C-reactive protein binds to phosphorylated carbohydrates

Fiona J. Culley, Katherine B. Bodman-Smith, Michael A. J. Ferguson2, Andrei V. Nikolaev3, Nalini Shantilal and John G. Raynes1

Immunology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK and 2Department of Biochemistry and 3Department of Chemistry, The University, Dundee DD1 4HN, UK

Received on May 24, 1999; revised on July 15, 1999; accepted on July 15, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
C-reactive protein (CRP) is a major acute phase protein in man. In order to more fully understand the physiological role of this serum protein, we have demonstrated high avidity binding for a defined chemically synthesized carbo­hydrate ligand which represents the repeating disaccharide of lipophosphoglycan, the major surface glycoconjugate of the unicellular parasite Leishmania donovani. Increasing the number of phosphorylated disaccharides in a molecule from one up to seven did not increase the avidity for CRP, however increasing this to 10 potential CRP binding sites did. In order to define the important features of this complex and variable structure for CRP binding we competed CRP binding to whole Leishmania parasites with amino, sulfated, phosphorylated, and unsubstituted monosaccharides, of which only phosphorylated monosaccharides were able to inhibit. Both the carbohydrate and the position of phosphorylation influenced the avidity for CRP. Synthetic oligosaccharides and phospho-oligosaccharides of various lengths and conformations were used to define the structural requirements for CRP recognition. The optimum structure for recognition of a single phosphate group was between two monosaccharide pyranose rings, and within a linear rather than a cyclic molecule. This stresses the importance of the interaction of the CRP binding site with both the carbohydrate and the phosphate group. CRP function may be mediated via the recognition of large arrays of phosphorylated carbohydrates as are characteristic of the surface of microorganisms.

Key words: pentraxin/lipophosphoglycan/Leishmania/galactose 6 phosphate/mannose 1 phosphate


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
C-reactive protein (CRP) and serum amyloid A are the major acute phase proteins (APPs) in man, the serum concentrations of which are dramatically upregulated following stimulation of hepatocytes by inflammatory cytokines. Although APPs are believed to be important in modulating inflammatory responses and in host defence, little is actually known of the roles that CRP plays in vivo. In vitro, CRP can act as an opsonin, both directly and by its ability to activate the classical pathway of complement (Kaplan and Volanakis, 1974Go; Volanakis and Kaplan, 1974Go; Mortensen et al., 1976Go; Mortensen and Duszkiewicz, 1977Go; Edwards et al., 1982Go; Kilpatrick and Volanakis, 1985Go).

CRP has been shown to bind to a wide range of ligands including damaged tissues, bacteria and yeasts (Kindmark, 1971Go; Richardson et al., 1991Go). Most known CRP binding is calcium dependent, however, calcium independent binding has also been reported. The only fully defined calcium dependent ligand for CRP is phosphorylcholine (PCh). PCh occurs on the C-polysaccharide of Streptococcus pneumoniae, the precipitation of which gave CRP its name, and also forms the polar head groups of the widespread phospholipids phosphatidylcholine and sphingomyelin found in cell membranes and which may become exposed following cell damage (Tillett and Francis, 1930Go; Volanakis and Kaplan, 1971Go; Volanakis and Wirtz, 1979Go).

CRP is a member of the pentraxin family of proteins, so-called because of their pentameric symmetry. The three-dimensional structure of CRP has been determined (Schrive et al., 1996Go), being made up of five identical noncovalently associated subunits, each of which possesses a calcium dependent ligand binding site, all arranged on the same face of the pentamer (Osmand et al., 1977Go; Roux et al., 1983Go). This suggests that ligands recognized by CRP with a high avidity will be those that occur in large arrays, such as on the surface of microorganisms, or highly repetitive molecules.

We believe that a fuller definition of the ligands recognized by CRP is essential for an understanding of its role in host defence and homeostasis. Previously, we reported that CRP bound to the surface of the promastigote form of the unicellular parasite Leishmania donovani, in a calcium dependent manner, where it causes the activation of complement (Culley et al., 1996Go, 1997). In addition to activating complement CRP caused a complement independent opsonization of the parasite into macrophages which adds CRP to the list of proteins which Leishmania can use to enter the macrophage. This occurred at normal plasma CRP concentrations (Culley et al., 1996Go). Evidence was presented that CRP bound to lipophosphoglycan (LPG) which forms the glycocalyx which coats the parasites. The binding was seen to LPG with poorly substituted repeating structure (e.g., L.mexicana, L.donovani) but not to those with large numbers of side-chains (e.g., L.major). A monoclonal antibody that recognized the repeating disaccharide (-6Galß1–4Man{alpha}1-PO3-H-)n up to 30 of which form the backbone of the LPG molecule, could inhibit binding to the LPG but not antibodies to other components of the LPG (Culley et al., 1996Go). This was the first description of recognition of a pathogen by CRP via carbohydrate structures rather than PCh. Since direct binding data was lacking in these studies we have confirmed our previous results using synthetic LPG oligosaccharides and defined the structures of the ligand that contribute toward the carbohydrate specificity of CRP.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CRP binding to repeating phosphorylated disaccharides
sPG1 is a synthetic phospho-oligosaccharide formed from repeating phosphorylated disaccharides, identical to those found in the backbone of L.donovani LPG, with an average of 10 repeats. The ability of sPG1 to precipitate CRP was assessed by standard methods in agarose. A visible precipitate formed between sPG1 and CRP (Figure 1), which could be stained for protein (not shown) directly demonstrating that CRP could bind to the phosphorylated disaccharide repeat structures. This did not occur in the presence of EDTA and therefore required the calcium dependent binding site of CRP. We then examined the effect of sPG1 on CRP binding to promastigotes in fluid phase assays. sPG1 was mixed with radiolabeled CRP and then incubated with L.donovani promastigotes. Binding was measured by centrifuging promastigotes through an oil layer and counting the associated CRP in a gamma counter. The increase of CRP pelleted in the presence of sPG1 was due to precipitation of the mixture, a conclusion supported by the observation that mixtures of solutions of sPG1 and CRP rapidly become opaque (unpublished observations). These studies confirmed that the repeating phosphorylated disaccharide of LPG was a novel ligand for CRP which had been implied from our previous competitive studies with monoclonal antibodies (Culley et al., 1996Go).



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Fig. 1. CRP binding to sPG1: precipitation in agarose gel. CRP (10.8 µg) and the synthetic carbohydrate sPG1 (7.5 µg) were added to adjacent wells in agarose gel in the presence (+Ca2+) or absence (+EDTA) of calcium and allowed to diffuse. The gel was photographed using backlighting from a light box. A visible precipitate appeared between the wells in the presence of calcium, which stained positively for protein. Precipitation did not occur in the absence of calcium.

 
Specificity of lectin activity of CRP
The specificity of the lectin activity of CRP was assessed by competing CRP binding to L.donovani promastigotes with phosphorylated and nonphosphorylated mono- and oligo-saccharides. Initial experiments were performed to demonstrate that CRP directly linked to alkaline phosphatase produced similar binding curves and similar rank order of inhibition to a system in which CRP binding was detected with an alkaline phosphatase conjugated goat IgG to human CRP or using radiolabeled CRP (data not shown). Experiments using this system also confirmed that CRP bound to the parasite ligand with high avidity (Kd ~1.5 x 10–11 M–1) which is similar to that obtained previously using radiolabeled CRP in fluid phase binding assays (Culley et al., 1996Go). This is consistent with observations that concentrations within the normal serum range give up to half maximal binding to promastigotes (Culley et al., 1996Go).

