Mutational analysis of the N-glycosylation sites of Duffy antigen/receptor for chemokines.
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.
Czerwinski
Marcin
M
Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wroclaw, Poland.
Kern
Joanna
J
Grodecka
Magdalena
M
Paprocka
Maria
M
Krop-Watorek
Anna
A
Wasniowska
Kazimiera
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004252:DNA Mutational Analysis; D004375:Duffy Blood-Group System / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D020014:K562 Cells; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics* / Q000276:immunology
C473745:ACKR1 protein, human; D000911:Antibodies, Monoclonal; D004375:Duffy Blood-Group System; D011956:Receptors, Cell Surface