©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation and Immunolocalization of Acyl-Coenzyme A:Cholesterol Acyltransferase in Mammalian Cells as Studied with Specific Antibodies (*)

(Received for publication, August 31, 1995; and in revised form, September 29, 1995)

Catherine C. Y. Chang (1) Jun Chen (1) Matthew A. Thomas (1) Dong Cheng (1) Veronica A. Del Priore (1) Roger S. Newton (2) Michael E. Pape (2) Ta-Yuan Chang (1)(§)

From the  (1)Dartmouth Medical School, Department of Biochemistry, Hanover, New Hampshire 03755 and (2)Warner-Lambert, Atherosclerosis Research, Division of Therapeutics, Parke-Davis Pharmaceutical Research Division, Ann Arbor, Michigan 48105

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the formation of intracellular cholesterol esters in various tissues. We recently reported the cloning and expression of human macrophage ACAT cDNA. In the current study, we report the production of specific polyclonal antibodies against ACAT by immunizing rabbits with the recombinant fusion protein composed of glutathione S-transferase and the first 131 amino acids of ACAT protein. Immunoblot analysis showed that the antibodies cross-reacted with a 50-kDa protein band from a variety of human cell lines. These antibodies immunodepleted more than 90% of detergent-solubilized ACAT activities from six different human cell types, demonstrating that the 50-kDa protein is the major ACAT catalytic component in these cells. In multiple human tissues examined, the antibodies recognized protein bands with various molecular weights. These antibodies also cross-reacted with the ACAT protein in Chinese hamster ovary cells. Immunoblot analysis showed that the ACAT protein contents in human fibroblast cells, HepG2 cells, or Chinese hamster ovary cells were not affected by sterol in the medium, demonstrating that the main mechanism for sterol-dependent regulation of ACAT activity in these cells is not change in ACAT protein content. As revealed by indirect immunofluorescent microscopy, the ACAT protein in tissue culture cells was located in the endoplasmic reticulum. This finding, along with earlier studies, suggests that cholesterol concentration in the endoplasmic reticulum may be the major determinant for regulating ACAT activity in the intact cells.


INTRODUCTION

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) (^1)is an intracellular enzyme that catalyzes the formation of cholesterol esters from cholesterol and fatty acyl coenzyme A. ACAT activities are present in a variety of tissues, including liver, intestines, adrenal gland, and aorta. ACAT participates in various physiological processes including cellular cholesterol homeostasis, hepatic lipoprotein assembly, and dietary cholesterol absorption. Under pathological conditions, cholesterol esters produced as ACAT reaction products accumulate as lipid droplets in macrophages and eventually cause foam cell formation in the atherosclerotic plaques (for review, see Chang et al. (1994) and references therein). ACAT is an integral membrane protein. Due to its sparse presence and its susceptibility to inactivation by various detergents, the enzyme has never been purified to homogeneity from any species. Recently, we reported the molecular cloning of human macrophage ACAT cDNA by functional complementation of a cell mutant (AC29) deficient in ACAT activity (Chang et al.(1993) and for review, see Chang et al.(1994)); mutant line AC29 was earlier isolated from mutagenized Chinese hamster ovary (CHO) cells (Cadigan et al., 1988, 1989). Expression of this cDNA in the form of recombinant baculovirus in insect cells, which are devoid of endogenous ACAT-like activity, resulted in abundant ACAT activity. The catalytic and regulatory properties of the insect cell-expressed ACAT activities were found to be essentially identical to those expressed in human tissue culture cells, thus demonstrating that ACAT cDNA encodes an essential catalytic component of an ACAT enzyme (Cheng et al., 1995).

An antipeptide antibody raised against a synthetic decapeptide deduced from the open reading frame of ACAT cDNA was produced (Cheng et al., 1995). This antibody was able to recognize the ACAT protein expressed in insect cells; however, our unpublished experiments showed that, due to its low titer, it failed to serve as a useful reagent in various human cells, which only expressed the ACAT protein at much lower levels. In addition, despite repeated attempts, we were not able to produce high titer anti-peptide antibodies against several other antigenic regions within the ACAT protein sequence. In the current study, we created a recombinant protein that is composed of bacterial glutathione S-transferase fused in frame with the first 131 amino acids of the predicted ACAT protein. We expressed this fusion protein in bacteria, purified it by SDS-PAGE, used the homogeneous protein to immunize the rabbits, and obtained high titer, specific antibodies against ACAT. These antibodies were affinity-purified and were employed in various immunological procedures to study ACAT in various human cells and in CHO cells. The results obtained demonstrate that in mammalian cells, the main mechanism for sterol-dependent regulation of ACAT activity is not by modulating the ACAT protein content. Additional results show that the ACAT protein is located in the endoplasmic reticulum. These results, along with earlier findings, suggest that the cholesterol concentration in the ER may be a major determinant for regulating ACAT activity in the intact cells.


