Understanding the recognition of Lewis X by anti-Le(x) monoclonal antibodies.
The recognition of the Le(x) antigen by the anti-Le(x) monoclonal antibody (mAb) SH1 was studied by ELISA using a panel of 4″-modified Le(x) analogues. We confirmed that these analogues maintained the stacked conformation adopted by natural Le(x) antigen using 1D ROESY experiments and measuring intramolecular distances. Our binding studies show that the 4-OH″ of galactose behaves as an H-bond donor to an electronegative amino acid side chain in the SH1 binding site. While removal of this H-bond leads to reduced inhibition, disturbing the hydrophobic α face of the β-galactosyl residue leads to complete loss of binding to SH1. We compared our results to the crystal structure of the Fab fragment of anti-Le(x) mAb 291-2G3-A complexed with Le(x) (PDB entry 1UZ8 ). While no H-bond involving the 4-OH″ was described, hydrophobic interactions between a tryptophan residue and the β-galactoside α face are observed. We conclude that the hydrophobic α face that is uniquely displayed by β-galactosyl residues is essential to the recognition of the Le(x) antigen by anti-Le(x) antibodies.
Department of Chemistry, University of Guelph , Guelph, Ontario, N1G 2W1, Canada.
Christopher J
CJ
Moore
France-Isabelle
FI
Auzanneau
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D018360:Crystallography, X-Ray; D004797:Enzyme-Linked Immunosorbent Assay; D005690:Galactose / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D005697:Galactosides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D016256:Lewis X Antigen / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000276:immunology; D017434:Protein Structure, Tertiary
D000911:Antibodies, Monoclonal; D005697:Galactosides; D007140:Immunoglobulin Fab Fragments; D016256:Lewis X Antigen; C007816:beta-galactoside; D005690:Galactose