An ELISA system was used to determine the specificity of CRP recognition of carbohydrates. Phosphorylated, sulfated, amino, and unsubstituted derivatives of galactose, mannose, glucose, and fructose were examined for their ability to inhibit CRP binding, in comparison to PCh. Figure 2 shows the results of competition with galactose sugars using L.donovani promastigotes immobilized on microtiter plates and an ELISA system to measure bound CRP. Monosaccharides were added to the promastigote-coated plates together with CRP at a range of concentrations. Phosphorylated monosaccharides were able to compete for CRP binding whereas unsubstituted, sulfated monosaccharides and amino sugars were poor inhibitors (less than 10% inhibition at 10 mM concentrations). Based on the concentration required for 50% inhibition, galactose 6-phosphate exhibited the highest relative avidity for CRP after PCh (Table I). Inhibition was not due to chelation of calcium by the phosphorylated carbohydrates. On titration of calcium into the assay from 0.3 to 10 mM, we demonstrated that the addition of calcium did not effect the ability of any of the inhibitors to reduce binding (data not shown).



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Fig. 2. Ligand specificity of CRP: Competitive ELISA with monosaccharides. L.donovani promastigotes were air dried onto a microtiter plate and CRP binding was measured in the presence of competing carbohydrates or PCh. Bound CRP was then detected by anti-CRP antibody and enzyme conjugated antibody. CRP binding is shown for a range of dilutions of competitor as percentage inhibition relative to positive control wells which contained no competitor (100%). Assays were performed in triplicate wells. The graph shows the results of a typical experiment as mean ± SEM. Solid circles, PCh; solid squares, galactose 6-phosphate; solid triangles, galactose 1-phosphate; open squares, galactosamine, x, galactose, O Galactose 6-sulfate.

 

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Table I. Inhibition of CRP binding by a range of monosaccharides
 
In order to determine the influence of structure and length of carbohydrates on the relative avidity of binding to CRP, a range of synthetic carbohydrates were tested for their ability to compete for CRP binding to L.donovani promastigotes immobilized onto microtiter plates. The first group of compounds tested (L1, L2, L5, and sPG1) were formed from varying numbers of repeats of the phosphorylated disaccharide structure found in LPG (Figure 3). The decenyl group alone had no effect on this assay (data not shown). The compounds fall into three groups in terms of their relative avidity for CRP: L1, which is not phosphorylated gave relatively poor inhibition, which was significantly lower than the other compounds tested (Student’s unpaired t-test, inhibition at 1 mM, L1 vs. L2 and L5, p < 0.05). The compounds L2 and L5 (with 1 and 3 phosphorylated disaccharide repeats, respectively) exhibited a higher avidity for CRP than L1 but did not differ significantly from each other in their avidity for CRP, despite the higher number of potential binding sites for CRP in L5. sPG1 (with 10 repeating phosphorylated disaccharide units) had an avidity significantly higher than the other compounds tested (at 0.1 mM, p < 0.05). This suggested that an increase in the number of potential binding sites on a molecule does not increase the avidity of CRP binding until a certain size of molecule has been reached. To test this hypothesis, we measured the avidity for CRP of a series of compounds formed only from phosphorylated mannose (Figure 4). The mannosyl phosphomannose, gave significantly higher avidity of binding to CRP than mannose 1-phosphate (at 1 mM, p < 0.05), demonstrating that the presence of carbohydrate groups either side of the phosphate can increase the avidity for CRP. The linear oligo(mannosyl phosphate), with 5–7 phosphate groups, was no better at inhibiting CRP binding than mannosyl phosphomannose (no significant difference up to 3 mM), in concurrence with the conclusions reached above. Mannobiose provided greater inhibition than mannose monosaccharide, suggesting that even in the absence of phosphate groups the carbohydrate can interact with the binding site of CRP, and that two carbohydrate units bind with a higher avidity than one.



