11
Biochimica et Biophysicu Acta, 1047 (1990) 11-18 Elsevier
BBALJP
53491
Physical and chemical characteristics of apolipoprotein A-I-lipid complexes produced by Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene T.M. Forte I, M.R. McCall I, S. Amacher ‘, R.W. Nordhausen ‘, J.L. Vigne ’ and J.B. Mallory ’ ’ Luwrenee Berkeley ~bo~a~o~, Unioers~~ of Cul~orn~o, Berkeley and ’ Cul~or~ia 3io~ech~oio~ Inc., Mountain View, CA (U.S.A.~
(Revised
Key words:
Apo~poprotein
(Received 5 March 1990) manuscript received 12 June 1990)
A-I; Chinese hamster ovary cells; Apolipoprotein Lecithin : cholesterol acyltransferase
A-I gene transfection;
Nascent
HDL;
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 f 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 I 1.21 g/ml. The latter complexes are composed of discs and vesicles as judged by electron microscopy and can be further separated by cohumr chromatography into three fractions: fraction I, mostly vesicles (60-260 nm) and large discs (B-20 nm diameter); fraction II, discs 14.2 f 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 dete~in~ by protein crossli~ing while large stnuztures 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 f 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 acyltransfear-deficient patients and those secreted by the human hepatoma line, Hep G2. The ability of the Chinese hamster ovary cell nascent HDLlike particles to undergo ~~sfo~ation in the presence of purified 1~ithin:cholesterol aeyltransferase indicates that they are functional particles.
Introduction High concentrations of plasma HDL and its major protein, apolipoprotein A-I, have been correlated with a reduced risk of premature cardiovascular disease [1,2]. Apolipoprotein A-I is thought to mediate the efflux of cholesterol from peripheral cells, thus playing an important role in the process of reverse cholesterol transport [3]. In the process of reverse cholesterol transport, cellular cholesterol taken up by HDL is esterified by lecithin : cholesterol acyltransferase and ultimately transferred by cholesteryl ester transfer protein to lower
Correspondence: Trudy M. Forte, Lawrence University of California, Donner Laboratory, Berkeley, CA 94720, U.S.A. ~5-2760/90/$03.50
Berkeley Laboratory, 1 Cyclotron Road,
0 1990 Elsevier Science Publishers
B.V. (Biomedical
density lipoproteins which are taken up and degraded by the liver. Apo~poprotein A-I is an activator of lecithin : cholesterol acyltransferase which is an important reactant in the reverse cholesterol transport mechanism. In vivo, the liver and intestine are the primary sites of apolipoprotein A-I synthesis. Recent studies have shown, however, that the human apo~poprotein A-I gene can also be expressed in nonhuman cells that normally do not express this gene. Chinese hamster ovary cells transfected with the 2.2 kb PstI region of the human apolipoprotein A-I gene construct containing the human metallot~onein gene promoter sequence secreted large quantities of mature apo~poprotein A-I [4]. A small portion of the secreted apolipoprotein A-I was apparently lipid-complexed, since it floated at a density of 1.125 g/ml. Mouse 3T3 cells transfected with Division)
12 the same human apolipoprotein A-I gene sequence but containing the mouse metallothionein gene promoter region also secreted apolipoprotein A-I when induced with zinc [5]. In these experiments, the secreted apolipoprotein A-I appeared to form apolipoprotein A-I-lipid complexes if the cells were first incubated with lipid emulsions. The human apolipoprotein A-I gene was also expressed in a mouse pituitary cell line (atT-20) where approx. 10% of the secreted protein was lipid complexed in the form of discoidal and small, spherical particles [6]. Ruiz-Opazo and Zannis [7] demonstrated that the human apolipoprotein A-I gene can be expressed in rat myogenic L6E9 cells without a metallothionein promoter if the entire pSV2 gpt 11 kilobase apolipoprotein A-I DNA sequence is transfected into the cell. Human apolipoprotein A-I gene transfection studies have clearly shown that nonhuman, nonhepatic cells are able to secrete this important HDL protein but relatively little is known about the chemical and physical properties of the lipid-associated particles produced by these transfected cells. The present study addresses these issues in Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene and also demonstrates that the HDL-like complexes are substrates for lecithin : cholesterol acyltransferase.
Methods Cell cultures. The stably transfected Chinese hamster ovary clone, CHO : pMTAIR143, was cultured as previously described by Mallory et al. [4]. Cells grown in Dulbecco’s modified Eagle’s/Coon F12 medium (1 : 1) containing 10% fetal bovine serum were pre-induced with 100 PM ZnSO,. When cells were confluent, the monolayers were rinsed with phosphate-buffered saline and then grown in serum-free medium in the presence of 15 mM Hepes, 80 PM ZnSO, and 30 FM FeSO,. Serum-free medium was harvested after 24 h and passed through a 0.2 pm filter in order to remove detached cells and cell debris. Medium was concentrated 20- to lOO-fold by ultrafiltration utilizing Amicon stirred cells with PM30 membranes at 4°C under nitrogen. Isolation of apolipoprotein A-I-lipid complexes. Concentrated medium was adjusted to density 1.21 g/ml by dialysis. The d s 1.21 g/ml material was isolated by ultracentrifugation in a Beckman 50.3 rotor (40000 t-pm, 48 h at 4°C). The top one ml containing the d < 1.21 g/ml material was aspirated from the 6-ml tube. The apolipoprotein A-I-lipid complexes of d < 1.21 g/ml were subfractionated by FPLC using a Superose 12 column (flow rate of 0.25 ml/mm). Samples were eluted with saline-Tris-EDTA (150 mM, 20 mM and 0.27 mM, respectively) buffer (pH 8.0); 0.5 ml fractions were collected.
Electrophoresis. Total d I 1.21 g/ml fractions and Superose column subfractions were analyzed by nondenaturing gradient gel electrophoresis (4-30% polyacrylamide precast gels, Pharmacia, Piscataway, NJ) in order to determine size distribution of complexes. Nondenaturing gels were electrophoresed according to the method of Nichols et al. [8]. Standards used to calibrate the gels consisted of the globular proteins thyroglobulin, apoferritin, lactate dehydrogenase, and albumin; peak positions reported in nanometers (nm) are based on the position of the globular standards. Peak positions reported as RF values represent migration distance of the complex (measured from top of gel) relative to the migration position of the bovine serum albumin standard. Gels were stained with Coomassie G250 and scanned at 603 nm with a RFT scanning densitometer (Transidyne Corp., Ann Arbor, MI). To verify presence of apolipoprotein A-I in the isolated d 2 1.21 g/ml fractions and Superose column fractions, samples were electrophoresed on SDS polyacrylamide gels (10%) and stained with Coomassie R250 according to Weber and Osborn [9]. Immunochemical identification of apolipoprotein A-I by Western blotting was carried out essentially as previously described by Thrift et al. [lo]. Electron microscopy. Fractions were examined by negative staining with 1% sodium phosphotungstate essentially as previously described [ll]. Analysis. Protein was determined by a modified Lowry procedure [12] with bovine serum albumin as the standard. Phospholipid was assayed according to Bartlett [13]. Free and esterified cholesterol were determined by gas-liquid chromatography as previously described
P41. Apolipoprotein A-I was quantitated by single radial immunodiffusion using plates and standards supplied by TAGO, Inc. (Burlingame, CA). Human serum was used as a reference with a lower level of sensitivity of 10 pg/ml apolipoprotein A-I. Chemical crosslinking of apolipoprotein A-I complexes was achieved by the dimethylsuberimidate dihydrochloride method described by Swaney and O’Brien [15]. Crosslinked complexes were delipidated and electrophoresed on SDS-polyacrylamide gels; crosslinked, purified apolipoprotein A-I was used as a standard. Lecithin : cholesterol acyltransferase assay. To determine whether d I 1.21 g/ml apolipoprotein A-I-lipid complexes serve as a substrate for lecithin : cholesterol acyltransferase, this fraction was incubated 18 hr in the presence of 1.5% human serum albumin and purified lecithin : cholesterol acyltransferase (kindly provided by Dr. A.V. Nichols and P.J. Blanche) at 37°C essentially as described by McCall et al [16]. The reaction was stopped by placing the sample on ice and adding p-hydroxymercuriphenylsulfonic acid (PHMPS) (3.5 mM final concentration). A control sample was incubated at 4°C in the presence of 3.5 mM PHMPS. Following
13 108
incubation, the density of the solution was adjusted to d 1.25 g/ml with solid NaBr and the d I 1.235 g/ml fraction isolated after ultracentrifugation (40000 rpm for 48 h at 4°C Beckman 50.3 rotor using adapters for 2-ml tubes). The top 0.35 ml was aspirated and the fractions were assayed for free and esterified cholesterol. Structure of the particles was assessed by electron microscopy.
nm..,...n& Rf
Results Recombinant expression of apolipoprotein A-I was achieved by inserting a 2.2 kilobase PstI human gene fragment into a vector regulated by the human metallothionein II promoter [4]. The apolipoprotein A-I genomic fragment included the entire gene and encompassed the 5’ untranslated region, including the TATA box, through the 3’ untranslated region, including the polyadenylation signal. Chinese hamster ovary cells transfected with this human apolipoprotein A-I expression vector secrete large amounts of apolipoprotein A-I (7.7 + 1.3 pg/ml, n = 3) into serum-free medium when induced by heavy metals. The apolipoprotein A-I protein represents 7.10 * 0.378 of the total secreted protein, thus indicating that apolipoprotein A-I represents a major secretory product of the transfected cells. Of the total secreted apolipoprotein A-I, 16.3 k 1.9% (n = 3) was isolated at d I 1.21 g/ml. Electron microscopy of this fraction reveals that it is morphologically very heterogeneous (Fig. 1) and consists of large and small discs, large vesicular structures, and varying sized round profiles. Nondenaturing gradient gel electrophoresis was carried out to determine whether discrete-sized subpopulations could be identified. Fig. 2 shows the apolipoprotein A-I particle distribution from two separate experiments. It is immediately apparent that, in agreement with the electron microscopic information, the cells produce a heterogeneous population of apolipoprotein A-I-lipid complexes. Although there are
Fig. 1. Electron micrograph of particles isolated from Chinese hamster ovary cell medium at d I 1.21 g/ml. Structures are heterogeneous in morphology and include small and large discs as well as vesicles. Bar marker represents 100 M-I.
4%
30%
Fig. 2. Densitometric scans of Chinese hamster ovary cell d 11.21 g/ml material from two separate experiments electrophoresed on 4-30% nondenaturing polyacrylamide gradient gels. Peak positions in nm are shown on top while lower numbers indicate corresponding RF values. The morphology of most particles is discoidal; hence the peak positions are only relative since globular proteins are used to calibrate the gels.
some differences in peak position and relative intensity of specific peaks, the patterns show a high degree of similarity. A prominent peak consistently bands at approx. 11.0 nm, while components banding at 9.0, 7.2 and 14.0 nm are common to both. The lower pattern possesses greater numbers of particles in the large pore region (greater than 20 nm) of the gel. Because of the size heterogeneity exhibited by the d I 1.21 g/ml fraction, this material was fractionated on a Superose column. The elution profile shown in Fig. 3A indicates that at least three components can be identified; these fractions labeled I-III represent 35.2%, 48.1% and 16.7%, respectively, of the recovered protein. Each of the fractions was electrophoresed on 4-30% nondenaturing gels in order to determine the size of particles within these fractions; a Western blot for
Fig. 3. Chinese hamster ovary cell apolipoprotein A-I complexes isolated by Superose column chromatography. Column fractions were pooled (I-III) as indicated in panel A. The corresponding nondenaturing gradient gel patterns are seen in panel B.The fractions along with unfractionated d < 1.21 g/ml material were first electrophoresed on 4-30% nondenaturing gradient gels and then electroluted to nitrocellulose paper. The latter was probed with antibody to apolipoprotein A-I.
14
A
B
C
D
ALB
Al
LYS
Fig. 4. Proteins from Superose column fractions analyzed on 10% SDS-PAGE gels: (A) Fraction I; (B) Fraction II; (C) Fraction III; and (D) standards consisting of albumin (alb), apolipoprotein A-I (A-I), and lysozyme (1~s). All samples were reduced with P-mercaptoethanol before application. The major band in Fractions I and II co-migrates with apolipoprotein A-I and accounts for 85% of the Coomassie staining material. The doublet in Fractions I and II migrating just above the albumin standard could be aggregated apolipoprotein A-I since it reacted positively with anti-apolipoprotein A-I in a Western blot.
apolipoprotein A-I distribution in the fractions is seen in Fig. 3B. Fraction I contains particles that band primarily at 14.0 and 16.5 nm; they correspond to the upper two bands seen in the unfractionated sample. Fraction II, which is the major apolipoprotein A-I-lipid complex, consists of two pro~nent bands at 9.0 and 11.0 nm. Fraction III has a slight band in the region of 7.2 nm but the majority of apolipoprotein A-I appears to migrate near the bottom of the gel, suggesting the presence of lipid-poor apolipoprotein A-I. The latter material is also in evidence in the total d II 1.21 g/ml fraction.
Fig. 5. Electron
micrographs of fraction be vesicular particles
The protein composition of the Superose column fractions was analyzed by 10% SDS-PAGE and the results are shown in Fig. 4. The major Coomassie staining band in each fraction co-migrates with apolipoprotein A-I (28.0 kDa); however, fractions I and II have an additional doublet band (approximate molecular weights 68.2 and 77.6 kDa) which represents 15% of the staining material. Western blots of 10% SDS-PAGE slab gels were probed with apolipoprotein A-I antibody and all bands stained positively for apolipoprotein A-I (data not shown), thus indicating that the doublet bands may represent aggregated and/or denatured forms of apolipoprotein A-I. The electron microscopic structure of the Superose column fractions are summarized in Fig. 5. Fraction I (Fig. 5A) contains numerous large vesicular and/or discoidal structures that range from 60-260 nm in size; some discs 18-20 nm in diameter are also present. Fraction II (Fig. 5B) contains discoidal particles with a mean diameter of 14.2 f 2.6 S.D. nm (4.2 f 0.4 nm thick); some are seen en face in the micrograph although most are in rouleaux. No identifiable structures were seen in fraction III, undoubtedly because the lipid-poor form of apolipoprotein A-I is not readily resolved by negative staining. Since almost 50% of the apolipoprotein A-I protein floating at d s 1.21 g/ml is in fraction II, the major apolipoprotein A-I-lipid complex is a relatively small, discoidal structure. The diameters of the discoidal particles determined by electron microscopy are larger than those estimated by gradient gel electrophoresis (Fig. 2) because the latter sizes assume a spherical particle morphology (i.e., standard globular proteins are used to calibrate the gels). The compositions of the isolated apolipoprotein A-Ilipid complexes are seen in Table I. The protein-to-lipid ratio increases with decreasing particle size. Cholesterol
I (A) and fraction II (B) complexes isolated by column chromatography. Large numbers of what appear isolate in fraction I, while those in fraction II are primarily discs. Bar represents 100 nm.
to
15 TABLE I composition (weight percent) of ~p~iipoprotei~-bipedcomplexes isointed by Superose column chromatography
Fraction
Protein
% Total weight phospholipid
Cholesterol
d I 1.21 g/ml
51.3 42.8 53.1 93.5
18.8 32.9 32.8 6.5
29.9 24.4 14.2 n.d. a
Fraction I Fraction II Fraction III a n.d., not detected.
