Studies of a murine monoclonal antibody directed against DARC: reappraisal of its specificity

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.

Figure 1. Inhibition of 2C3 MAb binding to ECD1-nuc.

The antibody was incubated with serially diluted DFEDVWNSSYG (circles) or DFEDVWNSSY(SO₃H)G (squares) peptides. Subsequent binding to ECD1-nuc was evaluated by ELISA as described in Materials and Methods. The difference in % of inhibition was statistically significant in the range of concentrations 3-60 nM (P < 0.05, two-tailed Mann-Whitney U test).

Figure 2. Comparison of binding of 2C3 MAb and 2C3 Fab to ECD1-nuc evaluated by ELISA.

The binding of 2C3 MAb (squares) and 2C3 Fab (circles) was evaluated by ELISA as described in Materials and Methods. The difference in binding is statistically significant at concentration 20 nM (P < 0.05, two-tailed Mann-Whitney U test).

Figure 3. STD-NMR analysis of the Fab-binding epitope.

The STD NMR (bottom) and reference ¹H NMR spectra of the peptide DFEDVWNSSYG (diagram) in the presence of Fab were acquired at 600 MHz and 25°C for samples (160μl, in a 3 mm tube) prepared in PBS made with ²H₂O, pH 7.5. The protein was irradiated at δ_H 9.5 ppm (on-resonance) and δ_H30 ppm (off-resonance) with a saturation time of 2 s. The excitation sculpting pulse sequence was used to suppress water signals. The oversized one-letter amino acid codes indicate the proton resonances that were enhanced in the STD experiments. The Greek letters and numbers designate the enhanced resonances of the aromatic systems. The unresolved resonances are grouped.

Figure 4. Three-dimensional structure of the peptide DFEDVWNSSYG (a) and peptides in which Val-25 is substituted by amino acids of different hydrophobicity (b-d).

Peptide structures were calculated in PEP-FOLD and then visualized and analyzed in Chimera software. The amino acid residues involved in the antibody binding are visualized in red, while the residue that was changed: Val (a), Ile (b), Leu (c) and Ala (d) is visualized in purple.