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Fig. 3. Relative avidity of CRP for synthetic fragments of L.donovani LPG. The synthetic carbohydrates L1({sigma}o{lambda}{iota}{delta} {sigma}{theta}{upsilon}{alpha}{rho}{varepsilon}{sigma}), L2 (x), L5 (open circles), and sPG1 ({sigma}o{lambda}{iota}{delta} {chi}{iota}{rho}{chi}{lambda}{varepsilon}{sigma}) were used to compete for binding of alkaline phosphatase conjugated CRP to promastigotes coated onto a microtiter plate. All assays were performed in triplicate. Data is from at least two identical experiments and is shown as average ± SEM.

 


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Fig. 4. CRP binding to phosphorylated mannan oligomers. Synthetic phosphorylated mannose oligomers were used to inhibit the binding of alkaline phosphatase conjugated CRP to promastigotes coated onto a microtiter plate. All assays were performed in triplicate. Data is from at least two identical experiments and is shown as average ± SEM. {Sigma}o{lambda}{iota}{delta} {tau}{rho}{iota}{alpha}{nu}{gamma}{lambda}{varepsilon}{sigma}, mannose 1-phosphate; open squares, mannobiose; {sigma}o{lambda}{iota}{delta} {chi}{iota}{rho}{chi}{lambda}{varepsilon}{sigma}, mannosyl phosphomannose; open circles, linear oligo(mannosyl phosphate); x, cyclic di(mannosyl phosphate); {sigma}o{lambda}{iota}{delta} {sigma}{theta}{upsilon}{alpha}{rho}{varepsilon}{sigma}, cyclic tri(mannosyl phosphate).

 
The cyclic di(mannosyl phosphate) and cyclic tri(mannosyl phosphate) were used to determine the influence of conformation on the avidity of CRP binding. The avidity of binding to CRP of the cyclic dimer was significantly reduced when compared to the linear molecules (at 1 mM, p < 0.05). However, the cyclic trimer was able to inhibit, with an avidity that was not significantly different to the linear compounds tested, up to 3.0 mM. Therefore, the conformation of the carbohydrate molecule to which the phosphate is attached also determines the avidity of CRP binding.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We had previously described the ability of CRP to recognize the repeating phosphorylated disaccharide structures of LPG, by competition for CRP binding to parasites using specific anti-LPG monoclonal antibodies (Culley et al., 1996Go). This was confirmed by the precipitation of CRP by sPG1, a synthetic phosphoglycan representing 10 of the repeating phosphorylated disaccharides of LPG. This demonstrates that these repeating units are sufficient for recognition of carbo­hydrate by CRP. We believe this accounts for the observation of the precipitation of CRP by components of Leishmania promastigote culture supernatants (Pritchard et al., 1985Go).

To determine the specificity of CRP binding, a range of monosaccharides, including phosphorylated, amino, and sulfated carbohydrates, were used to inhibit CRP binding to promastigotes and their relative avidity of binding was compared to PCh. Phosphorylation of the monosaccharides was necessary for this inhibition; sulfated, amino and unsubstituted monosaccharides were poor inhibitors (<10% inhibition at 10 mM). The monosaccharide itself and the position of the phosphate influenced the relative avidity of CRP for the monosaccharide. The highest avidity binding was shown by galactose 6-phosphate, and as this unit is a constituent of the repeating phosphorylated disaccharides of LPG, this may account for the ability of LPG to act as a good ligand for CRP.