content decreases as percentage protein increases; however, no cholesterol was detected in fraction III which is a lipid-poor fraction. Cholesterol associated with the apolipoprotein A-I-lipid complexes was all in the unesterified form which is consistent with the discoidal and vesicular morphology of the particles. In order to assess whether the apolipoprotein A-Ilipid complexes were formed with a specific number of apolipoprotein A-I molecules per particle, apolipoprotein A-I in fraction I and fraction II was crosslinked with dimethylsuberimidate and analyzed by SDS-PAGE (Fig. 6). Fraction I, which consists of vesicles and large discs, contains mainly six to seven apolipoprotein A-Is per particle. Fraction II, which contains primarily two forms of discoidal particles (Fig. 3), one that bands at 9 nm and the other at 11 nm, possesses primarily particles with three and four apolipoprotein A-Is. The former are most likely associated with the smaller-sized complex while the latter are probably associated with the larger particles since reported in vitro studies [17,18] with apolipoprotein A-I-phospholipid analogs have shown
Tetramer Trlmer Dimer
that complexes with RF values similar to those in fraction II contain two to four molecules of apolipoprotein A-I. Fraction III was not crosslinked because there was insufficient material for this purpose. Fraction III material banding at 7.1 nm is approx. 70000 molecular weight and thus must be at least a dimer of apo~poprotein A-I. The predominant apolipoprotein A-I-lipid complex produced by transfected Chinese hamster ovary cells is a discoidal particle similar in morphology and composition to discoidal HDL found in lecithin : cholesterol acyltransferase-deficient plasma [19], monkey liver perfusates [20] and Hep G2-conditioned medium [lo]. All of these discoidal structures have been shown to be substrates for lecithin : cholesterol acyltransferase [16, 19,211. In order to test whether apolipoprotein A-I-lipid complexes produced by transfected Chinese hamster ovary cells also serve as substrates for this enzyme, two samples of d < 1.21 g/ml fractions were incubated for 18 h in the presence of active lecithin : cholesterol acyltransferase and conversion of unesterified cholesterol to esterified cholesterol was determined. Control apolipoprotein A-I-lipid complexes incubated at 4’C in the absence of lecithin : cholesterol acyltransferase showed no conversion; however, samples incubated with active enzyme converted 18.9 and 21.1% of unesterified cholesterol to cholesteryl ester. The formation of cholesteryl ester was coupled with the transformation of discoidal particles into heterogeneous-sized (12.9 & 3.4 nm), core-cont~~ng particles (Fig. 7A and B). The histogram of particle size distribution (Fig. 7C) of the lecithin : cholesterol acyltransferase-converted products suggests that there may be three populations of particles. A major peak occurs between 11-12 nm which corresponds to the size range of HDL,, in mature plasma [22], a minor peak occurs at 8-9 nm corresponding to plasma HDL,, and HDL,, f22], and a major peak appears at 13-15 nm which is considerably larger than normal plasma HDL. The large vesicular structures present in unincubated samples do not appear to be altered during incubation. Discussion
Monomer
Std
Fr I
Fr II
Fig. 6. SDS-PAGE (4-30% gradient gel) pattern of crosslinked apolipoprotein A-I in fraction I (Fr I) and fraction II (Fr II). Samples were electrophoresed at two concentrations: Fr I at 15 and 7.5 pg protein per lane, and Fr II at 23 and 11.5 pg per lane. Crosslinked, purified plasma apolipoprotein A-I was used as the standard (Std).
It has previously been demonstrated that apolipoprotein A-I secreted by Chinese hamster ovary cells transfected with the human apo~poprotein A-I gene secrete fully processed apolipoprotein A-I that possesses little pro-apolipoprotein A-I [4]. This is in contrast to apolipoprotein A-I secreted by the human hepatoma line, Hep G2, which during a 24-hr incubation period produces apolipoprotein A-I which is 50% pro-apolipoprotein A-I [23]. The latter, however, does not prevent apolipoprotein A-I-lipid complex formation, thus, in hepatoma cell media, 30-40% of secreted apolipoprotein A-I is in the lipid associated form [23,24] whereas
16
30 i s! r p"
20 -
z t : 10 -
0 5.0
C
7.5
I 10.0
12.5
I 15.0
Diameter
17.5
I 20.0
22.5
2
.O
(nm)
Fig. 7. Electron micrograph of d < 1.235 g/ml apohpoprotein A-I-lipid complexes incubated at 4°C in the absence (A) and at 37°C in the presence (B) of purified 1ecithin:cholesterol acyltransferase. The smooth histogram (C) of particle diameters resulting after incubation with active lecithin : cholesterol acyltransferase suggests that particle size is heterogeneous and as many as three distinct components may be present.
in Chinese hamster ovary cell medium only 16% is lipid associated. Compared with hepatoma cells, transfected Chinese hamster ovary cells secrete high levels of apolipoprotein A-I (7% of total protein compared to 1% for hepatomas); such high apolipoprotein A-I secretory rates may outstrip the cellular lipoprotein assembly process if one assumes that the particles are assembled intracellularly. If, on the other hand, apolipoprotein A-I-lipid assembly occurs extracellularly, a major constraint might be the availability of membrane phospholipids and cholesterol that would be expected to contribute to complex formation. Apolipoprotein A-I-lipid complexes isolated from Chinese hamster ovary cell media are primarily dis-
coidal or ‘nascent’ HDL. Their morphology and size distribution is similar to discoidal HDL isolated from patients with lecithin : cholesterol acyltransferase deficiency where apolipoprotein A-I-containing discs have diameters of 11-28 nm [19]. By nondenaturing gradient gel analysis there is great similarity between that of Chinese hamster ovary cell nascent particles and nascent HDL particles isolated from Hep G2 medium [10,25]. A major difference lies in the presence of a particle banding at 7.4 nm in Hep G2 HDL and its absence in Chinese hamster ovary cell medium. The 7.4 nm particle from Hep G2 medium contains a core of cholesteryl ester [25] which is either derived from cellular pools of cholesteryl ester or by the action of lecithin : cholesterol
17 acyltransferase which is present in low levels in the medium [26]. Neither of these mechanisms appears to be operative in Chinese hamster ovary cell incubations. We had previously shown that 3T3 cells transfected with the human apolipoprotein A-I gene also secrete apolipoprotein A-I [5]. This cell line, however, required preincubation with phospholipid-rich lipid emulsions in order to obtain apolipoprotein A-I-lipid complexes. Nondenaturing gradient gel analysis suggests that 3T3 cells produce discoidal particles that band in the region between 9-12 nm [5] which is similar to the region in which Chinese hamster ovary cell Fraction II apolipoprotein A-I-lipid complexes have major bands. However, Western blots for apolipoprotein A-I also suggested the presence of a small, 7.4 nm particle in 3T3 cell media. Since not enough material was available for lipid analyses of complexes formed by 3T3 cells, it is unknown whether the complexes are lipid core-containing particles or whether they are unusually small, polarlipid complexes. Several in vitro studies have shown that apolipoprotein A-I-phospholipid or phospholipid plus cholesterol complexes formed by cholate dialysis produce discrete, reproducible structures stabilized by specific numbers of apolipoprotein A-I molecules [17,18,27,28]. In the present study, Fraction II particles banding at 9.0 nm isolated from Chinese hamster ovary cell medium fall within the size range of class 3 apolipoprotein A-I-egg yolk phosphatidylcholine complexes described by Nichols et al. [17,18]. Class 3 in vitro model complexes generally contained only two apolipoprotein A-I molecules per particle, whereas similar-sized