A monoclonal antibody (mAb-233) directed against an epitope in the nonglycosylated carboxyl-terminal region of human erythrocyte glycophorin A (GPA) was used in combination with metabolic labeling, the modification of N- and O-linked oligosaccharide processing by tunicamycin and monensin, and digestions with neuraminidase and O-glycanase to elucidate the pathway of GPA biogenesis in K562 human erythroleukemia cells. Cell-surface GPA is derived from two obligatory precursors in a stepwise manner. The initial GPA precursor has a M(r) of 27,000 and appears to contain one N-linked high mannose oligosaccharide chain. In tunicamycin-treated cells, the initial precursor is similar in size (M(r) = 24,000) to deglycosylated GPA from human erythrocytes. The 27-kDa initial precursor is rapidly converted to a transient 31-kDa intermediate by the addition of N-acetylgalactosamine residues to serine/threonine hydroxyl groups. Subsequent maturation involves the conversion of the high mannose chain to a complex-type oligosaccharide and the concomitant addition of galactose and sialic acid to internal N-acetylgalactosamine residues to extend the O-linked chains. These results define a single, stepwise processing pathway for the generation of all cell-surface GPA molecules and document for the first time the occurrence of both a unique initial precursor that contains a high mannose N-linked oligosaccharide chain but no O-linked sugars and a transient intermediate that appears to contain the same N-linked group and N-acetylgalactosamine at multiple serine/threonine residues. The properties of the intracellular GPA precursors and the relatively simple nature of the processing pathway reported herein contrast markedly with the characteristics of three intermediates and the complexity of two independent pathways in previously postulated schemes for GPA biogenesis (Gahmberg, C.G., Jokinen, M., Karhi, K.K., Kampe, O., Peterson, P.A., and Andersson, L.C. (1983) Methods Enzymol. 96, 281-298; Jokinen, M., Andersson, L.C., and Gahmberg, C.G. (1985) J. Biol. Chem. 260, 11314-11321).
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|State||Published - 1987|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology