Biogenesis of glycophorin A in K562 human erythroleukemia cells.

B Morrow,C S Rubin

doi:10.1016/s0021-9258(19)76498-9

Abstract

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 Mr 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 (Mr = 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).

Highlights

Immunoprecipitation of SolubilizedGPA from Metabolically LabeledCells-Membranes from 1 X lo7 [35S]methionine-labeledcells were resuspended in 0.5mlof mM Tris-HC1, pH 8, at 4 "C
The structural features of glycophorin A (GPA) indicate that this integral uration involves the conversion of the high mannose membrane protein[8]is translocated through the intracellular chain to a complex-type oligosaccharide and the concomitant addition of galactose and sialicacid to internal N-acetylgalactosamine residues to extend the 0linked chains
Will submit this work in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Sue Golding its progression from the rough endoplasmic reticulum to the plasma membrane canserve two relatedpurposes. Such studies will provide insights regarding the biogenesis of an erythrocyte-specific, plasma membrane protein that is functionally distinct from secreted hormonesr,eceptors, and viral

Summary

Immunoprecipitation of SolubilizedGPA from Metabolically Labeled

Cells-Membranes from 1 X lo7 [35S]methionine-labeledcells were resuspended in 0.5mlof mM Tris-HC1, pH 8, at 4 "C. Treatment of GPA with Neuraminidase, 0-Glycanase,and Endoglycosidase H-Washed immunoprecipitates containing GPA from 1 X lo7K562 cells (see above) were resuspended in 25 pl of 25 mM MES buffer, pH 6.0, containing 0.1% SDS and 1 mM calcium acetate and denatured by a 5-min incubation a t 100 "C. LectinAffinity Chromatography-Membranes from lo7 [35S]methionine-labeled K562 cells (see above) were extracted with 1.5 ml of 20 mM Tris-HCI, pH 8, containing 0.15 M NaCl and 0.8% Triton X-100 (buffer A) for 30 min at 0 "C. GPA precursors in the flow-through and thepeak of eluted radioactivity were immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography as described above. Incubations of the nitrocellulose blots with mAb-233, rabbit anti-mouse IgG, and 1251-proteinA and subsequent autoradiography were carried out as previously described [27]. Atkinson (Department of Developmental Biology and Cancer, Albert Einstein College of Medicine)

Glycophorin A Biogenesis

GlycophorinA Biogenesis

DISCUSSION