discoidal particles from Chinese hamster ovary cell medium contained three apolipoprotein A-I molecules. Although speculative, these differences may, in part, be the result of differences in types of phospholipids associated with the complexes where, in model systems, known purified phospholipids such as phosphatidylcho~ne were employed, whereas in Chinese hamster ovary cell complexes, phospholipid composition is unknown but is likely to be a combination of cell-derived phospholipids. The latter is presently under investigation. The somewhat larger Fraction II discs banding at 11.0 nm are similar to the class 4 and 5 complexes 1171 and, in agreement with this size, contain 4 apolipoprotein A-Is, Fraction I particles obtained from Chinese hamster ovary cell cultures fall within the region occupied by classes 7 and 8 in artificial complexes that contain five or more apo~poprotein molecules j17,28]. Discoidal HDL from Chinese hamster ovary cells have, on nondenaturing gradient gels, peaks in common with nascent HDL isolated from Hep G2 cell medium [lo] and lecithin : cholesterol acyltransferase-deficient plasma (Forte, T.M., unpublished observations). The presence of recurring, distinct size classes of apolipoprotein A-Icontaining discoidal complexes from disparate sources
including those from Chinese hamster ovary cells suggests that apolipoprotein A-I interaction with lipids plays an important role in determining size. Discrete molecular entities with specific numbers of apolipoprotein A-I are formed rather than a continuum of particle sizes. Nascent HDL from lecithin : cholesterol acyltransferase-deficient plasma [29], Hep G2 medium [16], and in vitro phospholipid-cholesterol-apolipoprotein A-I discoidal complexes [18,27] are excellent substrates for lecithin : cholesterol acyltransferase. HDL from plasma of lecithin : cholesterol acyltransferase-deficient patients undergo transesterification of cholesterol to cholesteryl ester which is associated with the transformation of 11.0-28.0 nm discs to spheres 10.7 nm diameter [29]. The mean particle diameter of transformed particles from Chinese hamster ovary cell products (12.9 nm) is somewhat larger than that reported for lecithin : cholesterol acyltransferase deficiency. This may in part be due to the use of patient’s total plasma and partially purified lecithin : cholesterol acyltransferase in these early incubation studies. Incubations involving whole plasma and partially purified lecithin : cholesterol acyltransferase would possess not only lipid transfer protein activity but also additional sources of free cholesterol for the lecithin : cholesterol acyltransferase reaction which may result in a more complete transfo~ation of nascent HDL. The large vesicular structures appearing in Chinese hamster ovary media did not appear to serve, in great degree, as substrates for lecithin : cholesterol acyltransferase since they persisted after 18-hr incubation with purified lecithin : cholesterol acyltransferase. Lack of lecithin : cholesterol acyltransferase-driven transformation of vesicles probably denotes a less favorable conformation of apolipoprotein A-I for activating lecithin : cholesterol acyltransferase activity. The poor reactivity of vesicular apolipoprotein A-I-phospho~pid substrates has previously been demonstrated by Chajek et al. [30], who suggested that the aqueous interior of vesicles, as opposed to the hydrophobic one in the discs, may be less favorable for incorporation of cholesteryl esters. It has also been suggested [31,32] that the higher apolipoprotein A-I-to-lipid molar ratio in discs as opposed to vesicles may provide a more favorable apolipoprotein A-I conformation for lecithin : cholesterol acyltransferase reactivity. In summary, the apolipoprotein A-I-lipid complexes isolated from media of Chinese hamster ovary cells transfected with the human apo~poprotein A-I gene form discoidal HDL particles that are indistinguishable from nascent HDL isolated from plasma lecithin : cholesterol acyltransferase-deficient patients and those isolated from Hep G2 culture media. The discoidal Chinese hamster ovary cell media apolipoprotein A-I complexes are also substrates for lecithin : cholesterol acyltrans-
18 ferase, thus suggesting that the transfected cells produce functional nascent HDL particles. Acknowledgements
We wish to thank Dr. Virgie Shore for her assistance on quantitation of apolipoprotein A-I, and Mary Lou Kurtz for preparation of the manuscript. This work was supported by NIH Program Project Grant HL 18574 and NRSA Training Grant HL 07279 from the National Heart, Lung, and Blood Institute of the National Institutes of Health, and was conducted at the Lawrence Berkeley Laboratory (Department of Energy contract DE-AC03-76SF00098 to the University of California). References
4
9 10 11
Miller, G.J. and Miller, N.E. (1975) Lancet i, 16-19. Gordon, T., Caste& W.P., Hjortland, M.C. and et al. (1977) Am. J. Med. 62, 707-714. Miller, N.E., La Ville, A. and Crook, D. (1985) Nature 314, 109-111. Mallory, J.B., Kushner, P.J., Protter, A.A., Cofer, C.L., Appleby, V.L., Lau, K., Schilling, J.W. and Vigne, J.-L. (1987) J. Biol. Chem. 262, 4241-4247. Lamon-Fava, S., Ordovas, J.M., Mandel, G., Forte, T.M., Goodman, R.H. and Schaefer, E.J. (1987) J. Biol. Chem. 262, 8944-8947. FennewaId, S.M., Hamilton, R.L., Jr., and Gordon, J.I. (1988) J. Biol. Chem. 263, 15568-15577. Ruiz-Opazo, N. and Zanms, V.I. (1988) J. Biol. Chem. 263, 1739-1744. Nichols, A.V., Blanche, P.J. and Gong, E.L. (1983) in CRC Handbook of Electrophoresis (Lewis, L. and Opplt, J., ed.), vol. III, pp. 29-47, CRC Press, Boca Raton, FL. Weber, K. and Osbom, M. (1969) J. Biol. Chem. 24444064412. Thrift, R.N., Forte, T.M., Cahoon, B.E. and Shore, V.G. (1986) J. Lipid Res. 27, 236-250. Forte, T.M. and Nordhausen, R.W. (1986) Methods Enzymol. 128, 442-457.
12 Markwell, M.A.K., Hass, S.M., Bieber, L.L. and Tolbert, N.E. (1978) Anal. B&hem. 87, 206-210. 13 Bartlett, G.R. (1959) J. Biol. Chem. 531, 233-236. 14 Hindriks, R.F., Wolthers, B.G. and Groen, A. (1977) Clin. Chim. Acta 74, 207-215. 15 Swaney, J.B. and O’Brien, K. (1978) J. Biol. Chem. 253,7069-7077. 16 McCall, M.R., Nichols, A.V., Blanche, P.J., Shore, V.G. and Forte, T.M. (1989) J. Lipid Res. 30, 1579-1589. 17 Nichols, A.V., Gong, E.L., Blanche, P.J. and Forte, T.M. (1983) B&him. Biophys. Acta 750, 353-364. 18 Nichols, A.V., Blanche, P.J., Gong, E.L., Shore, V.G. and Forte, T.M. (1985) Biochim. Biophys. Acta 834, 285-300. 19 Mitchell, CD., King, W.C., Applegate, K.R., Forte, T., Glomset, J.A., Norum, K.R. and Gjone, E. (1980) J. Lipid Res. 21,625-634. 20 Johnson, F.L., Babiak, J. and Rudel, L.L. (1986) J. Lipid Res. 27, 537-548. 21 Babiak, J., Tamachi, H., Johnson, F.L., Parks, J.S. and Rudel, L.L. (1986) J. Lipid Res. 27, 1304-1317. 22 Blanche, P.J., Gong, E.L., Forte, T.M., and Nichols, A.V. (1981) Biochim. Biophys. Acta 665, 408-419. 23 Forte, T.M., Nichols, A.V., Sehnek-Halsey, J., Caylor, L. and Shore, V.G. (1987) Biochim. Biophys. Acta 920, 185-194. 24 Forte, T.M., McCall, M.R., Knowles, B.B. and Shore, V.G. (1989) J. Lipid Res. 30, 817-829. 25 McCall, M.R., Forte, T.M. and Shore, V.G. (1988) J. Lipid Res. 29, 1127-1137. 26 Chen, C.-H., Forte, T.M., Cahoon, B.E., Thrift, R.N. and Albers, J.J. (1986) B&him. Biophys. Acta 877, 433-439. 21 Nichols, A.V., Gong, E.L., Blanche, P.J., Forte, T.M. and Shore, V.G. (1987) J. Lipid Res. 28, 719-732. 28 Gong, E.L., Nichols, A.V., Forte, T.M., Blanche, P.J. and Shore, V.G. (1988) Biochim. Biophys. Acta 961, 73-85. 29 Norum, K.R., Glomset, J.A., Nichols, A.V., Forte, T., Albers, J.J., King, W.C., Mitchell, C.D., Applegate, K.R., Gong, E.L., Cabana, V. and Gjone, E. (1975) Stand. J. Clin. Lab. Invest. 142, 31-55. 30 Chajek, T., Aron, L. and Fielding, C.J. (1980) Biochemistry 19, 3673-3677. 31 Matz, C.E. and Jonas, A. (1982) J. Biol. Chem. 257, 4541-4546. 32 Hefele WaId, J.E. and Jonas, A. (1989) Arteriosclerosis 9, 715a.
