Identification of heparin-binding sites in proteins by selective labeling.
Heparan sulfate proteoglycans are key regulators of complex molecular networks due to the interaction of their sugar chains with a large number of partner proteins, which in humans number more than 200 (Ori, A., Wilkinson, M. C., and Fernig, D. G. (2008) The heparanome and regulation of cell function: structures, functions and challenges. Front. Biosci. 13, 4309-4338). We developed a method to selectively label residues involved in heparin binding that matches the requirements for medium/high throughput applications called the "Protect and Label" strategy. This is based on the protection against chemical modification given by heparin/heparan sulfate to the residues located in the heparin-binding site. Thus, analysis of fibroblast growth factor-2 bound to heparin and incubated with N-hydroxysuccinimide acetate showed that lysines involved in the sugar binding are protected against chemical modification. Moreover following release from heparin, the protected lysine side chains may be specifically labeled with N-hydroxysuccinimide biotin. After protein digestion, the biotinylated peptides were readily isolated and identified by MALDI-Q-TOF mass spectrometry. The analysis of labeled peptides obtained from three well characterized heparin-binding proteins with very different heparin-binding sites, fibroblast growth factor-2, platelet factor-4, and pleiotrophin demonstrates the success of this new approach, which thus provides a rapid and reliable procedure to identify heparin-binding sites.
Ori
Alessandro
A
School of Biological Sciences and Centre for Glycobiology, University of Liverpool, Liverpool, United Kingdom.
Free
Paul
P
Courty
José
J
Wilkinson
Mark C
MC
Fernig
David G
DG
D000085:Acetates / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013388:Succinimides / Q000737:chemistry / Q000378:metabolism
D000085:Acetates; D019812:Heparan Sulfate Proteoglycans; D010455:Peptides; D011506:Proteins; D013388:Succinimides; D016222:Fibroblast Growth Factor 2; D006493:Heparin; D008239:Lysine; C001426:N-hydroxysuccinimide