Abstract
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.
Highlights
From the Section of Molecular Genetics, Cardiovascular Institute, Departments of Medicine and Biochemistry, Boston University Medical Center, Boston, Massachusetts 021I8
Thr-74,to GCT (Ala-74).Thenormalandmutant circulation [12, 13].Inaddition,ithas beenshown that apoCIII genes were placed under the control of lipoprotein uptake by hepatic tissues is stimulated by apoE
Theamount of mutant Our data show that apoCIII ismodified intracellularly with a apoCIII protein producedby C127 cell clones express- carbohydratechaincontaining 2 sialicacidresidues and is secretedexclusively in this form
Summary
That the intracellular glycosylation of apoCIII is not required for its intracellular transport andsecretion. Lackof glycosylation has no effect onthe Restriction enzymes were purchasedeitherfrom New England relative affinitiesof apoCIII for plasma very low den- Biolabs orBethesdaResearchLaboratories(BRL). The apoCIII, apoAI, and apoA-IV genes are closely linked. [35S]Methionine(1200 Ci/mmol), [Y-~’P]ATP(3000 Ci/ mmol), and [(u-~’P]~CT(P3000 Ci/mmol) were purchased from Du Pont-NewEnglandNuclear. Dulbecco’s modified Eagle’s medium (DMEM) and Dulbecco’s Eagle’s medium without methionine were purchasedfrom GIBCO.Calciumchloride (analar) was purchased from BritishDrugHouse(GallardSchlessinger). (7) and have been mapped on ltohneg arm of chromosome 11 quired for two-dimensional polyacrylamide gel electrophoresis have [8].In humans and mammalian species, the sites of apoCIII gene expression are theliver and the intestine[9,10,11]. Goat anti-human apoCIII was a gift from Dr. PeterHerbert, BrownUniversity,Providence, RI. This research was performed a t Housman Medical ResearchCenter of BostonUniversity Medical
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