Effects of O-linked glycosylation on the cell surface expression and stability of decay-accelerating factor, a glycophospholipid-anchored membrane protein.
Decay accelerating factor (DAF) is a glycophospholipid-anchored membrane glycoprotein that protects mammalian host cells from inadvertant complement lysis. The effects of inhibiting mucin-type O-glycosylation on the cell surface expression of DAF were studied by introducing an expression vector for human DAF into wild-type Chinese hamster ovary and ldlD cells. The ldlD cells express reversible defects in the addition of galactose and N-acetylgalactosamine (GalNAc) to oligosaccharide chains on glycoproteins and glycolipids. Mucin-type O-glycosylation of proteins is inhibited in ldlD cells and can be selectively corrected by the addition of GalNAc to the culture medium. The attachment of a phosphatidylinositol phospholipase C-sensitive glycolipid anchor to DAF and its efficient sorting to the cell surface in ldlD cells were independent of galactose and GalNAc additions to glycolipids and proteins. Attachment of galactose and GalNAc to DAF's glycolipid anchor were apparently not required for its normal function. However, in the absence of O-glycosylation DAF was proteolytically cleaved soon after reaching the cell surface, and a large fragment of DAF was released into the culture medium. This rapid proteolysis/release resulted in the expression of very low steady state levels of O-glycosylation-deficient DAF as measured by immunoblotting. These results, in conjunction with those obtained from studies of three other membrane glycoproteins expressed in ldlD cells, suggest that O-linked sugars on membrane glycoproteins may frequently play a role in determining the level of cell surface expression of these proteins.
Reddy
P
P
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Caras
I
I
Krieger
M
M
D000116:Acetylgalactosamine / Q000378:metabolism; D000818:Animals; D018958:CD55 Antigens; D002236:Carbohydrate Conformation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology*; D006017:Glycolipids / Q000378:metabolism*; D006031:Glycosylation; D006168:Guinea Pigs; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D013329:Structure-Activity Relationship; D014162:Transfection
D018958:CD55 Antigens; D006017:Glycolipids; D008565:Membrane Proteins; D000116:Acetylgalactosamine; D005690:Galactose