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Glycobiology Advance Access published online on February 6, 2003
Glycobiology, doi:10.1093/glycob/cwg043
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Submitted on September 8, 2002
Revised on January 2, 2003
Accepted on January 3, 2003

© 2003 Oxford University Press

ORIGINAL ARTICLES

Epitope mapping of Sialyl Lewisx bound to E-Selectin using saturation transfer difference NMR experiments

Meike Rinnbauer 1, Beat Ernst 2, Bea Wagner 2, John Magnani 3, Andrew J. Benie 1, Thomas Peters 1*

1 Institute of Chemistry, University of Luebeck, Ratzeburger Allee 160, D-23538 Luebeck
2 Institute of Molecular Pharmacy, Pharmacenter, University of Basel, Klingenbergstrasse 50, CH-4056 Basel, Switzerland
3 GlycoTech Corporation, 14915 Broschart Road, Rockville, MD 10850, USA

* To whom correspondence should be addressed. E-mail: thomas.peters{at}chemie.mu-luebeck.de.

Abstract

A complex between sialyl Lewisx ({alpha}-D-Neu5Ac-(2->3)-{beta}-D-Gal-(1->4)-[{alpha}-L-Fuc-(1->3)]-{beta}-D-GlcNAc-O-(CH2)8COOMe) and E-selectin was studied using saturation transfer difference (STD) NMR experiments. These experiments allow the identification of the binding epitope of a ligand at atomic resolution. A semi-quantitative analysis of STD-TOCSY spectra provides clear evidence that the galactose residue receives the largest saturation transfer. The protons H4 and H6 of the galactose residue are in especially close contact to the amino acids of the E-selectin binding pocket. The fucose residue also receives a significant saturation transfer. The GlcNAc and Neu5Ac residues with the exception of H3 and H3' of Neu5Ac were found to interact weakly with the protein surface. These findings are in excellent agreement with a recently published X-ray structure and with the earlier findings from syntheses and activity assays. To further characterize the binding pocket of E-selectin, an inhibitory peptide, Ac-TWDQLWDLMK-CONH2 was synthesized and the binding to E-selectin studied utilizing transfer NOESY (trNOESY) experiments. Finally, competitive trNOESY experiments were performed showing that the synthetic peptide is a competitive inhibitor of sialyl Lewisx.


STD NMR, transfer NOESY, E-selectin, sialyl Lewisx, epitope mapping
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