Purification of recombinant C-reactive protein mutants.

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.

The phosphocholine-binding pocket on C-reactive protein is necessary for initial protection of mice against pneumococcal infection.

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 Phe(66), Thr(76), and Glu(81) formed the phosphocholine-binding pocket, we constructed a CRP mutant F66A/T76Y/E81A in which the pocket was blocked by substituting Tyr for Thr(76). When compared with wild-type CRP, mutant CRP bound more avidly to phosphoethanolamine and could be purified by affinity chromatography using phosphoethanolamine-conjugated Sepharose. Mutant CRP did 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 25 µg or 150 µg 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. We conclude that the phosphocholine-binding pocket on CRP is necessary for CRP-mediated initial protection of mice against lethal pneumococcal infection.

Oct-1 acts as a transcriptional repressor on the C-reactive protein promoter.

C-reactive protein (CRP), a plasma protein of the innate immune system, is produced by hepatocytes. A critical regulatory region (-42 to -57) on the CRP promoter contains binding site for the IL-6-activated transcription factor C/EBPß. The IL-1ß-activated transcription factor NF-κB binds to a κB site located nearby (-63 to -74). The κB site overlaps an octamer motif (-59 to -66) which is the binding site for the constitutively active transcription factor Oct-1. Oct-1 is known to function both as a transcriptional repressor and as an activator depending upon the promoter context. Also, Oct-1 can regulate gene expression either by binding directly to the promoter or by interacting with other transcription factors bound to the promoter. The aim of this study was to investigate the functions of Oct-1 in regulating CRP expression. In luciferase transactivation assays, overexpressed Oct-1 inhibited (IL-6+IL-1ß)-induced CRP expression in Hep3B cells. Deletion of the Oct-1 site from the promoter drastically reduced the cytokine response because the κB site was altered as a consequence of deleting the Oct-1 site. Surprisingly, overexpressed Oct-1 inhibited the residual (IL-6+IL-1ß)-induced CRP expression through the promoter lacking the Oct-1 site. Similarly, deletion of the Oct-1 site reduced the induction of CRP expression in response to overexpressed C/EBPß, and overexpressed Oct-1 inhibited C/EBPß-induced CRP expression through the promoter lacking the Oct-1 site. We conclude that Oct-1 acts as a transcriptional repressor of CRP expression and it does so by occupying its cognate site on the promoter and also via other transcription factors by an as yet undefined mechanism.

Exposing a hidden functional site of C-reactive protein by site-directed mutagenesis.

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 Glu(42), 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.

Atherosclerosis-related functions of C-reactive protein.

C-reactive protein (CRP) is secreted by hepatocytes as a pentameric molecule made up of identical monomers, circulates in the plasma as pentamers, and localizes in atherosclerotic lesions. In some cases, localized CRP was detected by using monoclonal antibodies that did not react with native pentameric CRP but were specific for isolated monomeric CRP. It has been reported that, once CRP is bound to certain ligands, the pentameric structure of CRP is altered so that it can dissociate into monomers. Accordingly, the monomeric CRP found in atherosclerotic lesions may be a stationary, ligand-bound, by-product of a ligand-binding function of CRP. CRP binds to modified forms of low-density lipoprotein (LDL). The binding of CRP to oxidized LDL requires acidic pH conditions; the binding at physiological pH is controversial. The binding of CRP to enzymatically-modified LDL occurs at physiological pH; however, the binding is enhanced at acidic pH. Using enzymatically-modified LDL, CRP has been shown to prevent the formation of enzymatically-modified LDL-loaded macrophage foam cells. CRP is neither pro-atherogenic nor atheroprotective in ApoE⁻(/)⁻ and ApoB¹°°(/)¹°°Ldlr ⁻(/)⁻ murine models of atherosclerosis, except in one study where CRP was found to be slightly atheroprotective in ApoB¹°°(/)¹°°Ldlr ⁻(/)⁻ mice. The reasons for the ineffectiveness of human CRP in murine models of atherosclerosis are not defined. It is possible that an inflammatory environment, such as those characterized by acidic pH, is needed for efficient interaction between CRP and atherogenic LDL during the development of atherosclerosis and to observe the possible atheroprotective function of CRP in animal models.

Identification of acidic pH-dependent ligands of pentameric C-reactive protein.

C-reactive protein (CRP) is a phylogenetically conserved protein; in humans, it is 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 characteristic of inflammatory sites. We investigated the binding of fluid-phase CRP to six immobilized proteins: complement factor H, oxidized low-density lipoprotein, complement C3b, IgG, amyloid ß, 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 pH-treated 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.

Binding of the monomeric form of C-reactive protein to enzymatically-modified low-density lipoprotein: effects of phosphoethanolamine.

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 degrees 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 degrees 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.

Functional identification of novel activities: activity-based selection of proteins from complete proteomes.

The identification of proteins with desired activities, especially from complex samples such as plasma and whole blood, is a continual challenge. We have developed a technology platform called Functional Identification of Novel Activities (FIoNA) to discover desired protein activities from complex biological samples. FIoNA uses immobilized libraries of combinatorial peptide ligands to purify and concentrate essentially all of the components of a complex mixture on ligands synthesized on individual beads. No depletion or prefractionation of the starting material is performed before it is incubated with the library, and no a priori knowledge of the active protein or of the ligand to which it binds is required. Instead, the protein-loaded beads are individually evaluated en masse in disease- relevant assays to identify proteins possessing a desired function. Beads associated with the activity are selected, and the ligand is sequenced and resynthesized in bulk on the original backbone for purification and characterization of the active component. Here we illustrate the use of FIoNA in a cell proliferation assay to detect a growth factor present in conditioned cell medium at nanogram/milliliter concentrations. We also have selected beads associated with hydrolysis of nerve agent analogs in assays performed in 100,000-well microtiter plates.

