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

David J Hammond,Sanjay K Singh,James A Thompson,Bradley W Beeler,Antonio E Rusiñol,Michael K Pangburn,Lawrence A Potempa,Alok Agrawal

doi:10.1074/jbc.m110.142026

Abstract

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 β, 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.

Highlights

In normal healthy individuals, the median concentration of C-reactive protein (CRP) in the serum is 0.8 ␮g/ml; in acute phase, the concentration increases to 500 ␮g/ml or more [18]
At Acidic pH, CRP Binds to a Variety of Other Proteins Immobilized to Microtiter Plates—Fig. 1 shows the results of protein ligand-binding assays in which we determined the binding of CRP to immobilized factor H, Oxidized low density lipoprotein (oxLDL), C3b, IgG, A␤, and BSA, as a function of pH (7.0 – 4.6), at 25 and 37 °C
When only the immobilized proteins, and not CRP, were treated with acidic pH and the assay was performed at pH 7.0, CRP did not bind to immobilized proteins. These results indicated that CRP acquired the capability to bind to immobilized proteins when both CRP and immobilized proteins were exposed to acidic pH

Summary

Introduction

The median concentration of CRP in the serum is 0.8 ␮g/ml; in acute phase, the concentration increases to 500 ␮g/ml or more [18]. CRP is deposited at atherosclerotic lesions the circulating concentration of CRP increases only minimally in atherosclerosis [8, 22,23,24]. Because the pH at the sites of inflammation may be acidic [25,26,27,28,29,30,31,32,33,34,35], in this study, we investigated the binding specificities of CRP at acidic pH to define the functions of CRP at the sites of inflammation. We explored CRP-factor H interactions because of their possible involvement in the anti-pneumococcal functions of CRP observed in murine models of pneumococcal infection. In addition to factor H and oxLDL, we randomly selected four other proteins: complement component C3 fragment C3b, IgG, amyloid ␤ fragment 1–38 (A␤), and BSA, to explore the binding reactivities of CRP at acidic pH. We found that CRP, at acidic pH, bound to all six proteins including BSA, as if CRP recognized a general pattern created by these proteins that were immobilized to microtiter plates and exposed to acidic pH at 37 °C

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Conclusion