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J. Biol. Chem., Vol. 283, Issue 45, 30451-30460, November 7, 2008

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Complement Factor H Binds to Denatured Rather than to Native Pentameric C-reactive Protein^*

Svetlana Hakobyan, Claire L. Harris, Carmen W. van den Berg, Maria Carmen Fernandez-Alonso^¶, Elena Goicoechea de Jorge^¶, Santiago Rodriguez de Cordoba^¶, German Rivas^¶, Palma Mangione^||**, Mark B. Pepys^**, and B. Paul Morgan¹

From the Departments of Medical Biochemistry and Immunology and Pharmacology, School of Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom, the ^¶Departments of Molecular and Cellular Physiopathology and Protein Sciences, Centro de Investigaciones Biologicas and Ciber de Enfermedades Raras, 28040 Madrid, Spain, ^||Dipartimento di Biochimica, Universitá degli Studi di Pavia, I-27100 Pavia, Italy, and ^**the Centre for Amyloidosis and Acute Phase Proteins, Division of Medicine, Royal Free and University College Medical School, London, NW3 2PF United Kingdom

Binding 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.

Received for publication, May 12, 2008 , and in revised form, August 20, 2008.

^* The work was supported by Wellcome Trust Programme Grant 068590 (to B. P. M.), Wellcome Trust University Award 068823 (to C. L. H.), and Spanish Ministerio de Educacion y Cultura Grant SAF2005-00913 (to S. R. d.-C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Medical Biochemistry and Immunology, Henry Wellcome Bldg., School of Medicine, Cardiff University, Heath Park, Cardiff, CF14 4XN, UK. Tel.: 44-29-20687304; Fax: 44-29-20687303; E-mail: morganbp{at}cardiff.ac.uk.

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