Localization of sequence-determined neoepitopes and neutrophil digestion fragments of C-reactive protein utilizing monoclonal antibodies and synthetic peptides

Ying, Shan-Ching; Shephard, Elizabeth A.; Beer, Frederick C. de; Siegel, Joan N.; Harris, David C.H.; Gewurz, Benjamin E.; Fridkin, Mati; Gewürz, Henry

doi:10.1016/0161-5890(92)90205-c

Cited by 30 publications

(39 citation statements)

References 23 publications

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“…The HD2.4 mAb recognizes native pentameric CRP (62). The 3H12 mAb does not recognize native pentameric CRP because the epitope of 3H12 is hidden in the intersubunit contact zones of the pentamer; it recognizes modified and monomeric forms of CRP (15,63). At pH 7.2 ( Fig.…”

Section: The Binding Of Crp To Immobilized Proteins At Acidic Ph Is Nmentioning

confidence: 99%

Identification of Acidic pH-dependent Ligands of Pentameric C-reactive Protein

Hammond

Singh

Thompson

et al. 2010

Journal of Biological Chemistry

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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 ␤, 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.

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Section: The Binding Of Crp To Immobilized Proteins At Acidic Ph Is Nmentioning

confidence: 99%

Identification of Acidic pH-dependent Ligands of Pentameric C-reactive Protein

Hammond

Singh

Thompson

et al. 2010

Journal of Biological Chemistry

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“…The 9C9 mAb recognizes the C-terminal octapeptide of the CRP subunit, which is buried within the intersubunit contact regions of the nCRP structure and is expressed when the subunits are dissociated. The 8C10 mAb recognizes an epitope on the N-terminal 16-kDa fragment of the mCRP isoforms (33). This epitope is conformationally sensitive, requiring the single intrachain disulfide bond of the CRP subunit, in the mCRP isoform, to be intact.…”

Section: Antibodies and Chemicalsmentioning

confidence: 99%

Production of Modified C-Reactive Protein in U937-Derived Macrophages

Ciubotaru

Potempa

Wander

2005

Exp Biol Med (Maywood)

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Abstract:Plasma C-reactive protein (CRP) has been proposed to be a strong independent predictor for cardiovascular disease. This circulating form of CRP (native CRP or nCRP) is well described. Recently, the existence of a conformationally distinct isoform of CRP (modified CRP or mCRP) has been reported. The relevance of each CRP isoform to atherosclerotic disease is unknown. The purpose of this study was to examine the natural expression of CRP in undifferentiated, differentiated, and stimulated macrophages, cells known to contribute to atherogenesis in vivo, and to determine whether transcribed CRP was expressed as nCRP or mCRP. Macrophages were generated from U937 monocytes using phorbol 12-myristate 13-acetate. Differentiated macrophages were further stimulated with lipopolysaccharides (LPS). In undifferentiated, differentiated, and stimulated cells, CRP expression was assessed by reverse transcriptionpolymerase chain reaction, and CRP protein production was measured by fluorescence microscopy and flow cytometry (cellular CRP) or high-sensitivity enzyme-linked immunosorbent assay (secreted CRP). CRP transcript was minimally expressed in undifferentiated cells. Expression increased markedly in macrophages during differentiation and was not affected by LPS at 24 hrs. Cellular CRP protein increased in a time-dependent manner after LPS stimulation, and this induction was mediated via interleukin (IL)-6 and IL-1p. A small amount of secreted CRP was detected in the media of differentiated cells, but it was not significantly increased after LPS stimulation. Using specific monoclonal antibodies, our data indicate that cellular CRP is directly translated as the mCRP rather than the nCRP isomer. These results indicate that U937-derived macrophages are a good cell model to further study the production of mCRP under conditions relevant for the atherogenic process. Exp Biol Med 230:762-770, 2005 Article: Seven decades after its discovery, the acute-phase protein C-reactive protein (CRP) has emerged as a strong predictor for cardiovascular disease. These observations have accumulated as highly sensitive assays for the measurement of previously undetectable concentrations of circulating CRP became commercially available. Hence, low concentrations of CRP are sometimes denoted as high-sensitivity CRP (hs-CRP) to indicate the use of such assays. Recent studies conducted in healthy men and women indicate that plasma CRP concentrations as low as 1-3 gg/ml may