Previous studies of CRP binding to carbohydrate structures have been inconclusive as to the specificity of CRP binding under physiological conditions. Uhlenbruck and colleagues (Uhlenbruck et al., 1979Go, 1981) reported the ability of CRP to precipitate undefined galactans in a calcium dependent manner. At first this was assumed to be due to lectin activity of CRP, however this was later attributed to the presence of phosphate groups which were associated by an unknown linkage with the carbohydrates, and any lectin activity of CRP was dismissed (Soelter and Uhlenbruck, 1986Go). Köttgen and co-workers reported lectin activity of CRP specific for the terminal galactosyl components of plasma glycoproteins, including IgG (Köttgen et al., 1992Go). However, this required solid phase immobilisation of both CRP and ligand and a pH of 5–6, as the interaction was inhibited at physiological pH. Phosphate mediated recognition does not account for the ability of CRP to precipitate the depyruvylated capsular polysaccharide of type IV pneumococci (Heidelberger et al., 1972Go). This polysaccharide does not contain phosphorylcholine or any phosphate, rather it only shares 2-acetamido-2-deoxy-D-galactose with the C-polysaccharide. Therefore, this interaction may indeed result from recognition of the carbohydrate itself. Surface bound CRP, in the structurally altered neo-CRP form has been shown to function as a galactose specific receptor (Kempka et al., 1990Go; Egenhofer et al., 1993Go). Interestingly, in all cases a specificity for galactose or galactans was proposed.

The structures L2 and L5, which contain one and three LPG phosphorylated disaccharide units, respectively, showed no difference in their relative avidity for CRP. However, when the number of phosphorylated disaccharide repeats was increased to an average of 10 in sPG1, a significant increase in the avidity of binding to CRP was seen. It may be hypothesised that the compounds sPG1 but not L2 and L5 are of sufficient length to bind to two CRP subunits per molecule which would greatly increase the avidity of its interaction with CRP. The phosphorylated disaccharide repeats of LPG and sPG1 form a helix that can contract and expand like a spring, in the case of sPG1 from 56 Å to 100 Å (Homans et al., 1992Go). The diameter of a CRP subunit is ~70 Å, so it is theoretically possible that sPG1 could span two subunits to form either intra- or inter-subunit links (Schrive et al., 1996Go). A more detailed consideration of the interaction is not possible because of the variable conformation of the LPG.

Our studies also reveal something of the structural nature of CRP recognition of ligand. Unlike mannose binding protein which recognizes mannose via a calcium dependent carbo­hydrate recognition domain in which calcium interacts with mannose, CRP appears to resemble its homologue pentraxin serum amyloid P (SAP). SAP binds to the carbohydrate 4,6-O-(1-carboxyethylidene)-ß-D-galactopyranose mainly via calcium interactions with the charged carboxyethylidine group (Emsley et al., 1994Go). In fact, as discussed by Tennent and Pepys, recognition by SAP appears to be specific not for a particular pyranose ring but for the attached charged groups (Tennent and Pepys, 1994Go). Our studies suggest that this is generally true of pentraxin recognition of carbohydrates. We have shown that the presence of the phosphate group on monosaccharides is essential for recognition of carbohydrate and that the particular carbohydrate ring to which the phosphate is attached plays a less important role in recognition by CRP, although it does determine the relative avidity. The avidity of CRP for phosphorylated mannose was greatly increased by the addition of a second carbohydrate to the phosphate. This may be as a result of an alteration in the positioning of the phosphate group relative to the carbohydrates. Alternatively, the presence of the second carbohydrate may stabilise binding to CRP if the binding site of CRP is large enough to interact with a carbohydrate ring either side of the phosphate group. This conclusion is also supported by the fact that unphosphorylated mannobiose was better able to inhibit CRP binding than mannose monosaccharide, again suggesting that the CRP binding site may be large enough to accommodate two carbohydrate rings. Results obtained with cyclic carbohydrates demonstrate that the orientation of the carbohydrate relative to the phosphate group also has a profound influence on the recognition by CRP, the more linear the greater being the avidity.

Chelation of the calcium ions by phosphate appears to be essential for recognition of ligand by CRP. Therefore, CRP appears to be specific for phosphate groups, with the "carrier molecule," such as a galactose, mannose, or choline, acting to stabilize the interaction by forming bonds with adjacent amino acids at the ligand recognition site.

