Mutagenesis of the glycosylation site of human ApoCIII. O-linked glycosylation is not required for ApoCIII secretion and lipid binding.
We have used site-directed in vitro mutagenesis to alter the codon ACT of human apoCIII gene, specifying Thr-74, to GCT (Ala-74). The normal and mutant apoCIII genes were then placed under the control of the mouse metallothionein 1 promoter in a bovine papilloma virus vector and were used for cell transfection and selection of stable cell lines. Blotting analysis of RNA isolated from several independent cell clones showed that both the normal and mutant genes produced apoCIII mRNA in amounts larger than that found in human fetal liver. Pulse-chase analysis of cell clones expressing the normal and mutant apoCIII genes showed that only the normal apoCIII is modified intracellularly to produce a disialated form (apoCIIIs2). Cell clones expressing the normal apoCIII gene secrete exclusively the disialated form, whereas those expressing the mutant gene secrete the unmodified form. The amount of mutant apoCIII protein produced by C127 cell clones expressing the mutant gene was reduced as compared to that produced by the control cells. Density gradient ultracentrifugation analysis of the secreted apoCIII showed that the flotation properties of the secreted normal and mutant proteins were similar. These findings suggest that the intracellular glycosylation of apoCIII is not required for its intracellular transport and secretion. Furthermore, lack of glycosylation has no effect on the relative affinities of apoCIII for plasma very low density lipoproteins and high density lipoproteins.
Roghani
A
A
Department of Medicine, Boston University Medical Center, Massachusetts 02118.
Zannis
V I
VI
D000818:Animals; D053305:Apolipoprotein C-III; D001056:Apolipoproteins C / Q000235:genetics* / Q000378:metabolism; D015152:Blotting, Northern; D002460:Cell Line; D005796:Genes; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D009154:Mutation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection
D053305:Apolipoprotein C-III; D001056:Apolipoproteins C; D012333:RNA, Messenger; D008668:Metallothionein