C-Polysaccharide Bind to Phosphocholine And

The Journal of Immunology

A C-Reactive Protein Mutant That Does Not Bind to Phosphocholine and Pneumococcal C-Polysaccharide1

Alok Agrawal,2 Melanie J. Simpson, Steven Black, Marianne P. Carey, and David Samols

C-reactive protein (CRP), the major human acute-phase plasma protein, binds to phosphocholine (PCh) residues present in pneumococcal C-polysaccharide (PnC) of Streptococcus pneumoniae and to PCh exposed on damaged and apoptotic cells. CRP also binds, in a PCh-inhibitable manner, to ligands that do not contain PCh, such as fibronectin (Fn). Crystallographic data on CRP-PCh complexes indicate that Phe66 and Glu81 contribute to the formation of the PCh binding site of CRP. We used site- directed mutagenesis to analyze the contribution of Phe66 and Glu81 to the binding of CRP to PCh, and to generate a CRP mutant that does not bind to PCh-containing ligands. Five CRP mutants, F66A, F66Y, E81A, E81K, and F66A/E81A, were constructed, expressed in COS cells, purified, and characterized for their binding to PnC, PCh-BSA, and Fn. Wild-type and F66Y CRP bound to PnC with similar avidities, while binding of E81A and E81K mutants to PnC was substantially reduced. The F66A and F66A/E81A mutants did not bind to PnC. Identical results were obtained with PCh-BSA. In contrast, all five CRP mutants bound to Fn as well as did wild-type CRP. We conclude that Phe66 is the major determinant of CRP-PCh interaction and is critical for binding of CRP to PnC. The data also suggest that the binding sites for PCh and Fn on CRP are distinct. A CRP mutant incapable of binding to PCh provides a tool to assess PCh-inhibitable interactions of CRP with its other biologically significant ligands, and to further investigate the functions of CRP in host defense and inflammation. The Journal of Immunology, 2002, 169: 3217Ð3222.

-reactive protein (CRP),3 the prototypic human acute Thus, an investigation of the PCh binding site and the generation phase protein, belongs to a family of cyclic pentameric of a mutant incapable of binding to PCh are crucial to understand C proteins known as pentraxins, and is considered to play a the structure-function relationships of CRP. significant role in innate host defense and inflammation (1Ð3). The CRP is composed of five identical noncovalently bound subunits first described reactivity of CRP, which led to its discovery and of 206 aa with a molecular mass of ϳ23 kDa (1, 7). All five naming, was calcium-dependent binding to C-polysaccharide subunits have the same orientation in the pentamer, with a PCh (PnC) of the cell wall of Streptococcus pneumoniae (4, 5). Phos- binding site located on one face of each subunit (9). The PCh phocholine (PCh) residues present on PnC provide the major re- binding site consists of a hydrophobic pocket formed by residues 64 66 76 active group for the binding of CRP (6). Another member of the Leu , Phe , and Thr , and two calcium ions which are bound to pentraxin family, serum amyloid P (SAP), has a similar primary CRP by interactions with the side chains and main chain carbonyls and quaternary structure to CRP but binds PCh with lower affinity of amino acids from different parts of the primary structure (9). and with different stoichiometry (7Ð10). However, both CRP and Crystallographic analysis of CRP-PCh complexes (Fig. 1) has SAP bind to phosphoethanolamine (PEt) in a calcium-dependent demonstrated that the phosphate group of PCh directly coordinates with the two calcium ions (21). The choline moiety of PCh lies and comparable manner (11Ð13). CRP also binds various sub- within the hydrophobic pocket. The exposed face of Phe66 pro- stances that do not contain PCh, such as fibronectin (Fn), chroma- vides hydrophobic interactions with the methyl groups of choline, tin, and histones (14Ð16). Binding of CRP to all these ligands can while the side chain of Glu81, which is located on the other side of be inhibited by PCh, which has been interpreted to indicate par- the