Abstract
Duffy Antigen Receptor for Chemokines (DARC) plays multiple roles in human health as a blood group antigen, a receptor for chemokines and the only known receptor for Plasmodium vivax merozoites. It is the target of the murine anti-Fy6 monoclonal antibody 2C3 which binds to the first extracellular domain (ECD1), but exact nature of the recognized epitope was a subject of contradictory reports. Here, using a set of complex experiments which include expression of DARC with amino acid substitutions within the Fy6 epitope in E. coli and K562 cells, ELISA, surface plasmon resonance (SPR) and flow cytometry, we have resolved discrepancies between previously published reports and show that the basic epitope recognized by 2C3 antibody is 22FEDVW26, with 22F and 26W being the most important residues. In addition, we demonstrated that 30Y plays an auxiliary role in binding, particularly when the residue is sulfated. The STD-NMR studies performed using 2C3-derived Fab and synthetic peptide corroborated most of these results, and together with the molecular modelling suggested that 25V is not involved in direct interactions with the antibody, but determines folding of the epitope backbone.
Highlights
Duffy Antigen Receptor for Chemokines (DARC) is a seven-transmembrane domain glycoprotein present on red blood cells (RBC) and post-capillary endothelial cells or Purkinje cells
That phenotype, called Fy(a-b-) is caused by a single nucleotide polymorphism at position -67T>C in the GATA box of DARC gene promoter [1] and it was thought for decades that it was associated with resistance of red blood cells to infection by Plasmodium vivax merozoites [2]
As the role of monoclonal antibodies in biology and medicine becomes increasingly important, so does the precise evaluation of the epitopes being recognized by the antibodies
Summary
Duffy Antigen Receptor for Chemokines (DARC) is a seven-transmembrane domain glycoprotein present on red blood cells (RBC) and post-capillary endothelial cells or Purkinje cells. DARC plays an important role in human health, being a blood group antigen, the only known receptor for Plasmodium vivax parasite that causes malaria and a receptor for chemokines. DARC glycoprotein carries blood group antigens Fya and Fyb, which are encoded by two allelic genes, designated FYÃ01 and FYÃ02 respectively. That phenotype, called Fy(a-b-) is caused by a single nucleotide polymorphism at position -67T>C in the GATA box of DARC gene promoter [1] and it was thought for decades that it was associated with resistance of red blood cells to infection by Plasmodium vivax merozoites [2]. The crystal structure of DARC-PvDBP complex has been recently resolved [5]
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