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.

Robert D Williams,Jamie R Littlejohn,Trevor J Greenhough,Anthony A Fryer,Annette K Shrive,Jennifer A Moran,Harry M Williams

doi:10.3389/fimmu.2020.00115

Abstract

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.

Highlights

C-reactive protein (CRP) belongs to the family of proteins known as the pentraxins, which have been highly conserved throughout evolution and can be identified in species ranging from mammals and fish to the primitive Atlantic horseshoe crab, Limulus polyphemus [1]
Size exclusion chromatography was used to assess the level of dissociation of pentameric CRP into monomeric CRP in the presence of 1 mM ethylenediaminetetraacetic acid (EDTA) under a range of urea concentrations, each over a period of several weeks, and to purify out both pentameric human C-reactive protein (pCRP) and mCRP from the sample
There is no evidence in the elution traces of intermediate CRP aggregations comprising 2, 3, or 4 subunits. 5 ml fractions were collected throughout the elutions, the two fractions associated with each peak, for example 75–80 ml, 80–85 ml for pCRP, 90–95 ml, 95–100 ml for mCRP for the 3 M dissociation, being collected, pooled, and concentrated in the elution buffer for further characterization

Summary

Introduction

C-reactive protein (CRP) belongs to the family of proteins known as the pentraxins, which have been highly conserved throughout evolution and can be identified in species ranging from mammals and fish to the primitive Atlantic horseshoe crab, Limulus polyphemus [1]. It has been hypothesized that CRP exerts an alternative, pro-inflammatory role in disease development, with in vitro studies demonstrating up-regulation of cell adhesion molecules, activation of endothelial cells, deposition within atherosclerotic lesions and the increased production of inflammatory cytokines; all common features found in the pathophysiology of cardiovascular disease [8, 11,12,13,14,15]. This evidence has led to the development of a high sensitivity CRP assay which can accurately measure CRP levels

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