Artigo Revisado por pares

Mapping the Binding Areas of Human C-reactive Protein for Phosphorylcholine and Polycationic Compounds

2002; Elsevier BV; Volume: 277; Issue: 1 Linguagem: Inglês

10.1074/jbc.m106039200

ISSN

1083-351X

Autores

Reiko T. Lee, Isamu Takagahara, Yuan C. Lee,

Tópico(s)

Venomous Animal Envenomation and Studies

Resumo

We developed a fluorescence-based assay method for determining ligand binding activities of C-reactive protein (CRP) in solution. Using this method, we compared the phosphorylcholine (PC)- and polycation-based binding activities of human CRP. The PC-based binding required calcium, whereas a polycation (e.g.poly-l-lysine) was bound in the presence of either calcium or EDTA, the binding being stronger in the presence of EDTA. The published crystallographic structures of CRP and the CRP·PC complex show it to be a ring-shaped pentamer with a single PC-binding site per subunit facing the same direction. As expected from such a structure, binding affinity of a ligand increased tremendously when multiple PC residues were present on a macromolecular structure. In addition to PC-related structures, certain sugar phosphates (e.g.galactose 6-phosphate) are bound near the PC-binding site, and one of the sugar hydroxyl groups appears to interact with CRP. The best small ligands for the polycationic binding site were Lys-Lys and Lys4. Because of the presence of multiple Lys-Lys sequences, polylysines have tremendously enhanced affinity. Although PC inhibits both PC- and polycation-based binding, none of the amines that inhibit polylysine binding inhibits PC binding, suggesting that the PC and polycationic binding sites do not overlap. We developed a fluorescence-based assay method for determining ligand binding activities of C-reactive protein (CRP) in solution. Using this method, we compared the phosphorylcholine (PC)- and polycation-based binding activities of human CRP. The PC-based binding required calcium, whereas a polycation (e.g.poly-l-lysine) was bound in the presence of either calcium or EDTA, the binding being stronger in the presence of EDTA. The published crystallographic structures of CRP and the CRP·PC complex show it to be a ring-shaped pentamer with a single PC-binding site per subunit facing the same direction. As expected from such a structure, binding affinity of a ligand increased tremendously when multiple PC residues were present on a macromolecular structure. In addition to PC-related structures, certain sugar phosphates (e.g.galactose 6-phosphate) are bound near the PC-binding site, and one of the sugar hydroxyl groups appears to interact with CRP. The best small ligands for the polycationic binding site were Lys-Lys and Lys4. Because of the presence of multiple Lys-Lys sequences, polylysines have tremendously enhanced affinity. Although PC inhibits both PC- and polycation-based binding, none of the amines that inhibit polylysine binding inhibits PC binding, suggesting that the PC and polycationic binding sites do not overlap. C-reactive protein -phosphate aminoglycerol(3-amino-1,2-dihydroxypropane) 6-aminohexanoic acid aminohexanoylaminoethyl 3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid bovine serum albumin cholinephosphorylglycolaldehyde degree of polymerization diethylenetriaminepentaacetic acid 2-(N-morpholino)ethanesulfonic acid phosphorylcholine polyethylene glycol poly-l-lysine D-ribose N-[tris(hydoxymethyl)methyl]glycine galacturonic acid Human C-reactive protein (CRP)1 is a prototype acute-phase reactant. At the onset of infection or inflammation, the concentration of CRP may increase as much as 1000-fold. CRP acts as a defense molecule against some pathogens such as pneumococcal bacteria by initially binding to their surface components, e.g.phosphorylcholine (PC), and then activating the complement system or causing opsonization. The other major biological functions of CRP include removal of damaged cells and cell debris, wound healing, and immunomodulatory effect (1Kilpatrick J.M. Volanakis J.E. Immunol. Res. 1991; 10: 43-53Crossref PubMed Scopus (64) Google Scholar, 2Gewurz H. Zhang X.-H. Lint T.F. Curr. Opin. Immunol. 