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Published online before print April 9, 2004, 10.1110/ps.03516504
Protein Science (2004), 13:1356-1364. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Structural stability and heat-induced conformational change of two complement inhibitors: C4b-binding protein and factor H

Lena Kask1, Bruno O. Villoutreix2, Mårten Steen1, Bala Ramesh3, Björn Dahlbäck1 and Anna M. Blom1

1 Lund University, The Wallenberg Laboratory, Department of Clinical Chemistry, University Hospital Malmö, S-205 02 Malmö, Sweden
2 French National Institute of Health and Medical Research (INSERM) U428, University Paris V, Paris, France
3 Department of Biochemistry and Molecular Biology, Royal Free and University College Medical School, London NW3 PF, UK

Reprint requests to: Anna Blom, Lund University, Department of Clinical Chemistry, University Hospital Malmö, S-205 02 Malmö, Sweden; e-mail: Anna.Blom{at}klkemi.mas.lu.se; fax: (46) 40-33-70-44.

(RECEIVED November 17, 2003; FINAL REVISION January 23, 2004; ACCEPTED February 1, 2004)


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
The complement inhibitors C4b-binding protein (C4BP) and factor H (FH) both consist of complement control protein (CCP) domains. Here we examined the secondary structure of both proteins by circular dichroism and Fourier-transform infrared technique at temperatures ranging from 30°C–90°C. We found that predominantly {beta}-sheet structure of both proteins was stable up to 70°C, and that a reversible conformational change toward {alpha}-helix was apparent at temperatures ranging from 70°C to 90°C. The ability of both proteins to inhibit complement was not impaired after incubation at 95°C, exposure to extreme pH conditions, and storage at room temperature for several months. Similar remarkable stability was previously observed for vaccinia virus control protein (VCP), which is also composed of CCP domains; it therefore seems to be a general property of CCP-containing proteins. A typical CCP domain has a hydrophobic core, which is wrapped in {beta}-sheets and stabilized by two disulphide bridges. How the CCP domains tolerate harsh conditions is unclear, but it could be due to a combination of high content of prolines, hydrophobic residues, and the presence of two disulphide bridges within each domain. These findings are of interest because CCP-containing complement inhibitors have been proposed as clinical agents to be used to control unwanted complement activation that contributes to many diseases.

Keywords: complement inhibitors; structural stability; C4b-binding protein; factor H

Abbreviations: C4BP, C4b-binding protein • FH, factor H • CCP, complement control protein • CD, circular dichroism • FTIR, Fourier transform-infrared spectroscopy • PT, prothrombin • VCP, vaccinia virus complement control protein

Article published ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03516504.


   Introduction
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 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
The complement system is an important defense mechanism against microbial infections but also enhances acquired immunity and removes waste such as immune complexes and apoptotic cells (Walport 2001a, b). Activation of complement occurs in a cascade manner through three different pathways (the classical, alternative, and lectin routes). Because the complement system is very efficient and potentially harmful to its host, it must be tightly regulated. Thus there are several soluble and membrane-bound inhibitors that work on different levels of complement activation. Six of the inhibitors that work on the C3- and C5-convertase levels are encoded in the regulators of complement activation (RCA) gene cluster (Hourcade et al. 1989). These inhibitors are composed in part or entirely of complement control protein (CCP) domains [also called short consensus repeats (SCRs) or sushi domains].

