title: One-step immunopurification and lectinochemical characterization of the Duffy atypical chemokine receptor from human erythrocytes.
abstract: Duffy antigen/receptor for chemokines (DARC) is a glycosylated seven-transmembrane protein acting as a blood group antigen, a chemokine binding protein and a receptor for Plasmodium vivax malaria parasite. It is present on erythrocytes and endothelial cells of postcapillary venules. The N-terminal extracellular domain of the Duffy glycoprotein carries Fy(a)/Fy(b) blood group antigens and Fy6 linear epitope recognized by monoclonal antibodies. Previously, we have shown that recombinant Duffy protein expressed in K562 cells has three N-linked oligosaccharide chains, which are mainly of complex-type. Here we report a one-step purification method of Duffy protein from human erythrocytes. DARC was extracted from erythrocyte membranes in the presence of 1% n-dodecyl-β-D-maltoside (DDM) and 0.05% cholesteryl hemisuccinate (CHS) and purified by affinity chromatography using immobilized anti-Fy6 2C3 mouse monoclonal antibody. Duffy glycoprotein was eluted from the column with synthetic DFEDVWN peptide containing epitope for 2C3 monoclonal antibody. In this single-step immunoaffinity purification method we obtained highly purified DARC, which migrates in SDS-polyacrylamide gel as a major diffuse band corresponding to a molecular mass of 40-47 kDa. In ELISA purified Duffy glycoprotein binds anti-Duffy antibodies recognizing epitopes located on distinct regions of the molecule. Results of circular dichroism measurement indicate that purified DARC has a high content of α-helical secondary structure typical for chemokine receptors. Analysis of DARC glycans performed by means of lectin blotting and glycosidase digestion suggests that native Duffy N-glycans are mostly triantennary complex-type, terminated with α2-3- and α2-6-linked sialic acid residues with bisecting GlcNAc and α1-6-linked fucose at the core.
authors.affiliation: Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, 53-114, Wrocław, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Grodecka
authors.forename: Olivier
authors.initials: O
authors.lastname: Bertrand
authors.forename: Ewa
authors.initials: E
authors.lastname: Karolak
authors.forename: Marek
authors.initials: M
authors.lastname: Lisowski
authors.forename: Kazimiera
authors.initials: K
authors.lastname: Waśniowska
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D002942:Circular Dichroism / Q000379:methods; D004375:Duffy Blood-Group System / Q000737:chemistry / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004910:Erythrocyte Membrane / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000302:isolation & purification*; D019707:Receptors, Chemokine / Q000302:isolation & purification*
chemical_list: C473745:ACKR1 protein, human; D000911:Antibodies, Monoclonal; D004375:Duffy Blood-Group System; D037102:Lectins; D010446:Peptide Fragments; D011134:Polysaccharides; D011956:Receptors, Cell Surface; D019707:Receptors, Chemokine; D006026:Glycoside Hydrolases