Analysis of recombinant Duffy protein-linked N-glycans using lectins and glycosidases.
Duffy antigen is a glycosylated blood group protein acting as a malarial and chemokine receptor. Using glycosylation mutants we have previously demonstrated, that all three potential glycosylation sites of the Duffy antigen are occupied by N-linked oligosaccharide chains. In this study, wild-type Duffy glycoprotein and three mutants, each containing a single N-glycan, were used to characterize the oligosaccharide chains by lectin blotting and endoglycosidase digestion. The positive reaction of all the recombinant Duffy forms with Datura stramonium and Sambucus nigra lectins showed that each Duffy N-linked glycan contains Galbeta1-4GlcNAc units terminated by (alpha2-6)-linked sialic acid residues, typical of complex oligosaccharides. The reactivity with Aleuria aurantia and Lens culinaris lectins suggested the presence of (alpha1-6)-linked fucose at the N-glycan chitobiose core. The failure of the Galanthus nivalis and Canavalia ensiformis lectins to bind to any of the Duffy mutants or to the wild-type antigen indicated that none of the three Duffy N-glycosylation sites carries detectable levels of high-mannose oligosaccharide chains. Digestion of Duffy samples with peptide N-glycosidase F and endoglycosidase H confirmed the presence of N-linked complex oligosaccharides. Our results indicate that Duffy antigen N-glycans are mostly core-fucosylated complex type oligosaccharides rich in N-acetyllactosamine and terminated by (alpha2-6)-linked sialic acid residues.
Grodecka
Magdalena
M
Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.
Czerwiński
Marcin
M
Duk
Maria
M
Lisowska
Elwira
E
Waśniowska
Kazimiera
K
D001203:Ascomycota / Q000378:metabolism; D004375:Duffy Blood-Group System / Q000032:analysis* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D037102:Lectins / Q000378:metabolism*; D037121:Plant Lectins / Q000378:metabolism*; D010944:Plants / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism
D004375:Duffy Blood-Group System; D037102:Lectins; D037121:Plant Lectins; D011134:Polysaccharides; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C064549:lectin, Aleuria aurantia; D006026:Glycoside Hydrolases