Structure-function analysis of the extracellular domains of the Duffy antigen/receptor for chemokines: characterization of antibody and chemokine binding sites.
The Duffy antigen/receptor for chemokines (DARC), a seven-transmembrane glycoprotein carrying the Duffy (Fy) blood group, acts as a widely expressed promiscuous chemokine receptor. In a structure-function study, we analysed the binding of chemokines and anti-Fy monoclonal antibodies (mAbs) to K562 cells expressing 39 mutant forms of DARC with alanine substitutions spread out on the four extracellular domains (ECDs). Using synthetic peptides, we defined previously the Fy6 epitope (22-FEDVW-26), and we characterized the Fya epitope as the linear sequence 41-YGANLE-46. In agreement with these results, mutations of F22-E23, V25 and Y41, G42, N44, L45 on ECD1 abolished the binding of anti-Fy6 and anti-Fya mAbs to K562 cells respectively, Anti-Fy3 binding was abolished by D58-D59 (ECD1), R124 (ECD2), D263 and D283 (ECD4) substitutions. Mutations of C51 (ECD1), C129 (ECD2), C195 (ECD3) and C276 (ECD4 severely reduced anti-Fy3 and CXC-chemokine ligand 8 (CXCL-8) binding. CXCL-8 binding was also abrogated by mutations of F22-E23, P50 (ECD1) and D263, R267, D283 (ECD4). These results defined the Fya epitope and suggested that (1) two disulphide bridges are involved in the creation of an active chemokine binding pocket; (2) a limited number of amino acids in ECDs 1-4 participate in CXCL-8 binding; and (3) Fy3 is a conformation-dependent epitope involving all ECDs. We also showed that N-glycosylation of DARC occurred on N16SS and did not influence antibody and chemokine binding.
Tournamille
Christophe
C
INSERM U76, Institut National de la Transfusion Sanguine, Paris, France.
Filipe
Anne
A
Wasniowska
Kazimiera
K
Gane
Pierre
P
Lisowska
Elwira
E
Cartron
Jean-Pierre
JP
Colin
Yves
Y
Le Van Kim
Caroline
C
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D018925:Chemokines / Q000378:metabolism; D019743:Chemokines, CXC / Q000378:metabolism; D004375:Duffy Blood-Group System / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D018604:Epitope Mapping; D006031:Glycosylation; D006801:Humans; D020014:K562 Cells; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
C473745:ACKR1 protein, human; D000911:Antibodies, Monoclonal; D018925:Chemokines; D019743:Chemokines, CXC; D004375:Duffy Blood-Group System; D011956:Receptors, Cell Surface