FH. This was first assayed by an ELISA in the presence of 1 mM Ca2+ in VBS (pH 7.4). As shown in Fig. 3, heparin inhibited the binding of FH to CRP in a dose-dependent manner. Under the conditions used, 50% inhibition was achieved at ∼450 IU of heparin per ml.

FIGURE 3.

Inhibition of FH binding to CRP by heparin studied by ELISA. Microtiter plate wells were coated with CRP or BSA, samples with FH (1 μg/ml), and varying amounts of heparin were applied to the wells. The binding of FH is shown as percentage of binding without heparin. Results are mean values ± SEM from three separate experiments performed in duplicate wells.

FIGURE 3.

Inhibition of FH binding to CRP by heparin studied by ELISA. Microtiter plate wells were coated with CRP or BSA, samples with FH (1 μg/ml), and varying amounts of heparin were applied to the wells. The binding of FH is shown as percentage of binding without heparin. Results are mean values ± SEM from three separate experiments performed in duplicate wells.

Close modal

The effect of heparin on the binding of FH, FHL-1, and SCR 8–11 to CRP was also tested using the surface plasmon resonance assay. In accordance with the microtiter plate assay, heparin inhibited the binding of whole FH to CRP (Fig. 4,A). The association phase of the binding reaction was very weak at 170 IU/ml of heparin. A low-m.w. heparin (Fragmin) was also tested. Fragmin, like the unfractionated heparin, reduced the binding of FH to CRP in a dose-dependent manner (Fig. 4 B).

FIGURE 4.

Surface plasmon resonance analysis of the effect of heparin on the binding of FH or its fragments to CRP. FH, SCR 1–7 (FHL-1), and SCR 8–11 in 1/3 VBS with 0.67 mM Ca2+ were injected into a CRP-coupled Biacore flowcell. A, In accordance with the ELISA assay, heparin inhibited the binding of FH. B, The inhibition was also seen with the low-m.w. heparin Fragmin. C, Heparin inhibited dose-dependently the binding of FHL-1 to CRP and less efficiently the binding of SCR 8–11 (D).

FIGURE 4.

Surface plasmon resonance analysis of the effect of heparin on the binding of FH or its fragments to CRP. FH, SCR 1–7 (FHL-1), and SCR 8–11 in 1/3 VBS with 0.67 mM Ca2+ were injected into a CRP-coupled Biacore flowcell. A, In accordance with the ELISA assay, heparin inhibited the binding of FH. B, The inhibition was also seen with the low-m.w. heparin Fragmin. C, Heparin inhibited dose-dependently the binding of FHL-1 to CRP and less efficiently the binding of SCR 8–11 (D).

Close modal

As with FH, the association of FHL-1 with CRP was dose-dependently inhibited by heparin, and no binding could be seen when the heparin concentration reached 170 IU/ml (Fig. 4,C). As FHL-1 in contrast with FH only has one heparin binding site, the binding curves appeared more homogenous. In comparison, we examined the effect of heparin on binding of SCR 8–11 to CRP. As shown in Fig. 4 D, SCR 8–11 binding to CRP was also inhibited by heparin at 30–170 IU/ml. However, the inhibitory effect was not as marked as with FHL-1.

To analyze the possible functional consequences of the CRP-FH interactions, we tested the effects of CRP on FH binding to C3b and cofactor activity for factor I in C3b inactivation.

Biacore 2000 equipment was used for analyzing the effect of fluid phase CRP on FH-C3b interactions. First, we tested whether CRP could bind to surface-associated C3b by itself, but no binding was detected (Fig. 5,A). At a 4-fold molar excess of FH (65 μg/ml) over CRP (12 μg/ml), the latter had no effect on the FH-C3b-interaction (Fig. 5,B). At equimolar concentrations (CRP 52 μg/ml and FH 65 μg/ml), the binding of FH to surface-coupled C3b was slightly affected (Fig. 5,C). At a 4-fold molar excess of CRP (206 μg/ml) over FH (65 μg/ml), a reduction in the binding of FH to C3b was seen (Fig. 5 D).

FIGURE 5.

Biacore analysis of the effect of CRP on the FH-C3b-interaction. A, Lack of direct binding of CRP to C3b-coated chip and to the control flowcell (blank). B, FH in 1/3 VBS with or without CRP was injected into the C3b-coated flowcell. At a 4-fold molar excess of FH over CRP, the binding of FH to C3b was not affected. C, At equimolar concentrations, CRP (52 μg/ml) had a weak inhibitory effect on FH binding to C3b. D, A 4-fold molar excess of fluid phase CRP (206 μg/ml) over FH (65 μg/ml) partially inhibited the binding of FH to surface-associated C3b.