C4b-binding protein (C4BP) is a soluble inhibitor of the classical pathway of complement. C4BP is a polymeric protein, and its most common form present in plasma consists of seven {alpha}-chains and one {beta}-chain (Hillarp and Dahlbäck 1988). The {alpha}-chain contains eight CCP domains, and the {beta}-chain, three. The chains are connected together by their 60-amino acid C-terminal extensions, which follow the CCP domains. The C-terminal extensions form amphipathic helices and are held together by hydrophobic forces and two disulphide bridges for each chain (Kask et al. 2002). This particular assembly gives the protein a spider-like structure with multiple binding sites for C4b at the N-terminal end of each arm, which can be visualized by electron microscopy (Dahlbäck et al. 1983). C4BP inhibits the classical pathway, as it binds C4b in solution and thereby hinders the formation of the C3-convertase (C2aC4b), the crucial enzymatic complex required for the propagation of the complement cascade. C4BP also binds C4b when it is part of the C3-convertase, and therefore accelerates the natural decay of the complex. Furthermore, upon binding to C4BP, C4b becomes a substrate to the serine protease factor I (FI; Gigli et al. 1979). All {alpha}-chains are not able to bind C4b at the same time because of steric hindrance, but four C4b molecules can bind to one C4BP molecule simultaneously (Ziccardi et al. 1984). Because of this multivalent potency, C4BP down-regulates the classical pathway very effectively. C4BP also binds soluble C3b, a component of the alternative pathway C3-convertase, and presents it to FI for degradation (Blom et al. 2003). Other known ligands of the C4BP {alpha}-chains are heparin (Hessing et al. 1990), M-proteins of Streptococcus pyogenes (Johnsson et al. 1996), porins and pili of Neisseria gonorrhoeae (Blom et al. 2001), hemagglutinin on Bordetella pertussis (Berggard et al. 2001), and outer membrane protein A (ompA) on Escherichia coli (Prasadarao et al. 2002). Recently, {alpha}-chains of C4BP were also shown to bind to CD40 on B cells (Brodeur et al. 2003). Serum amyloid P component (SAP) binds to the central core of C4BP (Garcia de Frutos et al. 1995), and the vitamin K-dependent anticoagulant protein S binds to the {beta}-chain (Hillarp and Dahlbäck 1988).

Factor H (FH) is the major soluble inhibitor of the alternative pathway of complement. It binds C3b and hinders the formation of the alternative C3-convertase (C3bBb); it also accelerates the decay of this convertase and works as a cofactor to FI in the degradation of C3b (Weiler et al. 1976; Farries et al. 1990). Three distinct C3b-binding domains were located in the FH molecule in the N-terminal region, the middle region, and near the C terminus (Jokiranta et al. 2000). Other ligands that bind to FH are heparin (Blackmore et al. 1998), the acute-phase C-reactive protein (Jarva et al. 1999), adrenomedullin (Pio et al. 2001), and bacterial surface proteins and cell surface receptors (Horstmann et al. 1988; Hellwage et al. 1997; DiScipio et al. 1998). FH contains 20 CCP domains and is elongated and flexible, as shown by electron microscopy (DiScipio 1992).

A typical CCP domain is ~60 amino acids long and contains four cysteine residues, which form two disulphide bridges (cysI-cysIII, cysII-cysIV). CCP domains are also characterized by the invariant tryptophan (located between cys III and cys IV), highly conserved prolines, glycines, and hydrophobic residues. The peptide backbone wraps the hydrophobic core with antiparallel {beta}-strands. When CCP domains are arranged in series, they are separated by linkers that are variable in sequence and contain between two and seven residues (Kirkitadze and Barlow 2001).

Smith et al. (2002) investigated the unusual stability of the vaccinia virus complement control protein (VCP; four CCP domains) by exposing it to adverse conditions. In the present study we analyzed the structural and functional integrity of two human complement inhibitors: C4BP purified from plasma, a monomeric {alpha}-chain variant of C4BP (CCP1-8 without the C-terminal extension) and FH. We found that the unusual stability is a common property of CCP-containing proteins, which is an important observation as recombinant complement inhibitors have been proposed as a means of ameliorating the complement-mediated damage associated with a number of diseases.