Biochimica et Biophysicu Acta, 1047 (1990) 11-18 Elsevier
BBALJP
53491
Physical and chemical characteristics of apolipoprotein A-I-lipid complexes produced by Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene T.M. Forte I, M.R. McCall I, S. Amacher ‘, R.W. Nordhausen ‘, J.L. Vigne ’ and J.B. Mallory ’ ’ Luwrenee Berkeley ~bo~a~o~, Unioers~~ of Cul~orn~o, Berkeley and ’ Cul~or~ia 3io~ech~oio~ Inc., Mountain View, CA (U.S.A.~
(Revised
Key words:
Apo~poprotein
(Received 5 March 1990) manuscript received 12 June 1990)
A-I; Chinese hamster ovary cells; Apolipoprotein Lecithin : cholesterol acyltransferase
A-I gene transfection;
Nascent
HDL;
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 f 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 I 1.21 g/ml. The latter complexes are composed of discs and vesicles as judged by electron microscopy and can be further separated by cohumr chromatography into three fractions: fraction I, mostly vesicles (60-260 nm) and large discs (B-20 nm diameter); fraction II, discs 14.2 f 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 dete~in~ by protein crossli~ing while large stnuztures 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 f 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 acyltransfear-deficient patients and those secreted by the human hepatoma line, Hep G2. The ability of the Chinese hamster ovary cell nascent HDLlike particles to undergo ~~sfo~ation in the presence of purified 1~ithin:cholesterol aeyltransferase indicates that they are functional particles.
Introduction High concentrations of plasma HDL and its major protein, apolipoprotein A-I, have been correlated with a reduced risk of premature cardiovascular disease [1,2]. Apolipoprotein A-I is thought to mediate the efflux of cholesterol from peripheral cells, thus playing an important role in the process of reverse cholesterol transport [3]. In the process of reverse cholesterol transport, cellular cholesterol taken up by HDL is esterified by lecithin : cholesterol acyltransferase and ultimately transferred by cholesteryl ester transfer protein to lower
Correspondence: Trudy M. Forte, Lawrence University of California, Donner Laboratory, Berkeley, CA 94720, U.S.A. ~5-2760/90/$03.50
Berkeley Laboratory, 1 Cyclotron Road,
0 1990 Elsevier Science Publishers
B.V. (Biomedical
density lipoproteins which are taken up and degraded by the liver. Apo~poprotein A-I is an activator of lecithin : cholesterol acyltransferase which is an important reactant in the reverse cholesterol transport mechanism. In vivo, the liver and intestine are the primary sites of apolipoprotein A-I synthesis. Recent studies have shown, however, that the human apo~poprotein A-I gene can also be expressed in nonhuman cells that normally do not express this gene. Chinese hamster ovary cells transfected with the 2.2 kb PstI region of the human apolipoprotein A-I gene construct containing the human metallot~onein gene promoter sequence secreted large quantities of mature apo~poprotein A-I [4]. A small portion of the secreted apolipoprotein A-I was apparently lipid-complexed, since it floated at a density of 1.125 g/ml. Mouse 3T3 cells transfected with Division)
12 the same human apolipoprotein A-I gene sequence but containing the mouse metallothionein gene promoter region also secreted apolipoprotein A-I when induced with zinc [5]. In these experiments, the secreted apolipoprotein A-I appeared to form apolipoprotein A-I-lipid complexes if the cells were first incubated with lipid emulsions. The human apolipoprotein A-I gene was also expressed in a mouse pituitary cell line (atT-20) where approx. 10% of the secreted protein was lipid complexed in the form of discoidal and small, spherical particles [6]. Ruiz-Opazo and Zannis [7] demonstrated that the human apolipoprotein A-I gene can be expressed in rat myogenic L6E9 cells without a metallothionein promoter if the entire pSV2 gpt 11 kilobase apolipoprotein A-I DNA sequence is transfected into the cell. Human apolipoprotein A-I gene transfection studies have clearly shown that nonhuman, nonhepatic cells are able to secrete this important HDL protein but relatively little is known about the chemical and physical properties of the lipid-associated particles produced by these transfected cells. The present study addresses these issues in Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene and also demonstrates that the HDL-like complexes are substrates for lecithin : cholesterol acyltransferase.
Methods Cell cultures. The stably transfected Chinese hamster ovary clone, CHO : pMTAIR143, was cultured as previously described by Mallory et al. [4]. Cells grown in Dulbecco’s modified Eagle’s/Coon F12 medium (1 : 1) containing 10% fetal bovine serum were pre-induced with 100 PM ZnSO,. When cells were confluent, the monolayers were rinsed with phosphate-buffered saline and then grown in serum-free medium in the presence of 15 mM Hepes, 80 PM ZnSO, and 30 FM FeSO,. Serum-free medium was harvested after 24 h and passed through a 0.2 pm filter in order to remove detached cells and cell debris. Medium was concentrated 20- to lOO-fold by ultrafiltration utilizing Amicon stirred cells with PM30 membranes at 4°C under nitrogen. Isolation of apolipoprotein A-I-lipid complexes. Concentrated medium was adjusted to density 1.21 g/ml by dialysis. The d s 1.21 g/ml material was isolated by ultracentrifugation in a Beckman 50.3 rotor (40000 t-pm, 48 h at 4°C). The top one ml containing the d < 1.21 g/ml material was aspirated from the 6-ml tube. The apolipoprotein A-I-lipid complexes of d < 1.21 g/ml were subfractionated by FPLC using a Superose 12 column (flow rate of 0.25 ml/mm). Samples were eluted with saline-Tris-EDTA (150 mM, 20 mM and 0.27 mM, respectively) buffer (pH 8.0); 0.5 ml fractions were collected.
Electrophoresis. Total d I 1.21 g/ml fractions and Superose column subfractions were analyzed by nondenaturing gradient gel electrophoresis (4-30% polyacrylamide precast gels, Pharmacia, Piscataway, NJ) in order to determine size distribution of complexes. Nondenaturing gels were electrophoresed according to the method of Nichols et al. [8]. Standards used to calibrate the gels consisted of the globular proteins thyroglobulin, apoferritin, lactate dehydrogenase, and albumin; peak positions reported in nanometers (nm) are based on the position of the globular standards. Peak positions reported as RF values represent migration distance of the complex (measured from top of gel) relative to the migration position of the bovine serum albumin standard. Gels were stained with Coomassie G250 and scanned at 603 nm with a RFT scanning densitometer (Transidyne Corp., Ann Arbor, MI). To verify presence of apolipoprotein A-I in the isolated d 2 1.21 g/ml fractions and Superose column fractions, samples were electrophoresed on SDS polyacrylamide gels (10%) and stained with Coomassie R250 according to Weber and Osborn [9]. Immunochemical identification of apolipoprotein A-I by Western blotting was carried out essentially as previously described by Thrift et al. [lo]. Electron microscopy. Fractions were examined by negative staining with 1% sodium phosphotungstate essentially as previously described [ll]. Analysis. Protein was determined by a modified Lowry procedure [12] with bovine serum albumin as the standard. Phospholipid was assayed according to Bartlett [13]. Free and esterified cholesterol were determined by gas-liquid chromatography as previously described
P41. Apolipoprotein A-I was quantitated by single radial immunodiffusion using plates and standards supplied by TAGO, Inc. (Burlingame, CA). Human serum was used as a reference with a lower level of sensitivity of 10 pg/ml apolipoprotein A-I. Chemical crosslinking of apolipoprotein A-I complexes was achieved by the dimethylsuberimidate dihydrochloride method described by Swaney and O’Brien [15]. Crosslinked complexes were delipidated and electrophoresed on SDS-polyacrylamide gels; crosslinked, purified apolipoprotein A-I was used as a standard. Lecithin : cholesterol acyltransferase assay. To determine whether d I 1.21 g/ml apolipoprotein A-I-lipid complexes serve as a substrate for lecithin : cholesterol acyltransferase, this fraction was incubated 18 hr in the presence of 1.5% human serum albumin and purified lecithin : cholesterol acyltransferase (kindly provided by Dr. A.V. Nichols and P.J. Blanche) at 37°C essentially as described by McCall et al [16]. The reaction was stopped by placing the sample on ice and adding p-hydroxymercuriphenylsulfonic acid (PHMPS) (3.5 mM final concentration). A control sample was incubated at 4°C in the presence of 3.5 mM PHMPS. Following
13 108
incubation, the density of the solution was adjusted to d 1.25 g/ml with solid NaBr and the d I 1.235 g/ml fraction isolated after ultracentrifugation (40000 rpm for 48 h at 4°C Beckman 50.3 rotor using adapters for 2-ml tubes). The top 0.35 ml was aspirated and the fractions were assayed for free and esterified cholesterol. Structure of the particles was assessed by electron microscopy.
