Comparative epitope mapping with saturation transfer difference NMR of sialyl Lewis^a compounds and derivatives bound to a monoclonal antibody

The monoclonal antibody GSLA-2 recognizes the sialyl Lewis^a (sLe^a) epitope, which has an increased occurrence on mucin type glycoproteins of patients with colorectal carcinoma. GSLA-2 is therefore used in tumor diagnosis. To advance the understanding of this highly specific molecular recognition reaction, we have analyzed the binding epitope of sLe^a at atomic resolution using saturation transfer difference NMR. To compare, the binding epitopes of sialyl Lewis^x (sLe^x) and of four synthetic derivatives of sLe^a were explored. Surface plasmon resonance experiments furnished thermodynamic and kinetic data. It is observed that all pyranose rings of sLe^a are in contact with the protein surface, with the neuramic acid residue receiving the largest fraction of saturation transfer. In contrast, sLe^x binds very weakly, though specifically to GSLA-2, with a different binding epitope. This study provides a comprehensive picture of the recognition sLe^a and explains the exquisite specificity of the antibody.