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Acta Crystallographica Section C: Crystal Structure Communications (1)
Glycoconjugate Journal (1)
Lisowski, Marek (2)
Bertrand, Olivier (1)
Grodecka, Magdalena (1)
Karolak, Ewa (1)
Lis, Tadeusz (1)
Maciąg, Anna (1)
Makowski, Maciej (1)
Szlachcic, Anna (1)
Waśniowska, Kazimiera (1)
Wiktor, Maciej (1)
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One-step immunopurification and lectinochemical characterization of the Duffy atypical chemokine receptor from human erythrocytes
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 Fya/Fyb 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.
Duffy antigen; Immunopurification; Chemokine receptor; Glycoproteomics; N-glycans; Lectins
Two pentadehydropeptides with different configurations of the ΔPhe residues
Acta Crystallographica Section C: Crystal Structure Communications
Comparison of the crystal structures of two pentadehydropeptides containing ΔPhe residues, namely (Z,Z)-N-(tert-butoxycarbonyl)glycyl-α,β-phenylalanylglycyl-α,β-phenylalanylglycine (or Boc0–Gly1–ΔZPhe2–Gly3–ΔZPhe4–Gly5–OH) methanol solvate, C29H33N5O8·CH4O, (I), and (E,E)-N-(tert-butoxycarbonyl)glycyl-α,β-phenylalanylglycyl-α,β-phenylalanylglycine (or Boc0–Gly1–ΔEPhe2–Gly3–ΔEPhe4–Gly5–OH), C29H33N5O8, (II), indicates that the ΔZPhe residue is a more effective inducer of folded structures than the ΔEPhe residue. The values of the torsion angles ϕ and ψ show the presence of two type-III′ β-turns at the ΔZPhe residues and one type-II β-turn at the ΔEPhe residue. All amino acids are linked trans to each other in both peptides. β-Turns present in the peptides are stabilized by intramolecular 4→1 hydrogen bonds. Molecules in both structures form two-dimensional hydrogen-bond networks parallel to the (100) plane.
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