EXPERIMENTAL PROCEDURES

Materials

CHO cells, human skin fibroblast (Hf) cells, HepG2 cells, Caco-2 cells, A293 cells, A431 cells, melanoma A2058 cells, and human breast cancer MDA231 cells were routinely grown at 37 °C in a CO(2) incubator as monolayers in either F-12 (for CHO cells) or DMEM (for other cells) supplemented with 10% (except for Caco-2, which was 15%) fetal bovine serum (FBS). Cell detachment occurred through treatment with 0.25% trypsin solution (Life Technologies, Inc.) at 37 °C. THP-1 cells and lymphoblast cells were grown as suspension cultures in RPMI 1640 medium with 10% FBS. Media contained gentamicin as antibiotics. Hf cells were obtained as described previously (Cadigan and Chang, 1988); melanoma cells (A2058) and breast cancer cells (MDA231) were generous gifts from Dr. Connie Binckerkoff at Dartmouth Medical School. All other cell lines were obtained from ATCC. AC29-K1 cell is a stable transfectant cell clone expressing high level of human ACAT activity. It was cloned from populations of AC29 cells stably transfected with the plasmid pcDNA1-K1 (sense); the insert cDNA K1 encompasses the entire ACAT protein coding region (see Table IV in Chang et al.(1993)). Human LDL and delipidated FBS were prepared as described previously (Cadigan et al., 1988b; Chin and Chang, 1981). Egg phosphatidylcholine (PC) (Type XI), bovine serum albumin (BSA), control rabbit immunoglobulin (IgG), and most of the chemicals were from Sigma. The following reagents were obtained from the following companies: sodium cholate and deoxycholate, Calbiochem; 25-hydroxycholesterol, Steraloids; FITC-Guard, Testog; the fluorescent carbocyanine dye DiOC(6), Polysciences (it was stored as 0.5 mg/ml stock solution in alcohol in the dark at 4 °C); Texas red goat anti-rabbit antibody, Vector; prestained protein molecular weight markers, Life Technologies, Inc., Amersham Corp., and Bio-Rad; Immobilon-P membranes, Millipore; goat anti-rabbit immunoglobulins, Bio-Rad; the enhanced chemiluminescence reagent, Amersham Corp.; SDS and the AminoLink Sepharose affinity column, Pierce; TranS-label, ICN. The antisera against the GST-ACAT (1-131 amino acids) fusion protein were raised in rabbits by Cocalico Co. in Philadelphia, PA. The GST gene fusion system and protein A-Sepharose CL-4B were purchased from Pharmacia Biotech Inc. The human multiple tissue Western blot I (6.5-100 kDa), containing about 75 µg of protein/lane, was purchased from Clontech.

Methods

Standard molecular biology techniques (Sambrook et al., 1989) were used.

ACAT Enzyme Assay by the Reconstituted Vesicle Assay

This was performed essentially as described previously (Cadigan and Chang, 1988), with minor modifications. Cells were harvested by the hypotonic shock and scrapping method (Chang et al., 1981); the protein concentration of the broken homogenates was kept at 2-4 mg/ml. To solubilize the enzyme, a deoxycholate/PC stock solution in buffer A (50 mM Tris, 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, at pH 7.8) was added to the cell homogenates to obtain a final concentration of 10 mg/ml deoxycholate and 2 mg/ml PC (instead of 20 mg/ml deoxycholate and 4 mg/ml PC as used previously; with extracts prepared from various human cells, control experiments showed that 10 mg/ml deoxycholate and 2 mg/ml PC caused less ACAT enzyme inactivation while providing equally efficient ACAT enzyme solubilization); 15- (or 30-) µl aliquots of this solubilized extracts were then diluted into 100 (or 200) µl of cholesterol/PC vesicles with the cholesterol/PC molar ratio at 0.3 (cholesterol, 1.5 mg/ml; PC, 10 mg/ml). The cholesterol/PC vesicle was prepared according to the cholestyramine resin method (Shi et al., 1989). The cholestyramine was preequilibrated at pH 7.8 by washing twice with buffer A.

Cholesterol Esterification Rate in Intact Cells

[^3H]Oleate pulse in intact cells was carried out as described previously (Chang et al., 1986; Cadigan et al., 1988). The pulse time was 30 min. Assays were done in duplicate flasks.

Production of Antibodies against ACAT

In order to generate antibodies specific for ACAT, the N-terminal part of the ACAT protein (amino acid residues 1-131, about 14 kDa) was expressed as a fusion adduct with GST. To create the GST-ACAT construct, mutagenesis experiments were carried out (by using the Clontech transformer site-directed mutagenesis kit) with the human ACAT cDNA, 1.7 kilobases (Chang et al., 1993). Nucleotides 1397-1399 (ATG) were converted to CTA to create a SpeI site; nucleotides 1790-1792 (CTG) were converted to TTC to create an EcoRI site. The resultant plasmid DNA was digested by SpeI and filled in by the Klenow enzyme, and EcoRI digestion followed. The released fragment, which codes for ACAT protein amino acid residues 1-131, with the first amino acid being leucine instead of methionine, was gel-purified and then cloned into the GST fusion vector pGEX-3X (Pharmacia) to generate the in-frame fusion construct. The GST-ACAT fusion protein was induced in Escherichia coli by isopropyl-1-thio-beta-D-galactopyranoside addition. The resultant fusion protein (about 40 kDa, that constituted about 5-10% of total E. coli cellular protein) was purified by SDS-PAGE and electroeluted in buffer containing 0.2% SDS. About 340 µg of the purified protein was sent to Cocalico Co. to generate antiserum in two rabbits. The antigen was injected into rabbits as a solution in 0.2% SDS. The antisera were purified via GST-ACAT fusion protein affinity column chromatography. The affinity column was prepared by using the AminoLink Sepharose affinity column (Pierce), following the procedures described in the Pierce ImmunoPure Ag/Ab booklet (no. 44890). Around 2.8 mg of purified GST-ACAT protein was used for coupling the protein to the column. The coupling efficiency was about 50%. The column was used repeatedly for purifying the IgG from the antisera according to procedures as described by Pierce. The antibodies were eluted off the column with glycine buffer at pH 2.8. The affinity-purified antibodies were stored at 4 °C in 0.1 M Tris-glycine buffer, pH 7, under sterile conditions.