FIGURE 5.

Biacore analysis of the effect of CRP on the FH-C3b-interaction. A, Lack of direct binding of CRP to C3b-coated chip and to the control flowcell (blank). B, FH in 1/3 VBS with or without CRP was injected into the C3b-coated flowcell. At a 4-fold molar excess of FH over CRP, the binding of FH to C3b was not affected. C, At equimolar concentrations, CRP (52 μg/ml) had a weak inhibitory effect on FH binding to C3b. D, A 4-fold molar excess of fluid phase CRP (206 μg/ml) over FH (65 μg/ml) partially inhibited the binding of FH to surface-associated C3b.

Close modal

It has been shown earlier that the cofactor activity of FH for the factor I-mediated cleavage of C3b is located in SCRs 1–4 (12, 13). We wanted to examine whether CRP influenced the cofactor activity of FH in the fluid phase. In the cofactor assay (Fig. 6), it was found that CRP had no intrinsic cofactor activity itself, and, at concentration 10–100 μg/ml, CRP had no effect on the cofactor activity of FH (at 20 μg/ml) in the presence of Ca2+.

FIGURE 6.

Analysis of the fluid phase cofactor activity of FH in the presence of CRP. Radiolabeled C3b (125I-C3b; 80,000 cpm) was incubated (60 min, +37°C) with or without factors H (20 μg/ml) and I (30 μg/ml) and indicated amounts of CRP. The C3b cleavage products were separated by SDS-PAGE under reducing conditions and detected by autoradiography. FH was excluded from the sample in lane 4 to determine the intrinsic cofactor activity of CRP. Lane 3 is the positive control with C3b and factors H and I but without CRP. Lanes 1 and 2 represent negative controls. The mobilities of the α- and β-chains are indicated. The presence of CRP had no effect on the cofactor activity of FH, neither did CRP have any intrinsic cofactor activity. The difference between lanes 3 and 4–7 is due to a slightly different amount of C3b in the original sample.

FIGURE 6.

Analysis of the fluid phase cofactor activity of FH in the presence of CRP. Radiolabeled C3b (125I-C3b; 80,000 cpm) was incubated (60 min, +37°C) with or without factors H (20 μg/ml) and I (30 μg/ml) and indicated amounts of CRP. The C3b cleavage products were separated by SDS-PAGE under reducing conditions and detected by autoradiography. FH was excluded from the sample in lane 4 to determine the intrinsic cofactor activity of CRP. Lane 3 is the positive control with C3b and factors H and I but without CRP. Lanes 1 and 2 represent negative controls. The mobilities of the α- and β-chains are indicated. The presence of CRP had no effect on the cofactor activity of FH, neither did CRP have any intrinsic cofactor activity. The difference between lanes 3 and 4–7 is due to a slightly different amount of C3b in the original sample.

Close modal

CRP is one of the most widely used clinical markers of bacterial infection and severe tissue damage. In striking contrast, the main physiological function of CRP is still under discussion. To establish more firmly a connection between CRP and the complement system, we wanted to study the interactions of CRP with FH, the main regulator of the AP of complement. In the present study, we confirmed our preliminary finding that CRP binds to FH in the presence of Ca2+ (37) and observed that CRP, in fact, has two binding sites on FH. These sites were located to SCRs 7 and 8–11, which are distinct from the main C3b binding sites of FH (15, 16). CRP bound also to SCR 7 of FHL-1, a protein derived from an alternatively spliced transcript of the FH gene. The binding reactions were Ca2+-dependent, and binding to SCR 7 was dose-dependently inhibited by heparin. No significant effect of CRP on C activation in the fluid phase was observed (data not shown).

The direct binding of CRP to FH has been shown previously by radioimmunoassay and ELISA (37, 38). As CRP consists of five identical subunits, it is possible that one CRP molecule can bind simultaneously to multiple FH (or FHL-1) molecules. Importantly, the pentameric nature of CRP also allows it to react with at least two distinct ligands at the same time. Our data located the Ca2+-dependent CRP binding sites to SCR 7 of FH and FHL-1 and to SCRs 8–11 of FH. Because of instability of recombinant fragments representing SCR domains 12–14, we cannot exclude yet another binding site in this region. As FH thus has at least two binding sites for CRP, it could, in principle, be possible for one FH molecule to bind two CRP molecules, too. However, the close proximity of the two CRP binding sites on FH makes it more likely that a single CRP molecule binds to two sites on FH to increase the affinity of the interaction.