pocket, interacts with the positively charged quaternary nitro- ticipation of the PCh binding site on CRP in these interactions gen of choline (21). Previous mutational analyses of Thr76 in CRP (15Ð17). Once CRP is bound to a PCh-containing ligand or any have confirmed the significance of the hydrophobic pocket for PCh other suitable ligand, but not when bound to free PCh, it activates binding (22). the classical C pathway, a process initiated by binding of CRP In the present study, we have used site-directed mutagenesis to complexes to C1q, the first component of the pathway (18Ð20). produce CRP with mutations of Phe66 and Glu81. The goal was to determine the contribution of these two amino acids to PCh binding and to generate a CRP mutant incapable of binding to PCh- containing ligands. The results indicate that Phe66 is the major Department of Biochemistry, Case Western Reserve University, Cleveland, OH 66 81 44106 determinant of PCh binding, although both Phe and Glu participate. The F66A mutant CRP was found incapable of binding to Received for publication May 8, 2002. Accepted for publication July 17, 2002. PCh or PnC. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Materials and Methods This work was supported by National Institutes of Health Grant AG0246720. Construction of mutant CRP cDNA 2 Address correspondence and reprint requests to Dr. Alok Agrawal, Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, We constructed five CRP cDNA encoding F66A, F66Y, E81A, E81K, and OH 44106. E-mail address: [email protected] F66A/E81A mutants. The substitutions of Phe66 to Tyr and Glu81 to Lys 3 Abbreviations used in this paper: CRP, C-reactive protein; SAP, serum amyloid P; were based on the corresponding amino acids in SAP (7). The wild-type PnC, pneumococcal C-polysaccharide; PCh, phosphocholine; PEt, phosphoethano- (wt) CRP cDNA clone HLCRP-23 in the eukaryotic expression vector lamine; Fn, fibronectin; wt, wild type; CHO, Chinese hamster ovary. p91023 (23Ð25) was used as template for construction of mutant cDNA for

scribed previously (20, 26). The recombinant wt and all five mutant CRP were purified from culture media by a single affinity chromatography step using a PEt-conjugated agarose column (22). Briefly, 2 ml of culture media containing CRP was diluted to 8 ml in 0.1 M borate buffer saline (pH 8.3)

containing 5 mM CaCl2 and passed twice through a 1.0-ml column. After collecting the flow-through fractions and washing with the same buffer (8 ml), bound CRP was eluted with EDTA (10 mM)-containing borate buffer (8 ml). The wt, F66A, and F66A/E81A CRP were also purified by immunoaffinity chromatography using polyclonal anti-CRP Ab-conjugated agarose column for the purposes of recovering fractions containing concentrated CRP. The culture media (2 ml) containing CRP was diluted to 4 ml in TBS and passed twice through the immunoaffinity column (1.0 ml). After collecting the flow-through fractions and washing with the same buffer (8 ml), bound CRP was eluted with 50 mM glycine buffer (pH 3.0) and neutralized immediately with 1 M Tris (pH 9.0). All purified CRP preparations were immediately dialyzed against TBS. The polyclonal anti-CRP Ab was purified from rabbit anti-human CRP antiserum (Sigma-Aldrich, St. Louis, MO) by affinity chromatography on a CRP-conjugated agarose column. The conjugation of CRP or anti-CRP Ab to agarose was performed using the AminoLink Immobilization kit (Pierce). CRP ELISA The concentration of CRP was estimated using ELISA as described previously (20). Affinity-purified polyclonal anti-CRP was used as the capture Ab (2 ␮g/ml), and the monoclonal anti-CRP Ab HD2.4 (5 ␮g/ml) as reporter. The HD2.4 mAb (27) was affinity-purified from ascitic fluid gen- FIGURE 1. Crystal structure of CRP-PCh complex. The ViewerPro 4.2 erated from a hybridoma cell line obtained from American Type Culture software (Accelrys, San Diego, CA) was used to generate the ribbon dia- Collection (Manassas, VA). Standard curves were constructed with puri- gram