Identification of a neutral lipid core in a transiently expressed and secreted lipoprotein containing an apoB-48-like apolipoprotein.
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 (apoB-100) cDNA (nucleotides 1 to 6860, where nucleotide 129 is the start of translation). Northern blots of cellular message probed with apoB-48 showed abundant transcription of an apoB-48-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 apolipoprotein. This lipoprotein banded at a density of 1.11 g/ml in isopycnic NaBr gradients. Electron microscopy of the apoB-48-containing lipoproteins demonstrated spherical particles with an average diameter of 124A. A sedimentation rate of 8.4S was measured by sucrose gradient sedimentation. When the apoB-48-containing 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 particle 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. These data indicate that transfected HepG2 cells assembled and secreted lipoproteins possessing the same physical structure as naturally occurring lipoproteins.
Spring
D J
DJ
Department of Chemistry and Biochemistry, University of California, Los Angeles.
Lee
S M
SM
Puppione
D L
DL
Phillips
M
M
Elovson
J
J
Schumaker
V N
VN
D053283:Apolipoprotein B-48; D001055:Apolipoproteins B / Q000737:chemistry* / Q000235:genetics; D015152:Blotting, Northern; D002861:Chromobacterium / Q000201:enzymology; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008049:Lipase / Q000378:metabolism; D008055:Lipids / Q000737:chemistry*; D008074:Lipoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D010957:Plasmids; D011233:Precipitin Tests; D014407:Tumor Cells, Cultured; D014461:Ultracentrifugation
D053283:Apolipoprotein B-48; D001055:Apolipoproteins B; D008055:Lipids; D008074:Lipoproteins; D008049:Lipase