2.50
Hdl Handle:
http://hdl.handle.net/10541/56053
Title:
A new map of glycosaminoglycan and C3b binding sites on factor H.
Authors:
Schmidt, C; Herbert, A; Kavanagh, D; Gandy, C; Fenton, C; Blaum, B; Lyon, Malcolm; Uhrín, D; Barlow, P
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.
Affiliation:
Schools of Biological Sciences and Chemistry, University of Edinburgh, Edinburgh, UK.
Citation:
A new map of glycosaminoglycan and C3b binding sites on factor H. 2008, 181 (4):2610-9 J. Immunol.
Journal:
Journal of Immunology
Issue Date:
15-Aug-2008
URI:
http://hdl.handle.net/10541/56053
PubMed ID:
18684951
Type:
Article
Language:
en
ISSN:
1550-6606
Appears in Collections:
All Paterson Institute for Cancer Research; Medical Oncology

Full metadata record

DC FieldValue Language
dc.contributor.authorSchmidt, C-
dc.contributor.authorHerbert, A-
dc.contributor.authorKavanagh, D-
dc.contributor.authorGandy, C-
dc.contributor.authorFenton, C-
dc.contributor.authorBlaum, B-
dc.contributor.authorLyon, Malcolm-
dc.contributor.authorUhrín, D-
dc.contributor.authorBarlow, P-
dc.date.accessioned2009-03-17T17:20:00Z-
dc.date.available2009-03-17T17:20:00Z-
dc.date.issued2008-08-15-
dc.identifier.citationA new map of glycosaminoglycan and C3b binding sites on factor H. 2008, 181 (4):2610-9 J. Immunol.en
dc.identifier.issn1550-6606-
dc.identifier.pmid18684951-
dc.identifier.urihttp://hdl.handle.net/10541/56053-
dc.description.abstractHuman 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.en
dc.language.isoenen
dc.subject.meshBinding Sites-
dc.subject.meshChromatography, Affinity-
dc.subject.meshComplement C3b-
dc.subject.meshComplement Factor H-
dc.subject.meshComplement Pathway, Alternative-
dc.subject.meshComplement System Proteins-
dc.subject.meshGlycosaminoglycans-
dc.subject.meshHeparin-
dc.subject.meshHumans-
dc.subject.meshMagnetic Resonance Spectroscopy-
dc.subject.meshPeptide Fragments-
dc.subject.meshPeptide Mapping-
dc.subject.meshPolymers-
dc.subject.meshProtein Folding-
dc.subject.meshRecombinant Proteins-
dc.titleA new map of glycosaminoglycan and C3b binding sites on factor H.en
dc.typeArticleen
dc.contributor.departmentSchools of Biological Sciences and Chemistry, University of Edinburgh, Edinburgh, UK.en
dc.identifier.journalJournal of Immunologyen

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