Abstract
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.
Highlights
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
fibroblast growth factor-2 (FGF-2) was acetylated according to the conditions chosen in the absence or in the presence of heparin beads
This suggests a selective protection given by the sugar acetylation heparin biotinylation
Summary
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 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. From the ‡School of Biological Sciences and Centre for Glycobiology, University of Liverpool, Liverpool L69 7ZB, United Kingdom and §Laboratoire de Recherche sur la Croissance Cellulaire, la Reparation et la Regeneration Tissulaires (CRRET), CNRS UMR 7149, Universite Paris XII, 94010 Creteil Cedex, France They participate in the structural organization of the extracellular space [2, 3] and play an active role in molecular networks driving complex biological phenomena such as development (4 – 6), inflammation and immune response [7, 8], and disease [9]. Providing detailed structural and kinetic information about the interaction, all these methods are limited to the study of single protein-sugar interactions, and they cannot be translated into a high throughput format
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