of the x-ray crystal structure of CRP-PCh complex (21) obtained fied native CRP (6.25Ð200 ng/ml in TBS containing 0.1% BSA and 0.01% from Brookhaven Protein Data Bank (PDB:1B09). Only one of the five Nonidet P-40). HRP-conjugated goat anti-mouse IgG (Pierce) was used as subunits bound to PCh is shown. The side chains of Phe66 (blue) and Glu81 the secondary Ab, and the color was developed using the HRP substrate kit (brown) are highlighted. The two calcium ions are in black. Yellow, the (Bio-Rad, Hercules, CA). Color was measured at 405 nm in an ELISA phosphate group of PCh; red, the three methyl groups of choline; green, the plate reader (Molecular Devices, Menlo Park, CA). choline nitrogen. PCh binding assay Binding activity of CRP for PCh-containing ligands was evaluated by two assays using PCh-BSA or PnC in the solid phase as described previously F66A, F66Y, E81A, and E81K CRP. The F66A CRP cDNA was used as (20). PCh-BSA (9 mol PCh/mol BSA) was synthesized according to a a template to construct cDNA for the double mutant F66A/E81A. Muta- published method (28). PnC was purchased from Statens Seruminstitut genic oligonucleotides were obtained from Integrated DNA Technologies (Copenhagen, Denmark). Microtiter wells were coated with either PCh- (Coralville, IA). Mutagenesis was conducted using the QuikChange site- BSA or PnC at 10 ␮g/ml in TBS. Because CRP-PCh interaction requires directed mutagenesis kit (Stratagene, La Jolla, CA). Mutations were veri- calcium, the culture media or the purified wt and mutant CRP were diluted fied by nucleotide sequencing, using the services of the Core Facility of our to appropriate concentrations in calcium-containing buffer (TBS containing university. Two independent clones for each mutant were purified using the 0.1% BSA, 0.01% Nonidet P-40, and 5 mM CaCl2; TBS-Ca), and the same maxiprep plasmid purification kit (Qiagen, Valencia, CA), and used for buffer was used throughout the assay. Purified native CRP (6.25Ð200 ng/ subsequent protein expression. ml) was used to construct standard curves. The assays used HD2.4 mAb as a reporter and the wells were developed as in the ELISA. In some exper- Cell culture and transfection iments, binding curves were constructed by using serial dilutions of purified wt and mutant CRP, covering the concentration range from 1Ð1000 COS cells were used for transient expression of various mutant CRP ng/ml. clones, and Chinese hamster ovary (CHO) cells were used for stable expression of wt CRP. Cells were cultured in growth medium (RPMI 1640 EA4.1 binding assay medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 ␮g/ml Microtiter wells were coated with caprylic acid-purified anti-CRP mAb streptomycin) at 37¡C in a humidified atmosphere containing 5% CO2. For transient transfections, 5 ␮g of plasmid containing mutant CRP cDNA was EA4.1 at 10 ␮g/ml in TBS. Because EA4.1 mAb detects a calcium-depen- transfected into 2 ϫ 106 cells using the FuGENE 6 reagent (Roche, Indi- dent epitope on CRP (27, 29), the TBS-Ca buffer was used throughout the anapolis, IN). At 96 h posttransfection, culture media were collected to assay. The binding curves were constructed by using serial dilutions of determine expression of recombinant wt CRP. purified wt and mutant CRP, covering the concentration range from 10Ð For stable transfection, cells were cotransfected with a mixture of 10 ␮g 1000 ng/ml. Bound CRP was detected by using affinity-purified polyclonal wt CRP cDNA-p91023 construct and 2 ␮g pSV2neo vector (Invitrogen, anti-CRP Ab as reporter. Wells were developed with HRP-conjugated goat Carlsbad, CA) using the Lipofectin reagent (Life Technologies, Rockville, anti-rabbit IgG (Pierce) followed by the steps as in the ELISA. MD). The pSV2neo vector carrying neomycin resistance was used as the Fn binding assay helper plasmid because the vector p91023 does not harbor a drug selection marker (25). At 96 h posttransfection, stably transfected CHO cells were The binding of CRP to Fn was assessed as described