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C-reactive protein (CRP) is an acute phase protein in humans and rabbits that has the ability to bind a number of biologically important ligands including phosphocholine (PCh), histones, and polycations. In addition to this recognition function, ligand-complexed or aggregated CRP is capable of activating the classical complement pathway. We have generated two strains of transgenic mice in order to study CRP-binding to PCh and consequent complement activation. Based on crystallographic and mutagenesis studies in human CRP (huCRP), we mutated Phe66 and Glu81 in the rabbit CRP (rbCRP) gene and generated a strain of transgenic mice (F66Y/E81K), which expressed this variant form of rbCRP. We also mutated Tyr175 in rbCRP to generate transgenic mice which expressed a variant form of rbCRP (Y175A). In vitro, F66Y/E81K rbCRP purified from serum had dramatically reduced binding to PCh. Additionally F66Y/E81K rbCRP not only maintained its ability to bind polycations and histones, but also bound more avidly to specific histones and lysine polymers than wild type (wt) rbCRP. Y175A rbCRP was not able to activate complement when bound to pneumococcal C-polysaccharide (PnC), but was, along with F66Y/E81K and wild type rbCRP, able to activate complement when bound to a small lysine polymer or when directly adsorbed to a solid phase. This complement activation presumably occurs through the classical complement pathway as the three rbCRPs, adsorbed to a solid phase, bound C1q. Taken together, our results demonstrate that the PCh-binding and the polycation-binding sites on rbCRP are distinct but possibly overlapping. The conformational changes in the C1q-binding site of CRP to activate complement depend on the nature of the ligand and on the location of the ligand-binding site. [Copyright &y& Elsevier]

C-reactive protein (CRP) is present at sites of inflammation including amyloid plaques, atherosclerotic lesions, and arthritic joints. CRP, in its native pentameric structural conformation, binds to cells and molecules that have exposed phosphocholine (PCh) groups. CRP, in its non-native pentameric structural conformation, binds to a variety of deposited, denatured, and aggregated proteins, in addition to binding to PCh-containing substances. In this study, we investigated the effects of H2O2, a prototypical reactive oxygen species that is also present at sites of inflammation, on the ligand recognition function of CRP. Controlled H2O2 treatment of native CRP did not monomerize CRP and did not affect the PCh binding activity of CRP. In solid phase ELISA-based ligand binding assays, purified pentameric H2O2-treated CRP bound to a number of immobilized proteins including oxidized LDL, IgG, amyloid β peptide 1-42, C4bbinding protein, and factor H, in a CRP concentration- and ligand concentration-dependent manner. Using oxidized LDL as a representative protein ligand for H2O2-treated CRP, we found that the binding occurred in a Ca2+-independent manner and did not involve the PCh-binding site of CRP. We conclude that H2O2 is a biological modifier of the structure and ligand recognition function of CRP. Overall, the data suggest that the ligand recognition function of CRP is dependent on the presence of an inflammatory microenvironment. We hypothesize that one of the functions of CRP at sites of inflammation is to sense the inflammatory microenvironment, change its own structure in response but remain pentameric, and then bind to pathogenic proteins deposited at those sites. [ABSTRACT FROM AUTHOR]

C-reactive protein (CRP) is a phylogenetically conserved protein; in humans, it i~ present in the plasma and at sites of inflammation. At physiological pH, native pentameric CRP exhibits calcium-dependent binding specificity for phosphocholine. In this study, we determined the binding specificities of CRP at acidic pH, a chara:teristic of inflammatory sites. We investigated the binding of fluid-phase CRP to six immobilized proteins: complement factor H, oxidized low-density lipoprotein, complement C3b, EgG, amyloid β, and BSA immobilized on microtiter plates. At pH 7.0, CRP did not bind to any of these proteins, but, at pH ranging from 5.2 to 4.6, CRP bound to all six proteins. Acidic pH did not monomerize CRP but modified the pentameric structure, as determined by gel filtration, 1-anilinonaphthalene-8-sulfonic acid-binding fluorescence, and phosphocholine-binding assays. Some modifications in CRP were reversible at pH 7.0, for example, the phosphocholine-binding activity of CRP, which was reduced at acidic pH, was restored after pH neutralization. For efficient binding of acidic pHtreated CRP to immobilized proteins, it was necessary that the immobilized proteins, except factor H, were also exposed to acidic pH. Because immobilization of proteins on microtiter plates and exposure of immobilized proteins to acidic pH alter the conformation of immobilized proteins, our findings suggest that conformationally altered proteins form a CRP-ligand in acidic environment, regardless of the identity of the protein. This ligand binding specificity of CRP in its acidic pH-induced pentameric state has implications for toxic conditions involving protein misfolding in acidic environments and favors the conservation of CRP throughout evolution. [ABSTRACT FROM AUTHOR]

Abstract: Background: The 5 subunits of native pentameric C-reactive protein (CRP) are dissociated to generate the monomeric form of CRP (mCRP) in some in vitro conditions, both physiological and non-physiological, and also in vivo. Many bioactivities of mCRP generated by urea-treatment of CRP and of mCRP generated by mutating the primary structure of CRP have been reported. The bioactivities of mCRP generated by spontaneous dissociation of CRP are largely unexplored. Methods: We purified mCRP generated by spontaneous dissociation of CRP and investigated the binding of mCRP to enzymatically-modified low-density lipoprotein (E-LDL). Results: mCRP was approximately 60 times more potent than CRP in binding to E-LDL. In the presence of the small-molecule compound phosphoethanolamine (PEt), at 37°C, the binding of mCRP to E-LDL was enhanced <2-fold, while the binding of CRP to E-LDL was enhanced >10-fold. In contrast, PEt inhibited the binding of both CRP and mCRP to pneumococcal C-polysaccharide, another phosphocholine-containing ligand to which CRP and mCRP were found to bind. We have not investigated yet whether PEt alters the structure of CRP at 37°C. Conclusions: Combined data suggest that the targeting of CRP with the aim to monomerize CRP in vivo may be an effective approach to capture modified forms of LDL. [Copyright &y& Elsevier]

C1q is the first subcomponent of the classical complement pathway that can interact with a range of biochemically and structurally diverse self and nonself ligands. The globular domain of C1q (gC1q), which is the ligand-recognition domain, is a heterotrimeric structure composed of the C-terminal regions of A (ghA), B (ghB), and C (ghC) chains. The expression and functional characterization of ghA, ghB, and ghC modules have revealed that each chain has specific and differential binding properties toward C1q ligands. It is largely considered that C1q-ligand interactions are ionic in nature; however, the complementary ligand-binding sites on C1q and the mechanisms of interactions are still unclear. To identify the residues on the gC1q domain that are likely to be involved in ligand recognition, we have generated a number of substitution mutants of ghA, ghB, and ghC modules and examined their interactions with three selected ligands: IgG1, C-reactive protein (CRP), and pentraxin 3 (PTX3). Our results suggest that charged residues belonging to the apex of the gC1q heterotrimer (with participation of all three chains) as well as the side of the ghB are crucial for C1q binding to these ligands, and their contribution to each interaction is different. It is likely that a set of charged residues from the gC1q surface participate via different ionic and hydrogen bonds with corresponding residues from the ligand, instead of forming separate binding sites. Thus, a recently proposed model suggesting the rotation of the gC1q domain upon ligand recognition may be extended to C1q interaction with CRP and PTX3 in addition to IgG1. [ABSTRACT FROM AUTHOR]