Monomeric C-Reactive Protein in Serum With Markedly Elevated CRP Levels Shares Common Calcium-Dependent Ligand Binding Properties With an in vitro Dissociated Form of C-Reactive Protein
A monomeric form of C-reactive protein (CRP) which precipitates with cell wall pneumococcal C polysaccharide (CWPS) and retains the ability to reversibly bind to its ligand phosphocholine has been produced through urea-induced dissociation at an optimized concentration of 3 M urea over a 10 weeks pe...
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| Format: | Article |
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Frontiers Media S.A.
2020-02-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fimmu.2020.00115/full |
| _version_ | 1818351249111646208 |
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| author | Robert D. Williams Jennifer A. Moran Anthony A. Fryer Jamie R. Littlejohn Harry M. Williams Trevor J. Greenhough Annette K. Shrive |
| author_facet | Robert D. Williams Jennifer A. Moran Anthony A. Fryer Jamie R. Littlejohn Harry M. Williams Trevor J. Greenhough Annette K. Shrive |
| author_sort | Robert D. Williams |
| collection | DOAJ |
| description | A monomeric form of C-reactive protein (CRP) which precipitates with cell wall pneumococcal C polysaccharide (CWPS) and retains the ability to reversibly bind to its ligand phosphocholine has been produced through urea-induced dissociation at an optimized concentration of 3 M urea over a 10 weeks period. Dissociated samples were purified via size exclusion chromatography and characterized by western blot, phosphocholine affinity chromatography and CWPS precipitation. Human serum samples from patients with raised CRP levels (>100 mg/L as determined by the clinical laboratory assay) were purified by affinity and size exclusion chromatography and analyzed (n = 40) to determine whether circulating monomeric CRP could be detected ex vivo. All 40 samples tested positive for pentameric CRP via western blot and enzyme linked immunosorbent assay (ELISA) analysis. Monomeric C-reactive protein was also identified in all 40 patient samples tested, with an average level recorded of 1.03 mg/L (SE = ±0.11). Both the in vitro monomeric C-reactive protein and the human serum monomeric protein displayed a molecular weight of approximately 23 kDa, both were recognized by the same anti-CRP monoclonal antibody and both reversibly bound to phosphocholine in a calcium-dependent manner. In common with native pentameric CRP, the in vitro mCRP precipitated with CWPS. These overlapping characteristics suggest that a physiologically relevant, near-native monomeric CRP, which retains the structure and binding properties of native CRP subunits, has been produced through in vitro dissociation of pentameric CRP and also isolated from serum with markedly elevated CRP levels. This provides a clear route toward the in-depth study of the structure and function of physiological monomeric CRP. |
| first_indexed | 2024-12-13T18:34:44Z |
| format | Article |
| id | doaj.art-415272d7cd764e23b41e2e584f851a77 |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-12-13T18:34:44Z |
| publishDate | 2020-02-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-415272d7cd764e23b41e2e584f851a772022-12-21T23:35:24ZengFrontiers Media S.A.Frontiers in Immunology1664-32242020-02-011110.3389/fimmu.2020.00115510968Monomeric C-Reactive Protein in Serum With Markedly Elevated CRP Levels Shares Common Calcium-Dependent Ligand Binding Properties With an in vitro Dissociated Form of C-Reactive ProteinRobert D. Williams0Jennifer A. Moran1Anthony A. Fryer2Jamie R. Littlejohn3Harry M. Williams4Trevor J. Greenhough5Annette K. Shrive6School of Life Sciences, Keele University, Staffordshire, United KingdomSchool of Life Sciences, Keele University, Staffordshire, United KingdomDepartment of Clinical Biochemistry, Institute for Applied Clinical Sciences, University Hospitals of North Midlands, Keele University, Staffordshire, United KingdomSchool of Life Sciences, Keele University, Staffordshire, United KingdomSchool of Life Sciences, Keele University, Staffordshire, United KingdomSchool of