Mutational analysis of the N-glycosylation sites of Duffy antigen/receptor for chemokines

• Published in: Biochemical and biophysical research communications Vol. 356; no. 3; pp. 816 - 821
• Main Authors: Czerwinski, Marcin, Kern, Joanna, Grodecka, Magdalena, Paprocka, Maria, Krop-Watorek, Anna, Wasniowska, Kazimiera
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11.05.2007
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Antibodies, Monoclonal; Blotting, Western; Chemokine receptor; Chromatography, Affinity; DNA Mutational Analysis; Duffy antigen; Duffy Blood-Group System - chemistry; Duffy Blood-Group System - genetics; Duffy Blood-Group System - immunology; Electrophoresis, Polyacrylamide Gel; Glycosylation; Humans; K562 Cells; Molecular Sequence Data; N-Glycosylation; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - genetics; Receptors, Cell Surface - immunology; N-Glycosylation; Duffy antigen; Chemokine receptor
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2007.03.054

The Duffy antigen/receptor for chemokines (DARC) is a seven-transmembrane glycoprotein carrying the Duffy (Fy) blood group antigen. The polypeptide chain of DARC contains two NSS motifs at positions 16 and 27 and one NDS motif at position 33 that represent canonical sequences for efficient N-glycosylation. To verify whether all of these three sites are occupied by a sugar chain, we generated mutants in which potential N-glycosylation sites (AsnXSer) were removed by replacement of serine by alanine. Seven DARC glycosylation variants, missing one (S18A, S29A, S35A), two (S18A.S29A, S18A.S35A, S29A.S35A), or three (S18A.S29A.S35A) glycosylation sites, were obtained. cDNA encoding DARC mutants was cloned into the eukaryotic expression vector pcDNA3.1/myc-HisA and expressed in human K562 cells. Stable transfectants expressing wild-type or mutated forms of Duffy were then lysed, purified by metal-affinity chromatography, and subjected to Western blots with an anti-Duffy monoclonal antibody. The gel electrophoresis data indicate that all three canonical sites are used for sugar attachment.