PAGE and Immunoblotting Analysis

PAGE was performed essentially as described previously (Cheng et al., 1995). Samples were solubilized in buffer A containing 5% SDS without heating at elevated temperature. Thiol-reducing agent was not included in SDS-PAGE analysis. The primary antibodies used were the affinity-purified IgG against ACAT with a final concentration of 0.28 µg/ml in 1% milk.

Immunodepletion Experiments

Cells were harvested and solubilized as described under ``ACAT enzyme assay.'' The solubilized extract was held at 4 °C for 5 min and then centrifuged at 10,000 times g for 10 min to isolate the solubilized cell lysate from the nucleus and other particulate materials. The cell lysate (usually 450 µl), was incubated with 50 µl of 0.1 M Tris-glycine buffer, pH 7, containing indicated amount of primary antibodies in a microcentrifuge tube with constant shaking for 2 h at 4 °C. The immune complexes were mixed further for 1 h with 100 µl of protein A-Sepharose (diluted 1:1 with buffer A). The immunodepleted lysates and the immunoprecipitates were separated by centrifugation at 14,000 times g for 10 s. For SDS-PAGE, immune complexes were dissociated by adding 60 µl of 10% SDS prepared in gel-loading buffer without thiol-reducing reagent.

S Metabolic Labeling and Immunoprecipitation

Melanoma, AC29, and AC29-K1 cells were seeded in 6-well plates at approximately 1 times 10^5 cells/well. Cells were maintained in DMEM or F-12 medium with 10% FBS for 2 days. To label the cells, cells were washed twice with PBS and then incubated with 1 ml/well of methionine-free DMEM with 10% dialyzed FBS for 1 h at 37 °C. 100 µCi of [S]methionine (1152 Ci/mmol, TranS-label), or 10 µl of 3 mg/ml of cold methionine (for the mock-labeled cells) was added to each well. After 2 h in a 37 °C CO(2) incubator, the medium was removed, and the cells were washed twice with PBS and were lysed by the addition of 500 µl of 1% deoxycholate in buffer A. Cell lysates were transferred into microcentrifuge tubes. To preclear the lysate, 50 µl of protein A-Sepharose/buffer A (1:1) was added per tube, and the mixture was incubated at 4 °C for 30 min and then spun in a microcentrifuge at 4 °C for 2-5 min. The supernatants were transferred to new tubes. Aliquots were taken to measure the trichloroacetic acid-precipitable radioactivities. From the supernatants, equal amounts of radioactivity were aliquoted to new tubes. The volume of each tube was adjusted to 450 µl by adding 1% deoxycholate in buffer A. 2 µg of DM10 IgG was then added to each tube, and the mixture was incubated at 4 °C for 30-60 min. 20 µl of protein A-Sepharose/buffer A (1:1) was then added, and the mixture was incubated for another 30-60 min at 4 °C. The beads were collected by spinning the mixture in the microcentrifuge for 2 min and removing the supernatants. The beads were washed 3-5 times with 1% deoxycholate in buffer A. Proteins bound to the beads were extracted by the addition of 15-20 µl of 10% SDS to the beads and incubating the mixture at 37 °C for 15 min, followed by the addition of 15-20 µl of 2 times SDS-gel sample buffer and continuing the incubation at 37 °C for another 15-30 min. After the second incubation, the beads were removed by centrifugation, and the supernatants were split into two equal halves and loaded onto two 10% gels for SDS-PAGE analysis. One gel was fixed, dried, and exposed to x-ray film. The other gel was employed for immunoblot analysis.

Immunolocalization of ACAT by Confocal Fluorescent Microscopy

Melanoma cells were grown in DMEM supplemented with 10% FBS. For fixation and staining, the procedures described by Harlow and Lane(1988) were followed with minor modification. Cells were seeded on 22-mm square glass coverslips 1-2 days before staining. Cells were washed with PBS twice and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. The fixed cells were washed with PBS twice, permeabilized with cold methanol (prechilled at -20 °C) for 2 min at room temperature, and then washed with PBS and blocked with 1% BSA in PBS for 20-40 min. Cells were stained with 100 µl of 0.4 µg/ml IgG against ACAT (DM10) (in 1% BSA, 0.02% NaN(3), PBS) for 1 h followed by washing with 1% BSA, 0.02% NaN(3), PBS 4 times. Incubation of the secondary IgG (100 µl of 1 µg/ml Texas red goat anti-rabbit IgG) were carried out for 30-60 min, followed by four PBS washes. Cells were stained with 10 ng/ml of DiOC(6) in PBS for 2 min (Terasaki et al., 1984) and then washed with PBS 4 times. The coverslips were mounted, scanned, and viewed with Bio-Rad Confocal microscope (Type MRC 1000); the optical section chosen was at 0.36 µm/section.