Life Sciences, Keele University, Staffordshire, United KingdomSchool of Life Sciences, Keele University, Staffordshire, United KingdomA monomeric form of C-reactive protein (CRP) which precipitates with cell wall pneumococcal C polysaccharide (CWPS) and retains the ability to reversibly bind to its ligand phosphocholine has been produced through urea-induced dissociation at an optimized concentration of 3 M urea over a 10 weeks period. Dissociated samples were purified via size exclusion chromatography and characterized by western blot, phosphocholine affinity chromatography and CWPS precipitation. Human serum samples from patients with raised CRP levels (>100 mg/L as determined by the clinical laboratory assay) were purified by affinity and size exclusion chromatography and analyzed (n = 40) to determine whether circulating monomeric CRP could be detected ex vivo. All 40 samples tested positive for pentameric CRP via western blot and enzyme linked immunosorbent assay (ELISA) analysis. Monomeric C-reactive protein was also identified in all 40 patient samples tested, with an average level recorded of 1.03 mg/L (SE = ±0.11). Both the in vitro monomeric C-reactive protein and the human serum monomeric protein displayed a molecular weight of approximately 23 kDa, both were recognized by the same anti-CRP monoclonal antibody and both reversibly bound to phosphocholine in a calcium-dependent manner. In common with native pentameric CRP, the in vitro mCRP precipitated with CWPS. These overlapping characteristics suggest that a physiologically relevant, near-native monomeric CRP, which retains the structure and binding properties of native CRP subunits, has been produced through in vitro dissociation of pentameric CRP and also isolated from serum with markedly elevated CRP levels. This provides a clear route toward the in-depth study of the structure and function of physiological monomeric CRP.https://www.frontiersin.org/article/10.3389/fimmu.2020.00115/fullmonomeric C-reactive proteinserum C-reactive proteindissociated C-reactive proteincalcium-dependent ligand bindingphosphocholineC-polysaccharide |
| spellingShingle | Robert D. Williams Jennifer A. Moran Anthony A. Fryer Jamie R. Littlejohn Harry M. Williams Trevor J. Greenhough Annette K. Shrive Monomeric C-Reactive Protein in Serum With Markedly Elevated CRP Levels Shares Common Calcium-Dependent Ligand Binding Properties With an in vitro Dissociated Form of C-Reactive Protein Frontiers in Immunology monomeric C-reactive protein serum C-reactive protein dissociated C-reactive protein calcium-dependent ligand binding phosphocholine C-polysaccharide |
| title | Monomeric C-Reactive Protein in Serum With Markedly Elevated CRP Levels Shares Common Calcium-Dependent Ligand Binding Properties With an in vitro Dissociated Form of C-Reactive Protein |
| title_full | Monomeric C-Reactive Protein in Serum With Markedly Elevated CRP Levels Shares Common Calcium-Dependent Ligand Binding Properties With an in vitro Dissociated Form of C-Reactive Protein |
| title_fullStr | Monomeric C-Reactive Protein in Serum With Markedly Elevated CRP Levels Shares Common Calcium-Dependent Ligand Binding Properties With an in vitro Dissociated Form of C-Reactive Protein |
| title_full_unstemmed | Monomeric C-Reactive Protein in Serum With Markedly Elevated CRP Levels Shares Common Calcium-Dependent Ligand Binding Properties With an in vitro Dissociated Form of C-Reactive Protein |
| title_short | Monomeric C-Reactive Protein in Serum With Markedly Elevated CRP Levels Shares Common Calcium-Dependent Ligand Binding Properties With an in vitro Dissociated Form of C-Reactive Protein |
| title_sort | monomeric c reactive protein in serum with markedly elevated crp levels shares common calcium dependent ligand binding properties with an in vitro dissociated form of c reactive protein |
| topic | monomeric C-reactive protein serum C-reactive protein dissociated C-reactive protein calcium-dependent ligand binding phosphocholine C-polysaccharide |
| url | https://www.frontiersin.org/article/10.3389/fimmu.2020.00115/full |
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