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.
AuthorsBlaum, Bärbel S
Deakin, Jon A
Johansson, Conny M
Herbert, Andrew P
Barlow, Paul N
AffiliationEdinburgh Biomolecular NMR Unit, School of Chemistry and School of Biological Sciences, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, Scotland, United Kingdom.
MetadataShow full item record
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).
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 Soc
JournalJournal of the American Chemical Society
- Differences in the interaction of heparin with arginine and lysine and the importance of these basic amino acids in the binding of heparin to acidic fibroblast growth factor.
- Authors: Fromm JR, Hileman RE, Caldwell EE, Weiler JM, Linhardt RJ
- Issue date: 1995 Nov 10
- Site-specific N-glycan characterization of human complement factor H.
- Authors: Fenaille F, Le Mignon M, Groseil C, Ramon C, Riandé S, Siret L, Bihoreau N
- Issue date: 2007 Sep
- Identification of S-hydroxylysyl-methionine as the covalent cross-link of the noncollagenous (NC1) hexamer of the alpha1alpha1alpha2 collagen IV network: a role for the post-translational modification of lysine 211 to hydroxylysine 211 in hexamer assembly.
- Authors: Vanacore RM, Friedman DB, Ham AJ, Sundaramoorthy M, Hudson BG
- Issue date: 2005 Aug 12
- Investigation of the detoxification mechanism of formaldehyde-treated tetanus toxin.
- Authors: Thaysen-Andersen M, Jørgensen SB, Wilhelmsen ES, Petersen JW, Højrup P
- Issue date: 2007 Mar 8
- Investigation of lysine side chain interactions of interleukin-8 with heparin and other glycosaminoglycans studied by a methylation-NMR approach.
- Authors: Möbius K, Nordsieck K, Pichert A, Samsonov SA, Thomas L, Schiller J, Kalkhof S, Teresa Pisabarro M, Beck-Sickinger AG, Huster D
- Issue date: 2013 Nov