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The Binding of Factor H to a Complex of Physiological Polyanions and C3b on Cells Is Impaired in Atypical Hemolytic Uremic Syndrome¹

Viviana P. Ferreira^*, Andrew P. Herbert, Claudio Cortés^*, Kristi A. McKee^*, Bärbel S. Blaum, Stefan T. Esswein, Duan Uhrín, Paul N. Barlow, Michael K. Pangburn^* and David Kavanagh^2,

^* Department of Biochemistry, Center for Biomedical Research, University of Texas Health Science Center, Tyler, TX 75708; and Edinburgh Biomolecular NMR Unit, Schools of Chemistry and Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom

Factor H (fH) is essential for complement homeostasis in fluid-phase and on surfaces. Its two C-terminal domains (CCP 19–20) anchor fH to self-surfaces where it prevents C3b amplification in a process requiring its N-terminal four domains. In atypical hemolytic uremic syndrome (aHUS), mutations clustering toward the C terminus of fH may disrupt interactions with surface-associated C3b or polyanions and thereby diminish the ability of fH to regulate complement. To test this, we compared a recombinant protein encompassing CCP 19–20 with 16 mutants. The mutations had only very limited and localized effects on protein structure. Although we found four aHUS-linked fH mutations that decreased binding to C3b and/or to heparin (a model compound for cell surface polyanionic carbohydrates), we identified five aHUS-associated mutants with increased affinity for either or both ligands. Strikingly, these variable affinities for the individual ligands did not correlate with the extent to which all the aHUS-associated mutants were found to be impaired in a more physiological assay that measured their ability to inhibit cell surface complement functions of full-length fH. Taken together, our data suggest that disruption of a complex fH-self-surface recognition process, involving a balance of affinities for protein and physiological carbohydrate ligands, predisposes to aHUS.

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 This research was supported by American Heart Association National Scientist Development Grant 0735101N (to V.P.F.), D.K. is a Kidney Research U.K. Clinical Training Fellow, National Institutes of Health Grant DK-35081 (to M.K.P.), and Wellcome Trust Grant 078780/Z/05/Z (to D.U. and P.N.B.).

² Address correspondence and reprint requests to Dr. David Kavanagh, Edinburgh Biomolecular NMR Unit, Schools of Chemistry and Biological Sciences, Joseph Black Chemistry Building, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, United Kingdom. E-mail address: davidkavanagh{at}doctors.org.uk

³ Abbreviations used in this paper: aHUS, atypical hemolytic uremic syndrome; CCP, complement control protein domain; dp6, hexasaccharide; E_H, human erythrocyte; E_SC3b, C3b-coated sheep erythrocyte; fH, factor H; GAG, glycosaminoglycan; GMSA, gel mobility shift assay; HSQC, heteronuclear single quantum coherence; NHS, normal human serum; rH 19–20, recombinant domains 19–20 of factor H; RU, response unit; SA, streptavidin; SPR, surface plasmon resonance.