nm..,...n& Rf
Results Recombinant expression of apolipoprotein A-I was achieved by inserting a 2.2 kilobase PstI human gene fragment into a vector regulated by the human metallothionein II promoter [4]. The apolipoprotein A-I genomic fragment included the entire gene and encompassed the 5’ untranslated region, including the TATA box, through the 3’ untranslated region, including the polyadenylation signal. Chinese hamster ovary cells transfected with this human apolipoprotein A-I expression vector secrete large amounts of apolipoprotein A-I (7.7 + 1.3 pg/ml, n = 3) into serum-free medium when induced by heavy metals. The apolipoprotein A-I protein represents 7.10 * 0.378 of the total secreted protein, thus indicating that apolipoprotein A-I represents a major secretory product of the transfected cells. Of the total secreted apolipoprotein A-I, 16.3 k 1.9% (n = 3) was isolated at d I 1.21 g/ml. Electron microscopy of this fraction reveals that it is morphologically very heterogeneous (Fig. 1) and consists of large and small discs, large vesicular structures, and varying sized round profiles. Nondenaturing gradient gel electrophoresis was carried out to determine whether discrete-sized subpopulations could be identified. Fig. 2 shows the apolipoprotein A-I particle distribution from two separate experiments. It is immediately apparent that, in agreement with the electron microscopic information, the cells produce a heterogeneous population of apolipoprotein A-I-lipid complexes. Although there are
Fig. 1. Electron micrograph of particles isolated from Chinese hamster ovary cell medium at d I 1.21 g/ml. Structures are heterogeneous in morphology and include small and large discs as well as vesicles. Bar marker represents 100 M-I.
4%
30%
Fig. 2. Densitometric scans of Chinese hamster ovary cell d 11.21 g/ml material from two separate experiments electrophoresed on 4-30% nondenaturing polyacrylamide gradient gels. Peak positions in nm are shown on top while lower numbers indicate corresponding RF values. The morphology of most particles is discoidal; hence the peak positions are only relative since globular proteins are used to calibrate the gels.
some differences in peak position and relative intensity of specific peaks, the patterns show a high degree of similarity. A prominent peak consistently bands at approx. 11.0 nm, while components banding at 9.0, 7.2 and 14.0 nm are common to both. The lower pattern possesses greater numbers of particles in the large pore region (greater than 20 nm) of the gel. Because of the size heterogeneity exhibited by the d I 1.21 g/ml fraction, this material was fractionated on a Superose column. The elution profile shown in Fig. 3A indicates that at least three components can be identified; these fractions labeled I-III represent 35.2%, 48.1% and 16.7%, respectively, of the recovered protein. Each of the fractions was electrophoresed on 4-30% nondenaturing gels in order to determine the size of particles within these fractions; a Western blot for
Fig. 3. Chinese hamster ovary cell apolipoprotein A-I complexes isolated by Superose column chromatography. Column fractions were pooled (I-III) as indicated in panel A. The corresponding nondenaturing gradient gel patterns are seen in panel B.The fractions along with unfractionated d < 1.21 g/ml material were first electrophoresed on 4-30% nondenaturing gradient gels and then electroluted to nitrocellulose paper. The latter was probed with antibody to apolipoprotein A-I.
14
A
B
C
D
ALB
Al
LYS
Fig. 4. Proteins from Superose column fractions analyzed on 10% SDS-PAGE gels: (A) Fraction I; (B) Fraction II; (C) Fraction III; and (D) standards consisting of albumin (alb), apolipoprotein A-I (A-I), and lysozyme (1~s). All samples were reduced with P-mercaptoethanol before application. The major band in Fractions I and II co-migrates with apolipoprotein A-I and accounts for 85% of the Coomassie staining material. The doublet in Fractions I and II migrating just above the albumin standard could be aggregated apolipoprotein A-I since it reacted positively with anti-apolipoprotein A-I in a Western blot.
apolipoprotein A-I distribution in the fractions is seen in Fig. 3B. Fraction I contains particles that band primarily at 14.0 and 16.5 nm; they correspond to the upper two bands seen in the unfractionated sample. Fraction II, which is the major apolipoprotein A-I-lipid complex, consists of two pro~nent bands at 9.0 and 11.0 nm. Fraction III has a slight band in the region of 7.2 nm but the majority of apolipoprotein A-I appears to migrate near the bottom of the gel, suggesting the presence of lipid-poor apolipoprotein A-I. The latter material is also in evidence in the total d II 1.21 g/ml fraction.
Fig. 5. Electron
micrographs of fraction be vesicular particles
The protein composition of the Superose column fractions was analyzed by 10% SDS-PAGE and the results are shown in Fig. 4. The major Coomassie staining band in each fraction co-migrates with apolipoprotein A-I (28.0 kDa); however, fractions I and II have an additional doublet band (approximate molecular weights 68.2 and 77.6 kDa) which represents 15% of the staining material. Western blots of 10% SDS-PAGE slab gels were probed with apolipoprotein A-I antibody and all bands stained positively for apolipoprotein A-I (data not shown), thus indicating that the doublet bands may represent aggregated and/or denatured forms of apolipoprotein A-I. The electron microscopic structure of the Superose column fractions are summarized in Fig. 5. Fraction I (Fig. 5A) contains numerous large vesicular and/or discoidal structures that range from 60-260 nm in size; some discs 18-20 nm in diameter are also present. Fraction II (Fig. 5B) contains discoidal particles with a mean diameter of 14.2 f 2.6 S.D. nm (4.2 f 0.4 nm thick); some are seen en face in the micrograph although most are in rouleaux. No identifiable structures were seen in fraction III, undoubtedly because the lipid-poor form of apolipoprotein A-I is not readily resolved by negative staining. Since almost 50% of the apolipoprotein A-I protein floating at d s 1.21 g/ml is in fraction II, the major apolipoprotein A-I-lipid complex is a relatively small, discoidal structure. The diameters of the discoidal particles determined by electron microscopy are larger than those estimated by gradient gel electrophoresis (Fig. 2) because the latter sizes assume a spherical particle morphology (i.e., standard globular proteins are used to calibrate the gels). The compositions of the isolated apolipoprotein A-Ilipid complexes are seen in Table I. The protein-to-lipid ratio increases with decreasing particle size. Cholesterol
I (A) and fraction II (B) complexes isolated by column chromatography. Large numbers of what appear isolate in fraction I, while those in fraction II are primarily discs. Bar represents 100 nm.
to
15 TABLE I composition (weight percent) of ~p~iipoprotei~-bipedcomplexes isointed by Superose column chromatography
Fraction
Protein
% Total weight phospholipid
Cholesterol
d I 1.21 g/ml
51.3 42.8 53.1 93.5
18.8 32.9 32.8 6.5
29.9 24.4 14.2 n.d. a
Fraction I Fraction II Fraction III a n.d., not detected.