   Results
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 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 References
 
The CCP-containing proteins show conformational change when heated to 90°C during analysis by FTIR
The proteins that were studied are schematically shown in Figure 1Go and include complement inhibitors C4BP, C4BP CCP1-8, and FH, as well as prothrombin (PT), a zymogen of a serine protease central to coagulation cascade. First, the proteins were analyzed by Fourier transform-infrared spectroscopy (FTIR) at increasing temperatures. At 30°C and 50°C, all CCP-containing proteins showed similar secondary structure corresponding to {beta}-sheet (1635–1640 cm–1; Fig. 2Go), except for polymeric C4BP, which also generated a small {alpha}-helical signal (1658 cm–1) originating from the C-terminal extensions of each {alpha}-chain (Fig. 2AGo). At 70°C, the CCP-containing proteins began to show a shift toward {alpha}-helical structure (1651–1658 cm–1), which became very clear at 90°C. This temperature-induced shift toward {alpha}-helix was reversible, as seen in the spectra when the proteins were cooled down to 30°C (90–30). No significant precipitation or aggregation (1617 cm–1) was observed for the three CCP-containing proteins up to 70°C; however, at 90°C some precipitation was observed for C4BP (Fig. 2AGo) and C4BP CCP1-8 (Fig. 2BGo). At 90°C, the secondary structure was still present and appeared to be the major component in the signal. When another protein composed mainly of {beta}-sheet (PT) was analyzed, a discrete shift toward {alpha}-helix was apparent at 70°C and 90°C, but most importantly a large amount of precipitated/aggregated protein was detectable already at 70°C (Fig. 2DGo).



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  		Figure 1. Schematic representation of the proteins investigated. The major form of C4BP purified from plasma is a polymer of seven {alpha}-chains (eight CCP domains each) and a unique {beta}-chain (three CCP domains) held together by the C-terminal extension. FH purified from plasma is a monomer of 20 CCP domains, whereas C4BP CCP1-8 corresponds to a single arm of C4BP (monomer of eight CCP domains) and was expressed in HEK 293 cells. PT was used as a control protein and is a serine protease with one Gla-domain, and two kringle-domains followed by a serine-protease domain.

 


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  		Figure 2. Changes in secondary structure measured by FTIR. C4BP (A), C4BP CCP1-8 (B), and FH (C) were examined by FTIR at different temperatures. They all showed a high tolerance up to 70°C. At higher temperatures the signal is shifted toward {alpha}-helix. The shift in secondary structure is reversible for C4BP and FH when cooling the protein to 30°C (90–30). PT (D) was used as a control.

 
CD spectroscopy of the CCP-containing proteins shows a temperature-induced shift of the secondary structure from {beta}-sheet toward {alpha}-helix
In the circular dichroism (CD) analysis, both C4BP CCP1-8 and FH showed the typical CCP domain spectra characterized by the presence of a peak at 230 nm generated by the invariant tryptophan (Fig. 3B,CGo). Polymeric C4BP had a slightly different spectrum, as it also generates the {alpha}-helical signal from the C-terminal extensions (Fig. 3AGo). At 30°C and 50°C, there was no difference in the spectrum for the CCP-containing proteins, but at 70°C there was a marked shift toward {alpha}-helical signal, which remained at 90°C (Fig. 3A–CGo). There was a 10% increase of {alpha}-helix in the signal for the CCP-containing proteins when raising the temperature from 30°C to 90°C (Table 1Go). The CD spectra for PT also showed an increase of {alpha}-helical signal when raising the temperature, but to a lesser extent (5%, Fig. 3DGo, Table 1Go).



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  		Figure 3. Changes in secondary structure measured by CD spectrometry. Shown are spectra obtained for C4BP (A), C4BP CCP1-8 (B), FH (C), and PT (D) at various temperatures. Each spectrum was collected six times, and the mean values for each wavelength were calculated.

 

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  		Table 1. Percent {alpha}-helix in the CD-signal at different temperatures
 
The CCP-containing proteins tolerate heating at 95°C for at least 10 min without losing their functional activity
The functional activity of the CCP-containing proteins after heating to 95°C for different time intervals was examined by a functional assay, in which we assessed the ability of each protein to act as a cofactor to the serine protease factor I in the cleavage of activated complement factors C3b or C4b. The cofactor activity can be determined from the amount of degradation fragments of radiolabeled C4b or C3b appearing after incubation. After 10 min of incubation at 95°C, FH (Fig. 4CGo) was as equally good a cofactor as freshly thawed protein. Polymeric C4BP (Fig. 4AGo) and C4BP CCP1-8 (Fig. 4BGo) were somewhat less resistant to heating at 95°C. Even after 20 min at 95°C, cofactor activity was observed for FH (Table 2Go).



