Abstract
The human asialoglycoprotein receptor (ASGP-R) is a membrane glycoprotein of 46,000 Da which possesses two N-linked oligosaccharide chains (Schwartz, A. L., and Rup, D. (1983) J. Biol. Chem. 258, 11249-11255). In order to examine the role of N-linked oligosaccharides in the biosynthesis, intracellular routing, and function of the ASGP-R, we have used Hep G2 cells, which have a large number of ASGP-R, and two inhibitors of glycosylation, swainsonine and tunicamycin. In the presence of swainsonine, newly synthesized ASGP-R is a 43,000-Da species which is endoglycosidase H-sensitive, appears on the Hep G2 cell surface, and specifically binds 125I-asialoorosomucoid (ASOR). In the presence of tunicamycin newly synthesized ASGP-R is a 34,000-Da nonglycosylated species which appears on the Hep G2 cell surface where it specifically binds 125I-ASOR. There is no major effect on subsequent uptake and degradation of 125I-ASOR in cells whose ASGP-R was synthesized in the presence of tunicamycin. The turnover of ASGP-R synthesized in the presence of either swainsonine or tunicamycin is not significantly altered from that found for the normal 46,000-Da species. Thus, it appears that the two N-linked oligosaccharide chains of the human ASGP-R do not play a major role in the intracellular routing, turnover, or function of ASGP-R.
Highlights
Plasma glycoproteins whose terminal sialic acid has been examined by the use of inhibitors of glycosylation (20, 22) as removed, exposing the penultimate galactose residues, well as by examination of cells exhibiting mutations in the are cleared from the circulatiobny liverparenchy- steps of oligosaccharide processing (24, 25)
Thisis accomplishedvia receptor-mediated role of the two N-linked oligosaccharide chains in the biosynendocytosis (1).The receptor is the hepatic ASGP-R1 and it thesis,intracellulartransport,routingtothe cell surface, is specific for galactose-terminal oligosaccharides of glycopro- turnover, and functionof the human asialoglycoprotein receptor (ASGP-R),in the HepG2 hepatoma cell line we have used the inhibitors of glycosyla
Swainsonine blocks the processing of glycoproteins containingN-linked complex oligosaccharides atthestep catalyzed by Golgi mannosidase I1 (26) resulting in the formation of hybrid oligosaccharide chains (27)
Summary
Since it is known that the ASGP-R is a glycoprotein conabc d e fghi taining N-linked oligosaccharides (4), inhibitors of glycosylation were utilized toassessthe role thattheN-linked oligosaccharides might play in ASGP-R biosynthesis, turn-. Functional measurementscould oligosaccharide side chains on ASGP-R function was examthen be made a t a time when essentially all of the receptors ined by measuring specific binding, uptake, and degradation present were synthesized in the presence or absence of the of "'1-ASOR in cells preincubated with and without tunicaappropriatedrug. Oligosaccharideside chains of the human ASGP-R do not the absenceof N-linked oligosaccharide side chains does not play a major role in ASGP-R intracellular transport, turnover, significantly alter ASGP-R targetintgo the cell surface, turn- ligand binding, or receptor-mediated endocytosis. Factors thamt ay determine thebiologic a major role in the routing to the cell surface or function of importance of the oligosaccharide side chains for a particular the human ASGP-R, these findings cannotbe generalized to glycoprotein include the extent of glycosylation, the primary other glycoproteins.
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