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.