Temporal aspects of O-glycosylation and cell surface expression of ascites sialoglycoprotein-1, the major cell surface sialomucin of 13762 mammary ascites tumor cells

We have investigated the biosynthesis and cell surface expression of the major cell surface sialomucin (ascites sialoglycoprotein-1 (ASGP-1] of 13762 rat mammary ascites tumor cells by pulse or pulse-chase metabolic labeling combined with precipitation with peanut agglutinin and alkaline borohydride elimination or proteolytic fragmentation. The minimum time for initial glycosylation was estimated from the time required for the protein to acquire the ability to bind to peanut agglutinin to be less than 5 min. Moreover, when cells were labeled with threonine for 5 min and the ASGP-1 isolated by peanut agglutinin precipitation, 3% of the labeled threonine could be converted to 2-aminobutyric acid by alkaline borohydride elimination of the carbohydrate, indicating that at least 3% of the threonines of ASGP-1 are O-glycosylated within 5 min of polypeptide synthesis. The minimum time between the final glycosylation reactions in the cell and appearance of ASGP-1 at the cell surface was determined by trypsinizing galactose- or glucosamine-labeled cells at timed intervals after labeling to occur within 5-10 min of labeling. Both labeled glucosamine and galactosamine appeared in ASGP-1 fragments within 5 min, but the amount of labeled galactosamine was less than the amount of labeled glucosamine until after 20 min, when the 1:1 equilibrium ratio was reached. The half-time for appearance of glucosamine-labeled ASGP-1 at the cell surface was found to be greater than 4 h. The minimum time required from synthesis of the ASGP-1 polypeptide to appearance at the cell surface was determined by leucine labeling and proteolysis to be 70-80 min. These combined studies suggest a continuum of O-linked oligosaccharide initiation events extending over most of the period of ASGP-1 biosynthesis and transit from the endoplasmic reticulum to the cell surface.