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  		Figure 4. C4b/C3b-degradation assay after incubation of cofactor proteins at 95°C. The cofactors were incubated for 3, 5, 10, and 20 min at 95°C and kept at 4°C overnight. They were then mixed with FI, C3met, or C4met and trace amounts of I125-labeled C3b or C4b, incubated for 2 h at 37°C, and separated on a 10%–15% gradient polyacrylamide gel. Separated proteins were visualized by a Phosphor-Imager. The formation of the cleavage products, C4d for C4b and the 68-kD and 43-kD bands for C3b, are the measure of cofactor activity.

 

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  		Table 2. Cofactor activity in degradation assay after harsh treatment
 
The functional activity of PT was tested in a prothrombinase assay, during which PT is converted into thrombin by factor Xa in the presence of cofactors: factor Va and phospholipids. The formation of thrombin was then measured by a chromogenic substrate. PT was heated at 95°C for 3, 5, 10, and 20 min before performing the prothrombinase assay. The activity of the protein decreased down to 23% after 20 min of incubation at 95°C (Fig. 5Go).



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  		Figure 5. Prothrombin activity after heating for different time periods. Prothrombin was analyzed in the prothrombin assay after being subjected to 95°C for 3, 5, 10, and 20 min.

 
C4BP, C4BP CCP1-8, and FH may be left at room temperature for 6 mo without losing their activity
The CCP-containing proteins were left at room temperature for up to 1 mo before performing a degradation assay to test their functional activity. There is no difference in cofactor activity for polymeric C4BP, C4BP CCP1-8 (Fig. 6AGo), and FH (Fig. 6BGo) compared to freshly thawed proteins. The proteins were equally active even after a 6-mo incubation at room temperature (data not shown).



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  		Figure 6. Degradation assay after prolonged incubation of the cofactors at room temperature. C4BP, C4BP CCP1-8 (A), and FH (B) were left at room temperature for 6 h, 24 h, 48 h, 72 h, 1 wk and 1 mo. Their activity was then tested in the degradation assay.

 
The CCP-containing proteins tolerate a wide range of pH conditions
C4BP, C4BP CCP1-8, and FH were subjected to various pH conditions for 30 min. The pH of the solution was reversed to physiological before the proteins were tested in the degradation assay. Polymeric C4BP (Fig. 7AGo), C4BP CCP1-8 (Fig. 7BGo), and FH (Fig. 7CGo) retained their cofactor activity after being subjected to most of the tested conditions. All proteins tolerated treatment by all pH conditions except for pH 13. Even after 30 min of incubation in 3 M HCl, all proteins functioned as well as the untreated protein as cofactors (Table 2Go).



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  		Figure 7. Degradation assay after incubation of the cofactors in solutions with pH ranging from 1 to 13. The proteins were incubated for 30 min at various pH levels, then washed with 50 mM Tris-HCl, 150 mM NaCl (pH 7.4) in concentrators until physiological pH was reached. The proteins were tested for cofactor activity in the degradation assay.

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
Our present study was inspired by the investigation of the structural and functional integrity of the viral complement inhibitor VCP in adverse conditions. VCP was found to be very stable when exposed to high temperatures, prolonged incubation at room temperature, and various pH conditions (Smith et al. 2002). As VCP has the potential to be used clinically to control complement activation, it is favorable that the protein is able to withstand these conditions. However, clinical use of VCP may be prevented by the fact that VCP is a viral protein and may evoke strong immune response and production of neutralizing antibodies. In that respect, fragments of human inhibitors (perhaps with enhanced ability to inhibit complement) would be a better option, and CCP-containing proteins are now in trials as potential therapeutic agents. Because the VCP molecule is composed entirely of CCP domains, we decided to investigate whether the stability is a common property of CCP-containing proteins. We focused our study on two important soluble human complement control proteins: FH and C4BP.