In the long FH molecule, the functionally important domains for cofactor activity and decay acceleration (SCRs 1–4) (12, 19) and at least the two major binding sites for C3b at the N and C terminus (16) are distinct from the observed CRP binding sites. In accordance, CRP did not affect the cofactor activity of FH in a fluid phase functional assay. The effect of fluid phase CRP on the binding of FH to solid phase C3b was dependent on the relative concentrations of the different factors. High amounts of CRP relative to FH reduced the binding of FH to C3b. Thus, although fluid phase CRP may not directly bind to the C3b binding sites on FH, it could interfere with the C3b-FH interaction, e.g., by restricting the mobility or access of FH molecules to surface-associated C3b. Alternatively, the CRP and C3b binding sites in the middle part of FH may partially overlap.

Upon its formation, C3b may bind to any nearby surface, including “self” cells. AP nonactivating surfaces are typically coated by polyanions, like sialic acid, heparan sulfate, or other glycosaminoglycans (7, 8, 9, 26). FH may recognize nonactivator surfaces with its heparin binding sites in SCRs 7, 19–20 (24, 25), and possibly in a region near SCR 13 (23). The heparin binding domain SCR 7 is present in both FH and FHL-1 (24). In the present study, we observed that both unfractionated and low-m.w. heparin inhibited dose-dependently the binding of FH, FHL-1 and, to a lesser extent, of SCR 8–11 to CRP. This suggested a common, or at least a partially overlapping, binding site within SCR 7 for heparin and CRP. As a consequence, CRP could affect the recognition function and FH-and FHL-1-mediated control of AP activation and lead to a reduced AP inhibition on normal cells. On the other hand, the binding of CRP to SCRs 8–11 is only weakly inhibited by heparin, indicating that these two interactions are of different nature.

The ability of CRP to bind to phosphocholine and chromatin on one hand and to FH and FHL-1 on the other suggests that a key physiological function of CRP could be to restrict C-mediated inflammation induced by damaged self cells, e.g., in areas of injury or ischemia. During tissue damage, many usually unexposed structures, like phospholipids, mitochondria, and chromatin, become released from injured and apoptotic cells (30). Modified cells and many of the intracellular structures are able to induce an inflammatory response, e.g., through activation of the CP of complement. On the other hand, these structures offer binding sites for CRP, and CRP can itself bind C1q and activate the CP. Sublytic C attack has been shown to reveal binding sites for CRP (44). Recently, CRP was found to colocalize on the surface of infarcted myocardial cells along with C3 and C4 (45). CRP has also been shown to bind to perturbed (“flip-flopped”) cell membranes, where phospholipids, particularly phosphoserine, from the inner membrane leaflet become exposed (30, 46). However, excessive complement activation induces an inflammatory reaction and may lead to increased and unwanted tissue damage. Binding of FH with the help of CRP could inhibit AP amplification and prevent an excessive inflammatory response at a time when clearance has been initiated and healing in general should begin.

Yet an additional consequence of targeting of FH and FHL-1 by CRP to sites of tissue damage is that this, by lowering the level of inhibitors, may allow an enhanced activation of the AP amplification loop in the fluid phase. Such an unleashing of the AP could be useful in temporarily boosting AP-mediated anti-microbial defense. Thus, CRP could act in the prevention of C-mediated damage to self tissues and simultaneously provide stronger AP activity against invading microbes. In the case of aged pneumococci, which expose the CRP-binding C-polysaccharide, binding of FH may, however, inhibit AP activation. Whether pneumococci by this mechanism utilize CRP to evade C attack remains to be investigated.

In conclusion, we have shown in this work that CRP binds to the SCR domain 7 of FH and FHL-1 and domains 8–11 of FH in a calcium-dependent manner. The binding was partially inhibited by heparin. Since binding of CRP did not significantly impair the functional activity of FH, we suggest that the primary function of CRP is to target suppression of AP activation and accelerated inactivation of C3b to iC3b to sites of tissue injury. CRP can redistribute and concentrate FH and FHL-1, e.g., to areas of ischemia where clearance of damaged tissue by CR3-receptor carrying macrophages needs to be enhanced. On the other hand, redistribution of FH away from the fluid phase could allow a temporarily stronger activity of the AP against invading bacteria and other microorganisms during the acute phase reaction.

We thank Pharmacia & Upjohn for the Fragmin used in this study.

1

This work was supported by the Academy of Finland, the Sigrid Jusélius Foundation, the Heart Foundation, and the University of Helsinki. Funding by the Deutsche Forschungsgemeinschaft (Zi 432) is kindly acknowledged.

3

Abbreviations used in this paper: CP, classical pathway of complement; CRP, C-reactive protein; FH, factor H; AP, alternative pathway of complement; SCR, short consensus repeat; FHL-1, FH-like protein 1; VBS, veronal buffered saline.

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