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A New Map of Glycosaminoglycan and C3b Binding Sites on Factor H¹

Christoph Q. Schmidt^*, Andrew P. Herbert^*, David Kavanagh^*, Carina Gandy^*, Christopher J. Fenton^*, Bärbel S. Blaum^*, Malcolm Lyon, Duan Uhrín^* and Paul N. Barlow^2,*

^* Schools of Biological Sciences and Chemistry, University of Edinburgh, Edinburgh, U.K.; and Cancer Research U.K. Department of Medical Oncology, University of Manchester, Christie Hospital National Health Service Trust, Manchester, U.K.

Human complement factor H, consisting of 20 complement control protein (CCP) modules, is an abundant plasma glycoprotein. It prevents C3b amplification on self surfaces bearing certain polyanionic carbohydrates, while complement activation progresses on most other, mainly foreign, surfaces. Herein, locations of binding sites for polyanions and C3b are reexamined rigorously by overexpressing factor H segments, structural validation, and binding assays. As anticipated, constructs corresponding to CCPs 7–8 and 19–20 bind well in heparin-affinity chromatography. However, CCPs 8–9, previously reported to bind glycosaminoglycans, bind neither to heparin resin nor to heparin fragments in gel-mobility shift assays. Introduction of nonnative residues N-terminal to a construct containing CCPs 8–9, identical to those in proteins used in the previous report, converted this module pair to an artificially heparin-binding one. The module pair CCPs 12–13 does not bind heparin appreciably, notwithstanding previous suggestions to the contrary. We further checked CCPs 10–12, 11–14, 13–15, 10–15, and 8–15 for ability to bind heparin but found very low affinity or none. As expected, constructs corresponding to CCPs 1–4 and 19–20 bind C3b amine coupled to a CM5 chip (K_ds of 14 and 3.5 µM, respectively) or a C1 chip (K_ds of 10 and 4.5 µM, respectively). Constructs CCPs 7–8 and 6–8 exhibit measurable affinities for C3b according to surface plasmon resonance, although they are weak compared with CCPs 19–20. Contrary to expectations, none of several constructs encompassing modules from CCP 9 to 15 exhibited significant C3b binding in this assay. Thus, we propose a new functional map of factor H.

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 D.U. and P.N.B. were funded by the Wellcome Trust (078780/Z/05/Z); C.Q.S. acknowledges the support of the Darwin Trust of Edinburgh.

² Address correspondence and reprint requests to Dr. Paul N. Barlow, Joseph Black Chemistry Building, University of Edinburgh, Edinburgh EH9 3JJ, U.K. E-mail address: Paul.Barlow{at}ed.ac.uk

³ Abbreviations used in this paper: fH, complement factor H; CCP, complement control protein; GAG, glycosaminoglycan; GMSA, gel-mobility shift assay; HSQC, heteronuclear single quantum coherence; NMR, nuclear magnetic resonance; RCA, regulators of complement activation; RU, response units; SPR, surface plasmon resonance.