Comparative Epitope Mapping with Saturation Transfer Difference NMR of Sialyl Lewis^a Compounds and Derivatives Bound to a Monoclonal Antibody

Abstract

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.

Mechanistic Insight into Formation of Oxo‐Iron(IV) Porphyrin π‐Cation Radicals from Enzyme Mimics of Cytochrome P450 in Organic Solvents

“…It is interesting to note that using K D values obtained from the STD NMR titrations as described in the accompanying paper24 yields k on and k off rate constants through CORCEMA calculations (Table S4) that correspond very well with surface plasmon resonance data obtained for the binding of sialyl Lewis a to a monoclonal antibody 35. In the present study k on and k off values were obtained by fitting experimental and calculated trNOE curves.…”

From the Ganglioside GQ1b? to Glycomimetic Antagonists of the Myelin-Associated Glycoprotein (MAG)

“…milli-to micromolar affinities. [23] This also proved true for MAG with a 180 micromolar affinity for tetrasaccharide 4, the binding epitope of GQ1bα (Fig. 3).…”

Modern High Resolution NMR for the Study of Structure, Dynamics and Interactions of Biological Macromolecules

“…Protons in immediate contact with the receptor experience the strongest saturation while protons further away from the contact site are less affected [321,326,327]. Reliable interface mapping requires a relatively high off-rate of the ligand and a large excess of ligand over receptor.…”