title: 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 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.
authors.affiliation: Department of Pharmacology, Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Hammond
authors.forename: Sanjay K
authors.initials: SK
authors.lastname: Singh
authors.forename: James A
authors.initials: JA
authors.lastname: Thompson
authors.forename: Bradley W
authors.initials: BW
authors.lastname: Beeler
authors.forename: Antonio E
authors.initials: AE
authors.lastname: Rusiñol
authors.forename: Michael K
authors.initials: MK
authors.lastname: Pangburn
authors.forename: Lawrence A
authors.initials: LA
authors.lastname: Potempa
authors.forename: Alok
authors.initials: A
authors.lastname: Agrawal
mesh_terms: D000143:Acids / Q000737:chemistry; D019943:Amino Acid Substitution; D016229:Amyloid beta-Peptides / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002097:C-Reactive Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002522:Chlorocebus aethiops; D003179:Complement C3b / Q000378:metabolism; D017242:Complement Factor H / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000378:metabolism; D008024:Ligands; D008077:Lipoproteins, LDL / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D012710:Serum Albumin, Bovine / Q000378:metabolism
chemical_list: D000143:Acids; D016229:Amyloid beta-Peptides; D007074:Immunoglobulin G; D008024:Ligands; D008077:Lipoproteins, LDL; C099252:oxidized low density lipoprotein; D012710:Serum Albumin, Bovine; D003179:Complement C3b; D017242:Complement Factor H; D002097:C-Reactive Protein; D002118:Calcium