Identification of the Fy6 epitope recognized by two monoclonal antibodies in the N-terminal extracellular portion of the Duffy antigen receptor for chemokines.
The epitope Fy6 recognized by two monoclonal antibodies (i3A and BG6), which inhibit binding of chemokines to the Duffy antigen, was characterized by means of peptides synthesized on pins (Epitope Scanning Kit) and deletion mutagenesis. Both antibodies showed very similar specificities. They recognized a linear epitope, the essential portion of which was the heptapeptide Gln-Leu-Asp-Phe-Glu-Asp-Val comprising amino acid residues 21-27, located between two glycosylation sites of the Duffy protein. All the amino acid residues of the epitope, except Glu, were essential for antibody binding, since they could not be replaced by any other amino acid residues or by only one or two. The Glu residue could be replaced by most other amino acid residues, and its replacement by 10 amino acid residues gave a distinct increase in the antibody binding. The results were in full agreement with the finding that the mutant of the Duffy antigen, lacking amino acid residues 23-25 (-Asp-Phe-Glu-), did not bind the i3A antibody, but bound the anti-Fy3 monoclonal antibody similarly to the wild type of the Duffy antigen. The apparent affinity constants of both anti-Fy6 antibodies were determined by surface plasmon resonance, using immunopurified Duffy protein as a ligand.
Waśniowska
K
K
Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.
Blanchard
D
D
Janvier
D
D
Wang
Z X
ZX
Peiper
S C
SC
Hadley
T J
TJ
Lisowska
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000913:Antibodies, Protozoan / Q000276:immunology; D000953:Antigens, Protozoan; D002352:Carrier Proteins / Q000276:immunology* / Q000378:metabolism*; D018925:Chemokines / Q000378:metabolism*; D004375:Duffy Blood-Group System / Q000276:immunology; D000939:Epitopes / Q000032:analysis* / Q000276:immunology*; D008565:Membrane Proteins / Q000276:immunology*; D008969:Molecular Sequence Data; D010961:Plasmodium / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding / Q000276:immunology; D011487:Protein Conformation; D015800:Protozoan Proteins; D011956:Receptors, Cell Surface / Q000276:immunology* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D002352:Carrier Proteins; D018925:Chemokines; D004375:Duffy Blood-Group System; C066001:Duffy antigen binding protein, Plasmodium; D000939:Epitopes; D008565:Membrane Proteins; D015800:Protozoan Proteins; D011956:Receptors, Cell Surface