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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]

Human C-reactive protein (CRP) protects mice from lethal Streptococcus pneumoniae infection when injected into mice within the range of 6 h before to 2 h after the administration of pneumococci. Because CRP binds to phosphocholine-containing substances and subsequently activates the complement system, it has been proposed that the antipneumococcal function of CRP requires the binding of CRP to phosphocholine moieties present in pneumococcal cell wall C-polysaccharide. To test this proposal experimentally, in this study, we utilized a new CRP mutant incapable of binding to phosphocholine. Based on the structure of CRP-phosphocholine complexes, which showed that Phe66, Thr76, and Glu81 formed the phosphocholine-binding pocket, we constructed a CRP mutant F66A/T76Y/E81A in which the pocket was blocked by substituting Tyr for Thr76. When compared with wild-type CRP, mutant CRP bound more avidly to phosphoethanolamine and could be purified by affinity chromatography using phosphoethanolamine-conjugated Sepharose. MutantCRPdid not bind to phosphocholine, C-polysaccharide, or pneumococci. Mutant CRP was free in the mouse serum, and its rate of clearance in vivo was not faster than that of wild-type CRP. When either 25μgor 150μg of CRP was administered into mice, unlike wild-type CRP, mutant CRP did not protect mice from lethal pneumococcal infection. Mice injected with mutant CRP had higher mortality rates than mice that received wild-type CRP. Decreased survival was due to the increased bacteremia in mice treated with mutant CRP.Weconclude that the phosphocholine-binding pocket on CRP is necessary for CRP-mediated initial protection of mice against lethal pneumococcal infection. [ABSTRACT FROM AUTHOR]

C-reactive protein (CRP) is a cyclic pentameric protein whose major binding specificity, at physiological pH, is for substances bearing exposed phosphocholine moieties. Another pentameric form of CRP, which exists at acidic pH, displays binding activity for oxidized LDL (ox-LDL). The ox-LDL-binding site in CRP, which is hidden at physiological pH, is exposed by acidic pH-induced structural changes in pentameric CRP. The aim of this study was to expose the hidden ox-LDL-binding site of CRP by site-directed mutagenesis and to generate a CRP mutant that can bind to ox-LDL without the requirement of acidic pH. Mutation of Glu42, an amino acid that participates in intersubunit interactions in the CRP pentamer and is buried, to Gln resulted in a CRP mutant (E42Q) that showed significant binding activity for ox-LDL at physiological pH. For maximal binding to ox-LDL, E42Q CRP required a pH much less acidic than that required by wild-type CRP. At any given pH, E42Q CRP was more efficient than wild-type CRP in binding to ox-LDL. Like wild-type CRP, E42Q CRP remained pentameric at acidic pH. Also, E42Q CRP was more efficient than wild-type CRP in binding to several other deposited, conformationally altered proteins. The E42Q CRP mutant provides a tool to investigate the functions of CRP in defined animal models of inflammatory diseases including atherosclerosis because wild-type CRP requires acidic pH to bind to deposited, conformationally altered proteins, including ox-LDL, and available animal models may not have sufficient acidosis or other possible modifiers of the pentameric structure of CRP at the sites of inflammation. [ABSTRACT FROM AUTHOR]