previously (14, 17) selected by growth for 2 wk in the growth medium supplemented with with some modifications reported earlier (23). In brief, microtiter wells increasing concentrations (0.4Ð1.0 mg/ml) of Geniticin-418. A single were coated with 2 ␮g/ml Fn (Roche) in PBS. The binding curves were transfectant producing wt CRP was isolated by a series of subcloning steps. constructed by using serial dilutions of purified wt, and mutant CRP, cov- The stable transfectant, starting from 2 ϫ 106 cells, secreted ϳ6 mg CRP/l ering the concentration range from 10Ð5000 ng/ml in PBS (pH 5.0) con- suspension culture in 1 mo. taining 0.01% Nonidet P-40 and 2% polyethylene glycol-6000. Affinity- purified rabbit anti-CRP was used as reporter to detect bound CRP. The Purification of CRP wells were developed as in the EA4.1 binding assay. Native human CRP was purified from ascitic fluid by calcium-dependent Gel filtration and SDS-PAGE affinity chromatography on a PCh-conjugated agarose column (Pierce, Rockford, IL) followed by HPLC anion-exchange chromatography on a Gel filtration of wt and mutant CRP was conducted by HPLC on a Super- MonoQ column (Amersham Pharmacia Biotech, Piscataway, NJ) as de- ose 12 PC 3.2/30 column (Amersham Pharmacia Biotech) as described The Journal of Immunology 3219 previously (23). The affinity-purified CRP preparations (50 ␮l, 0.4Ð1.2 ␮g depending upon the concentrations of the stocks) were injected into the column and eluted with TBS at a flow rate of 40 ␮l/min. Twenty fractions (3 min each) were collected and CRP-containing fractions were located by ELISA. Purified native CRP was used as molecular size standard. SDS- PAGE of purified CRP (10 ␮l, 80Ð240 ng depending upon the concentrations of the stocks) was performed under reducing conditions. Results Construction, expression, and PCh-binding activity of CRP All mutant CRP cDNA were expressed successfully following transient transfections in COS cells, as determined by ELISA. The PCh binding assay, using PCh-BSA as a ligand, was performed on culture media containing wt and mutant CRP (Fig. 2). The wt and F66Y CRP bound to PCh-BSA with similar avidities, as indicated by the ratio of CRP concentrations measured by the PCh binding assay to that measured by ELISA. This ratio reflects specific ap- FIGURE 3. Purification of wt and mutant CRP by affinity chromatog- parent avidity of individual CRP species for PCh-BSA. The bind- raphy on a PEt-agarose column. The culture media (2 ml), containing in- ing of E81A CRP to PCh-BSA was reduced by more than half dicated amount of CRP (shown in parentheses), diluted to 8 ml in a cal- compared with wt CRP. The three other mutants, F66A, E81K, and cium-containing buffer were passed through a 1.0-ml column. After collecting the flow-through fractions and washing with the same buffer, F66A/E81A failed to bind PCh-BSA. Two independent clones for bound CRP was eluted with EDTA-containing buffer. ELISA was per- each mutant gave identical results. formed on all fractions (ϳ0.8 ml each) to determine CRP-containing fractions. A representative of two experiments is shown. Purification of CRP by PEt-affinity chromatography To exclude the possibility that the culture media may interfere with the binding of CRP to PCh-BSA, we purified wt and mutant CRP PCh interactions because all the mutant CRP bound to from the culture media of transiently transfected COS cells. The PEt-agarose. purification profiles from affinity chromatography on a PEt-conjugated agarose column are shown in Fig. 3. The wt, F66Y, E81A, Dose-response curves of binding of purified CRP to PCh-BSA and E81K CRP bound to PEt-agarose in the presence of calcium and PnC and could be eluted by EDTA, producing similar elution profiles. PCh binding assays using purified CRP were performed using two Interestingly, F66A and F66A/E81A mutants bound to PEt poorly, different PCh-containing ligands: PCh-BSA (Fig. 4A) and PnC as indicated by the shallow peaks in the unbound fractions, but (Fig. 4B). The wt (native