Biosynthesis of the major human red cell sialoglycoprotein, glycophorin A. O-Glycosylation.
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.
D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006021:Glycophorins / Q000096:biosynthesis* / Q000302:isolation & purification; D006026:Glycoside Hydrolases; D006027:Glycosides / Q000378:metabolism; D006801:Humans; D007951:Leukemia, Myeloid; D008715:Methionine / Q000097:blood; D012795:Sialoglycoproteins / Q000096:biosynthesis*; D013462:Sulfur Radioisotopes
D006021:Glycophorins; D006027:Glycosides; D012795:Sialoglycoproteins; D013462:Sulfur Radioisotopes; D008715:Methionine; D006026:Glycoside Hydrolases