RESULTS

Characterization of the Antibodies against ACAT by Immunoblot Analysis

As detailed under ``Experimental Procedures,'' a fusion protein composed of bacterial glutathione S-transferase and the first 131 amino acids of the predicted ACAT protein was produced, purified by SDS-PAGE, and injected into two rabbits. Both rabbits eventually produced high titer polyclonal antibodies against ACAT (designated as DM9 and DM10 antibodies). These antibodies were affinity-purified using the GST-ACAT fusion protein as the affinity ligand. Previously, immunoblot analysis using an anti-peptide antibody against a synthetic decapeptide within the ACAT protein sequence showed that the apparent molecular weight of the Sf9 cell expressed ACAT protein in SDS-PAGE is around 50 kDa (the major band) or 56 kDa (the minor band) (Cheng et al., 1995). The same result was obtained when the DM9 antibodies were used in immunoblot analysis (Fig. 1, lane 10). The apparent size(s) of the ACAT protein deviated significantly from its estimated size of 64.8 kDa deduced from the open-reading frame analysis of ACAT cDNA. As discussed earlier (Cheng et al., 1995), protein sequence analysis revealed that ACAT protein is a hydrophobic protein with a strongly basic isoelectric point (9.78). These unusual features may cause the protein to migrate abnormally in SDS-PAGE. The specificity of the fusion protein antibodies is evident from the observation that they did not react with any protein in cell lysates prepared from ACAT-deficient mutant AC29 cells (lanes 3 and 8). In contrast, they reacted with a rather broad protein band with an apparent size of about 50 kDa in lysates prepared from transfectant AC29-K1 cells, which stably expressed high levels of human ACAT activity in CHO cells (Chang et al., 1993) (Table 1). In addition, these antibodies specifically reacted with a 50-kDa protein band in lysates prepared from six different human cell lines (Fig. 1, lanes 1, 2, 4-7). In other experiments not shown, the same result was obtained in lysates of Epstein Barr virus-transformed human lymphoblast cells and lysates of phorbol ester-activated THP-1 macrophage cells. On the whole, the relative intensities of the 50-kDa protein band (Fig. 1) correlated well with the relative ACAT activities (measured by the reconstituted vesicle assay) found in extracts of these cells (Table 1).


Figure 1: Immunoblot of ACAT protein from various cell lines. Various indicated cell lines were grown as described under ``Experimental Procedures.'' Aliquots of 300 µg of protein/lane were loaded onto lanes 1-8. For lane 9, 100 µg of protein was loaded. For lane 10, 5 µg of protein was loaded. Samples in lanes 1-9 were freshly prepared from SDS lysates of cell monolayers. The sample in lane 10 was prepared from frozen cell pellets of Sf9 cells infected with recombinant ACAT virus (Cheng et al., 1995). To prepare fresh cell extracts for the immunoblotting experiment, cells were seeded at a density of 0.75 times 10^6 cells/well in 6-well dishes for 48 h. To harvest, cells were rinsed in 4 times 5 ml of PBS at 4 °C and dissolved in 100 µl of 7% SDS, followed by incubation of the cell lysates at 37 °C for 30 min. After protein concentration were determined, samples were loaded for SDS-PAGE analysis. The immunoblotting analysis was performed as described under ``Experimental Procedures.'' The autoradiogram presented here is representative of three independent experiments. The same results were obtained with or without thiol reducing agents present in SDS-PAGE analysis.





The size of the ACAT protein (the 50-kDa protein band) described here and previously (Cheng et al., 1995) was based on the prestained protein molecular weight standards from Bio-Rad. We have found that if the prestained molecular weight standards from a different company (Life Technologies Inc. or Amersham Corp.) were used, the apparent size of the ACAT protein was considerably smaller (around 45 kDa). For convenience, this band is operationally designated as the 50 kDa band. In the infected Sf9 cell lysates, in addition to the 50/56-kDa protein bands, the antibodies also reacted with the 110 and the 220 kDa bands (Fig. 1, lane 10; values in sizes were only approximate estimations). These additional bands, presumably as a result of ACAT protein dimerization or tetramerization, were previously detectable in infected Sf9 cell lysates by an ACAT anti-peptide antibody (Cheng et al., 1995). We found that these immunoreactive bands, as well as additional bands with even higher molecular weight(s), could also be detected as minor bands in lysates from various human cell lines. The same results were obtained with or without thiol reducing agents present in SDS-PAGE analysis. These higher molecular weight bands could be minimized by preparing fresh cell extracts in SDS at room temperature and then performing SDS-PAGE analysis within 1 day of sample preparation.

In lysates prepared from A431 cells, the DM9 antibodies reacted with an additional protein band at about 60 kDa (lane 2) with less intensity. When the antibodies produced from a different rabbit (DM10 antibodies) were used, only the 50 kDa band was recognized in immunoblot analysis (two independent experiments). Therefore it is unlikely that the 60 kDa protein band in A431 cells maybe an isoform of ACAT protein; its identity is unknown at present. Other than this difference, the DM10 antibodies gave the same specific signal as the DM9 antibodies did in all the other cell lysates described in Fig. 1. For the rest of the experiments reported in this manuscript, only the DM10 antibodies were used. In lysates prepared from AC29-K1 cells, the DM9 antibodies reacted with an additional protein band with an apparent size of 35 kDa (Fig. 1, lane 9). The same result was reproducibly seen when DM10 antibodies were used. The existence of the 35 kDa band will be described further in Fig. 3and Fig. 4.