1995; 7: 54-64Crossref PubMed Scopus (261) Google Scholar, 3Szalai A.J. Agrawal A. Greenbough T.J. Volanakis J.E. Immunol. Res. 1997; 16: 127-136Crossref PubMed Scopus (90) Google Scholar). Such a diverse range of biological activity of CRP is matched by a wide range of ligand with which it interacts. The known ligands can be classified into three major groups. They are 1) compounds that contain phosphorylcholine or related structures, 2) polycationic compounds such as poly-l-lysine and protamine sulfate, and 3) carbohydrates that containd-galactose-related structures.CRP is a member of the pentraxin family and, thus, is a homopentamer whose subunits associate non-covalently side by side to form a wreath-like structure (4Shrive A.K. Cheetham G.M.T. Holden D. Myles D.A.A. Turnell W.G. Volanakis J.E. Pepys M.B. Bloomer A.C. Greenhough T.J. Nat. Struct. Biol. 1996; 3: 346-354Crossref PubMed Scopus (293) Google Scholar). Because all subunits face the same direction, the surface of each side of the wreath has distinct binding characteristics. It is known that the conformation of CRP changes depending on the presence or absence of calcium (5Kilpatrick J.M. Kearney J.F. Volanakis J.E. Mol. Immun. 1982; 19: 1159-1165Crossref PubMed Scopus (49) Google Scholar). In addition, the soluble form of CRP and CRP bound to a plastic surface or cell surface (so-called neo-CRP) present different epitopes (6Ying S.-C. Gewurz H. Kinoshita C.M. Potempa L.A. Siegel J.N. J. Immun. 1989; 143: 221-228PubMed Google Scholar), and therefore, there is a potential for altered binding specificity (7Kempka G. Roos P.H. Kolb-Bachofen V. J. Immun. 1990; 144: 1004-1009PubMed Google Scholar).We describe here a sensitive assay method for the ligand binding activities of CRP in the solution phase. The assay is based on polyethylene glycol (PEG) precipitation of CRP complexed with Eu-labeled, high affinity ligands. This method allows measurement of the binding activity of all three ligand groups, namely PC-, polycationic-, and galactose-based binding, using a similar assay strategy and the same detection method. We describe in this paper a detailed mapping of the PC-binding site and polycationic binding site of human CRP and the relationship between the two types of ligand.RESULTSEffect of pH on Ligand BindingThe binding activity of CRP for Eu-PC40-Et-BSA was remarkably constant in the pH range of 4.5–8.5 in the presence of CaCl2 (Fig. 2) and showed a slight decrease in the bound amount at pH 9. On the acidic side, however, there was a sharp drop in the binding activity below pH 4.5, reaching essentially no activity at pH 4. The pH dependence of Eu-PL binding was quite similar, with a sharp drop in the binding activity between pH 4.5 and 4.0 (not shown). This sudden loss of activity on the acidic side may be because of dissociation of the pentameric organization of CRP.Dependence of Ligand Binding on CalciumThe conformation of CRP is known to change depending on the presence or absence of calcium ion (5Kilpatrick J.M. Kearney J.F. Volanakis J.E. Mol. Immun. 1982; 19: 1159-1165Crossref PubMed Scopus (49) Google Scholar). The binding activity of CRP for Eu-PC40-Et-BSA without added calcium was about 60% of the maximal binding activity, which occurred at around 10 mmcalcium, and there was a gradual increase in the bound Eu-PC40-Et-BSA between 0 and 10 mm of added CaCl2. Perhaps the amount of calcium in the CRP sample and buffer is sufficient to mostly maintain the calcium-bound conformation of CRP without any added calcium. The bound amount of Eu-PL in the presence of 0–10 mm calcium did not seem to change much.To investigate if calcium is truly required for the binding, binding activity was measured in the presence of EDTA. As mentioned under "Experimental Procedures," EDTA competes Eu off the labeled macromolecules so that they had to be reloaded with Eu after filtration. Using this modified assay method, we found that CRP does not bind Eu-PC40-Et-BSA in the presence of EDTA (0.5 mm), since the fluorescence recovery was essentially the same whether CRP was present or not. This observation is in agreement with the prevailing idea that the PC-dependent binding to CRP requires calcium.As to Eu-PL, DiCamelli et al. (16DiCamelli R. Potempa L.A. Siegel J. Suyehira L. Petras K. Gewurz H. J. Immun. 1980; 125: 1933-1938PubMed Google Scholar) report that the binding of PL (measured by nephelometry) was inhibited in the presence of calcium. However, PL apparently can be bound by CRP in the presence of calcium if PC is also present (17Potempa L.A. Siegel J.N. Gewurz H. J. Immunol. 1981; 127: 1509-1514PubMed Google Scholar). We carried out two parallel assays of CRP binding Eu-PL in the presence of EDTA and in the presence of calcium at the Eu-PL and CRP concentrations of 185 and 380 nm, respectively. The bound amount in the presence of EDTA was 0.274 million counts/s (Mcps) as compared with 0.11 Mcps in the presence of 2 mm calcium, suggesting that Eu-PL binds better to the calcium-free conformation. To find out if this increase in the bound amount is because of an increased number of binding sites or because of increased affinity, IC50 of PL (DP 240) was determined in the presence of 0.5 mm EDTA and in the presence of 2 mm CaCl2 using the same washing/reloading protocol. IC50 in the presence of EDTA was 150 nm as compared with 600 nm in the presence of calcium, indicating that Eu-PL bound tighter to CRP in the calcium-free conformation. It should be noted, however, that the IC50 determined in the presence of calcium by this modified protocol is about four times higher than that obtained in the standard assay. Presumably the higher values resulted from excessive manipulations of washing and loading in the modified assay.Inhibition AssaysAt least two separate series of inhibition assay were carried out for each inhibitor. Inhibition curves generally had an appearance similar to those of PC9-Et-BSA and PC-glycerol shown in Fig. 1. In each case, 10–90% inhibition was observed within 2 decades of inhibitor concentration. The only exception to this was PC, which showed a very steep inhibition curve (Fig. 1). This may mean that there is cooperative inhibitory effect among five subunits of CRP,i.e. binding of the first PC molecule somehow facilitates the subsequent binding events.Inhibition of the Eu-PC40-Et-BSA BindingPolyvalent InhibitorsAs shown in TableI, binding affinity of the short-armed, PC-substituted BSA derivatives (PC-Et-BSA) was highly dependent on the PC content, showing a 42-fold increase in affinity when the PC content was increased by 6-fold. In contrast, there was only a small increase in affinity of the long-armed derivatives (PC-AhAe-BSA) beyond the PC content of 27 mol/mol. Affinity even higher than the PC-BSA derivatives could be attained when PC residues were on the flexible backbone of polylysine (DP 240). However, we found that the high inhibitory potency of PC-PLs is dependent not only on the PC content but also on its polycationic nature because when the three PC-PL preparations wereN-acetylated to eliminate the polycationic nature, the affinity of all three so-modified PC-PLs decreased. For instance, PC75-PL, which has roughly 165 unmodified amino groups, is an ∼4-fold stronger inhibitor than the N-acetylated counterpart, whereas PC139-PL, which has fewer free amino groups, was only a 2-fold better inhibitor than the NAc-PC139-PL. Thus, NAc-PC-PLs not only had lower overall affinities, but their affinities were more dependent on the PC content than PC-PLs.Table IInhibition of the Eu-PC40-Et-BSA binding to CRP by multivalent, PC-containing inhibitorsInhibitorPC contentIC50mol/molμmPC-Et-BSA 91.50 210.20 400.102 570.036PC-AhAe-BSA 270.098 410.085 450.070PC-PL 750.0281140.0121390.0105NAc-PC-PL 750.1081140.0681390.024 Open table in a new tab Small InhibitorsThe IC50 value for PC of 40 μm obtained by us (TableII) is quite comparable with what was reported by others, e.g. 42.5 μm (18Young N.M. Williams R.E. J. Immun. 1978; 121: 1893-1898PubMed Google Scholar) and 75 μm (19Volanakis J.E. Kaplan M.H. Proc. Soc. Exp. Biol. Med. 1971; 136: 612-614Crossref PubMed Scopus (323) Google Scholar). These values are slightly higher thanKD of 18 μm determined by capillary electrophoresis (20Heegaard H.H. Robey F.A. J. Immunol. Methods. 1993; 166: 103-110Crossref PubMed Scopus (44) Google Scholar). A KD value as low as 3 μm was obtained using a less direct enzyme-linked immunosorbent assay method (21Tanaka T. Robey F.A. J. Immunol. Methods. 1983; 65: 333-341Crossref PubMed Scopus (19) Google Scholar). The relative IC50 values were also comparable; the values for PC, PC-glycerol, and phosphorylethanolamine were 1:18:32 in our assay and 1:22:55 by Volanakis and Kaplan (19Volanakis J.E. Kaplan M.H. Proc. Soc. Exp. Biol. Med. 1971; 136: 612-614Crossref PubMed Scopus (323) Google Scholar). Shown also in Table II are IC50values of a number of other phosphate-containing compounds and related structures. These non-choline-containing phosphates can be divided into three groups: 1) compounds with the binding affinity comparable with Pi (IC50 20 mm): Glc-6-P and Man-6-P; 2) compounds with ∼10-fold higher affinity than Pi: glycerol phosphate, Gal-6-P, Rib-5-P, 2-deoxy-d-ribose-5-P as well as AMP and dAMP; 3) compounds that have very poor inhibitory potency: α-Glc-1-P and α-Gal-1-P. Interestingly, Gal-6-sulfate, although quite similar in structure to Gal-6-P (Fig. 3), was totally inactive at 50 mm. As will be described under "Discussion," the phosphate group of PC is held in the binding site by direct coordination to a pair of calcium ions. The failure of Gal-6-sulfate to bind suggests that somehow sulfate cannot make strong coordination bonds to calcium ions. Galacturonic acid (at 47 mm) was also totally ineffective as an inhibitor. However, serum amyloid P-component, another pentraxin structurally homologous to CRP, binds methyl 4,6-(1-carboxyethylidene)-β-d-galactopyranoside (Fig. 3) via its carboxylate interacting with calcium atoms much the same way as phosphate does in CRP (22Elmsley J. White H.E. O'Hara B.P. Oliva G. Srinivasan N. Tickle I.J. Blundell T.L. Pepys M.B. Wood S.P. Nature. 1994; 367: 338-345Crossref PubMed Scopus (414) Google Scholar). Perhaps in the case of galacturonic acid the carboxylate group is unfavorably oriented for the calcium chelation, probably because of close proximity of carboxylate to the sugar ring.Table IIInhibition of the Eu-PC40-Et-BSA binding to CRP by small, PC-related and other phosphate-containing moleculesInhibitorIC50μmPC40 ± 10PC-glycerol550PE1270 ± 130Glycerophosphate4500Phosphate24000 ± 1000Gal-6-P1900 ± 300Glc-6-P20000 ± 3000Man-6-P16000 ± 0Rib-5-P2000 ± 5002-Deoxy-Rib-5-P1650 ± 50AMP3100 ± 400dAMP3100 ± 400Galα1-PNI (40,000)2-aNI, not inhibitory at the concentration shown in parentheses.Glcα1-PNI (11,000)2-aNI, not inhibitory at the concentration shown in parentheses.2-bGlcα1-P gave 27% inhibition at 45,000 μm.Manα1-P30000 ± 01-Hexanoylphosphatidylcholine1180 ± 201-Decanoylphosphatidylcholine1500 ± 5001-Decanoyl-2-acetyl phosphatidylcholine2300 ± 500PE, phosphorylethanolamine.2-a NI, not inhibitory at the concentration shown in parentheses.2-b Glcα1-P gave 27% inhibition at 45,000 μm. Open table in a new tab Figure 3Structures of galactose derivatives with negative charge(s) on the C-6 position. GalA, galacturonic;MOβDG, methyl 4,6-(1-carboxyethylidene)-β-d-galactopyranoside.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Lysolecithin in the lipid vesicle or on the cell surface is known to be bound by CRP, whereas totally acylated lecithin is not (23Volanakis J.E. Narkates A.J. J. Immun. 1981; 126: 1820-1825PubMed Google Scholar). To assess the inhibitory potency of the individual lysolecithin molecule in solution as well as to assess the importance of having free β-OH for binding, two lysolecithins, each with one short fatty acid chain (C6 and C10) and 1-decanoyl-2-acetyl-phosphatidylcholine, were tested. All three phosphatidylcholine derivatives were soluble in water and expected to remain dispersed as monomers because of short acyl chains and the presence of a zwitterionic PC residue. The IC50values of these acylated phosphatidylcholine derivatives were all in 1–2 mm range, only slightly higher than that of PC-glycerol (0.5 mm), suggesting that the presence of free β-OH is not a requirement for binding. Perhaps, as suggested by others, physical disposition of lysolecithin on the membrane and/or disruption of membrane structure caused by the presence of lysolecithin are the reasons for preferential binding of lysolecithin-containing vesicles. The compounds with amino groups, Lys (50 mm), Arg (11 mm), Lys-Lys (8 mm), and Lys4 (4 mm), choline chloride (30 mm) as well as PL of DP 240 (5.6 μm) and