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C-reactive protein (CRP) performs two recognition functions that are relevant to cardiovascular disease. First, in its native pentameric conformation, CRP recognizes molecules and cells with exposed phosphocholine (PCh) groups, such as microbial pathogens and damaged cells. PCh-containing ligand-bound CRP activates the complement system to destroy the ligand. Thus, the PCh-binding function of CRP is defensive if it occurs on foreign pathogens because it results in the killing of the pathogen via complement activation. On the other hand, the PCh-binding function of CRP is detrimental if it occurs on injured host cells because it causes more damage to the tissue via complement activation; this is how CRP worsens acute myocardial infarction and ischemia/reperfusion injury. Second, in its nonnative pentameric conformation, CRP also recognizes atherogenic low-density lipoprotein (LDL). Recent data suggest that the LDL-binding function of CRP is beneficial because it prevents formation of macrophage foam cells, attenuates inflammatory effects of LDL, inhibits LDL oxidation, and reduces proatherogenic effects of macrophages, raising the possibility that nonnative CRPmay show atheroprotective effects in experimental animals. In conclusion, temporarily inhibiting the PCh-binding function of CRP along with facilitating localized presence of nonnative pentameric CRP could be a promising approach to treat atherosclerosis and myocardial infarction. There is no need to stop the biosynthesis of CRP. [ABSTRACT FROM AUTHOR]

Abstract: C-reactive protein (CRP) that has been conserved throughout evolution is a host-defense molecule. Its attraction towards phosphocholine-ligands, such as modified low-density lipoprotein, and apoptotic cells leads to the “masking” of these substances that have the capabilities to otherwise engage in deleterious activities. Complement activation by CRP complexes and the modulation by CRP of complement activation by its ligands add up to its beneficial effects. In the presence of CRP, production of membrane-damaging last product of the complement pathway is arrested. CRP is currently serving as an indicator of cardiovascular diseases, but to pinpoint the role of CRP in atherosclerosis, a drug that can lower cholesterol levels, but not the CRP levels, is needed for experimentation. [Copyright &y& Elsevier]

C-reactive protein (CRP) is an evolutionarily conserved protein, a component of the innate immune system, and an acute phase protein in humans. In addition to its raised level in blood in inflammatory states, CRP is also localized at sites of inflammation including atherosclerotic lesions, arthritic joints and amyloid plaque deposits. Results of in vivo experiments in animal models of inflammatory diseases indicate that CRP is an anti-pneumococcal, anti-atherosclerotic, anti-arthritic and an anti-amyloidogenic molecule. The mechanisms through which CRP functions in inflammatory diseases are not fully defined; however, the ligand recognition function of CRP in its native and non-native pentameric structural conformations and the complement-activating ability of ligand-complexed CRP have been suggested to play a role. One tool to understand the structure-function relationships of CRP and determine the contributions of the recognition and effector functions of CRP in host defense is to employ site-directed mutagenesis to create mutants for experimentation. For example, CRP mutants incapable of binding to phosphocholine are generated to investigate the importance of the phosphocholine-binding property of CRP in mediating host defense. Recombinant CRP mutants can be expressed in mammalian cells and, if expressed, can be purified from the cell culture media. While the methods to purify wild-type CRP are well established, different purification strategies are needed to purify various mutant forms of CRP if the mutant does not bind to either calcium or phosphocholine. In this article, we report the methods used to purify pentameric recombinant wild-type and mutant CRP expressed in and secreted by mammalian cells. [ABSTRACT FROM AUTHOR]

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]

Abstract: Background: C-reactive protein (CRP) is an acute phase plasma protein. An important binding specificity of CRP is for the modified forms of low-density lipoprotein (LDL) in which the phosphocholine-binding sites of CRP participate. CRP, however, does not bind to native LDL. Methods: We investigated the interaction of CRP with native LDL using sucrose density gradient ultracentrifugation. Results: We found that the blocking of the phosphocholine-binding sites of CRP with phosphoethanolamine (PEt) converted CRP into a potent molecule for binding to native LDL. In the presence of PEt, CRP acquired the ability to bind to fluid-phase purified native LDL. Because purified native LDL may undergo subtle modifications, we also used whole human serum as the source of native LDL. In the presence of PEt, CRP bound to native LDL in serum also. The effect of PEt on CRP was selective for LDL because PEt-complexed CRP did not bind to high-density lipoprotein in the serum. Conclusions: The pharmacologic intervention of endogenous CRP by PEt-based compounds, or the use of exogenously prepared CRP-PEt complexes, may turn out to be an effective approach to capture native LDL cholesterol in vivo to prevent the development of atherosclerosis. [Copyright &y& Elsevier]