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. 2007 Mar;120(3):404–411. doi: 10.1111/j.1365-2567.2006.02516.x

Structural and functional comparison of native pentameric, denatured monomeric and biotinylated C-reactive protein

Karolina E Taylor 1, Carmen W van den Berg 1
PMCID: PMC2265887  PMID: 17163961

Abstract

There are many controversies surrounding the biological activities of native C-reactive protein (nCRP) and its various modified forms such as monomerized and biotinylated CRP (mCRP and bCRP). No simple methods have been described to distinguish among these forms. By adapting established electrophoresis methods, we have developed a useful quality control method with which we have investigated the structural and functional characteristics of these forms of CRP. Under all electrophoresis conditions, biotinylation altered the electrophoretic mobility of CRP. nCRP was sensitive to sodium dodecyl sulphate (SDS)-induced monomerization, and only mCRP was susceptible to digestion by trypsin or neutrophil-derived serine proteases. bCRP and mCRP but not nCRP bound to cells, suggesting that chemical modification by biotin and denaturation had altered the structural integrity of CRP. Neither nCRP nor mCRP had the ability to induce secretion of chemokines, nor did they increase intracellular adhesion molecule 1 (ICAM-1) expression in endothelial cells.

Keywords: C-reactive protein, endothelial cells, polyacrylamide electrophoresis

Introduction

C-reactive protein (CRP) is an acute-phase protein which can have both pro- and anti-inflammatory effects.1 CRP is a member of the pentraxin family of proteins and in its native form (nCRP) is composed of five identical subunits, non-covalently linked to form a symmetrical disc of approximately 115 kDa.2 One side of the pentamer participates in binding ligands such as phosphorylcholine (PC), and the other side binds effector molecules such as C1q.3,4 nCRP can dissociate from its pentameric form into its monomeric subunits (mCRP) under a variety of conditions including low pH,5 absence of calcium,6 increased temperature5 and urea chelation.7

There are many controversies surrounding certain areas of CRP biology, including whether or not CRP plays a role in cardiovascular disease (reviewed by Scirica and Morrow8), which is the active form (nCRP or mCRP) and whether or not nCRP binds to the cell surface. nCRP was reported to directly activate human endothelial cells by inducing adhesion molecule expression.9 However, we and others have since then shown that many of the effects attributed to nCRP were caused by contaminating artefacts, such as lipopolysaccharide (LPS) and sodium azide, in commercial CRP preparations.10–15 Recently, Khreiss et al. reported that mCRP but not nCRP was the inducer of endothelial cell activation;16 however, Devaraj et al. reported the reverse.17 Monoclonal antibodies (mAbs) have been described that recognize specific epitopes only expressed on the dissociated CRP;7 however, these antibodies are not commercially available. In some studies, CRP has been labelled with, for example, biotin and it is important to establish that such modifications have not altered the characteristics of CRP, for example binding to ligands and cells. When using methods that modify (e.g. by biotinylation), monomerize (e.g. by heating or urea/ethylenediaminetetraacetic acid (EDTA) chelation) or degrade (e.g. by use of trypsin or neutrophil-derived enzymes) CRP, it is necessary to be able to validate the identity of the resulting protein. We therefore developed simple methods to distinguish modified CRP from nCRP. We investigated how temperature affects dissociation of nCRP and studied the enzyme susceptibility and binding characteristics of nCRP, mCRP and biotinylated CRP (bCRP). Furthermore, we assessed the biological consequences of CRP modifications on interaction of CRP with endothelial cells.

