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.
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Abstract |
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Key words: pentraxin/lipophosphoglycan/Leishmania/galactose 6 phosphate/mannose 1 phosphate
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Introduction |
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CRP has been shown to bind to a wide range of ligands including damaged tissues, bacteria and yeasts (Kindmark, 1971; Richardson et al., 1991
). 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, 1930
; Volanakis and Kaplan, 1971
; Volanakis and Wirtz, 1979
).
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., 1996), 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., 1977
; Roux et al., 1983
). 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., 1996, 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., 1996
). 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 (-6Galß14Man
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., 1996
). 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.
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Results |
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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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Discussion |
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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., 1979, 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, 1986
). Köttgen and co-workers reported lectin activity of CRP specific for the terminal galactosyl components of plasma glycoproteins, including IgG (Köttgen et al., 1992
). However, this required solid phase immobilisation of both CRP and ligand and a pH of 56, 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., 1972
). 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., 1990
; Egenhofer et al., 1993
). 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., 1992). 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., 1996
). 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., 1994). 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, 1994
). 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.
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Materials and methods |
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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., 1992) 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 TrisHCl (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 phosphataseconjugated 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.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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