content decreases as percentage protein increases; however, no cholesterol was detected in fraction III which is a lipid-poor fraction. Cholesterol associated with the apolipoprotein A-I-lipid complexes was all in the unesterified form which is consistent with the discoidal and vesicular morphology of the particles. In order to assess whether the apolipoprotein A-Ilipid complexes were formed with a specific number of apolipoprotein A-I molecules per particle, apolipoprotein A-I in fraction I and fraction II was crosslinked with dimethylsuberimidate and analyzed by SDS-PAGE (Fig. 6). Fraction I, which consists of vesicles and large discs, contains mainly six to seven apolipoprotein A-Is per particle. Fraction II, which contains primarily two forms of discoidal particles (Fig. 3), one that bands at 9 nm and the other at 11 nm, possesses primarily particles with three and four apolipoprotein A-Is. The former are most likely associated with the smaller-sized complex while the latter are probably associated with the larger particles since reported in vitro studies [17,18] with apolipoprotein A-I-phospholipid analogs have shown
Tetramer Trlmer Dimer
that complexes with RF values similar to those in fraction II contain two to four molecules of apolipoprotein A-I. Fraction III was not crosslinked because there was insufficient material for this purpose. Fraction III material banding at 7.1 nm is approx. 70000 molecular weight and thus must be at least a dimer of apo~poprotein A-I. The predominant apolipoprotein A-I-lipid complex produced by transfected Chinese hamster ovary cells is a discoidal particle similar in morphology and composition to discoidal HDL found in lecithin : cholesterol acyltransferase-deficient plasma [19], monkey liver perfusates [20] and Hep G2-conditioned medium [lo]. All of these discoidal structures have been shown to be substrates for lecithin : cholesterol acyltransferase [16, 19,211. In order to test whether apolipoprotein A-I-lipid complexes produced by transfected Chinese hamster ovary cells also serve as substrates for this enzyme, two samples of d < 1.21 g/ml fractions were incubated for 18 h in the presence of active lecithin : cholesterol acyltransferase and conversion of unesterified cholesterol to esterified cholesterol was determined. Control apolipoprotein A-I-lipid complexes incubated at 4’C in the absence of lecithin : cholesterol acyltransferase showed no conversion; however, samples incubated with active enzyme converted 18.9 and 21.1% of unesterified cholesterol to cholesteryl ester. The formation of cholesteryl ester was coupled with the transformation of discoidal particles into heterogeneous-sized (12.9 & 3.4 nm), core-cont~~ng particles (Fig. 7A and B). The histogram of particle size distribution (Fig. 7C) of the lecithin : cholesterol acyltransferase-converted products suggests that there may be three populations of particles. A major peak occurs between 11-12 nm which corresponds to the size range of HDL,, in mature plasma [22], a minor peak occurs at 8-9 nm corresponding to plasma HDL,, and HDL,, f22], and a major peak appears at 13-15 nm which is considerably larger than normal plasma HDL. The large vesicular structures present in unincubated samples do not appear to be altered during incubation. Discussion
Monomer
Std
Fr I
Fr II
Fig. 6. SDS-PAGE (4-30% gradient gel) pattern of crosslinked apolipoprotein A-I in fraction I (Fr I) and fraction II (Fr II). Samples were electrophoresed at two concentrations: Fr I at 15 and 7.5 pg protein per lane, and Fr II at 23 and 11.5 pg per lane. Crosslinked, purified plasma apolipoprotein A-I was used as the standard (Std).
It has previously been demonstrated that apolipoprotein A-I secreted by Chinese hamster ovary cells transfected with the human apo~poprotein A-I gene secrete fully processed apolipoprotein A-I that possesses little pro-apolipoprotein A-I [4]. This is in contrast to apolipoprotein A-I secreted by the human hepatoma line, Hep G2, which during a 24-hr incubation period produces apolipoprotein A-I which is 50% pro-apolipoprotein A-I [23]. The latter, however, does not prevent apolipoprotein A-I-lipid complex formation, thus, in hepatoma cell media, 30-40% of secreted apolipoprotein A-I is in the lipid associated form [23,24] whereas
16
30 i s! r p"
20 -
z t : 10 -
0 5.0
C
7.5
I 10.0
12.5
I 15.0
Diameter
17.5
I 20.0
22.5
2
.O
(nm)
Fig. 7. Electron micrograph of d < 1.235 g/ml apohpoprotein A-I-lipid complexes incubated at 4°C in the absence (A) and at 37°C in the presence (B) of purified 1ecithin:cholesterol acyltransferase. The smooth histogram (C) of particle diameters resulting after incubation with active lecithin : cholesterol acyltransferase suggests that particle size is heterogeneous and as many as three distinct components may be present.
in Chinese hamster ovary cell medium only 16% is lipid associated. Compared with hepatoma cells, transfected Chinese hamster ovary cells secrete high levels of apolipoprotein A-I (7% of total protein compared to 1% for hepatomas); such high apolipoprotein A-I secretory rates may outstrip the cellular lipoprotein assembly process if one assumes that the particles are assembled intracellularly. If, on the other hand, apolipoprotein A-I-lipid assembly occurs extracellularly, a major constraint might be the availability of membrane phospholipids and cholesterol that would be expected to contribute to complex formation. Apolipoprotein A-I-lipid complexes isolated from Chinese hamster ovary cell media are primarily dis-
coidal or ‘nascent’ HDL. Their morphology and size distribution is similar to discoidal HDL isolated from patients with lecithin : cholesterol acyltransferase deficiency where apolipoprotein A-I-containing discs have diameters of 11-28 nm [19]. By nondenaturing gradient gel analysis there is great similarity between that of Chinese hamster ovary cell nascent particles and nascent HDL particles isolated from Hep G2 medium [10,25]. A major difference lies in the presence of a particle banding at 7.4 nm in Hep G2 HDL and its absence in Chinese hamster ovary cell medium. The 7.4 nm particle from Hep G2 medium contains a core of cholesteryl ester [25] which is either derived from cellular pools of cholesteryl ester or by the action of lecithin : cholesterol
17 acyltransferase which is present in low levels in the medium [26]. Neither of these mechanisms appears to be operative in Chinese hamster ovary cell incubations. We had previously shown that 3T3 cells transfected with the human apolipoprotein A-I gene also secrete apolipoprotein A-I [5]. This cell line, however, required preincubation with phospholipid-rich lipid emulsions in order to obtain apolipoprotein A-I-lipid complexes. Nondenaturing gradient gel analysis suggests that 3T3 cells produce discoidal particles that band in the region between 9-12 nm [5] which is similar to the region in which Chinese hamster ovary cell Fraction II apolipoprotein A-I-lipid complexes have major bands. However, Western blots for apolipoprotein A-I also suggested the presence of a small, 7.4 nm particle in 3T3 cell media. Since not enough material was available for lipid analyses of complexes formed by 3T3 cells, it is unknown whether the complexes are lipid core-containing particles or whether they are unusually small, polarlipid complexes. Several in vitro studies have shown that apolipoprotein A-I-phospholipid or phospholipid plus cholesterol complexes formed by cholate dialysis produce discrete, reproducible structures stabilized by specific numbers of apolipoprotein A-I molecules [17,18,27,28]. In the present study, Fraction II particles banding at 9.0 nm isolated from Chinese hamster ovary cell medium fall within the size range of class 3 apolipoprotein A-I-egg yolk phosphatidylcholine complexes described by Nichols et al. [17,18]. Class 3 in vitro model complexes generally contained only two apolipoprotein A-I molecules per particle, whereas similar-sized