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The biosynthesis of the major human red cell sialoglycoprotein, glycophorin A, was studied in the erythroleukemia cell line K562 with emphasis on O-glycosylation. The cells were pulse-chase labeled with [35S] methionine, and either directly immune precipitated with anti-glycophorin A antiserum or detergent-solubilized extracts first passed through columns containing the N-acetylgalactosamine-specific lectin from Helix pomatia or the glucose/mannose specific lectin from lentil beans. From the sugar-eluted fractions anti-glycophorin A antiserum was used to identify precursor molecules. After 5 min of labeling the first glycophorin A precursors were seen. The largest had an apparent molecular weight of 37,000, and bound to lentil lectin-Sepharose, but not to H. pomatia lectin-Sepharose. The lentil lectin-reactive glycophorin A molecules increased to Mr = 39,000 during chase and obtained sialic acids after 9 min of chase reflecting terminal N- and O-glycosylation. After 5-6 min of labeling two H. pomatia-interacting glycophorin A precursors with apparent molecular weights of 24,000 and 30,000 were obtained. These did not bind to lentil lectin-Sepharose. During chase also these molecules increased in size to Mr = 39,000. The immune precipitation of all antiglycophorin A-reactive precursor molecules was inhibited by purified red cell glycophorin A. The carboxylic ionophore, monensin, caused the accumulation of incompletely O-glycosylated glycophorin A molecules, which bound to H. pomatia lectin-Sepharose. These were degraded by treatment with endo-beta-N-acetylglucosaminidase H reflecting incomplete processing of the N-glycosidic oligosaccharide.

The human continuous leukemia cell line K562 synthesizes and expresses on its surface the major red cell sialoglycoprotein, glycophorin A. Glycophorin A contains 1 N-glycosidic and 15 O-glycosidic oligosaccharides, which are attached to known sites on the polypeptide chain. By immune precipitation with specific anti-glycophorin A antiserum of radioactively labeled cells followed by polyacrylamide gel electrophoresis, we have been able to study its biosynthesis in considerable detail. The synthesis of the N-glycosidic oligosaccharide of glycophorin A is inhibited by the antibiotic tunicamycin, while the O-glycosidic oligosaccharides are not affected. Incomplete glycophorin A, lacking the N-glycosidic oligosaccharide, is apparently incorporated normally into the surface membrane, but the total amount of glycophorin A is decreased. Thus, N-glycosylation is not necessary for externalization of glycophorin A.

We have found that the human leukemic cell line K562 (Lozzio, C.B., and Lozzio, B.B. (1975) Blood 45, 321-334) synthesizes a surface membrane glycoprotein which is identical or closely similar to the major red cell sialoglycoprotein, glycophorin A. The protein can be precipitated by specific anti-glycophorin A antiserum both from surface-labeled and metabolically labeled K562 cells. Cyanogen bromide cleavage of glycophorin A from red cells and the K562 cell protein gives apparently identical fragments, and the glycopeptides and oligosaccharides obtained after Pronase and mild alkaline treatment are closely similar. An antiserum made against intact K562 cells and absorbed with normal human white blood cells precipitated surface-labeled glycophorin A from erythrocytes. The amount of glycophorin A per cell in erythrocytes and K562 cells was very similar when determined by radioimmunoassay. The K562 cells contained blood group MN activity when tested with rabbit anti-M and anti-N sera. When incubated at 37 degrees C with rabbit anti-glycophorin A F(AB)2 fragments and fluorescent sheep anti-rabbit IgG, partial redistribution of glycophorin A (patching and capping) was seen in K562 cells but not in erythrocytes.