Materials and methods

Reagents and buffers

Phorbol myristic acid (PMA), phenylmethylsulphonyl fluoride (PMSF), 4-(2-aminoethyl)benzene sulfonyl fluoride hydrochloride (AEBSF), 1,10-phenanthroline, E64 and monoclonal anti-CRP (CRP8) were obtained from Sigma (Poole, UK), and Ez-Link-Sulfo-NHS-LC-Biotin was obtained from Pierce (Tattenhall, UK). Tumour necrosis factor (TNF)-α was obtained from Peprotech (London, UK). Elastase inhibitor II, commercial recombinant CRP and rabbit anti-CRP were obtained from Calbiochem (Nottingham, UK), 2·5% trypsin and Streptavidin Alexa Fluor-488 (Strep-Al488) were obtained from Invitrogen (Paisley, UK), goat anti-mouse immunoglobulin G (IgG)-horse radish peroxidase (HRPO) (GAM-HRPO) and goat anti-rabbit IgG-HRPO (GAR-HRPO) were obtained from Stratech (Soham, UK), and goat anti-mouse IgG-fluorescein isothiocyanate (FITC) (GAM-FITC) was obtained from Dako (Ely, UK). Buffers used were phosphate-buffered saline (PBS), which consisted of 3 mm Na2HPO4, 2·5 mm Na2HPO4 and 145 mm NaCl, pH 7·4; fluorescence-activated cell sorter (FACS) buffer, which consisted of 1% bovine serum albumin (BSA) and 0·1% NaN3 in PBS; cell binding buffer, which consisted of 20 mm Tris, 140 mm NaCl, 2 mm CaCl2, 1% BSA and 0·01% NaN3, pH 7·5; storage buffer, which consisted of 20 mm Tris, 140 mm NaCl and 2 mm CaCl2, pH 7·5; and EDTA buffer, which consisted of 20 mm Tris, 140 mm NaCl and 10 mm EDTA, pH 7·5.

Cell culture

Human umbilical vein endothelial cells (HUVEC) were harvested and cultured as described previously.13

Modifications of native CRP

Recombinant human CRP was purified from tissue culture supernatants of CHO cells transfected with the cDNA encoding human CRP as described previously.11

Monomerization

mCRP was generated by urea/EDTA chelation of nCRP as described previously.7 mCRP was also generated by heat treatment, which involved heating nCRP (1 mg/ml in storage buffer) at 70° for 1 hr. The two mCRPs were structurally and functionally indistinguishable.7

Biotinylation

nCRP in PBS was biotinylated according to the manufacturer's instructions (Pierce) using a 20-fold molar biotin excess.

Digestion by neutrophil-derived enzymes

Neutrophils were isolated as described previously.18 Neutrophil protease preparation and proteolysis of CRP were carried out according to the methods of Shephard et al.19 Neutrophils (4 × 107 cells/ml Krebs/HEPES buffer18) were freeze-thawed (−20°) three times, or incubated in the presence of PMA (10 ng/ml; 30 min at 37°) to release granule constituents. Neutrophils were removed by centrifugation. Cell lysates and enzyme-rich cell-free supernatant (S/N), produced from 5 × 105 neutrophils, were incubated (18 hr at 37°) with CRP (5 µg), in a total volume of 25 µl (in storage buffer). For inhibition experiments, 10 mm EDTA, 10 µm 1,10-phenanthroline, 10 µm PMSF, 1·2 mm AEBSF, 10 µm E64 and 50 µg/ml elastase inhibitor II were added.

Trypsin digestion

nCRP and mCRP, at 50 µg/ml in storage buffer, were incubated (1 hr at 37°) with trypsin at various molar ratios (calculated using the molarity of the mCRP).

Polyacrylamide gel electrophoresis

Polyacrylamide gels were prepared according to the method of Laemmli.20 For SDS-containing gels 12·5% gels were used, and for native polyacrylamide electrophoresis (PAGE) 10% gels were used. Protein samples were mixed with an equal volume of sample buffer. Samples were only heated (10 min at 90°) when indicated. For denaturing SDS-PAGE, sample buffer was supplemented with 10 mm dithiothreitol (DTT). In some experiments SDS levels in all solutions were reduced to 1/20th of normal levels. For native electrophoresis, SDS was omitted from all solutions. Electrophoresis was carried out at 30 mA/0·75 mm gel in a Hoeffer MightySmall (Amersham, Little Chalfont, UK) electrophoresis unit using tap water for cooling. Proteins were visualized using Coomassie brilliant blue staining or western blotting.

Western blotting

After electrophoresis, proteins were transferred onto nitrocellulose, blocked with 5% milk/PBS and incubated with polyclonal or monoclonal anti-CRP (both 1/1000), followed by GAM-HRPO or GAR-HRPO (1/1000 in 5% milk/PBS), and developed with Pierce ECL Western Blotting Substrate (Pierce). Blots were exposed to CL-Xposure film (Pierce).

CRP binding to cells

HUVEC, at 107 cells/ml in cell-binding buffer, were incubated with CRP or buffer only, for 30 min at 37°. All subsequent incubations and wash steps were carried out in cell-binding buffer at 4°. Cells were washed, resuspended at 3 × 106 cells/ml and incubated with mAb anti-CRP (5 µg/ml) followed by GAM-FITC (1/100) or Strep-Al488 (1/100), and fixed in 1% paraformaldehyde/PBS and analysed on a FACSCalibur (Becton Dickinson, San Jose, CA).

Incubation of HUVEC with CRP and analysis of cell activation

HUVEC were incubated for 24 hr with culture media supplemented with CRP from different sources or buffer control as described previously.13 HUVEC were subsequently analysed for surface intracellular adhesion molecule 1 (ICAM-1) expression using flow cytometry and cell media interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 content was measured by enzyme-linked immunosorbent assay (ELISA), as described previously.13

Results

Electrophoretic mobility of pentameric, monomeric and biotinylated CRP

Migration of nCRP, bCRP and mCRP under different electrophoretic conditions was compared using methods based on the SDS-PAGE method described by Laemmli.20 When the sample heating was omitted, nCRP ran as a broad smear with an apparent molecular weight of 30–80 kDa, while mCRP ran at a relative molecular mass (Mr) of 23 kDa, close to its expected molecular weight (Fig. 1a). Heating the samples to 90° resulted in denaturation of the nCRP and a Mr similar to that of mCRP was observed (Fig. 1b). Using 1/20th SDS in all buffers compared with the original method resulted in apparent Mr values of 150 kDa for nCRP and 50 kDa for mCRP (Fig. 1c). Under native conditions, nCRP ran at a high Mr; however, mCRP could not be detected (Fig. 1d). When nCRP was biotinylated, its electrophoretic mobility changed under most conditions tested. bCRP ran at a higher Mr than nCRP and mCRP, irrespective of whether the sample was heated or not (Figs 1a and b). Under conditions in which less (Fig. 1c) or no (Fig. 1d) SDS was used, bCRP ran at a lower Mr than nCRP. A small amount of protein with a lower Mr (Fig. 1a) or higher Mr (Fig. 1b–d) was observed in the bCRP preparations, which was more pronounced after detection by western blotting (Fig. 3c). The change in electrophoretic mobility of bCRP was dependent on the amount of biotin used (not shown). Only under completely denaturing conditions did all CRP preparations run at a similar Mr of 23 kDa (Fig. 1e).

Figure 1.

Figure 1

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Analysis of native, biotinylated and urea/ethylenediaminetetraacetic acid (EDTA)-chelated C-reactive protein (CRP) under different electrophoretic conditions. Native CRP (N), biotinylated CRP (B) and monomerized CRP (M), mixed with sample buffer, and with or without heating or reduction, as indicated, were subjected to polyacrylamide electrophoresis (PAGE) [with or without sodium dodecyl sulphate (SDS) (standard SDS PAGE conditions) or with 1/20th SDS (1/20 of standard SDS) as indicated]. Gels were stained with Coomassie brilliant blue. (a) Not heated, + SDS; (b) heated, + SDS; (c) not heated, 1/20th SDS; (d) not heated, no SDS; (e) heated, reduced, + SDS. Results are representative of at least three experiments.

Figure 3.

Figure 3

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Susceptibility of C-reactive protein (CRP) forms to enzymatic digestion. Native CRP (a) and monomerized CRP (b; generated by heat treatment) at 50 µg/ml were incubated for 1 hr at 37° with different trypsin:CRP molar ratios (as indicated) in the presence of 2 mm CaCl2. (c) Native CRP (N), biotinylated CRP (B) and monomerized CRP (M; generated by heat treatment) were incubated with freeze/thaw (F) or phorbol myristic acid (PMA) (P)-activated neutrophil supernatants, or buffer (C) for 18 hr at 37°. (d) Monomerized CRP, generated by heat treatment, was incubated with the supernatant of PMA-stimulated neutrophils in the presence or absence of various inhibitors for 18 hr at 37°. Lane 1, monomerized CRP control incubated in the absence of enzymes. Other lanes, neutrophil supernatants plus: (lane 2) buffer control; (lane 3) 10 mm ethylenediaminetetraacetic acid (EDTA); (lane 4) 10 µm 1,10-phenanthroline; (lane 5) 10 µm phenylmethylsulphonyl fluoride (PMSF); (lane 6) 1·2 mm 4-(2-aminoethyl)benzene sulfonyl fluoride hydrochloride (AEBSF); (lane 7) 10 µm E64; (lane 8) 50 µg/ml elastase inhibitor II. Proteins were analysed on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) (without heating) followed by western blot analysis using a monoclonal (a, b) or a polyclonal (c, d) anti-CRP antibody. Results are representative of at least three experiments.

Effect of heat on stability of CRP

CRP was subjected to heating at different temperatures using thin-walled polymerase chain reaction (PCR) tubes and a PCR machine with a peltier heating block. Analysis by SDS-PAGE with 1/20th SDS showed that nCRP was largely resistant to monomerization at 65°, and only at 70° was nearly all nCRP monomerized (Fig. 2a). A time–course of nCRP heating at 70° showed that CRP monomerization had already begun within 10 min of incubation, but only neared completion after 1 hr (Fig. 2b).

Figure 2.

Figure 2

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Heat stability of C-reactive protein (CRP). Native CRP (100 µg/ml) was incubated at different temperatures in a polymerase chain reaction (PCR) machine with a peltier heating block for 1 hr (a) or at 70° for various periods of time (b), mixed with sample buffer and, without heating, run on polyacrylamide electrophoresis (PAGE) gels containing 1/20th sodium dodecyl sulphate (SDS). Gels were stained with Coomassie brilliant blue. Results are representative of at least three experiments.

Only monomeric CRP is sensitive to enzymatic digestion

The sensitivity of nCRP and mCRP to tryptic digestion was assessed and, as shown in Fig. 3(a), nCRP was nearly completely resistant to tryptic digestion, while mCRP could be completely digested although a very high trypsin:CRP ratio was required (Fig. 3b). The nCRP preparation used in this experiment also contained a small amount of mCRP, which was digested during the incubation.

Neutrophils are a source of many proteolytic enzymes and it has been shown that neutrophil-derived enzymes are capable of digesting 125I-labelled CRP.19 nCRP, bCRP and mCRP were exposed to freeze/thaw lysates of neutrophils and supernatants of neutrophils stimulated with the phorbol ester PMA. nCRP and bCRP were resistant to neutrophil-derived proteolytic enzymes, while mCRP was digested by neutrophil cell lysates and by supernatants of PMA-stimulated neutrophils but not by supernatants of neutrophils incubated in buffer alone (Fig. 3c). Addition of protease inhibitors showed that EDTA and the serine esterase inhibitors PMSF and AEBSF and also the specific neutrophil elastase inhibitor II inhibited the degradation of mCRP (Fig. 3d).

Native CRP does not bind to HUVEC

The ability of CRP to bind to human umbilical vein endothelial cells (HUVEC) was investigated by flow cytometry. No binding of nCRP (Fig. 4a) to HUVEC could be detected; however, both mCRP (Fig. 4b) and bCRP (Figs 4c and d) bound to the cells. Binding of bCRP was detected using antibodies specific for CRP (Fig. 4c) as well as Alexa488-labelled streptavidin (Fig. 4d).

Figure 4.

Figure 4

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Comparison of native, biotinylated and monomeric C-reactive protein (CRP) binding to human umbilical cord endothelial cells (HUVEC). HUVEC were incubated with different forms of CRP (100 µg/ml): (a) native CRP; (b) monomeric CRP [generated by urea/ethylenediaminetetraacetic acid (EDTA) chelation]; (c) and (d) biotinylated CRP. Cells were washed and analysed by flow cytometry for the binding of CRP using a monoclonal anti-CRP antibody and goat anti-mouse immunoglobulin fluorescein isothiocyanate (GAM-FITC) (a–c) or Strep-Al488 (d). Results are expressed as fluorescence histograms, with thin, medium and thick lines representing background fluorescence, buffer control and cells incubated with CRP, respectively. Results are representative of at least three experiments carried out in triplicate.

Neither native nor monomeric CRP activates endothelial cells

The ability of CRP monomerized by urea/EDTA chelation to induce secretion of the chemokines IL-8 and MCP-1 and induce the expression of the adhesion molecule ICAM-1 in HUVEC was assessed. As shown in Fig. 5, neither mCRP nor nCRP induced secretion of these chemokines, nor did they induce ICAM-1. These cell activation events could only be induced by undialysed commercial CRP preparations (Cal); these events are not caused by CRP itself, but, as we have previously shown, are caused by contaminants, including azide and endotoxin, of these commercial CRP preparations.13

Figure 5.

Figure 5

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Comparison of the effects of C-reactive protein (CRP) from different sources on human umbilical cord endothelial cell (HUVEC) activation. HUVEC were incubated with CRP buffer control, 10 ng/ml tumour necrosis factor (TNF)-α, or 100 µg/ml of native CRP (nCRP), monomerized CRP (mCRP) [urea/ethylenediaminetetraacetic acid (EDTA)-chelated], Calbiochem recombinant CRP (Cal) or dialysed Calbiochem CRP (DCal). After 24 hr, cell culture media were analysed for interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 content by enzyme-linked immunosorbent assay (ELISA) and HUVEC were analysed for intracellular adhesion molecule (ICAM)-1 expression by flow cytometry. Results are expressed as mean IL-8 or MCP-1 concentrations or median fluorescence intensities (MFI) ± standard error of the mean for experiments carried out in triplicate. Results are representative of at least three experiments. Data points marked with asterisks were significantly different from the control treatment (P < 0·001).

Discussion

We have adapted PAGE conditions to distinguish among native, monomeric and biotinylated CRP. By omitting the sample heating step and reducing the percentage of SDS in all buffers to 1/20th of the amount described in the original method,20 nCRP and mCRP could easily be visualized and distinguished. Under these conditions, nCRP ran as a tight band, while under normal SDS conditions nCRP ran as a smear, suggesting partial denaturation by SDS. Nearly all electrophoresis conditions showed that biotinylation of CRP modified its electrophoretic mobility (Fig. 1), suggesting that a structural alteration had occurred. Biotinylation also generated some CRP species with an altered Mr; the nature of these products is unknown but they may represent partially denatured CRP, as they were susceptible to enzymatic digestion. Under native PAGE conditions, mCRP could not be detected, possibly because of insolubility problems. Standard SDS-PAGE techniques have been employed previously to compare nCRP and mCRP,7 nCRP and FITC-conjugated CRP21 and different forms of mCRP,5,22 but all forms of CRP displayed identical protein bands with Mr between 22 and 25 kDa. However, in these studies, CRP samples were all heated prior to analysis and, as we have shown here, sample heating negates any difference otherwise observed. None of the electrophoresis conditions gave a near accurate Mr for nCRP; under the 1/20th SDS or native conditions, the charge of the protein itself may affect electrophoretic behaviour and molecular weight markers and CRP preparations may not run according to their real molecular weight.

CRP was found to be relatively resistant to heat denaturation and only completely monomerized after 1 hr of incubation at 70° (Fig. 2). While we carried out the heat-induced monomerization in the presence of calcium, a previous study reported that denaturation had already occurred after 5 min of incubation at 63°;5 however, this study was carried out in the absence of calcium, which will make CRP more unstable.6 In one study, a commercial calcium-containing CRP preparation was heated for 1 hr at 65° to demonstrate the validity of the results;23 however, denaturation of this CRP preparation was not verified and our results suggest that heating at 65° would be insufficient to cause nCRP degradation.

Several studies have investigated the susceptibility of CRP to enzymatic digestion24–26 and, while plasma calcium concentrations were typically above 2 mm, some of these studies were carried out in the absence of calcium.19,27–29 We have shown here that nCRP is very resistant to enzymatic digestion (Fig. 3a); mCRP, however, was susceptible to digestion by neutrophil-derived enzymes (Fig. 3c) and to trypsin digestion, but only at a very high CRP:trypsin molar ratio (Fig. 3b). This corroborates findings by Ying et al., who showed that CRP degradation products were generated by the action of trypsin only after heat/SDS denaturation of CRP.29 In several in vitro studies, the authors mention the use of trypsinization of commercial calcium-containing nCRP preparations as a control to demonstrate that the effects they observed on endothelial cells were attributable to nCRP.30,31 Here we have shown that nCRP in the presence of 2 mm CaCl2 (as provided by commercial suppliers) is resistant to tryptic digestion, which suggests that trypsinization of nCRP as a control procedure to remove its activity is an unsound method. Shepard et al. showed that neutrophil lysates and supernatants of phorbol ester-stimulated neutrophils were able to digest calcium-free 125I-labelled nCRP.32 Our data show that calcium-containing nCRP preparations are not susceptible to degradation by neutrophil-derived enzymes (Fig. 3c) and that only mCRP and low Mr forms of bCRP can be digested. Shepard et al.'s method of iodination may have resulted in destabilization of nCRP, and this together with the absence of calcium in their experiments may have made their CRP more susceptible to enzymatic cleavage. Shephard et al. identified the neutrophil-derived enzymes responsible for the degradation of CRP as serine proteases,19 while Ying et al. showed that nCRP in a calcium-containing buffer was very resistant to enzymatic cleavage, and observed some digestion of CRP by purified neutrophil-derived elastase in the presence of calcium and only at a very high CRP:elastase ratio (2:1).29 We have shown here that the serine proteases PMSF and AEBSF and the neutrophil-specific elastase II inhibitor inhibited the cleavage of mCRP, strongly suggesting that the enzyme responsible is elastase. EDTA also prevented the degradation of mCRP by neutrophil-derived enzymes, which is consistent with the observation that elastase requires calcium to function.33 Shepard et al. showed that neutrophil-derived enzymatic cleavage of CRP resulted in fragments with biological activities; however, commonly a 30-fold molar excess over nCRP was required to induce cell activation.34,35 Considering that only mCRP is susceptible to enzymatic degradation, it is questionable if in vivo sufficient levels of peptides could be generated to have any biological effect.

Using flow cytometry, we found that bCRP and mCRP but not nCRP bound to HUVEC. Binding of bCRP to HUVEC is probably an artefact of the biotin-induced modification of CRP. The binding of urea/EDTA-chelated mCRP to filamentous structures in the cell cytoplasm of fixed endothelial cells has previously been reported.36 We have shown for the first time that urea/EDTA-chelated mCRP also binds to the cell surface. When the binding of nCRP to cells is investigated, it is therefore very important to check that the nCRP does not contain a small amount of mCRP, which would yield artefactual results. Using the PAGE methods described in Fig. 1, the presence of mCRP in nCRP preparations could be easily detected. It has been reported that mCRP binds the Fc receptor CD16;37 however, CD16 is not expressed by HUVEC, and it remains to be determined whether mCRP and bCRP bind to a particular cell surface molecule. We have shown here that biotin modifies the structural and binding characteristics of nCRP and thus is unsuitable to use as an nCRP labelling reagent, and research using bCRP should be interpreted with caution. This may also have consequences for studies in which other chemical modifications of CRP have been used to investigate the binding and biological properties of CRP.

Two studies have compared the ability of nCRP and mCRP to activate endothelial cells but reported conflicting results. While Khreiss et al. reported that mCRP was more potent than nCRP in endothelial cell activation,16 in a similar study Devaraj et al. showed the reverse.17 However, we demonstrated here that mCRP, like nCRP, had no effect on IL-8 and MCP-1 release and ICAM-1 expression by HUVEC (Fig. 5). We and others previously showed that contamination with endotoxin and possibly other bacterial products is responsible for many effects on human endothelial cells attributed to CRP.10,13 The most likely explanation for the discrepancies between the studies by Khreiss et al. and Devaraj et al. and our study is contamination of their CRP preparations with bacterial products. The mCRP preparation of Khreiss et al. was expressed in Escherichia coli and was only 95% pure.22 A higher level of contamination with endotoxin or other bacterial products of their mCRP compared with their nCRP may have been responsible for their observations. Similarly, Devaraj et al. used nCRP expressed in E. coli, which, to generate mCRP, they subsequently urea/EDTA-chelated and extensively dialysed. We have shown that extensive dialysis of commercial recombinant CRP preparation results in a loss of endothelial cell activating ability (see Taylor et al.13 and Fig. 5). Extensive dialysis of the mCRP used by Devaraj et al. to remove the urea and EDTA may thus similarly have resulted in loss of contaminating agents and this CRP preparation may therefore have lost its ability to activate endothelial cells. A recent paper by Oroszlan et al.38 showed that neither azide-free commercial nCRP (from human serum) nor recombinant monomeric CRP was able to induce cell activation events (including ICAM-1 expression and IL-8 secretion), which corroborates our findings.

In conclusion, we have carried out a comprehensive investigation of the structural and functional aspects of native pentameric, denatured monomeric and biotinylated CRPs. We have developed simple methods that can distinguish among the different forms of CRP, which provide a readily available quality control tool to assess the monomerization, denaturation or enzymatic inactivation of CRP, which is useful for studies in which inactivated CRP is required as a control reagent or in which the biological activities of native, denatured or modified CRP are investigated. Modification of nCRP with biotin, urea or heat affected its electrophoretic mobility and increased its ability to bind to cells but none of the forms of CRP has the ability to activate endothelial cells. Furthermore, it was found that nCRP is a stable molecule that is very resistant to proteolytic degradation. Only mCRP was digested by trypsin or neutrophil-derived serine proteases. Further investigations showed that neutrophil-derived elastase was probably responsible for the degradation.

Acknowledgments

This study was financially supported by the British Heart Foundation.

Glossary

Abbreviations

CRP

C-reactive protein

DTT

dithiothreitol

ELISA

enzyme-linked immunosorbent assay

FITC

fluorescein isothiocyanate

HRPO

horse radish peroxidase

HUVEC

human umbilical cord endothelial cells

ICAM-1

intracellular adhesion molecule 1

IL

interleukin

LPS

lipopolysaccharide

MCP-1

monocyte chemotactic protein 1

MFI

median fluorescence intensity

PAGE

polyacrylamide electrophoresis

PC

phosphoryl choline

PMA

phorbol myristic acid

S/N

supernatant

SDS

sodium dodecyl sulphate

TNF-α

tumour necrosis factor α

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