One of the functions of APPs is believed to be a role in innate defence against infection. Carbohydrate recognition is central to innate immunity and it is believed that serum lectins, such as MBP, act by direct agglutination and opsonization of the invading organism or by recruiting the complement cascade. In fulfilment of the requirements of a molecule involved in innate immunity, CRP can direct effector functions such as phago­cytosis and activation of the complement cascade. We have described how CRP can recognize large arrays of phosphorylated carbohydrates, a characteristic of the surface of many pathogens. In particular CRP exhibits a specificity for galactose 6-phosphate and that a high repeat number as found in Leishmania LPG, increases avidity of CRP for ligand. Therefore, CRP may provide a first line of defence in the recognition of infectious organisms by the innate immune system. Further determination of the ligands recognized by CRP and the effects of ligand recognition on the interaction of CRP with effector mechanisms should reveal more about the roles of this serum protein in host defence and homeostasis.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Reagents
CRP was extracted from acute phase human serum by avidity chromatography using PCh-Sepharose beads and purified by ion exchange chromatography and gel filtration (Volanakis et al., 1978Go). Anti-CRP antibodies, were purified from immunised goat serum by affinity chromatography on CRP-Sepharose. CRP was radiolabeled using carrier free Na125I (100mCi/ml) (Amersham) and N-bromosuccinimide (Sigma) (Sinn et al., 1988Go). CRP was directly conjugated to alkaline phosphatase using a standard single step glutaraldehyde method (Avrameas, 1969Go). L.donovani (LV9) promastigote cultures were established from isolated amastigotes and were maintained in vitro by serial passage at 25°C, in Medium 199 (Life Technologies) containing 10% fetal calf serum (Life Technologies), 2 mM glutamine (Sigma), 1 mM pyruvate (Sigma), 10mM HEPES (Life Technologies), 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies). Cultures were seeded at 5 x 105 per ml and were used at late stationary phase. All monosaccharides used were sodium salts (Sigma). The following synthetic oligosaccharides and phospho-oligosaccharides were used: sPG1, H-(6Galß1–4Man{alpha}1-PO3 H•NH3) n -OH•NH3, where average n = 10 (Nikolaev et al., 1995aGo); L1, Galß1–4Man{alpha}1-O-decenyl (Nikolaev et al., 1995bGo); L2, Galß1–4Man{alpha}1–PO3H-O-Dec, Et3NH+ salt (Nikolaev et al., 1996Go); L5, Man{alpha}1–2Man{alpha}1-PO3H-(-6Galß1–4Man{alpha}PO3H-)2-Galß1–4Man{alpha}1-Dec,Et3NH+ salt (Nikolaev et al., 1995bGo); mannobiose, Man{alpha}1–2Man; mannosyl phosphomannose, Man{alpha}1-PO3H-6Man{alpha}1–OCH3, Et3NH+ salt (Nikolaev et al., 1989Go); linear oligo(mannosyl phosphate), H-(6Man{alpha}1-PO3H-)n-OH, n=5–7, NH4+ salt (Nikolaev et al., 1992Go); cyclic di(mannosyl phosphate), (6-Man{alpha}1-PO3H-)2, NH4+ salt (Nikolaev et al., 1992Go); cyclic tri(mannosyl phosphate), (6-Man{alpha}1-PO3H)3, NH4+ salt (Nikolaev et al., 1992Go).

The carbohydrates sPG1, L1, L2, and L5 were formed from varying numbers of the repeating phosphorylated (or nonphosphorylated, for L1) disaccharides of L.donovani LPG. The mannosyl phosphomannose and the linear oligo(mannosyl phosphate) are synthetic fragments of the yeast exophosphomannan of Hansenula capsulata. The cyclic di- and tri-(mannosyl phosphate)s were isolated as side products at the chemical preparation (Nikolaev et al., 1992Go) of the linear oligo(mannosyl phosphate).

