Mutational analysis of the N-glycosylation sites of Duffy antigen/receptor for chemokines |
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Authors: | Czerwinski Marcin Kern Joanna Grodecka Magdalena Paprocka Maria Krop-Watorek Anna Wasniowska Kazimiera |
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Affiliation: | Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wroclaw, Poland. |
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Abstract: | 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. |
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Keywords: | Duffy antigen N-Glycosylation Chemokine receptor |
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