The human red blood cell sialoglycoprotein, glycophorin A (GpA), contains a 'mucin-like' extensively O-glycosylated extracellular domain which carries the MN blood group antigens. We have revised the sites of O-glycosylation in the extracellular domain of GpA by automated solid-phase Edman degradation, which allowed positive identification and quantitation of O-glycosylated Ser and Thr residues, as well as the single N-glycosylation site. One N-linked and 16 O-linked sites were identified. Carbohydrate was absent on Ser1, Ser14, Ser15, Ser23, Thr28 and Thr58 in GpA. We propose that the glycosyltransferases present in erythrocytes recognize specific flanking sequences around potential O-glycosylation sites. All 16 O-glycosylation sites are explained on the basis of four motifs. Three motifs are associated with Thr-glycosylation: Xaa-Pro-Xaa-Xaa where at least one Xaa = Thr; Thr-Xaa-Xaa-Xaa where at least one Xaa = Thr; Xaa-Xaa-Thr-Xaa where at least one X = Arg or Lys. The fourth motif is associated with Ser-glycosylation: Ser-Xaa-Xaa-Xaa where at least one Xaa = Ser. These simple rules explain the glycosylation (or lack of it) on 21 of 22 Ser/Thr in the extracellular domain of GpA.
