The major human red cell sialoglycoprotein, glycophorin A, is structurally one of the best known membrane proteins. Until recently, however, nothing was known about its biosynthesis. We found in 1979 that the K562 cell line is erythroid and synthesizes glycophorin A. Glycophorin A mRNA isolated from K562 cells directed in vitro the synthesis of a protein with an apparent molecular weight of 19500. That exceeded the molecular weight of the apoprotein with 5000, indicating the presence of a signal peptide. Glycophorin A synthesized in vitro in the presence of dog pancreatic membranes had an apparent molecular weight of 37000. This precursor bound to lentil lectin but not to Helix pomatia lectin. Pulse-chase experiments in vivo showed that initially a 37000 molecular weight protein, which bound to lentil lectin, was synthesized which gradually was replaced by a 39000 molecular weight protein. Glycosylation was completed in about 10 min and the protein appeared at the cell surface after 25 min. Inhibition of glycosylation of the single K-glycosidic oligosaccharide ty tunicamycin did not affect the migration of the protein to the cell surface. Using the N-acetylgalactosamine specific lectin from Helix pomatia coupled to Sepharose two early N-acety]galactosamine-containing O-giycosylated glycophorin A precursors with apparent molecular weights of 24000 and 27000 were identified. O-glycosylation took place in the presence of carboxylic ionophore monensin, but the protein stained intracellularly. The relationship between the 37000 and 24000–27000 molecular weight proteins remains unclear but the results indicate that the synthesis of O-glycosylated glycoproteins is more complex than generally believed and may involve early uncharacterized precursors.
KeywordsSerine Polypeptide Trypsin Methionine Oligosaccharide
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