CRP binding to sPG1
Three milliliters of 1% (w/v) agarose (Life Technologies) in Tris-buffered saline (10 mM Tris–HCl (BDH), 0.15M NaCl (BDH), 0.5 mM Ca2+, pH 8.0) (TBSC) with or without 10 mM EDTA, was heated and pipetted onto a horizontal glass plate. When the agarose had set, small holes were punched into it and 5 µl of 2.4 mg/ml CRP and 1.5 mg/ml sPG1 were added to adjacent holes. Plates were placed in a humidified box at 4°C for 72 h. A visible precipitate was recorded by photographing plates on a light box. To detect protein, plates were dried with layers of Whatman paper followed by air drying and stained for 5 min in 12.5 mg/ml Coomassie blue R-250, 5:1:4 (v/v) methanol:acetic acid:dH20, and then destained in 5:1:4 (v/v) methanol:acetic acid:dH20.

The relative avidity of CRP binding to monosaccharides: 125I-CRP binding to promastigotes
Monosaccharides were used to compete for CRP binding to ligand. Monosaccharides, sPG1 and PCh (Sigma) were diluted in a 2-fold dilution series in 1% (w/v) BSA, 0.05% (w/v) sodium azide, TBS; 10 µl of 25 µg/ml radiolabeled CRP was mixed with 15 µl of inhibitor, layered onto 150 µl of a (40:60 v/v) dinonyl phthalate (Fisher Scientific)/dibutryl phthalate (BDH) oil layer in a microcentrifuge tube and incubated at 37°C for 1 h. All assays were performed in triplicate. A positive control which contained no competitor and a negative control which contained 10 mM EDTA were also performed. Nine day old promastigote cultures were washed and resuspended at 7.5 x 107 /ml and 25 µl was added to each tube. After 1 h, the promastigotes were centrifuged through the oil layer, the pellets cut off, and the associated CRP counted in a gamma counter.

The relative avidity of CRP binding to carbohydrates: detection of CRP binding by ELISA
Nine day old L.donovani promastigotes were washed, fixed in 4% (w/v) formaldehyde, RPMI 1640, washed, and resuspended in RPMI and coated onto microtiter plates (Nunc Maxisorb) at 5 x 105 in 50 µl per well by air drying overnight at room temperature. Wells were blocked with 200 µl 1% (w/v) BSA, PBS for 4 h at room temperature and then washed twice with PBS; 25 µl of a range of concentrations of carbohydrate was added per well, followed by 25 µl of 30 µg/ml CRP, both in TBSC, and plates were incubated for 2 h at room temperature. PCh was also used to compete for binding and a positive control, which included no carbohydrate and a negative control, which included 10 mM EDTA, were performed on each plate. Assays were performed in triplicate. CRP binding was detected by two methods, which did not differ in terms of final results obtained (data not shown). For experiments with monosaccharides, plates were incubated with goat anti-CRP (1:100) and rabbit anti-goat- alkaline phosphatase conjugate (1:1000) (Sigma) in TBSC for 1 h at room temperature washing three times with TBSC, between each stage. For all subsequent reported experiments, alkaline phosphatase–conjugated CRP was added at final concentration of 15µg/ml. After washing, plates were developed using 1mg/ml of p-nitrophenyl phosphate (Sigma) in 15 mM Na2CO3, 35 mM NaHCO3, pH 9.6 (bicarbonate buffer) containing 2 mM MgCl2 and the OD read at 405 nm (ref. 490 nm). The OD of CRP binding in the absence of inhibitor was considered to be 100% binding and that of the EDTA control 0%.

In order to determine the effects of calcium concentration on the carbohydrate inhibition of CRP binding, CaCl2 was added to the assay at a final concentration of 0.3, 1, and 3 mM and binding measured as above.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This work was supported by grants from the Medical Research Council and the Wellcome Trust. The research of A.V.N. was supported by an International Research Scholar’s award from the Howard Hughes Medical Institute.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CRP, C-reactive protein; LPG, Leishmania lipophosphoglycan; PCh, phosphorylcholine; TBS, Tris-buffered saline; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; SAP, serum amyloid P; APP, acute phase protein; MBP, mannan binding protein.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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