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Glycobiology Advance Access originally published online on February 6, 2003
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Glycobiology, 2003, Vol. 13, No. 6 435-443
© 2003 Oxford University Press

Epitope mapping of sialyl Lewisx bound to E-selectin using saturation transfer difference NMR experiments

Meike Rinnbauer2, Beat Ernst3, Bea Wagner3, John Magnani4, Andrew J. Benie2 and Thomas Peters1,2

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

Received on September 8, 2002; revised on January 2, 2003; accepted on January 3, 2003

A complex between sialyl Lewisx ({alpha}-D-Neu5Ac-[2->3]- ß-D-Gal-[1->4]-[{alpha}-L-Fuc-(1->3)]-ß-D-GlcNAc-O-[CH2]8 COOMe) and E-selectin was studied using saturation transfer difference (STD) nuclear magnetic resonance (NMR) experiments. These experiments allow the identification of the binding epitope of a ligand at atomic resolution. A semiquantitative analysis of STD total correlation spectroscopy 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 nuclear Overhauser effect spectroscopy (trNOESY) experiments. Finally, competitive trNOESY experiments were performed, showing that the synthetic peptide is a competitive inhibitor of sialyl Lewisx.

1 To whom correspondence should be addressed; e-mail: thomas.peters{at}chemie.mu-luebeck.de


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