Biosynthetic maturation of an ascites tumor cell surface sialomucin. Evidence for O-glycosylation of cell surface glycoprotein by the addition of new oligosaccharides during recycling.
Previous biosynthetic studies of the ascites 13762 rat mammary adenocarcinoma cell surface sialomucin ASGP-1 (ascites sialoglycoprotein-1) showed that it is synthesized initially as a poorly glycosylated immature form, which is converted to a larger premature form (t1/2 30 min) and more slowly to the mature glycoprotein (t1/2 greater than 4 h). In the present study O-glycosylation of ASGP-1 polypeptide is shown to occur in two phases: an early phase complete in less than 30 min, which corresponds to the synthesis of the premature form, and a later phase that continues for hours and corresponds to the synthesis of the mature form. Pulse-chase labeling studies indicate that 95% of the ASGP-1 has moved to the cell surface in 2 h. Since transit to the cell surface is faster than the slow phase of addition of new oligosaccharides, some new oligosaccharides must be added after ASGP-1 has reached the cell surface. Initiation of new oligosaccharides on cell surface ASGP-1 was demonstrated directly using a biotinylation procedure to identify cell surface molecules. Glucosamine labeling of biotinylated ASGP-1 was shown to occur on galactosamine residues, which are linked to the polypeptide, establishing the addition of new oligosaccharides to the cell surface molecules. Finally, resialylation studies indicate that ASGP-1 rapidly recycles through a sialylating compartment. From these results we propose that ASGP-1 reaches the cell surface in an incompletely glycosylated state and that additional oligosaccharides are added to the glycoprotein in a second process involving recycling.
Hull
S R
SR
Department of Cell Biology and Anatomy, University of Miami School of Medicine, Florida 33101.
Sugarman
E D
ED
Spielman
J
J
Carraway
K L
KL
D000230:Adenocarcinoma / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism; D001201:Ascites; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D006031:Glycosylation; D008325:Mammary Neoplasms, Experimental / Q000276:immunology / Q000378:metabolism*; D055264:Mucin-4; D009363:Neoplasm Proteins / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D012795:Sialoglycoproteins / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism
D000951:Antigens, Neoplasm; D055264:Mucin-4; D009363:Neoplasm Proteins; D009844:Oligosaccharides; D012795:Sialoglycoproteins; D013912:Threonine