Reduction in infectivity of endogenous transmissible spongiform encephalopathies present in blood by adsorption to selective affinity resins.

BACKGROUND: Transmissible spongiform encephalopathies (TSE) can be contracted through blood transfusion. Selective adsorption of the causative agent from donated blood might be one of the best ways of managing this risk. In our study, affinity resin L13, which reduces brain-derived infectivity spiked into human red blood cell concentrate by around 4 log(10)ID(50), and its equivalent, L13A, produced on a manufacturing scale, were assessed for their ability to remove TSE infectivity endogenously present in blood. METHODS: 500 mL of scrapie-infected hamster whole blood was leucoreduced at full scale before passage through the affinity resins. Infectivity of whole blood, leucoreduced whole blood (challenge), and the recovered blood from each flow-through was measured by limiting dilution titration. FINDINGS: Leucoreduction removed 72% of input infectivity. 15 of 99 animals were infected by the challenge, whereas none of the 96 or 100 animals inoculated with the final flow-throughs from either resin developed the disease after 540 days. The limit of detection of the bioassay was 0.2 infectious doses per mL. The overall reduction of the challenge infectivity was more than 1.22 log10ID. The results showed removal of endogenous TSE infectivity from leucoreduced whole blood by affinity ligands. The same resins adsorb normal and abnormal prion protein from human infections with variant, sporadic, and familial Creutzfeldt-Jakob disease, in the presence of blood components. INTERPRETATION: TSE affinity ligands, when incorporated into appropriate devices, can be used to mitigate the risks from TSE-infected blood, blood products, and other materials exposed to TSE infectivity.

Rarity gives a charm: evaluation of trace proteins in plasma and serum.

Since plasma potentially contacts every cell as it circulates through the body, it may carry clues both to diagnosis and treatment of disease. It is commonly expected that the growing ability to detect and characterize trace proteins will result in discovery of novel therapeutics and biomarkers; however, the familiar, super-abundant plasma proteins remain a fundamental stumbling block. Furthermore, robust validation of proteomic data is a sometimes overlooked but always necessary component for the eventual development of clinical reagents. This review surveys some of the uses of typical and atypical low-abundance proteins, current analytical methods, existing impediments to discovery, and some innovations that are overcoming the challenges to evaluation of trace proteins in plasma and serum.

A fluorogenic substrate for detection of organophosphatase activity.

A new fluorogenic substrate for the specific detection of organophosphatase (OPase) activity has been designed and evaluated. Our results indicate that 7-diethylphospho-6,8-difluor-4-methylumbelliferyl (DEPFMU) is hydrolyzed specifically by the OPases, mammalian serum paraoxonase and bacterial organophosphorus hydrolase (OPH). The apparent K(m) of DEPFMU is 29 microM for OPH and 91 and 200 microM for the PON1 L(55)R(192) and PON1 L(55)Q(192) isoforms of human paraoxonase, respectively. DEPFMU-based assay systems are 10-100 times more sensitive for OPH and mammalian paraoxonase detection than existing methods. Importantly, DEPFMU is poorly hydrolyzed by both serum and cellular phosphatases and, therefore, may be used as part of a robust and sensitive assay for detecting not only purified, but also highly impure, preparations of OPase such as blood samples. The superior sensitivity of DEPFMU makes it potentially useful in the search for new enzymes that may hydrolyze nerve poisons such as sarin, soman, and VX, monitoring the decontamination of organophosphates (OPs) by OPH and determining serum paraoxonase activity which appears to be important for protection against atherosclerosis, sepsis, and OP toxicity.

Therapeutic potential of the plasma proteome.

Plasma contains numerous and diverse proteins with existing and potential therapeutic value. Plasma has been used clinically as both a source of purified derivatives for treating diseases such as hemophilia, and as a diagnostic medium. Recent research directed towards mining plasma's true potential takes advantage of state-of-the-art proteomic analytical methods to develop multi-protein, disease-specific biomarker panels to improve the reliability and specificity of diagnostics. Recombinant production and chromatographic purification methods are increasing the yield and safety of traditional plasma derivatives. Emerging cell-based technologies are being applied to discover novel protein activities and identify epitope-specific antibodies that may have clinical promise.

Affinity purification of fibrinogen using a ligand from a peptide library.

An affinity resin containing the peptide ligand Phe-Leu-Leu-Val-Pro-Leu (FLLVPL) has been developed for the purification of fibrinogen. The ligand was identified by screening a solid-phase combinatorial peptide library using an immunostaining technique. The specific binding of fibrinogen to the ligand has been characterized by isothermal calorimetry and adsorption isotherms and is dominated by both hydrophobic interactions and ionic interactions with the N-terminal free amino group. The effective association constant of fibrinogen was substantially higher when the peptide was immobilized on the resin than in solution; moreover, it increased with increasing peptide density, suggesting a cooperative binding effect. A low ionic strength buffer at pH 4 was used successfully to elute adsorbed fibrinogen from the column with high purity, retention of factor XIII crosslinking activity, and minimal, if any, loss of biological function. This general approach to ligand selection and characterization can be used to develop peptide ligands for the affinity purification of diverse proteins on a large scale.