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“…Prepared samples were analyzed using a dot-enzyme-linked immunofiltration assay system (dot-ELIFA) in which test samples were adsorbed onto nitrocellulose immobilized in a Fast-Titer TM gasketed housing unit (Pierce Chemical). After washing and blocking excess nitrocellulose sites, the following mAbs described in Ying et al [36,37] were used as primary antibodies to react with immobilized antigen-mAb 1D6 (specific for native-CRP) and mAbs 3H12, 7A8 and 8C10 (all specific for different epitopes of mCRP). After appropriate washing steps, goat or horse antimouse IgG-horseradish peroxidase conjugate was used as a secondary reagent, and assays were quantified using a peroxidase substrate [38].…”

Section: Quantitation Of the Native-crp And Mcrp Antigens Expressed Amentioning

confidence: 99%

“…Native-CRP and mCRP antigens in LUVETs and MLV preparations were identified and quantified using monoclonal antibodies (mAbs), obtained from Dr. Henry Gewurz, specific for epitopes expressed on either molecular form of CRP [36,37]. Prepared samples were analyzed using a dot-enzyme-linked immunofiltration assay system (dot-ELIFA) in which test samples were adsorbed onto nitrocellulose immobilized in a Fast-Titer TM gasketed housing unit (Pierce Chemical).…”

Section: Quantitation Of the Native-crp And Mcrp Antigens Expressed Amentioning

confidence: 99%

Inhibition of Mouse Mammary Adenocarcinoma (EMT6) Growth and Metastases in Mice by a Modified Form of C-Reactive Protein

Kresl

Potempa

Anderson³

et al. 1999

Tumor Biol

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Mice were injected in the hind limb with a mouse mammary adenocarcinoma cell line, EMT6, and tumor growth at the primary site as well as the incidence of lung metastases were measured. Groups of animals were treated with the acute-phase reactant C-reactive protein, (native-CRP), or a conformationally modified form of CRP (mCRP) made by dissociating CRP subunits under chelating, denaturing conditions. Each form of CRP was injected (intravenously) through the tail vein, encapsulated in large unilamellar lipid vesicles made by an extrusion technique (LUVETs). mCRP was also injected without the LUVET carrier. Mice not treated, or treated with LUVETs alone, exhibited both progressive tumor growth at the primary site and a high incidence of metastatic lung tumors quantified at necropsy. Treatment with native-CRP encapsulated in LUVETs had little or no effect on either tumor growth or metastases. Treatment with mCRP, however, alone or encapsulated in LUVETs, effectively slowed or stopped the progression of tumor growth, and in some mice, showed a decrease in tumor size. After cessation of mCRP injections, tumor growth resumed at a rate comparable to that measured in untreated animals. Fifty to 85% of mice treated with mCRP or mCRP in LUVETs developed necrotic lesions at the primary tumor site within 24–48 h following the initial injection of protein. Furthermore, at necropsy, only 6% of mice treated with mCRP in LUVETs and 40% of mice treated with mCRP alone showed evidence of lung metastases compared to 67–80% of animals in no-treatment, native-CRP in LUVETs and in LUVET control group animals. These results show that the prototypic acute-phase reactant, CRP, has therapeutic anticancer and antimetastatic activity only when the native pentameric subunit structure is dissociated to form the mCRP conformer.

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Localization of sequence-determined neoepitopes and neutrophil digestion fragments of C-reactive protein utilizing monoclonal antibodies and synthetic peptides

Cited by 30 publications

References 23 publications

Identification of Acidic pH-dependent Ligands of Pentameric C-reactive Protein

Identification of Acidic pH-dependent Ligands of Pentameric C-reactive Protein

Production of Modified C-Reactive Protein in U937-Derived Macrophages

Inhibition of Mouse Mammary Adenocarcinoma (EMT6) Growth and Metastases in Mice by a Modified Form of C-Reactive Protein

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