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

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 ␤, 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.

Regulated conformation changes in C-reactive protein orchestrate its role in atherogenesis

“…Interestingly, the acidic pH frequently found in inflamed tissues can also boost the binding capacity of CRP even in the absence of pentamer dissociation [83]; however, after binding to immobilized ligands at a low pH, a prominent exposure of the mCRP epitope has been observed [83]. These results further support the idea that interactions of CRP with the inflammatory microenvironment are critical for the expression of enhanced bioactivities.…”

The Phosphocholine-binding Pocket on C-reactive Protein Is Necessary for Initial Protection of Mice against Pneumococcal Infection

“…Alternatively, 25 g of CRP double mutant might not have been protective as opposed to 150 g. Because administering a higher dose of CRP allows CRP to stay longer in the host, we hypothesize that CRP mutants, when given at the beginning of the experiment, undergo remodeling to gain other ligand binding properties, as yet undefined, during their stay in the host, and that may be responsible for the protection of mice from infection independent of the PCh binding activity of CRP. This hypothesis also provides insight into why CRP does not protect mice from infection if administered later and how CRP can be engineered so that it can protect mice from infection even if administered during the late stages of infection (33,51). However, this is just a hypothesis because we have no data.…”

“…PCh Binding Assay-Binding activity of CRP for PCh was evaluated by using PCh-conjugated BSA and PnC (Statens Serum Institut) as the ligands, as described previously (7,33). Microtiter wells (96-well plates) were coated with 10 g/ml PCh-BSA or PnC in TBS, overnight at 4°C.…”

“…Purification of Native WT CRP-WT CRP was purified from discarded human pleural fluid by affinity chromatography on a PCh-Sepharose column (Pierce) followed by ion-exchange chromatography on a MonoQ column (GE Healthcare) and gel filtration chromatography on a Superose 12 column (GE Healthcare), as described previously (33), and stored frozen. On The PDB coordinates of mutant CRP were generated from the PDB file 1B09 using SYBYL (Tripos, Inc.).…”

Purification of recombinant C-reactive protein mutants

“…CRP, in a Ca 2+ -dependent interaction, also binds to phosphoethanolamine (PEt) (Agrawal et al, 2002;Mikolajek et al, 2011). In its alternate pentameric structural conformation, CRP binds to deposited, aggregated and immobilized proteins including amyloid β peptide, oxidized low-density lipoprotein and complement regulator factor H, in a Ca 2+ -independent manner (Agrawal, 2013;Agrawal et al, 2014;Hakobyan et al, 2008;Hammond et al, 2010;Singh et al, 2012;Suresh et al, 2004). The significance of the recognition function of CRP in its alternate structural pentameric conformation in host defense is unknown.…”