Use of a mutant cell line to study the kinetics and function of O-linked glycosylation of low density lipoprotein receptors.
A rapidly reversible defect in protein O-glycosylation exhibited by a line of mutant Chinese hamster ovary (CHO) cells was used to study the kinetics and function of O-glycosylation of the low density lipoprotein (LDL) receptor. The mutant line, genotype LDLD, cannot synthesize UDP-N-acetylgalactosamine under normal culture conditions and, therefore, cannot add mucin-type O-linked oligosaccharides to proteins. The UDP-N-acetylgalactosamine pools in LDLD cells can be filled rapidly when N-acetylgalactosamine is added to the culture medium, thus restoring normal synthesis of O-linked carbohydrates. Pulse-chase metabolic labeling experiments were used to show that (i) the first step in the O-glycosylation of LDL receptors can occur posttranslationally; (ii) after O-linked sugar-deficient LDL receptors reach the cell surface, they are not subject to subsequent O-linked sugar addition, suggesting that they do not return to compartments in which O-glycosylation takes place; (iii) O-linked carbohydrate chains on the LDL receptor itself are required for normal stability and function; and (iv) the instability of the O-linked sugar-deficient LDL receptor is due to proteolytic cleavage and the release into the medium of the bulk of the NH2-terminal extracellular domain of the receptor. It appears that O-glycosylation of the LDL receptor and several other cell surface glycoproteins permits stable cell-surface expression by preventing proteolytic cleavage of the extracellular domains of these proteins.
Kozarsky
K
K
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Kingsley
D
D
Krieger
M
M
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011499:Protein Processing, Post-Translational; D011973:Receptors, LDL / Q000235:genetics* / Q000378:metabolism
D008562:Membrane Glycoproteins; D011973:Receptors, LDL