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Complement factor H binds to denatured rather than to native pentameric C-reactive protein

AuthorsHakobyan, Svetlana; Harris, Claire L.; Van Den Berg, Carmen W.; Fernández-Alonso, M. Carmen ; Goicoechea de Jorge, Elena ; Rodríguez de Córdoba, Santiago ; Rivas, Germán ; Mangione, Palma; Pepys, Mark B.; Morgan, B. Paul
Issue Date7-Nov-2008
PublisherAmerican Society for Biochemistry and Molecular Biology
CitationJournal of Biological Chemistry 283:30451-30460(2008)
AbstractBinding of the complement regulatory protein, factor H, to C-reactive protein has been reported and implicated as the biological basis for association of the H402 polymorphic variant of factor H with macular degeneration. Published studies utilize solid-phase or fluid-phase binding assays to show that the factor H Y402 variant binds C-reactive protein more strongly than H402. Diminished binding of H402 variant to C-reactive protein in retinal drusen is posited to permit increased complement activation, driving inflammation and pathology. We used well validated native human C-reactive protein and pure factor H Y402H variants to test interactions. When factor H variants were incubated with C-reactive protein in the fluid phase at physiological concentrations, no association occurred. When C-reactive protein was immobilized on plastic, either non-specifically by adsorption in the presence of Ca2+ to maintain its native fold and pentameric subunit assembly or by specific Ca2+-dependent binding to immobilized natural ligands, no specific binding of either factor H variant from the fluid phase was observed. In contrast, both factor H variants reproducibly bound to C-reactive protein immobilized in the absence of Ca2+, conditions that destabilize the native fold and pentameric assembly. Both factor H variants strongly bound C-reactive protein that was denatured by heat treatment before immobilization, confirming interaction with denatured but not native C-reactive protein. We conclude that the reported binding of factor H to C-reactive protein results from denaturation of the C-reactive protein during immobilization. Differential binding to C-reactive protein, thus, does not explain association of the Y402H polymorphism with macular degeneration.
Complement (C)2 is the principle effector in the innate host defense against infection and also contributes to tissue damaging inflammatory reactions, processing and clearance of immune complexes and regulation of the immune response (1). Activation is controlled by a group of C regulatory proteins that includes factor H (fH), the plasma protein responsible for regulating the alternative pathway, an amplification loop that is pivotal in physiological and pathological activation of C (2). fH is a single-chain serum glycoprotein of 150 kDa consisting of a string of 20 homologous units, each of about 60 amino acids, called short consensus repeats (SCR). Deficiency of fH causes unregulated consumption of C with secondary deficiency of C3 (3). Although fH is a plasma protein, many of its activities occur on the cell membrane. Indeed, mutations in the carboxyl terminus of fH that affect membrane binding capacity cause defective C regulation at the membrane and are associated with the renal disease, atypical hemolytic uremic syndrome (4). Within the fH molecule there are multiple binding sites for C3b, heparin, and several other ligands (5). An interaction of fH with the plasma pentraxin molecule, C-reactive protein (CRP), the classical major acute phase reactant, was first reported almost 25 years ago. Mold et al. (6) showed that CRP coating of pneumococci had divergent effects, increasing activation of the classical pathway while inhibiting the alternative pathway; this latter effect was fH-dependent and associated with a doubling of fH binding to the CRP-coated pneumococcus. The same group later provided direct evidence of fH binding to plastic-immobilized CRP, although when the assay format was reversed and fluid phase CRP was offered to immobilized fH, less than 1% of added CRP bound (7). In a surface plasmon resonance study, when CRP was covalently immobilized on the chip surface through amine coupling in the absence of Ca2+, calcium-dependent binding of the recombinant fH fragments comprising both SCR 7 and SCRs 8–11 was seen (8).
The seventh SCR of fH is a “hotspot” for ligand interactions with reported binding to heparin, CRP, and streptococcal M-protein (9). When a fH polymorphism, resulting in a single amino acid change (Y402H) in SCR7 was implicated as a powerful risk factor for age-related macular degeneration (AMD) (10–12), there was immediately intense interest in exploring differences in ligand binding between these polymorphic variants that might explain the disease association. Several groups have lately reported that the fH Y402 and H402 variants show differential binding affinity for CRP (13–17). Each used similar methods with CRP directly immobilized on a surface (ELISA plate or surface plasmon resonance chip) and either intact fH variants or recombinant fragments in the fluid phase. All concluded that both fH variants bound CRP and that the H402 variant bound less well than the Y402 variant. C proteins are present in drusen, the hallmark of the retinal pathology of AMD, but there is controversy about CRP, some reporting that it is absent (18), whereas others find it in the retinal layers and drusen deposits and more abundantly in H402 homozygotes than Y402 homozygotes (19). These observations have led to the suggestion that binding of fH to CRP in the retina limits C activation and the H402 variant because it binds less well and permits more C activation, thus, predisposing to disease. This has potentially important therapeutic implications, especially with the recent development of the first specific inhibitor of CRP ligand binding and pathogenic function in vivo (20). As a first step toward evaluation of this suggestion, we have investigated the interaction between CRP and fH under conditions in which CRP retains its native fold and homopentameric assembly of protomers and which are, thus, most likely to be of physiological and pathophysiological relevance. Here we report that there is no interaction between fH and intact native CRP in the fluid phase or with CRP immobilized directly to plastic in the presence of Ca2+ or immobilized via its specific Ca2+-dependent binding to its natural ligands. fH binds CRP only after the CRP has been denatured, either before immobilization or by coating onto plastic in the absence of Ca2+. We conclude that the reported binding of fH to CRP is likely caused by denaturation of CRP, which has long been known to result from its adsorption to plastic surfaces in the non-physiological absence of Ca2+ (21, 22) or by other structural modifications of CRP resulting from its covalent immobilization. Alternative mechanisms for the strong association of the fH H402 variant with AMD should be sought
Description10 páginas, 4 figuras -- PAGS nros. 30451-30460
Publisher version (URL)http://dx.doi.org/10.1074/jbc.M803648200
Appears in Collections:(CIB) Artículos
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