or recombinant) and F66Y CRP bound to enough could be purified for use in the solid-phase PCh binding PCh-BSA and PnC in a dose-dependent manner and produced es- assays. Variation in the peaks for unbound CRP and eluted CRP sentially overlapping curves, indicating that their PCh-binding ac- was due to variations in fraction size and flow rate. Thus, the tivities did not differ from each other. Substitution of Glu81 with substitution of Phe66 with Ala abolished the binding of CRP to Ala reduced binding to both the ligands and the reduction was PCh, but did not abolish the binding of CRP to PEt. Because CRP more pronounced toward PnC. The substitution of Glu81 with Lys also binds to other phosphate monoesters such as dAMP (30), we substantially reduced binding to both the ligands. F66A and the performed affinity chromatography of various CRP species on a F66A/E81A CRP mutants did not bind to either PCh-BSA or PnC dAMP-conjugated agarose column (Sigma-Aldrich). The results throughout the dose-response range. Each mutant CRP was puri- (data not shown) were similar to those obtained from chromatog- fied from two independent transfection experiments and was as- raphy on PEt-agarose column. Assuming that the PEt- and the PCh sayed three times. The data shown in this study represent CRP binding sites of CRP are same, we conclude that the presence of purified by PEt-affinity chromatography and that obtained for one culture media in the binding assays does not interfere with CRP-

FIGURE 2. Binding of wt and mutant CRP to PCh-BSA. Microtiter wells were coated with PCh-BSA. Culture media containing various CRP FIGURE 4. Dose-response curves of the binding of various CRP to species were diluted to a concentration of ϳ50 ng/ml in TBS-Ca buffer and PCh-BSA and PnC. Microtiter wells were coated with either PCh-BSA (A) added to the wells. Bound CRP was detected using the HD2.4 mAb as or PnC (B). Increasing concentrations of purified wt and mutant CRP in reporter. Values shown on the y-axis represent the ratio of the concentra- TBS-Ca buffer were added to the wells. Bound CRP was detected by using tion of CRP measured by the PCh binding assay over that measured by the the HD2.4 mAb as reporter. Native wt CRP was purified from ascitic fluid ELISA run in parallel, using native CRP to generate standard curves in and the recombinant wt CRP was purified from a CHO cell line. A repre- both assays. sentative of three experiments is shown. 3220 PCh BINDING SITE OF CRP clone. CRP from the second clone gave similar results. There was no difference in the PCh-binding activity of either wt CRP or any of the mutant CRP, purified by either PEt-affinity or immunoaffinity chromatography (data not shown).

Characterization of PCh binding site of CRP using an mAb We also investigated the relative avidities of various CRP for the mAb EA4.1 (Fig. 5). The binding of this mAb to CRP is calcium- dependent, and can be inhibited by PCh, indicating that EA4.1 binds at or near the PCh binding site (27). As shown, the wt, F66Y, and E81A CRP bound to EA4.1 in a dose-dependent manner and produced overlapping curves, indicating that their EA4.1-binding epitopes, and hence the PCh binding sites, are almost identical. Substitution of Phe66 with Ala or the substitution of Glu81 with Lys drastically decreased binding to EA4.1. The double mutant F66A/E81A was similar to the single mutant F66A in binding to EA4.1 mAb. Therefore, both Phe66 and Glu81 are parts of the FIGURE 6. Dose-response curves of the binding of CRP to Fn. Micro- EA4.1-binding epitopes on CRP. Because the binding of F66A and titer wells were coated with Fn. Increasing concentrations of purified wt E81K CRP mutant to EA4.1 reflected their PCh-binding activity, and mutant CRP were added to the wells. Bound CRP was detected by we interpret the data to indicate and confirm the presence of a using polyclonal anti-CRP Ab as reporter. A representative of three exper- structurally altered PCh binding site in these CRP mutants. iments is shown.

