Lysine and arginine side chains in glycosaminoglycan-protein complexes investigated by NMR, cross-linking, and mass spectrometry: a case study of the factor H-heparin interaction.

2.50
Hdl Handle:
http://hdl.handle.net/10541/109421
Title:
Lysine and arginine side chains in glycosaminoglycan-protein complexes investigated by NMR, cross-linking, and mass spectrometry: a case study of the factor H-heparin interaction.
Authors:
Blaum, Bärbel S; Deakin, Jon A; Johansson, Conny M; Herbert, Andrew P; Barlow, Paul N; Lyon, Malcolm; Uhrín, Dusan
Abstract:
We have used the interaction between module 7 of complement factor H (CFH approximately 7) and a fully sulfated heparin tetrasaccharide to exemplify a new approach for studying contributions of basic side chains to the formation of glycosaminoglycan (GAG)-protein complexes. We first employed HISQC and H(2)CN NMR experiments to monitor the side-chain resonances of lysines and arginines in (15)N, (13)C-labeled protein during titrations with a fully sulfated heparin tetrasaccharide under physiological conditions. Under identical conditions and using (15)N-labeled protein, we then cross-linked tetrasaccharide to CFH approximately 7 and confirmed the 1:1 stoichiometry by FT-ICR-MS. We subsequently characterized this covalent protein-GAG conjugate by NMR and further MS techniques. MALDI-TOF MS identified protein fragments obtained via trypsin digestion or chemical fragmentation, yielding information concerning the site of GAG attachment. Combining MS and NMR data allowed us to identify the side chain of K405 as the point of attachment of the cross-linked heparin oligosaccharide to CFH approximately 7. On the basis of the analysis of NMR and MS data of the noncovalent and cross-linked CFH approximately 7-tetrasaccharide complexes, we conclude that the K446 side chain is not essential for binding the tetrasaccharide, despite the large chemical shift perturbations of its backbone amide (15)N and (1)H resonances during titrations. We show that R444 provides the most important charge-charge interaction within a C-terminal heparin-binding subsite of CFH approximately 7 whereas side chains of R404, K405, and K388 are the predominant contributors to an N-terminal binding subsite located in the immediate vicinity of residue 402, which is implicated in age-related macular degeneration (AMD).
Affiliation:
Edinburgh Biomolecular NMR Unit, School of Chemistry and School of Biological Sciences, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, Scotland, United Kingdom.
Citation:
Lysine and arginine side chains in glycosaminoglycan-protein complexes investigated by NMR, cross-linking, and mass spectrometry: a case study of the factor H-heparin interaction. 2010, 132 (18):6374-81 J Am Chem Soc
Journal:
Journal of the American Chemical Society
Issue Date:
12-May-2010
URI:
http://hdl.handle.net/10541/109421
DOI:
10.1021/ja1000517
PubMed ID:
20394361
Type:
Article
Language:
en
ISSN:
1520-5126
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorBlaum, Bärbel Sen
dc.contributor.authorDeakin, Jon Aen
dc.contributor.authorJohansson, Conny Men
dc.contributor.authorHerbert, Andrew Pen
dc.contributor.authorBarlow, Paul Nen
dc.contributor.authorLyon, Malcolmen
dc.contributor.authorUhrín, Dusanen
dc.date.accessioned2010-08-10T16:05:43Z-
dc.date.available2010-08-10T16:05:43Z-
dc.date.issued2010-05-12-
dc.identifier.citationLysine and arginine side chains in glycosaminoglycan-protein complexes investigated by NMR, cross-linking, and mass spectrometry: a case study of the factor H-heparin interaction. 2010, 132 (18):6374-81 J Am Chem Socen
dc.identifier.issn1520-5126-
dc.identifier.pmid20394361-
dc.identifier.doi10.1021/ja1000517-
dc.identifier.urihttp://hdl.handle.net/10541/109421-
dc.description.abstractWe have used the interaction between module 7 of complement factor H (CFH approximately 7) and a fully sulfated heparin tetrasaccharide to exemplify a new approach for studying contributions of basic side chains to the formation of glycosaminoglycan (GAG)-protein complexes. We first employed HISQC and H(2)CN NMR experiments to monitor the side-chain resonances of lysines and arginines in (15)N, (13)C-labeled protein during titrations with a fully sulfated heparin tetrasaccharide under physiological conditions. Under identical conditions and using (15)N-labeled protein, we then cross-linked tetrasaccharide to CFH approximately 7 and confirmed the 1:1 stoichiometry by FT-ICR-MS. We subsequently characterized this covalent protein-GAG conjugate by NMR and further MS techniques. MALDI-TOF MS identified protein fragments obtained via trypsin digestion or chemical fragmentation, yielding information concerning the site of GAG attachment. Combining MS and NMR data allowed us to identify the side chain of K405 as the point of attachment of the cross-linked heparin oligosaccharide to CFH approximately 7. On the basis of the analysis of NMR and MS data of the noncovalent and cross-linked CFH approximately 7-tetrasaccharide complexes, we conclude that the K446 side chain is not essential for binding the tetrasaccharide, despite the large chemical shift perturbations of its backbone amide (15)N and (1)H resonances during titrations. We show that R444 provides the most important charge-charge interaction within a C-terminal heparin-binding subsite of CFH approximately 7 whereas side chains of R404, K405, and K388 are the predominant contributors to an N-terminal binding subsite located in the immediate vicinity of residue 402, which is implicated in age-related macular degeneration (AMD).en
dc.language.isoenen
dc.subject.meshAmino Acid Sequence-
dc.subject.meshArginine-
dc.subject.meshComplement Factor H-
dc.subject.meshCross-Linking Reagents-
dc.subject.meshHeparin-
dc.subject.meshHumans-
dc.subject.meshLysine-
dc.subject.meshMagnetic Resonance Spectroscopy-
dc.subject.meshMass Spectrometry-
dc.subject.meshModels, Molecular-
dc.subject.meshMolecular Sequence Data-
dc.subject.meshProtein Binding-
dc.subject.meshProtein Multimerization-
dc.subject.meshProtein Structure, Quaternary-
dc.subject.meshSolutions-
dc.subject.meshTrypsin-
dc.titleLysine and arginine side chains in glycosaminoglycan-protein complexes investigated by NMR, cross-linking, and mass spectrometry: a case study of the factor H-heparin interaction.en
dc.typeArticleen
dc.contributor.departmentEdinburgh Biomolecular NMR Unit, School of Chemistry and School of Biological Sciences, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, Scotland, United Kingdom.en
dc.identifier.journalJournal of the American Chemical Societyen

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