discoidal particles from Chinese hamster ovary cell medium contained three apolipoprotein A-I molecules. Although speculative, these differences may, in part, be the result of differences in types of phospholipids associated with the complexes where, in model systems, known purified phospholipids such as phosphatidylcho~ne were employed, whereas in Chinese hamster ovary cell complexes, phospholipid composition is unknown but is likely to be a combination of cell-derived phospholipids. The latter is presently under investigation. The somewhat larger Fraction II discs banding at 11.0 nm are similar to the class 4 and 5 complexes 1171 and, in agreement with this size, contain 4 apolipoprotein A-Is, Fraction I particles obtained from Chinese hamster ovary cell cultures fall within the region occupied by classes 7 and 8 in artificial complexes that contain five or more apo~poprotein molecules j17,28]. Discoidal HDL from Chinese hamster ovary cells have, on nondenaturing gradient gels, peaks in common with nascent HDL isolated from Hep G2 cell medium [lo] and lecithin : cholesterol acyltransferase-deficient plasma (Forte, T.M., unpublished observations). The presence of recurring, distinct size classes of apolipoprotein A-Icontaining discoidal complexes from disparate sources
including those from Chinese hamster ovary cells suggests that apolipoprotein A-I interaction with lipids plays an important role in determining size. Discrete molecular entities with specific numbers of apolipoprotein A-I are formed rather than a continuum of particle sizes. Nascent HDL from lecithin : cholesterol acyltransferase-deficient plasma [29], Hep G2 medium [16], and in vitro phospholipid-cholesterol-apolipoprotein A-I discoidal complexes [18,27] are excellent substrates for lecithin : cholesterol acyltransferase. HDL from plasma of lecithin : cholesterol acyltransferase-deficient patients undergo transesterification of cholesterol to cholesteryl ester which is associated with the transformation of 11.0-28.0 nm discs to spheres 10.7 nm diameter [29]. The mean particle diameter of transformed particles from Chinese hamster ovary cell products (12.9 nm) is somewhat larger than that reported for lecithin : cholesterol acyltransferase deficiency. This may in part be due to the use of patient’s total plasma and partially purified lecithin : cholesterol acyltransferase in these early incubation studies. Incubations involving whole plasma and partially purified lecithin : cholesterol acyltransferase would possess not only lipid transfer protein activity but also additional sources of free cholesterol for the lecithin : cholesterol acyltransferase reaction which may result in a more complete transfo~ation of nascent HDL. The large vesicular structures appearing in Chinese hamster ovary media did not appear to serve, in great degree, as substrates for lecithin : cholesterol acyltransferase since they persisted after 18-hr incubation with purified lecithin : cholesterol acyltransferase. Lack of lecithin : cholesterol acyltransferase-driven transformation of vesicles probably denotes a less favorable conformation of apolipoprotein A-I for activating lecithin : cholesterol acyltransferase activity. The poor reactivity of vesicular apolipoprotein A-I-phospho~pid substrates has previously been demonstrated by Chajek et al. [30], who suggested that the aqueous interior of vesicles, as opposed to the hydrophobic one in the discs, may be less favorable for incorporation of cholesteryl esters. It has also been suggested [31,32] that the higher apolipoprotein A-I-to-lipid molar ratio in discs as opposed to vesicles may provide a more favorable apolipoprotein A-I conformation for lecithin : cholesterol acyltransferase reactivity. In summary, the apolipoprotein A-I-lipid complexes isolated from media of Chinese hamster ovary cells transfected with the human apo~poprotein A-I gene form discoidal HDL particles that are indistinguishable from nascent HDL isolated from plasma lecithin : cholesterol acyltransferase-deficient patients and those isolated from Hep G2 culture media. The discoidal Chinese hamster ovary cell media apolipoprotein A-I complexes are also substrates for lecithin : cholesterol acyltrans-
18 ferase, thus suggesting that the transfected cells produce functional nascent HDL particles. Acknowledgements
We wish to thank Dr. Virgie Shore for her assistance on quantitation of apolipoprotein A-I, and Mary Lou Kurtz for preparation of the manuscript. This work was supported by NIH Program Project Grant HL 18574 and NRSA Training Grant HL 07279 from the National Heart, Lung, and Blood Institute of the National Institutes of Health, and was conducted at the Lawrence Berkeley Laboratory (Department of Energy contract DE-AC03-76SF00098 to the University of California). References
4
9 10 11
Miller, G.J. and Miller, N.E. (1975) Lancet i, 16-19. Gordon, T., Caste& W.P., Hjortland, M.C. and et al. (1977) Am. J. Med. 62, 707-714. Miller, N.E., La Ville, A. and Crook, D. (1985) Nature 314, 109-111. Mallory, J.B., Kushner, P.J., Protter, A.A., Cofer, C.L., Appleby, V.L., Lau, K., Schilling, J.W. and Vigne, J.-L. (1987) J. Biol. Chem. 262, 4241-4247. Lamon-Fava, S., Ordovas, J.M., Mandel, G., Forte, T.M., Goodman, R.H. and Schaefer, E.J. (1987) J. Biol. Chem. 262, 8944-8947. FennewaId, S.M., Hamilton, R.L., Jr., and Gordon, J.I. (1988) J. Biol. Chem. 263, 15568-15577. Ruiz-Opazo, N. and Zanms, V.I. (1988) J. Biol. Chem. 263, 1739-1744. Nichols, A.V., Blanche, P.J. and Gong, E.L. (1983) in CRC Handbook of Electrophoresis (Lewis, L. and Opplt, J., ed.), vol. III, pp. 29-47, CRC Press, Boca Raton, FL. Weber, K. and Osbom, M. (1969) J. Biol. Chem. 24444064412. Thrift, R.N., Forte, T.M., Cahoon, B.E. and Shore, V.G. (1986) J. Lipid Res. 27, 236-250. Forte, T.M. and Nordhausen, R.W. (1986) Methods Enzymol. 128, 442-457.
12 Markwell, M.A.K., Hass, S.M., Bieber, L.L. and Tolbert, N.E. (1978) Anal. B&hem. 87, 206-210. 13 Bartlett, G.R. (1959) J. Biol. Chem. 531, 233-236. 14 Hindriks, R.F., Wolthers, B.G. and Groen, A. (1977) Clin. Chim. Acta 74, 207-215. 15 Swaney, J.B. and O’Brien, K. (1978) J. Biol. Chem. 253,7069-7077. 16 McCall, M.R., Nichols, A.V., Blanche, P.J., Shore, V.G. and Forte, T.M. (1989) J. Lipid Res. 30, 1579-1589. 17 Nichols, A.V., Gong, E.L., Blanche, P.J. and Forte, T.M. (1983) B&him. Biophys. Acta 750, 353-364. 18 Nichols, A.V., Blanche, P.J., Gong, E.L., Shore, V.G. and Forte, T.M. (1985) Biochim. Biophys. Acta 834, 285-300. 19 Mitchell, CD., King, W.C., Applegate, K.R., Forte, T., Glomset, J.A., Norum, K.R. and Gjone, E. (1980) J. Lipid Res. 21,625-634. 20 Johnson, F.L., Babiak, J. and Rudel, L.L. (1986) J. Lipid Res. 27, 537-548. 21 Babiak, J., Tamachi, H., Johnson, F.L., Parks, J.S. and Rudel, L.L. (1986) J. Lipid Res. 27, 1304-1317. 22 Blanche, P.J., Gong, E.L., Forte, T.M., and Nichols, A.V. (1981) Biochim. Biophys. Acta 665, 408-419. 23 Forte, T.M., Nichols, A.V., Sehnek-Halsey, J., Caylor, L. and Shore, V.G. (1987) Biochim. Biophys. Acta 920, 185-194. 24 Forte, T.M., McCall, M.R., Knowles, B.B. and Shore, V.G. (1989) J. Lipid Res. 30, 817-829. 25 McCall, M.R., Forte, T.M. and Shore, V.G. (1988) J. Lipid Res. 29, 1127-1137. 26 Chen, C.-H., Forte, T.M., Cahoon, B.E., Thrift, R.N. and Albers, J.J. (1986) B&him. Biophys. Acta 877, 433-439. 21 Nichols, A.V., Gong, E.L., Blanche, P.J., Forte, T.M. and Shore, V.G. (1987) J. Lipid Res. 28, 719-732. 28 Gong, E.L., Nichols, A.V., Forte, T.M., Blanche, P.J. and Shore, V.G. (1988) Biochim. Biophys. Acta 961, 73-85. 29 Norum, K.R., Glomset, J.A., Nichols, A.V., Forte, T., Albers, J.J., King, W.C., Mitchell, C.D., Applegate, K.R., Gong, E.L., Cabana, V. and Gjone, E. (1975) Stand. J. Clin. Lab. Invest. 142, 31-55. 30 Chajek, T., Aron, L. and Fielding, C.J. (1980) Biochemistry 19, 3673-3677. 31 Matz, C.E. and Jonas, A. (1982) J. Biol. Chem. 257, 4541-4546. 32 Hefele WaId, J.E. and Jonas, A. (1989) Arteriosclerosis 9, 715a.