Figure 3: A, immunodepletion of ACAT activity from human melanoma cell lysates. The immunodepletion experiment was performed as described under ``Experimental Procedures.'' For each sample, 0.5 ml of cell lysates at 2 mg of protein/ml were immunodepleted with either nonspecific rabbit IgG (open circles) or IgG against ACAT (DM10) (closed circles) at various indicated quantities. The depleted lysates were aliquoted at 30 µl/assay for measuring ACAT activity remaining in the supernatants according to method described under ``Experimental Procedures.'' The values shown are the averages of duplicate assays from the same cell lysates, and vary within 10% of the mean. B, immunodepletion of ACAT activities from various cell lysates. Various cell lysates as indicated were prepared for the immunodepletion experiment and for ACAT activity measurement as described in A. C, immunoblot analysis after immunodepletion of AC29-K1 cell lysates. After the immunodepletion experiment described in B, 7% of the immunodepleted AC29-K1 cell lysates or 6% of the corresponding immunoprecipitated pellets per sample were used for immunoblot analysis according to ``Experimental Procedures.''




Figure 4: Immunoprecipitation of various [S]methionine-labeled cell lysates. Melanoma, AC29, and AC29-K1 cells were labeled with [S]methionine, or were mock-labeled with nonradioactive methionine. The cell lysates were mixed with buffer alone or were mixed with other lysates as indicated. To prepare the mixture, 300 µl of each cell lysate or buffer was aliquoted. Each labeled lysate contained 3.9 times 10^5 cpm/µl. The lysate mixtures underwent the immunoprecipitation process as described under ``Experimental Procedures.'' The immunoprecipitated samples were separated by 10% SDS-PAGE and were detected by immunoblot (A) or were analyzed by autoradiography (B). Lane 1, S-labeled melanoma cell lysate mixed with unlabeled AC29-K1 cell lysate; lane 2, S-labeled melanoma cell lysate mixed with S-labeled AC29 cell lysate; lane 3, S-labeled melanoma cell lysate mixed with 1% deoxycholate in buffer A; lane 4, S-labeled AC29-K1 cell lysate mixed with 1% deoxycholate in buffer A; lane 5, S-labeled AC29 cell lysate mixed with equal volume of 1% deoxycholate in buffer A; lane 6, unlabeled AC29-K1 cell lysate mixed with equal volume of 1% deoxycholate in buffer A were used for immunoprecipitation without including the primary antibody (DM10).



To find out whether the DM10 antibodies reacted with the ACAT protein in CHO cells, we prepared lysates from ACAT-deficient mutant AC29 cells (which expressed no ACAT activity in vitro) and from parental 25-RA cells (which expressed normal ACAT activity) (Cadigan et al., 1989). Fig. 2A shows that the DM10 antibodies reacted with a protein with an apparent size of 47 kDa (which is slightly smaller than the human ACAT protein) in lysates of 25-RA cells. In contrast, such signal was not produced in lysates of AC29 cells.


Figure 2: Immunoblot (panel A) and ACAT activity measurement (panel B) in various CHO cell lines. In panel A, aliquots of either 100 µg (1times) or 200 µg (2times) of protein/lane from various indicated CHO cell lines grown in F-12 with 10% FBS were immunoblotted with IgG against ACAT (DM10). In panel B, cells grown in parallel were harvested for measuring ACAT activity using the reconstituted vesicle assay as described under ``Experimental Procedures.''



Characterization of the Antibodies against ACAT by Immunodepletion Analysis

We found that neither the DM9 nor the DM10 antibodies caused any detectable inhibition in ACAT activity when the antibodies were incubated with the cell extracts from various human and CHO cells (results not shown). To further test the specificity of these antibodies, we performed immunodepletion experiments. To establish this procedure, the ACAT enzyme, an integral membrane protein, needs to be solubilized in detergent with retention of biological activity. We found that the ACAT activities from various human tissue culture cells (described in Fig. 1) could all be solubilized in 1% deoxycholate, similar to what we previously reported using the human fibroblast cells and CHO cells (Cadigan and Chang, 1988). Control experiments showed that the solubilized ACAT activity gradually lost activity with time; when kept at 4 °C for 3-4 h, about 20-40% of the total solubilized ACAT activities were lost. We developed a protocol (described under ``Experimental Procedures'') for immunodepletion of ACAT from deoxycholate solubilized cell extracts. The process took less than 3.5 h to complete. Fig. 3A shows that the DM10 antibodies caused significant immunodepletion of ACAT activity from human melanoma cell lysates; at 10 µg of IgG/0.5-ml sample, more than 90% of measurable ACAT activity could be removed from the supernatant. In contrast, nonspecific rabbit IgG caused little effect in depleting the ACAT activity. Fig. 3B shows that the DM10 antibodies were also efficient in immunodepleting ACAT activities (by more than 90%) from lysates prepared from CHO cells or from AC29-K1 cells, which expressed human ACAT activity in AC29 cells. Fig. 3C shows the immunoblot analysis of the depleted lysates and the immunoprecipitates of AC29-K1 cell lysates with increasing amount of DM10 antibodies (up to 10.8 µg of IgG/0.5-ml sample). These results show that the intensity of the 50 kDa protein band diminished from the supernatants, with the corresponding band emerged in the precipitates, in a manner dependent on antibody dosage. Moreover, comparing the results in Fig. 3, B and C, shows that the intensities of the 50 kDa protein band remaining in the supernatants correlated well with the residual ACAT activities remaining in the supernatants. Control experiment showed that nonspecific rabbit IgG (up to 10.8 µg/0.5-ml sample) did not cause any detectable depletion of the 50 kDa band from the supernatant (result not shown). These results show that the DM10 antibodies specifically reacted with the human ACAT protein with an apparent size of 50 kDa. As shown in Fig. 3C, in addition to the 50 kDa protein band, the DM10 antibodies also depleted a 35 kDa protein band from the solubilized lysates in a dose-dependent manner; this additional band was also detectable in immunoblot analysis (Fig. 1, lane 9).

