Recognition of Lewis X by Anti-Lex Monoclonal Antibody IG5F6

mAbs directed toward the Lewis X (Le(x)) determinant have been shown to display different specificities, depending on the presentation of Le(x) to the immune system. Of interest is the murine anti-Le(x) mAb IG5F6, generated against the O chain polysaccharide of Helicobacter pylori that contains polymeric Le(x) structures. The mAb was found to have a higher affinity for polymeric Le(x) over monomeric Le(x) In this study, we explore the recognition of monomeric Le(x) by IG5F6 using a panel of Le(x) 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 Le(x) Ag, indicating that all three residues are essential in the recognition of Le(x) by mAb IG5F6. We explored the involvement of 4''-OH of d-Gal in the binding with IG5F6 using a panel of 4''-modified Le(x) analogues. Although the 4''-OH is only involved in a weak polar interaction, we conclude that the D-Gal residue in Le(x) 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 Le(x) Thus, anti-Le(x) mAbs SH1 and IG5F6 bind to Le(x) in different manners, even though the hydrophobic patch displayed by the beta-galactoside in Le(x) is essential in both cases for their binding to Le(x).