Physical and chemical characteristics of apolipoprotein A-I-lipid complexes produced by Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene.
Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene linked to the human metallothionein gene promoter region secrete large quantities of apolipoprotein A-I (7.1 +/- 0.4% total secreted protein) in the presence of zinc. Approx. 16% of the secreted apolipoprotein A-I is complexed with lipid and can be isolated ultracentrifugally at d less than or equal to 1.21 g/ml. The latter complexes are composed of discs and vesicles as judged by electron microscopy and can be further separated by column chromatography into three fractions: fraction I, mostly vesicles (60-260 nm) and large discs (18-20 nm diameter); fraction II, discs 14.2 +/- 2.6 nm diameter; and fraction III, nonresolvable by electron microscopy. The latter fraction is extremely lipid-poor (94% protein, 6% phospholipid); in contrast, the protein, phospholipid and unesterified cholesterol content for the other fractions are 43, 33 and 24%, respectively, for fraction I and 53, 33 and 14%, respectively, for fraction II. Fraction II particles contain three and four apolipoprotein A-Is per particle as determined by protein crosslinking while large structures in fraction I contain primarily six to seven apolipoprotein A-Is per particle. Following incubation with purified lecithin: cholesterol acyltransferase, discoidal particles were transformed into apparent spherical particles 12.9 +/- 3.4 nm diameter; this transformation coincided with 19-21% conversion of unesterified cholesterol to esterified cholesterol. The apolipoprotein A-I-lipid complexes isolated from Chinese hamster ovary cell media are similar to nascent HDL found in plasma of lecithin:cholesterol acyltransferase-deficient patients and those secreted by the human hepatoma line, Hep G2. The ability of the Chinese hamster ovary cell nascent HDL-like particles to undergo transformation in the presence of purified lecithin:cholesterol acyltransferase indicates that they are functional particles.
Forte
T M
TM
Lawrence Berkeley Laboratory, University of California 94702.
McCall
M R
MR
Amacher
S
S
Nordhausen
R W
RW
Vigne
J L
JL
Mallory
J B
JB
D000818:Animals; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002784:Cholesterol / Q000032:analysis; D002845:Chromatography; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D046911:Macromolecular Substances; D008668:Metallothionein / Q000235:genetics; D008854:Microscopy, Electron; D010053:Ovary; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism; D010743:Phospholipids / Q000032:analysis; D011401:Promoter Regions, Genetic / Q000235:genetics; D011506:Proteins / Q000032:analysis; D014162:Transfection; D014461:Ultracentrifugation
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D008055:Lipids; D046911:Macromolecular Substances; D010743:Phospholipids; D011506:Proteins; D008668:Metallothionein; D002784:Cholesterol; D007862:Phosphatidylcholine-Sterol O-Acyltransferase