histones (400 μm) did not inhibit the PC-dependent binding at the highest concentration tested, which is indicated in parenthesis.Inhibition of the Eu-PL BindingPolyvalent InhibitorsTable IIIlists the inhibitory potency of all the polyvalent inhibitors tested. As observed for Eu-PC40-Et-BSA binding, the inhibitory potency of PL is highly dependent on the number of cationic groups present, which is proportional to the molecular weight. A comparison of PL of DP 16 and DP 240 shows that an increase in DP of 15-fold resulted in the affinity increase of 1000-fold. Poly-l-Arg and poly-l-ornithine, although tested at only one molecular size, seem to inhibit at the concentration comparable to polylysines. Among the basic proteins tested, only a lysine-rich histone preparation (enriched in H-1) had a measurable IC50 (220 μg/ml), although its affinity was much weaker than polylysine of the comparable size, i.e. DP-60 PL (13 kDa) had IC50 of 23 μg/ml. PC40-Et-BSA could inhibit the Eu-PL binding but much less effectively (IC50 = 2.7 μm) than it inhibited the Eu-PC40-Et-BSA binding (IC50 = 0.1 μm).Table IIIInhibition of the Eu-PL (DP-240) binding to CRP by poly-(basic amino acid) and basic proteinsInhibitorDegree of polymerizationMolecular massIC50μg/mlμmkDaPoly-L-Lys163.4480100601322.81.75240505 ± 1.50.1 ± 0.03Poly-L-Arg20038.312.30.32Poly-L-Orn1693315.50.47Histone3-aHistones II-AS and III-S are both preparations from calf thymus. II-AS contains 11.7% Lys and 14.7% Arg, whereas III-S is a lysine-rich preparation containing 27% Lys and 2% Arg. II-AS143-bApproximate molecular mass assuming mostly composed of histones, H2A, H2B, H3, and H4.NI (300)3-cNI, not inhibitory at the concentration shown in parentheses.NI (21) III-S223-dApproximate molecular mass assuming mostly composed of histone, H1.237 ± 1711 ± 0.8Lysozyme14NI (460)NI (33)3-a Histones II-AS and III-S are both preparations from calf thymus. II-AS contains 11.7% Lys and 14.7% Arg, whereas III-S is a lysine-rich preparation containing 27% Lys and 2% Arg.3-b Approximate molecular mass assuming mostly composed of histones, H2A, H2B, H3, and H4.3-c NI, not inhibitory at the concentration shown in parentheses.3-d Approximate molecular mass assuming mostly composed of histone, H1. Open table in a new tab Small InhibitorsBasic amino acids, Lys, Arg, and His, all showed a large affinity enhancement effect in the intermediate concentration range as shown in Fig. 4, the maximum bound amount being as high as 200% that bound in the absence of inhibitor. The reason for this interesting phenomenon is unclear, but to find out which groups are responsible for this effect, we tested various substituted Lys derivatives. As shown in TableIV, the presence of both unmodified α-carboxylic acid and α-amino group may be responsible, sinceNα-acetyl-Lys (slight activation) and Lys methyl ester had normal inhibition curves, whereasNε-acetyl Lys gave the activation (190%). A neutral amino acid, Ala, gave activation, although only up to 140%. Of all the amino-containing compounds tested, only Lys-Lys and Lys4 had sub-mm IC50 values, whereas all other amines were much poorer inhibitors.Figure 4Enhancement of Eu-PL binding by certain amino acids. ▪, l-Lys; ▴, l-Arg.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IVInhibition of the Eu-PL (DP-240) binding to CRP by basic moleculesInhibitorIC50μmLys-Lys370 ± 50Lys4230 ± 30Lys, Arg, HisEnh4-aA transient increase in the bound amount of ligand does not allow accurate IC50determination.Nε-Acetyl-LysEnhLys methyl ester15,000 ± 3,000Nα-Acetyl-Lys13,000 ± 1,0001,2-Diaminoethane62,0001,6-Diaminohexane17,0001,8-Diaminooctane3,500 ± 5001,12-Diaminododecane27,000 ± 0Octylamine20,000 ± 4,000Choline chloride20,000 ± 4,000PC4,700 ± 7004-a A transient increase in the bound amount of ligand does not allow accurate IC50determination. Open table in a new tab Human C-reactive protein (CRP)1 is a prototype acute-phase reactant. At the onset of infection or inflammation, the concentration of CRP may increase as much as 1000-fold. CRP acts as a defense molecule against some pathogens such as pneumococcal bacteria by initially binding to their surface components, e.g.phosphorylcholine (PC), and then activating the complement system or causing opsonization. The other major biological functions of CRP include removal of damaged cells and cell debris, wound healing, and immunomodulatory effect (1Kilpatrick J.M. Volanakis J.E. Immunol. Res. 1991; 10: 43-53Crossref PubMed Scopus (64) Google Scholar, 2Gewurz H. Zhang X.