First we used FTIR and CD spectroscopy to analyze the secondary structure of C4BP and FH at various temperatures. The FTIR and CD experiments both showed that C4BP and FH tolerate heating up to 70°C without any change in their secondary structure, which is mainly {beta}-sheet. When increasing the temperature to 90°C, a shift toward {alpha}-helix was seen, which was a 10% increase in the CD-spectroscopy signal. We then studied the functional activity of C4BP and FH as cofactors to FI after being exposed to high temperatures, various pH and room temperature for several weeks. As determined by the degradation assay, most of these conditions were well tolerated by both C4BP and FH. We also analyzed PT to compare the stability of the CCP-containing proteins with another abundant plasma protein. PT is a zymogen of the serine protease thrombin and is composed mostly of {beta}-sheet structure when analyzed by FTIR and CD spectroscopy. The CD spectrum of PT after heating to 90°C showed an increase of {alpha}-helix in the signal but to a lesser extent than that observed for the CCP-containing proteins. Most importantly, a large amount of PT precipitate appeared already at 70°C when the protein was analyzed by FTIR. The activity of PT determined in the prothrombinase assay decreased to 23% after 20 min incubation at 95°C.

Proteins that can tolerate very high temperatures, that is, thermophiles, do not have any particular amino acid sequence or posttranslational modifications different from other proteins. However, some common features have been found to increase the resistance of proteins to high temperature. Increased content of hydrophobic residues with branched side chains, a high amount of prolines, internal packing, loop shortening, metal binding, a high amount of hydrogen-bonds, salt bridges, and glycosylation all raise the melting temperature of a protein (Kumar and Nussinov 2001). All of these determinants are associated with a decrease in flexibility, but flexibility can also increase a protein’s thermal stability and is significant for catalytic activity. The balance between flexibility and rigidity gives the protein its dynamical properties (Fitter et al. 2001). Several of the common features of thermophilic proteins can be found in C4BP and FH. They both have many buried hydrophobic residues inside the CCP domain. Comparing the first eight CCP domains, C4BP has 26% hydrophobic residues and FH has 19% (23% for total FH). C4BP and FH also contain many proline residues. C4BP has been referred to as a prolinerich protein (Matsuguchi et al. 1989) and contains 9% proline residues, compared to 7% in FH and 5% in PT. The two disulphide bridges in each CCP domain may also be a reason for the high stability of these proteins. C4BP contains 6% cysteine residues, FH 7%, and PT 3%. These bridges most likely protect the whole protein from being completely denatured and probably enable the reversible conformational change that we have observed.

The conformational change, from {beta}-sheet to {alpha}-helix, observed clearly for FH and the monomeric variant of C4BP at 90°C was observed previously for immunoglobulins (Vermeer and Norde 2000). This heat-induced conformational change may be ascribed to balancing the energy consumption. Upon denaturation, part of the hydrophobic interior of a protein is exposed to the solution. Instead of losing energy due to the presence of exposed hydrophobic residues, these residues form hydrogen bonds within the polypeptide, which induces the formation of {alpha}-helices. The induction of {alpha}-helices has been experimentally observed for proteins that were absorbed on hydrophobic surfaces (Zoungrana et al. 1997; Vermeer et al. 1998).

It has been proposed that certain residues in a protein can act as gatekeepers and protect them from denaturation (Thirumalai et al. 2003). Otzen et al. (2000) proposed that these residues are often charged and block aggregation by an electrostatic mechanism. Several electropositive clusters were found on the {alpha}-chain of C4BP in a computer-based homology model of C4BP (Villoutreix et al. 1998), which could be the reason for its nonaggregating properties. Similar positively charged clusters were observed for other complement inhibitors, and some of them were proposed to form binding sites for negatively charged regions of C4b and C3b. One explanation for the fact that complement inhibitors are usually composed of a number of CCP domains could be that some of these CCPs position binding sites, some form the actual binding site, and some prevent aggregation.

Unwanted complement activation contributes to pathogenesis of many acute and chronic diseases such as ischemia/reperfusion injury, rheumatoid arthritis, glomerulonephritis, multiple sclerosis, Alzheimer’s disease, and prion disease (Morgan and Walport 1991). There is as yet no complement inhibitor that can be used clinically, but recombinant complement inhibitors have been proposed for such a purpose. Our results concerning the unusual stability of CCP-containing proteins support this strategy.


   Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Expression and purification of proteins
C4BP, FH, and FI were purified from human plasma as described (Crossley and Porter 1980; Dahlbäck 1983; Blom et al. 2003). The monomeric C4BP variant with eight CCP domains of the {alpha}-chain (C4BP CCP1-8), but no C-terminal extension, was constructed by introducing a stop codon after CCP 8 with the Quikchange point mutagenesis kit (Stratagene) using the primer: 5'AAG TGT GAG TGG TAG ACC CCC GAA GGC 3'. The mutant was expressed in human embryonic kidney 293 cells (ATCC no. 1573-CRL). Expressed protein was purified by affinity chromatography using a monoclonal antibody directed against CCP1 of the {alpha}-chain. Recombinant C4BP was used in the CD analyses and in the degradation assay after exposure to high temperature, and was purified as the monomeric variant of the {alpha}-chain. Human prothrombin (PT) was purchased from Kordia, and recombinant factor V was expressed as described (Steen and Dahlbäck 2002).

C4b- and C3b-like molecules (C4met and C3met) were made by incubation of purified C4 or C3 (Andersson et al. 1991) with 100 mM methylamine (pH 8.0) for 2 h at 37°C, and subsequent dialysis against 100 mM Tris-HCl (pH 7.5), 150 mM NaCl. C3b and C4b were purchased from Advanced Research Technologies.

Fourier transform-infrared (FTIR) analysis of temperature sensitivity
Infrared spectra of C4BP (60 mg/mL), the monomeric C4BP mutant (C4BP CCP1-8, 36 mg/mL), FH (50 mg/mL), and PT (105 mg/mL) were recorded on a Perkin-Elmer 1750 Fourier transform infrared spectrometer equipped with a triglycine sulphate detector. The proteins were dialysed against deionized H2O and freeze-dried. Before the proteins were analyzed, they were reconstituted in deuterated buffer (0.01M PBS/D2O, pD 7.4). Spectral data were acquired from a 10-µL volume gas-tight CaF2 cell (path length 6 µm). The spectrometer was continuously purged with dry air to eliminate water vapor absorptions from the spectral region of interest. For each sample, 200 scans were signal-averaged at a resolution of 4 cm–1 and at 30°, 50°, 70°, and 90°C and at 40°, 60°, and 80°C (data not shown). The proteins were cooled down to 30°C directly after exposure to 90°C. Absorbance spectra of the samples were obtained by digital subtraction of a spectrum of PBS/D2O buffer recorded under identical conditions to that of the sample spectrum, so that a straight baseline was observed in the 2100–1800 cm–1 region. Detailed analysis of the amide I band was carried out using a second derivative procedure. Second derivative spectra were calculated using GRAMS Derivative function with a 13-data point Savitzky-Golay smoothing window (Grams Research Paragon 1000 FTIR, version 3.01A Level II, Driver Version 1.03.)

Circular dichroism (CD) analysis of temperature sensitivity
FH was dialysed against 10 mM Na-phosphate (pH 7.4). To avoid precipitation, C4BP CCP1-8 was dialysed against 50 mM NaF, and C4BP against 150 mM NaF. CD spectra were recorded (C4BP 0.13 mg/mL, C4BP CCP1-8 0.53 mg/mL, FH 0.24 mg/mL, PT 0.96 mg/mL) on a J-720 spectropolarimeter from Jasco at 30°, 50°, 70°, and 90°C. Quartz cuvettes (from Hellma) with a 1-mm path length were used. All spectra were recorded from 250 nm to 190 nm with a scan-speed of 20 nm/min, response time 8 sec, and a step resolution of 1 nm. To improve the signal-to-noise ratio, the spectrum was accumulated six times.

The amount of {alpha}-helix in the signal was evaluated by the method of Scholtz et al. (1991), which focuses on the ellipticity measured at 222 nm.

