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*
Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki and the Helsinki University Central Hospital, Helsinki, Finland; and
Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 analysis of CRP-FH interactions
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.
Microtiter plate binding assays
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).
Cofactor assay for C3b inactivation
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% ß-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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1B).
Ca2+ by itself gave only a weak signal (Fig. 1B). The reverse binding of fluid-phase CRP to
surface-coupled FH was also observed in the presence of
Ca2+ (Fig. 1C), and EDTA inhibited
this binding (Fig. 1D). 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).
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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. 2E) 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.
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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.
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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. 4B).
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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. 4D,
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. The effects of CRP on FH function
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. 5A). 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. 5B). At equimolar
concentrations (CRP 52 µg/ml and FH 65 µg/ml), the binding of FH to
surface-coupled C3b was slightly affected (Fig. 5C). 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. 5D).
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), 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+.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hanna Jarva, Haartman Institute, P.O. Box 21 (Haartmaninkatu 3), FIN-00014 University of Helsinki, Helsinki, Finland. E-mail:
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.
Received for publication March 17, 1999. Accepted for publication July 22, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 H. K. Meier-Ewert, P. M. Ridker, N. Rifai, N. Price, D. F. Dinges, and J. M. Mullington Absence of Diurnal Variation of C-Reactive Protein Concentrations in Healthy Human Subjects Clin. Chem., March 1, 2001; 47(3): 426 - 430. [Abstract] [Full Text] [PDF]
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- and ß-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.