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

Smolarek D, Hattab C, Buczkowska A, Kaczmarek R, Jarząb A, Cochet S, de Brevern AG, Lukasiewicz J, Jachymek W, Niedziela T, Grodecka M, Wasniowska K, Colin Aronovicz Y, Bertrand O, Czerwinski M - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Glycoconjugate Immunochemistry Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland; INSERM, UMR_S1134, F-75015 Paris, France; Institut National de la Transfusion Sanguine, F-75015 Paris, France; Division of Transplantation Immunology and Mucosal Biology, MRC Centre for Transplantation, King's College London, Guy's Hospital, London, United Kingdom.

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.

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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 δH 9.5 ppm (on-resonance) and δH30 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.
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pone.0116472.g003: 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 δH 9.5 ppm (on-resonance) and δH30 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.

Mentions: The role of structural elements within the peptide that contribute to the Fab-binding epitope were investigated by STD NMR [34, 35] using synthetic peptide DFEDVWNSSYG. We found that the enhanced resonances in the STD NMR spectrum belonged predominantly to protons ε3, ζ2, ζ3, η2 and δ1 of the Trp-26 residue (the enhanced resonances are indicated in Fig. 3). Minor enhancements of the resonances of protons ε1/ε2 and δ1/δ2 of the Phe residue were also observed. The enhanced resonance at ~7.12 ppm could not be assigned unequivocally as protons δ1/δ2 of Tyr residue and proton ζ3 of the Trp were not resolved. Minute enhancements of Tyr protons ε1/ε2 were only noticeable when the on-resonance irradiation frequency was set to 9.5 ppm. Additionally, no substantial enhancements were observed in the region of signals from aliphatic side chains. Interestingly, no enhancements of valine (V) CH3 proton resonance were observed. No enhancement of Val-35 STD signal gives evidence of either the lack of binding or too strong binding and low off rate value or insufficient energy transfer from the protein to this residue due to the unfavorable conformation causing the distance too large for STD signal observation. The STD-NMR experiments confirmed that the immunodominant epitope recognized by the Fab is limited predominantly to the Trp residue within the peptide. However, the intensities of the enhanced signals observed in the STD NMR spectrum were low. This could be explained by the tight binding of the Fab to the peptide and presumably small off rates (koff) resulting in an inefficient saturation transfer to ligand.


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

Smolarek D, Hattab C, Buczkowska A, Kaczmarek R, Jarząb A, Cochet S, de Brevern AG, Lukasiewicz J, Jachymek W, Niedziela T, Grodecka M, Wasniowska K, Colin Aronovicz Y, Bertrand O, Czerwinski M - PLoS ONE (2015)

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 δH 9.5 ppm (on-resonance) and δH30 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4338028&req=5

pone.0116472.g003: 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 δH 9.5 ppm (on-resonance) and δH30 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.
Mentions: The role of structural elements within the peptide that contribute to the Fab-binding epitope were investigated by STD NMR [34, 35] using synthetic peptide DFEDVWNSSYG. We found that the enhanced resonances in the STD NMR spectrum belonged predominantly to protons ε3, ζ2, ζ3, η2 and δ1 of the Trp-26 residue (the enhanced resonances are indicated in Fig. 3). Minor enhancements of the resonances of protons ε1/ε2 and δ1/δ2 of the Phe residue were also observed. The enhanced resonance at ~7.12 ppm could not be assigned unequivocally as protons δ1/δ2 of Tyr residue and proton ζ3 of the Trp were not resolved. Minute enhancements of Tyr protons ε1/ε2 were only noticeable when the on-resonance irradiation frequency was set to 9.5 ppm. Additionally, no substantial enhancements were observed in the region of signals from aliphatic side chains. Interestingly, no enhancements of valine (V) CH3 proton resonance were observed. No enhancement of Val-35 STD signal gives evidence of either the lack of binding or too strong binding and low off rate value or insufficient energy transfer from the protein to this residue due to the unfavorable conformation causing the distance too large for STD signal observation. The STD-NMR experiments confirmed that the immunodominant epitope recognized by the Fab is limited predominantly to the Trp residue within the peptide. However, the intensities of the enhanced signals observed in the STD NMR spectrum were low. This could be explained by the tight binding of the Fab to the peptide and presumably small off rates (koff) resulting in an inefficient saturation transfer to ligand.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Glycoconjugate Immunochemistry Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland; INSERM, UMR_S1134, F-75015 Paris, France; Institut National de la Transfusion Sanguine, F-75015 Paris, France; Division of Transplantation Immunology and Mucosal Biology, MRC Centre for Transplantation, King's College London, Guy's Hospital, London, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus