Glycosylation of human apolipoprotein E. The carbohydrate attachment site is threonine 194.
The glycosylation of human apolipoprotein (apo) E was examined with purified plasma apoE and apoE produced by transfected cell lines. The carbohydrate attachment site of plasma apoE was localized to a single tryptic peptide (residues 192-206). Sequence analysis and amino sugar analysis of this peptide derived from asialo-, monosialo-, or disialo-apoE indicated that the carbohydrate moiety is attached only to Thr194 in monosialo- and disialo-apoE and that asialo-apoE is not glycosylated. Mammalian cells that normally do not express apoE were transfected with human apoE plasmid expression vectors to test the utilization of potential carbohydrate attachment sites and the role of apoE glycosylation in secretion. Site-specific mutants of apoE, designed to eliminate or alter glycosylation sites, were expressed in HeLa cells by acute transfection. Apolipoprotein E(Thr194----Ala) was secreted exclusively as the asialo isoform, confirming that Thr194 is the site of carbohydrate attachment in these cells and indicating that glycosylation of apoE is not essential for secretion. Apolipoprotein E(Thr194----Asn,Gly196----Ser), which introduces a potential site for N-glycosylation at position 194, was secreted with a higher apparent molecular weight than native, O-glycosylated apoE. Studies with tunicamycin indicated that this apoE was N-glycosylated at Asn194. Stably transfected cell lines expressing human apoE were prepared from wild-type Chinese hamster ovary (CHO) cells and from CHO ldlD cells, which are defective in glycosylation. The transfected wild-type cells secreted multiply sialylated apoE. The transfected ldlD cells also secreted high levels of apoE even in the absence of glycosylation, which confirms that glycosylation is not essential for secretion of apoE.
Wernette-Hammond
M E
ME
Gladstone Foundation Laboratories for Cardiovascular Disease, San Francisco, California 94140-0608.
Lauer
S J
SJ
Corsini
A
A
Walker
D
D
Taylor
J M
JM
Rall
S C
SC
D000818:Animals; D001057:Apolipoproteins E / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics; D002460:Cell Line; D005822:Genetic Vectors; D006031:Glycosylation; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D010957:Plasmids; D011487:Protein Conformation; D015183:Restriction Mapping; D013912:Threonine; D014162:Transfection
D001057:Apolipoproteins E; D013912:Threonine