Nature of the 35-kDa Protein Band in AC29-K1 Cells

To address the question as to whether the 35 kDa protein band detected in the AC29-K1 cell lysate was due to proteolysis of the 50-kDa ACAT protein in vitro, we performed radioimmunoprecipitation experiments. Melanoma cells, AC29-K1 cells, or AC29 cells were metabolically labeled with [S]methionine for 2 h. The cell lysates were prepared in 1% deoxycholate and then mixed in various manners as indicated. The mixed lysates were subject to radioimmunoprecipitation analysis as described under ``Experimental Procedures.'' Fig. 4B shows the resulting autoradiography of this experiment. Comparison of lane 4 (AC29-K1 cell lysate) and lane 5 (AC-29 cell lysate) indicates that the 50-kDa protein and the 35-kDa protein were the two specifically labeled bands detected in AC29-K1 cells. Lane 3 shows that the 50-kDa protein was the only specifically labeled band detected in melanoma cells. Mixing the labeled melanoma cell lysate with the unlabeled AC29-K1 cell lysate (or with the unlabeled AC-29 cell lysate) did not produce the labeled 35-kDa protein (lanes 1 and 2). These results indicate that the 35-kDa protein was not due to proteolysis in vitro of the 50-kDa protein during the radioimmunoprecipitation experiment. To serve as the control, Fig. 4A shows the result of immunoblot analysis of lysates used in Fig. 4B. These results also suggest that the immunoprecipitation procedure caused significant aggregation/dimerization of the 50-kDa ACAT protein in the melanoma cell and the AC29-K1 cell lysates.

We next performed the immunodepletion experiment in cell lysates prepared from CHO (25-RA) cells. Fig. 5A shows that DM10 antibodies depleted more than 85% of the ACAT activity from the solubilized lysates; this experiment also showed that the ACAT activity disappeared from the supernatants and emerged from the immunoprecipitates in a manner dependent on antibody dosage: when 12.5 µg of IgG/sample was used, as much as 60% of total measurable ACAT activity could be detectable from the immunoprecipitates. In contrast, the nonspecific IgG caused virtually no depletion in ACAT activity in the solubilized lysate (Fig. 5B). A similar result (not shown) was obtained when cell lysates prepared from wild-type CHO cells were used. This result, together with the result shown in Fig. 2, shows that DM10 antibodies specifically reacted with the CHO ACAT protein with an apparent size of 47 kDa.


Figure 5: Immunodepletion of ACAT activity from CHO cell lysates. Lysates of 25-RA cells were used for immunodepletion experiment with various amounts of IgG either against ACAT (panel A) or nonspecific rabbit (panel B). Aliquots of 6% of the immunodepleted lysates or 6% of the corresponding immunoprecipitated pellets were resuspended in 120 µl of preformed PC/cholesterol vesicles and subjected to ACAT enzyme assay in duplicates.



Immunodepletion of ACAT from Various Human Cell Lines

To address the question as to whether the 50-kDa protein (Fig. 1) is the major catalytic component of ACAT in various human cell lines, we performed immunodepletion experiments in lysates prepared from six different human cell lines (skin fibroblast (Hf) cells, adrenal tumor SW13 cells, A293 embryonic kidney tumor cells, HepG2 liver tumor cells, A431 epithelial carcinoma cells, and melanocyte tumor cells). Fig. 6shows that DM10 antibodies efficiently depleted more than 90% of measurable ACAT activities from all of the cell lysates tested; in contrast, the nonspecific IgG elicited either no effect, or caused slight activation of ACAT activities.


Figure 6: Immunodepletion of ACAT activities from lysates of various human cell lines. Cell lysates were prepared from various indicated cell lines for immunodepletion experiment as described under ``Experimental Procedures.'' 0.45-ml aliquots of about 2 mg/ml of cell lysates were immunodepleted with 1) buffer only (0.1 M Tris-glycine, pH 7.0) (open bars), 2) buffer with 10.5 µg of nonspecific rabbit IgG (hatched bars), or 3) buffer with 10.5 µg of IgG against ACAT (DM10) (solid bars). The ACAT activities remaining in the immunodepleted lysates were assayed in duplicates as described in Fig. 3.



