Expression of ApoE gene in Chinese hamster cells with a reversible defect in O-glycosylation. Glycosylation is not required for apoE secretion.
The effects of O-glycosylation on the synthesis and secretion of apolipoprotein E (apoE, a glycoprotein with O- but not N-linked sugars) were studied with a UDP-galactose/UDP-N-acetylgalactosamine 4-epimerase-deficient cell mutant (ldlD cells) which expresses a reversible defect in protein O-glycosylation. Under normal culture conditions the mutant ldlD cells cannot add N-acetylgalactosamine (GalNAc) to proteins. GalNAc is the first sugar of mucin-type O-linked oligosaccharides attached to the protein. This O-glycosylation defect is rapidly corrected when GalNAc is added to the culture medium. These cells also require external sources of galactose for the addition of this sugar to O-linked and other oligosaccharides. A bovine papilloma virus-based expression vector for human apoE and the human metallothionein 1A gene were transfected into ldlD cells, and apoE-expressing cell clones resistant to CdCl2 were selected and used in the present studies. The structure and secretion of apoE in these cells were examined by immunoprecipitation and one- and two-dimensional gel electrophoresis and autoradiography. The synthesis, rate, and extent of secretion of apoE were unaffected by O-glycosylation (GalNAc-independent). In the presence of both galactose and GalNAc, multiple apoE isoforms were synthesized in ldlD cells as a result of variation in the extent of sialylation. ApoE sialylation was dependent on the addition of galactose as well as GalNAc to the extracellular medium, suggesting that addition of galactose to the nascent oligosaccharide chains was required for the addition of sialic acid.
Zanni
E E
EE
Department of Medicine, Boston University Medical Center, Massachusetts 02118.
Kouvatsi
A
A
Hadzopoulou-Cladaras
M
M
Krieger
M
M
Zannis
V I
VI
D000116:Acetylgalactosamine; D000818:Animals; D001057:Apolipoproteins E / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005690:Galactose / Q000502:physiology; D005700:Galactosyltransferases / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010053:Ovary; D014162:Transfection
D001057:Apolipoproteins E; D005700:Galactosyltransferases; D000116:Acetylgalactosamine; D005690:Galactose