title: Expression of human apolipoprotein E but not that of apolipoprotein A-I by mouse C127 cells is associated with increased secretion of lipids in the form of vesicles and discs.
abstract: Transfected mouse mammary-derived cells (C127) expressing human apolipoprotein (apo) E (C127E) were used a) to determine whether the lipid-binding character of apoE is sufficient to promote its assembly with lipid to form lipoprotein-like particles when expressed by cells not normally expressing apolipoproteins; b) to characterize the secreted complexes in terms of morphology, size, and composition; and finally c) to determine whether apoE or apoA-I gene expression by these transfected cells has any effect on the levels and the profiles of the synthesized and secreted lipids. The findings of the present study demonstrate that: a) as determined by density gradient ultracentrifugation and gel filtration chromatography, about 20% of the secreted [35S]methionine-labeled apoE expressed by C127E cells is lipid-associated. b) Negative-stain electron microscopic analysis of the lipid-protein complexes recovered in the lipoprotein fractions (d less than 1.21 g/ml) revealed that approximately 13% of the total population of particles were discs (16 +/- 5 nm mean diameter and 4-6 nm thick), resembling nascent high density lipoproteins (HDL). The majority of the particles however (greater than 82%) appeared vesicular with varying diameters (48 +/- 40 nm mean diameter). The discoidal and the vesicular appearance of the particles secreted by C127E cells is consistent with the composition of lipids. These consisted mostly of surface lipids, phospholipids (45 +/- 18%), diacylglycerols (36 +/- 17%), and free cholesterol (17 +/- 7%) (by weight). c) Expression of apoE by C127E cells was associated with an increased release of [35S]methionine-labeled protein and [3H]glycerol-labeled lipid (3- to 5- and 4- to 8-fold, respectively) compared to nontransfected C127 cells. Expression of mutant apoE or normal apoA-I, however, was not associated with increased release of the major lipid classes compared to the parent C127 cells, strongly suggesting that this character of C127E cells is specific to apoE expression. The release of lipids by C127E cells could be reduced considerably by the addition of the metabolic inhibitors, colchicine or cycloheximide (10 and 1 microM, respectively), suggesting that lipid release by C127E cells is an active process requiring both protein synthesis and functional secretory mechanisms. Taken together the findings suggest that apoE expression by C127 cells promotes the formation of nascent discoidal lipoprotein-like particles and enhances the release of vesicular lipids, possibly by promoting shedding of cell plasma membrane fragments.
authors.affiliation: Department of Biophysics, Boston University School of Medicine, Housman Medical Center, MA 02118-2394.
authors.forename: H
authors.initials: H
authors.lastname: Herscovitz
authors.forename: D
authors.initials: D
authors.lastname: Gantz
authors.forename: A M
authors.initials: AM
authors.lastname: Tercyak
authors.forename: V I
authors.initials: VI
authors.lastname: Zannis
authors.forename: D M
authors.initials: DM
authors.lastname: Small
mesh_terms: D000818:Animals; D016632:Apolipoprotein A-I / Q000096:biosynthesis / Q000378:metabolism*; D001057:Apolipoproteins E / Q000096:biosynthesis / Q000378:metabolism*; D002458:Cell Fractionation; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002999:Clone Cells; D003470:Culture Media / Q000737:chemistry; D006801:Humans; D008051:Lipid Bilayers; D050356:Lipid Metabolism; D046911:Macromolecular Substances; D008325:Mammary Neoplasms, Experimental; D051379:Mice; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D016632:Apolipoprotein A-I; D001057:Apolipoproteins E; D003470:Culture Media; D008051:Lipid Bilayers; D046911:Macromolecular Substances