Immunoblot Analysis of Various Human Tissue Samples

To detect the presence of ACAT protein in various human tissues, immunoblot analysis was performed with a multiple tissue Western blot (purchased from Clontech). The result (Fig. 7) showed that the immunoreactive materials as recognized by the ACAT antibody were present in all six tissues examined. In the liver, the antibodies mainly reacted with a 50-kDa protein band. The same protein band could be detected in other tissues including lung, kidney, and heart. In the heart, skeletal muscle as well as the brain, the antibodies also reacted with a protein band with an apparent molecular mass of about 35 kDa. In addition, the antibodies recognized protein(s) with higher molecular mass(es) (about 100 kDa and 170 kDa) in all tissues examined except in the liver.


Figure 7: Immunoblot analysis of multiple human tissue samples. Immunoblot analysis was performed with the human multiple tissue Western blot I purchased from Clontech. Each lane contained 75 µg of protein. Arrow indicates the 50-kDa protein band.



Regulation of ACAT by Sterol in Mammalian Cells

We used DM10 antibodies to examine the regulation of the ACAT protein content by sterol in Hf cells, HepG2 cells, and in CHO cells. Fig. 8A shows that in Hf cells (left panel), adding LDL or 25-hydroxycholesterol to culture medium caused very large increase in cholesteryl oleate synthesis rate (79-fold with LDL and 6-fold with oxysterol). In HepG2 cells (right panel), the activation by sterol occurred similarly but with less magnitude. Additional dishes of these cells grown in parallel were prepared for immunoblot analysis. Fig. 8B shows that the ACAT protein contents in Hf cells (part 1) or in HepG2 cells (part 2) were not significantly affected by exposing these cells to sterol in the growth medium.


Figure 8: Activation of cholesteryl ester synthesis by LDL or by 25-hydroxycholesterol (A) and immunoblot analysis of ACAT protein content (B) in Hf cells or in HepG2 cells. Confluent cultures of Hf cells and HepG2 cells, each grown in a 75-cm^2 flask, were split by trypsin and plated evenly into nine 25-cm^2 flasks and grown in DMEM supplemented with 10% FBS for 72 h. The monolayers (nearly confluent at this stage) were rinsed with PBS, fed with 4 ml/flask of DMEM with 10% delipidated FBS for 48 h. Cells were fed with 4 ml/flask of the same medium supplemented without sterol (open bars), with LDL (160 µg/ml) (hatched bars), or with 25-hydroxycholesterol (10 µg/ml; delivered from a 10 mg/ml stock solution) (light gray bars). For each flask, the final alcohol concentration in the medium was adjusted to 0.1%. 12 h later, cells were employed for measuring cholesterol esterification rate in intact cells as described under ``Experimental Procedures'' (panel A) or for immunoblot analysis (panel B). For immunoblot analysis, aliquots of 100 µg (1 times), 150 µg (1.5 times), or 300 µg (3 times) protein of cell lysates were used per lane.



We have examined wild-type CHO cells and 25-RA cells, which are mutant CHO cells resistant to down-regulation by sterol (Chang and Limanek, 1980) by immunoblot analysis, and found that the ACAT protein contents in these two cell types were very similar; the ACAT protein in either cell type was not significantly affected by serum lipids in the growth medium (results not shown).

Immunolocalization of ACAT in Human Cells

We used confocal microscopy and the DM10 antibodies as the probe to perform indirect immunofluorescent studies in tissue culture cells. Three cell types were used: human melanoma cells, human skin fibroblast cells, and CHO cells expressing human ACAT (AC29-K1). A typical result, performed in melanoma cells, shows (Fig. 9A) that the antibodies stained the nuclear envelope region as well as the entire reticulate network; the reticulate network was significantly vesicularized, probably due to fixation of cells with paraformaldehyde (Terasaki et al., 1984). The rather uniformly distributed staining pattern throughout the reticulate network shows that the ACAT protein is not preferentially enriched near the plasma membrane region. The same group of melanoma cells were co-stained with the fluorescent dye DiOC(6), which stains the mitochondria and the ER network, with mitochondria being the more highly stained structures and distinguishable from the ER network (Terasaki et al., 1984). The DiOC(6) ER staining pattern (Fig. 9B) extensively overlapped with the ACAT antibody staining pattern (Fig. 9A), demonstrating that the ACAT protein is mainly localized in the ER. (^2)Using human fibroblast cells and AC29-K1 cells, we have obtained similar ACAT staining patterns. We have also found that growing these three cell types in 10% delipidated FBS medium for 48 h did not cause detectable alteration in ACAT protein staining pattern in these cells (results not shown). Thus, in these cells, sterol did not cause detectable translocation of ACAT from the ER to other cellular compartment(s).


Figure 9: Immunolocalization of ACAT by confocal fluorescent microscopy. Melanoma cells were fixed and stained as described under ``Experimental Procedures.'' Cells were viewed with the red laser beam to visualize the Texas red secondary antibody-stained ACAT protein (A) or viewed with green laser beam to visualize the staining pattern of the fluorescent dye DiOC(6) (which stains mitochondria and endoplasmic reticulum) (B). Control experiments showed that without antibodies against ACAT (DM10) as the primary antibodies, or with nonspecific rabbit IgG as the primary antibodies; no discrete staining pattern could be obtained when cells were viewed with the red laser beam.