Effect of mutated PCh binding sites on CRP-Fn interaction filtration column was similar to that of native CRP. The data are Because it was previously shown that PCh inhibits CRP-Fn inter- shown only for wt, F66A, and F66A/E81A CRP. The latter two are action, and it was proposed that CRP binds to Fn via the PCh the ones that did not bind PCh. These results demonstrated that binding site (17), we tested the CRP mutants for binding to Fn mutant CRP had the same size as native CRP, indicating that they (Fig. 6). All of the mutants bound to Fn as well as did the native all had a pentameric structure. SDS-PAGE analysis (Fig. 7B)of and recombinant wt CRP, suggesting that Phe66 and Glu81 do not purified CRP showed a single band and demonstrated that the ap- participate, directly or indirectly, in the formation of the Fn bind- parent molecular mass of the subunits of wt and mutant CRP was ing site. The observed differences between the binding curves for also identical with that of native CRP. Pentameric CRP-containing each CRP were within experimental error. The experiment shown fractions from the gel filtration chromatography were again tested in this study used F66A and F66A/E81A CRP mutants which were for binding to PnC and the results (data not shown) were compa- purified by immunoaffinity chromatography. A second experiment rable to the results obtained from affinity-purified CRP and from using PEt-affinity purified F66A and F66A/E81A mutant CRP in CRP in the media. Thus, F66A CRP mutant is pentameric and does the range of 10Ð200 ng/ml gave similar results (data not shown). not bind PCh or PnC. Assessment of the overall structure of CRP Discussion 66 81 We performed gel filtration chromatography to assess the pentam- The results indicate that both Phe and Glu participate in the 66 eric nature of the affinity-purified mutant CRP (Fig. 7A), although formation of the PCh binding site, although Phe is the major the results of the Fn binding assay, combined with the finding that determinant of CRP-PCh and CRP-PnC interactions. The PCh all CRP mutants bound PEt in a calcium-dependent manner, indi- binding site of CRP contains a hydrophobic pocket, two calcium 81 cated that the structure of the CRP mutants was likely to be similar ions, and Glu , which is located on the other side of the hydro- 64 66 to native CRP. The elution profile of all CRP species from the gel phobic pocket. The hydrophobic pocket consists of Leu , Phe , and Thr76 (9). The exposed face of Phe66 provides hydrophobic interactions with the methyl groups of choline, while the side chain of Glu81 interacts with the positively charged quaternary nitrogen of choline (21). The contrasting PCh-binding activities of F66A and F66Y CRP mutants, combined with previously reported findings on the mutational analysis of Thr76 (22), indicate that the topology of the hydrophobic pocket and the interaction between Phe66 and the methyl groups of choline are more important than the interaction between Glu81 and choline nitrogen for binding of CRP to PCh-containing ligands. The importance of the methyl groups of choline in CRP-PCh interaction has been indicated previously by hapten inhibition experiments (30, 31). It has been shown that PCh, which contains three methyl groups, is ϳ50-fold more powerful an inhibitor of CRP-PnC interaction than PEt, which contains no methyl groups (30, 31), although PCh and PEt are structural analogs (13), and presumably both bind to the same FIGURE 5. Binding of CRP to EA4.1 mAb. Microtiter wells were site on CRP. coated with EA4.1 mAb. Increasing concentrations of purified wt and mu- Our findings on the mutational analysis of the PCh binding site tant CRP in TBS-Ca buffer were added to the wells. Bound CRP was are also informative about the carbohydrate binding site of CRP. detected by using polyclonal anti-CRP Ab as reporter. A representative of CRP binds to depyruvylated pneumococcal type IV capsular poly- two experiments is shown. saccharide that does not contain PCh (31). Because galactose is the The Journal of Immunology 3221