-H. Lint T.F. Curr. Opin. Immunol. 1995; 7: 54-64Crossref PubMed Scopus (261) Google Scholar, 3Szalai A.J. Agrawal A. Greenbough T.J. Volanakis J.E. Immunol. Res. 1997; 16: 127-136Crossref PubMed Scopus (90) Google Scholar). Such a diverse range of biological activity of CRP is matched by a wide range of ligand with which it interacts. The known ligands can be classified into three major groups. They are 1) compounds that contain phosphorylcholine or related structures, 2) polycationic compounds such as poly-l-lysine and protamine sulfate, and 3) carbohydrates that containd-galactose-related structures. CRP is a member of the pentraxin family and, thus, is a homopentamer whose subunits associate non-covalently side by side to form a wreath-like structure (4Shrive A.K. Cheetham G.M.T. Holden D. Myles D.A.A. Turnell W.G. Volanakis J.E. Pepys M.B. Bloomer A.C. Greenhough T.J. Nat. Struct. Biol. 1996; 3: 346-354Crossref PubMed Scopus (293) Google Scholar). Because all subunits face the same direction, the surface of each side of the wreath has distinct binding characteristics. It is known that the conformation of CRP changes depending on the presence or absence of calcium (5Kilpatrick J.M. Kearney J.F. Volanakis J.E. Mol. Immun. 1982; 19: 1159-1165Crossref PubMed Scopus (49) Google Scholar). In addition, the soluble form of CRP and CRP bound to a plastic surface or cell surface (so-called neo-CRP) present different epitopes (6Ying S.-C. Gewurz H. Kinoshita C.M. Potempa L.A. Siegel J.N. J. Immun. 1989; 143: 221-228PubMed Google Scholar), and therefore, there is a potential for altered binding specificity (7Kempka G. Roos P.H. Kolb-Bachofen V. J. Immun. 1990; 144: 1004-1009PubMed Google Scholar). We describe here a sensitive assay method for the ligand binding activities of CRP in the solution phase. The assay is based on polyethylene glycol (PEG) precipitation of CRP complexed with Eu-labeled, high affinity ligands. This method allows measurement of the binding activity of all three ligand groups, namely PC-, polycationic-, and galactose-based binding, using a similar assay strategy and the same detection method. We describe in this paper a detailed mapping of the PC-binding site and polycationic binding site of human CRP and the relationship between the two types of ligand. RESULTSEffect of pH on Ligand BindingThe binding activity of CRP for Eu-PC40-Et-BSA was remarkably constant in the pH range of 4.5–8.5 in the presence of CaCl2 (Fig. 2) and showed a slight decrease in the bound amount at pH 9. On the acidic side, however, there was a sharp drop in the binding activity below pH 4.5, reaching essentially no activity at pH 4. The pH dependence of Eu-PL binding was quite similar, with a sharp drop in the binding activity between pH 4.5 and 4.0 (not shown). This sudden loss of activity on the acidic side may be because of dissociation of the pentameric organization of CRP.Dependence of Ligand Binding on CalciumThe conformation of CRP is known to change depending on the presence or absence of calcium ion (5Kilpatrick J.M. Kearney J.F. Volanakis J.E. Mol. Immun. 1982; 19: 1159-1165Crossref PubMed Scopus (49) Google Scholar). The binding activity of CRP for Eu-PC40-Et-BSA without added calcium was about 60% of the maximal binding activity, which occurred at around 10 mmcalcium, and there was a gradual increase in the bound Eu-PC40-Et-BSA between 0 and 10 mm of added CaCl2. Perhaps the amount of calcium in the CRP sample and buffer is sufficient to mostly maintain the calcium-bound conformation of CRP without any added calcium. The bound amount of Eu-PL in the presence of 0–10 mm calcium did not seem to change much.To investigate if calcium is truly required for the binding, binding activity was measured in the presence of EDTA. As mentioned under "Experimental Procedures," EDTA competes Eu off the labeled macromolecules so that they had to be reloaded with Eu after filtration. Using this modified assay method, we found that CRP does not bind Eu-PC40-Et-BSA in the presence of EDTA (0.5 mm), since the fluorescence recovery was essentially the same whether CRP was present or not. This observation is in agreement with the prevailing idea that the PC-dependent binding to CRP requires calcium.As to Eu-PL, DiCamelli et al. (16DiCamelli R. Potempa L.A. Siegel J. Suyehira L. Petras K. Gewurz H. J. Immun. 1980; 125: 1933-1938PubMed Google Scholar) report that the binding of PL (measured by nephelometry) was inhibited in the presence of calcium. However, PL apparently can be bound by CRP in the presence of calcium if PC is also present (17Potempa L.A. Siegel J.N. Gewurz H. J. Immunol. 1981; 127: 1509-1514PubMed Google Scholar). We carried out two parallel assays of CRP binding Eu-PL in the presence of EDTA and in the presence of calcium at the Eu-PL and CRP concentrations of 185 and 380 nm, respectively. The bound amount in the presence of EDTA was 0.274 million counts/s (Mcps) as compared with 0.11 Mcps in the presence of 2 mm calcium, suggesting that Eu-PL binds better to the calcium-free conformation. To find out if this increase in the bound amount is because of an increased number of binding sites or because of increased affinity, IC50 of PL (DP 240) was determined in the presence of 0.5 mm EDTA and in the presence of 2 mm CaCl2 using the same washing/reloading protocol. IC50 in the presence of EDTA was 150 nm as compared with 600 nm in the presence of calcium, indicating that Eu-PL bound tighter to CRP in the calcium-free conformation. It should be noted, however, that the IC50 determined in the presence of calcium by this modified protocol is about four times higher than that obtained in the standard assay. Presumably the higher values resulted from excessive manipulations of washing and loading in the modified assay.Inhibition AssaysAt least two separate series of inhibition assay were carried out for each inhibitor. Inhibition curves generally had an appearance similar to those of PC9-Et-BSA and PC-glycerol shown in Fig. 1. In each case, 10–90% inhibition was observed within 2 decades of inhibitor concentration. The only exception to this was PC, which showed a very steep inhibition curve (Fig. 1). This may mean that there is cooperative inhibitory effect among five subunits of CRP,i.e. binding of the first PC molecule somehow facilitates the subsequent binding events.Inhibition of the Eu-PC40-Et-BSA BindingPolyvalent InhibitorsAs shown in TableI, binding affinity of the short-armed, PC-substituted BSA derivatives (PC-Et-BSA) was highly dependent on the PC content, showing a 42-fold increase in affinity when the PC content was increased by 6-fold. In contrast, there was only a small increase in affinity of the long-armed derivatives (PC-AhAe-BSA) beyond the PC content of 27 mol/mol. Affinity even higher than the PC-BSA derivatives could be attained when PC residues were on the flexible backbone of polylysine (DP 240). However, we found that the high inhibitory potency of PC-PLs is dependent not only on the PC content but also on its polycationic nature because when the three PC-PL preparations wereN-acetylated to eliminate the polycationic nature, the affinity of all three so-modified PC-PLs decreased. For instance, PC75-PL, which has roughly 165 unmodified amino groups, is an ∼4-fold stronger inhibitor than the N-acetylated counterpart, whereas PC139-PL, which has fewer free amino groups, was only a 2-fold better inhibitor than the NAc-PC139-PL. Thus, NAc-PC-PLs not only had lower overall affinities, but their affinities were more dependent on the PC content than PC-PLs.Table IInhibition of the Eu-PC40-Et-BSA binding to CRP by multivalent, PC-containing inhibitorsInhibitorPC contentIC50mol/molμmPC-Et-BSA 91.50 210.20 400.102 570.036PC-AhAe-BSA 270.098 410.085 450.070PC-PL 750.0281140.0121390.0105NAc-PC-PL 750.1081140.0681390.024 Open table in a new tab Small InhibitorsThe IC50 value for PC of 40 μm obtained by us (TableII) is quite comparable with what was reported by others, e.g. 42.5 μm (18Young N.M. Williams R.E. J. Immun. 1978; 121: 1893-1898PubMed Google Scholar) and 75 μm (19Volanakis J.E. Kaplan M.H. Proc. S

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