Recognition of Lewis X by Anti-Lex Monoclonal Antibody IG5F6.

mAbs directed toward the Lewis X (Lex) determinant have been shown to display different specificities, depending on the presentation of Lex to the immune system. Of interest is the murine anti-Lex mAb IG5F6, generated against the O chain polysaccharide of Helicobacter pylori that contains polymeric Lex structures. The mAb was found to have a higher affinity for polymeric Lex over monomeric Lex In this study, we explore the recognition of monomeric Lex by IG5F6 using a panel of Lex analogues in which N-acetyl-d-glucosamine, l-fucose, or d-galactose (D-Gal) are replaced with d-glucose and/or l-rhamnose. Our studies show that all analogues were weaker inhibitors than the Lex Ag, indicating that all three residues are essential in the recognition of Lex by mAb IG5F6. We explored the involvement of 4″-OH of d-Gal in the binding with IG5F6 using a panel of 4″-modified Lex analogues. Although the 4″-OH is only involved in a weak polar interaction, we conclude that the D-Gal residue in Lex is primarily involved in aromatic stacking interactions with the Ab binding site. We compared these results to our work with mAb SH1. Although stacking interactions between D-Gal and an aromatic residue was also suggested for SH1, an H-bond involving the 4″-OH was identified that is not found in the binding of IG5F6 to Lex Thus, anti-Lex mAbs SH1 and IG5F6 bind to Lex in different manners, even though the hydrophobic patch displayed by the β-galactoside in Lex is essential in both cases for their binding to Lex.