nificant PCh-containing molecules including enzymatically degraded low density lipoprotein (36), membrane phospholipids like phosphatidylcholine and sphingomyelin (18), and pulmonary surfactant lipids (37). In addition, CRP has been shown to bind to damaged and necrotic cells (38Ð41) and to apoptotic cells (42), probably as a result of binding to exposed PCh moieties. CRP has the capacity to bind to a variety of non-PCh ligands also, including chromatin, histones and small nuclear ribonucleoproteins (15, 16, 43, 44), polycations (45), and Fn (14). Binding of CRP to most of these ligands is PCh inhibitable. This suggested an important func- tional role of the PCh binding site in binding to these ligands. However, our finding that Fn does not bind CRP via the PCh binding site raises the need to reevaluate the precise role of the PCh binding site in the interactions of CRP to its known ligands including the non-PCh ligands. CRP binds to several bacterial species (46) including S. pneumoniae (47), Hemophilus influenzae (48), and Neisseriae spp. (49) most likely through PCh groups. The binding of CRP to S. pneumoniae has been shown to be PCh inhibitable (47). CRP also binds, in a calcium-dependent manner, to fungi (50), yeast (51), and certain parasites such as Plasmodium falciparum (52) and Leishmania donovani (53) through either PCh or non-PCh ligands FIGURE 7. Pentameric structure of mutant CRP. A, Gel filtration of on their surfaces. In mouse models of bacterial infections, human affinity-purified wt, F66A, and F66A/E81A CRP was performed on a CRP has been shown to be protective against infection with S. HPLC Superose 12 PC column. The arrows indicate the void volume (V ), 0 pneumoniae and Salmonella typhimurium, perhaps due to binding and the positions of peaks for native CRP (118 kDa) and albumin (66 kDa). of CRP to bacteria through PCh groups present on their surface B, Silver-stained gel of various CRP species after reducing SDS-PAGE. (54Ð57). In animal models of myocardial infarction, CRP is found deposited on myocardial cells within the infarcted area (58). Ad- only common group between PnC and depyruvylated pneumococ- ministration of human CRP into a rat model of myocardial infarc- cal type IV capsular polysaccharide, it has been proposed that CRP tion resulted in deposition of CRP on the surfaces of damaged interacts with PnC via galactose residues as well (31). However, it myocardial cells in and around the infarct (59) and enhanced the was shown that the binding of CRP to carbohydrates occurs with extent of damage by C activation. Based on these findings, it has much lower avidity than binding to PCh (30Ð33). Because the been proposed that the PCh binding site could be used as a ther- F66A and F66A/E81A mutant CRP did not differentiate between apeutic target in coronary heart disease. We propose that experi- PCh-BSA and PnC for binding, we propose that the PCh binding ments involving F66A CRP mutant in in vitro binding studies with site also participates in binding of CRP to carbohydrate moieties. bacteria and the experiments involving administration of F66A This conclusion is supported by the fact that CRP-carbohydrate CRP mutant in the animal models of human diseases would shed interactions are also calcium-dependent and PCh inhibitable (31Ð light on the role of PCh binding site of CRP. The mutants reported 33). Indeed in SAP, it has been shown that both PEt and the car- in this paper, available for use in in vivo experiments, will help bohydrates bind to a common site (8). prove if the binding of CRP to bacteria via PCh groups is required The finding that the CRP mutants F66A and F66A/E81A, inca- for protection, and if deposition of CRP on damaged cells and pable of binding to PCh, bound to Fn was unexpected because PCh subsequent C activation depend on an intact PCh binding site. is known to inhibit CRP-Fn interaction (17). Our results indicate Taken together, our results demonstrate that Phe66 is the major that the PCh binding and Fn binding sites on CRP are distinct. determinant of CRP-PCh interaction and is critical for binding of Because the binding of CRP to Fn is inhibited by high concentra- CRP to PnC. The data also suggest that PCh binding and Fn bind- tions of calcium (14, 17) that would probably result in occupancy ing sites on CRP are distinct. A CRP mutant, F66A, incapable of of both the calcium binding sites, we hypothesize that the binding binding to PCh provides a tool to assess PCh-inhibitable interac- of Fn requires amino acids in CRP that bind calcium ions. There- tions of CRP with its other biologically significant ligands in vitro, fore, the inhibition of CRP-Fn interaction by PCh (17) in the pres- and to investigate the functions of CRP in host defense and in- ence of calcium could be due to occupancy of the calcium binding flammation in vivo. sites by calcium. The binding of CRP to Fn is similar to the binding of CRP to polycations; both being inhibited by calcium (30, Acknowledgments 34). Our data are consistent with the proposal that the Fn and We thank Dr. John Volanakis for anti-CRP mAb EA4.1 and Dr. Irving polycation binding sites on CRP are overlapping (14, 23) and that Kushner and Dr. Raman Bhasker for critical reading of the manuscript. the PCh binding and polycation binding sites are distinct (30, 34). 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