title: A new map of glycosaminoglycan and C3b binding sites on factor H.
abstract: 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(d)s of 14 and 3.5 microM, respectively) or a C1 chip (K(d)s of 10 and 4.5 microM, 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.
authors.affiliation: Schools of Biological Sciences and Chemistry, University of Edinburgh, Edinburgh, UK.
authors.forename: Christoph Q
authors.initials: CQ
authors.lastname: Schmidt
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Herbert
authors.forename: David
authors.initials: D
authors.lastname: Kavanagh
authors.forename: Carina
authors.initials: C
authors.lastname: Gandy
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Fenton
authors.forename: Bärbel S
authors.initials: BS
authors.lastname: Blaum
authors.forename: Malcolm
authors.initials: M
authors.lastname: Lyon
authors.forename: Dusan
authors.initials: D
authors.lastname: Uhrín
authors.forename: Paul N
authors.initials: PN
authors.lastname: Barlow
mesh_terms: D001665:Binding Sites / Q000276:immunology; D002846:Chromatography, Affinity; D003179:Complement C3b / Q000737:chemistry / Q000378:metabolism*; D017242:Complement Factor H / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003170:Complement Pathway, Alternative / Q000276:immunology; D003165:Complement System Proteins / Q000737:chemistry / Q000378:metabolism; D006025:Glycosaminoglycans / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D000071228:Polyelectrolytes; D011108:Polymers / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D003179:Complement C3b; D017242:Complement Factor H; D003165:Complement System Proteins; D006025:Glycosaminoglycans; D006493:Heparin; D010446:Peptide Fragments; D000071228:Polyelectrolytes; D011108:Polymers; D011994:Recombinant Proteins