Degradation assay
Four hundred nM C4met was mixed with plasma-purified C4BP (0.44 µM) or C4BP CCP1-8 (3.4 µM); 1.1 µM of C3met was mixed with FH (1.2 µM). Trace amounts of I125-labeled C4b or C3b were then added, followed by 0.19 µM factor I. The mixture was incubated at 37°C for 2 h and terminated by the addition of sample buffer (0.5 M Tris-HCl [pH 6.8], 15% glycerol, 0.005% Coomassie G-250, 4% SDS) for electrophoresis with reducing agent (dithiothreitol, 10 mM). The samples were incubated at 95°C for 3 min and applied to a 10%–15% SDS gradient gel. The proteins were visualized using a PhosphorImager (Molecular Dynamics/Amersham Pharmacia Biotech), and the bands were quantified with ImageQuant 1.11.

Temperature sensitivity
The proteins were kept in Eppendorf tubes in a heating block at 95°C for 3, 5, 10, and 20 min and thereafter left in at 4°C overnight. Wild-type C4BP (0.44 µM), C4BP CCP1-8 (3.4 µM), and FH (1.2 µM) were then tested in the degradation assay.

Prothrombinase assay
Prothrombin (6.25 µM in 50 mM Tris-HCl, 150 mM NaCl [pH 7.4]) was kept at 95°C for 3, 5, 10, and 20 min. The conditions for the prothrombinase assay were 50 µM of small unilamellar phospholipid vesicles (PS/PC, 10/90, mol/mol), FXa (5 nM), and prothrombin (0.25 µM, which is around Km; Steen et al. 2002). Prothrombin, FXa, and phospholipid vesicles were preincubated at 37°C, and the thrombin generation was started by the addition of preheated FVa. The reactions were stopped after 1 min by 1/40 dilution in ice-cold EDTA buffer. The generated thrombin was quantified using the chromogenic substrate S-2238.

Shelf-life
C4BP (1.2 mg/mL), C4BP CCP1-8 (0.71 mg/mL), and FH (0.5 mg/mL) in 50 mM Tris-HCl, 150 mM NaCl (pH 7.4) were stored at room temperature in Eppendorf tubes. After 6 h, 24 h, 48 h, 72 h, 1 wk, 1 mo, and 6 mo, aliquots (4.2 µL, 7 µL, and 9 µL, respectively) were taken and stored at –20°C until tested in the degradation assay.

pH sensitivity
Aliquots of C4BP, C4BP CCP1-8, and FH were incubated in Eppendorf tubes in 1 mL of water adjusted to the desired pH (1, 3, 5, 7, 9, 11, 13) or 3M of HCl for 30 min at room temperature. The samples were then transferred to concentrators (Amicon, cut-off 30 kD for C4BP and FH and 10 kD for C4BP CCP1-8) and washed with 50 mM Tris-HCl, 150 mM NaCl, pH 7.4 until physiological pH was reached. Aliquots of C4BP (0.44 µM), C4BP CCP1-8 (3.4 µM), and FH (1.2 3M) were then tested in the degradation assay. The fresh sample was treated the same way.


   Acknowledgments
 
We thank Sara Linse (Lund Institute of Technology) for assistance with the CD spectroscopy. This study was supported by grants from the Swedish Research Council, Greta and Johan Kock’s Trust, Österlunds Trust, Crafoord Trust, Royal Physiographic Society in Lund, Clas Groschinsky’s Trust, King Gustav V’s 80th Anniversary Foundation, Thelma Zoegas Trust, Professor Nanna Svartz Trust, Magnus Bergvalls Trust, Albert Påhlsson’s Trust, and research grants from the University Hospital in Malmö.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Andersson, M., Hanson, A., Englund, G., and Dahlbäck, B. 1991. Inhibition of complement components C3 and C4 by cadralazine and its active metabolite. Eur. J. Clin. Pharmacol. 40: 261–265.[CrossRef][Medline]

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A. P. Sjoberg, L. A. Trouw, F. D. G. McGrath, C. E. Hack, and A. M. Blom
Regulation of complement activation by C-reactive protein: targeting of the inhibitory activity of c4b-binding protein.
J. Immunol., June 15, 2006; 176(12): 7612 - 7620.
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