Identification of a neutral lipid core in a transiently expressed and secreted lipoprotein containing an apo

The presence of core lipids in lipoproteins expressed and secreted by transfected HepG2 cells was demonstrated by measuring the densities of these lipoproteins before and after treatment with a bacterial lipase specific for neutral lipids. HepG2 cells were reproducibly transfected with pRSV/B48, containing a truncated human apolipoprotein B-100 (apoB100) cDNA (nucleotides 1 to 6860, where nucleotide 129 is the start of translation). Northern blots of cellular message probed with apoB48 showed abundant transcription of an apoB48-sized message as well as endogenous apoB-100 message. When grown in the presence of [35S] methionine, pRSV/B48-transfected cells secreted lipoproteins containing an apoB-48-like a p e lipoprotein. This lipoprotein banded at a density of 1.1 1 g/ml in isopycnic NaBr gradients. Electron microscopy of the apoB-48-containing lipoproteins dempstrated spherical particles with an average diameter of 124A. A sedimentation rate of 8.4s was measured by sucrose gradient sedimentation. When the apoB4&ontaining particles were treated with a bacterial lipase (from Chromobacterium viscosum), shown to hydrolyze triglycerides and cholesteryl esters but not phospholipids, their density increased to 1.18 g/ml, consistent with removal of core lipids. When the secreted lipoprotein was modeled as a spherical partical containing a single molecule of apoB-48, a triglyceride-filled core, and a surface monolayer of phospholipid and protein, the hydrodynamic properties were consistent with the observed sedimentation coefficient, buoyant densities before and after lipase treatment, and the diameter as seen with the electron microscope. I1p These data indicate that transfected HepGZ cells assembled and secreted lipoproteins pos sessing the same physical structure as naturally occurring lipoproteins.-Spring, D.J., SM. Lee, D.L. Puppione, M. Phillips, J. Elovson, and V.N. Schumaker. Identification of a neutral lipid core in a transiently expressed and secreted lipoprotein containing an apoE48-like apolipoprotein. J. Lipid Res. 1992. 33: 233-240. Supplementary key words transfected HepG2 cells bacterial lipase Various lengths of apolipoprotein B (apoB) have been expressed recently in a variety of cell types, including COS cells, a rat hepatoma cell line ( M A RH7777), a mouse mammary cell line (C-127), and a human hepatoma cell line (HepG2) (1-3). In McARH7’777 cells, Yao, et al. (4) have expressed a series of carboxyl-terminally truncated forms of human apoB, and have shown that these are secreted as lipoproteins; moreover, when the logarithm of the length of apoB was plotted as a function of the buoyant density of the lipoprotein, an inverse, linear function was obtained. They have also shown that the size of the secreted particles increases with apoB length. These data, however, do not define the actual shape and lipid composition of the secreted particles, leaving open the question of whether these truncated apoB peptides have been assembled into spherical lipoproteins containing a nonpolar lipid core, or whether they are disk-shaped particles containing a phospholipid bilayer rimmed with apoB, similar to the apoB disk-shaped lipoproteins reported by Hadzopoulou-Cladaras et al. (2). In the present study, we demonstrate that transfected HepG2 cells secrete an apoM&ontaining particle of the same size and density as that found by Yao et al. (4) in transfected rat hepatoma cells. In addition, we demonstrate that this particle is spherical by electron microscopy, that it has the appropriate sedimentation coefficient for its size, and that it contains a core of hydrophobic lipids susceptible to hydrolysis by a bacterial lipase that hydrolyses triglycerides and cholesteryl esters, but not phospholipids. Abreviations: apoB, apolipoprotein B BHT, butylated hydroxytoluene; cDNA, complementary deoxyribonucleic acid; EDTA, ethylenediaminetetraacetic acid HEPES, n-2-hydroxyethylpiperazine-N’-Zethanesulfonic acid; LDL, low density lipoprotein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; rpm, revolutions per minute; rRNA, ribosomal ribonucleic acids; SDS, sodium dodecylsulfate; Tris, tris(hydroxymethy1)aminomethane; Tris-HC1, Tris hydrochloride; VLDL, very low density lipoprotein. ‘To whom correspondence should be addressed. Journal of Lipid Research Volume 33, 1992 233 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 0, 2013 w w w .j.org D ow nladed fom Together these data indicate that certain cell culture expression systems, utilizing transfected apoB cDNA, are capable of assembling and secreting lipoproteins that are structurally similar to naturally occurring lipoproteins. Enzymatic methods for the analysis of triglycerides, cholesterol, and cholesteryl esters are widely used for the determination of the lipid composition of purified plasma lipoproteins (5). In these assays, a nonionic detergent is added to the lipoprotein preparation presumably disrupting the lipoprotein structure and exposing the core lipids to the action of the enzymes used in the analysis. Here we report that the neutral lipids in the intact lipoproteins are readily accessible to the lipase from Chromobuctm’um viscosum in the absence of detergent. It is also shown that this enzyme cleaves both triglycerides and cholesteryl esters, but no measurable phospholipid. Because the secreted lipoprotein containing the apoB-48-like apolipoprotein has a density similar to the high density lipoproteins (HDL), i t is difficult to purify; therefore, colorimetric analyses performed after enzymatic hydrolysis would not distinguish between HDL neutral lipids and those present in the apoB containing particles. Here an alternate approach was used: measuring the density difference of the apoB-containing lipoproteins before and after lipase treatment demonstrated the presence of neutral lipids, which is the hallmark of the naturally occurring apoB-containing lipoproteins. EXPERIMENTAL PROCEDURES