Identification of Heparin-binding Sites in Proteins by Selective Labeling*
AUTOR(ES)
Ori, Alessandro
FONTE
The American Society for Biochemistry and Molecular Biology
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2758754Documentos Relacionados
- Augmented production of heparin-binding mitogenic proteins by preadipocytes from massively obese persons.
- Identification and Analysis of a Novel Heparin-Binding Glycoprotein Encoded by Human Herpesvirus 7
- In vivo labeling of L-type Ca2+ channels by fluorescent dihydropyridines: evidence for a functional, extracellular heparin-binding site.
- Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2 Capsids†
- Molecular mapping of the heparin-binding exosite of thrombin.