DISCUSSION

The ACAT cDNA K1 was isolated from the cDNA library prepared from the phorbol ester-activated THP-1 macrophage cells (Chang et al., 1993). By immunoblot analysis, the apparent size of the major form of the ACAT protein expressed in Sf9 cells or in CHO cell mutant AC29 is about 50 kDa. To address the question as to whether different form(s) of the ACAT enzyme may exist in different human cell lines, we used high titer specific antibodies against ACAT to perform immunoblot analysis and immunodepletion experiments. Among the cell lines employed, the HepG2 cell has been used extensively as a model for hepatocyte (for review, see Javitt(1990)). The SW13 cell is a cell line derived from adrenal cortex and has been used to study cholesterol metabolism (Sarria et al., 1992). The Caco-2 cell is a human colon tumor cell line; when reaching confluence, it differentiates and expresses intestinal enterocyte functions (Trotter and Storch, 1993; Field et al., 1995). The phorbol ester-activated THP-1 cell is a human macrophage cell (Via et al., 1989). The A431 cell is an epidermoid carcinoma cell line that over-expresses epidermal growth factor receptor and the LDL receptor (for a review, see Stoscheck and Carpenter(1983)). The A293 cell is a human adenoviral DNA-transformed cell line derived from primary human embryonic kidney; this cell line expresses the adenoviral T antigen (Graham et al., 1978) and has been used extensively as a recipient cell line for DNA transfection studies. Our results showed that the 50-kDa protein is present in all of these cell lines. In addition, the immunodepletion study led us to conclude that the 50-kDa protein is the major catalytic component of measurable ACAT activity in six different cell lines tested. It is still possible that isoform(s) of ACAT protein, distinctly different from the 50-kDa protein described here, may exist in other cell types that were not tested in this study. This possibility can best be addressed by producing ACAT gene knockout mice, and testing for possible residual ACAT-like enzyme activity in these mice. Previously, using the coding region of human ACAT cDNA as the probe, Northern analysis showed that the K1 gene transcripts expressed in various human tissues exhibited four different sizes (at 3.1, 4.0, 4.9, and 7.6 kilobases, respectively) (Chang et al., 1993). Multiple ACAT transcripts were also found in various rabbit tissues (Pape et al., 1995). Our current results demonstrate that in various human cells, multiple ACAT mRNAs give rise to only one single protein product (the 50-kDa protein).

In addition to the 50-kDa protein, the presence of a 35-kDa protein could be reproducibly detected in lysates prepared from AC29-K1 cells, which are CHO cells highly expressing the human ACAT (Fig. 1, Fig. 3, and Fig. 4). To examine the origin of the 35-kDa protein, we developed a protocol to perform the radioimmunoprecipitation experiment. The results showed that the 35-kDa protein could not be produced from the 50-kDa protein by proteolysis in vitro during the immunoprecipitation process. It is possible that the 35-kDa protein may be derived from the 50-kDa protein as the result of proteolysis in intact AC29-K1 cells. In the future, radioactive pulse-chase experiment followed by immunoprecipitation will be performed to test this possibility. The result of multiple human tissue immunoblot analysis showed that the ACAT antibody cross-reacted with proteins in all six tissues examined. Depending on the tissue, the apparent molecular weights of the cross-reacting material were about 50, 35, 100, and/or 170 kDa. It is possible that the 100-kDa protein and the 170-kDa protein detected in these tissues may be the result of dimerization/aggregation of the 50-kDa protein during sample preparation(s). According to the protocol provided by the supplier (Clontech), the human tissue homogenates were solubilized by heating at 90 °C for 5 min in buffer containing 2% SDS. We have found that the heating step would cause aggregation of the human ACAT protein in SDS-PAGE analysis (result not shown). We also do not know whether the 35-kDa protein detected in the heart, skeletal muscle, and brain bears any relationship with the 35-kDa protein detected in the AC29-K1 cells. Further experiments using samples carefully prepared from fresh tissues are needed to pursue these observations.

The current results also demonstrate that the DM10 antibodies cross-reacted with the wild-type and the 25-RA CHO cell ACAT, with an apparent size of 47 kDa; the antibodies did not cross-react with this protein band in AC29 cell lysate. Our previous results showed that in Northern analysis, using the coding region of human ACAT cDNA as the probe, we could detect a cross-reacting signal (at 3 kilobases) in poly(A) mRNAs of 25-RA cells, while we could not detect the same signal in poly(A) mRNAs of AC29 cells (Chang et al., 1993) (Fig. 3). Thus, it is likely that the AC29 cell contains deletion mutation(s) such that it does not produce any ACAT message or any ACAT protein.

Earlier studies in various tissue culture cells, such as human fibroblasts and CHO cells, showed that the rate of cholesterol ester synthesis and ACAT activity from cell extracts are greatly activated by the addition of LDL or by oxygenated sterols such as 25-hydroxycholesterol (Goldstein et al., 1974; Brown et al., 1975; Doolittle and Chang, 1982b). The activation of ACAT by sterol could not be prevented by protein synthesis inhibitors such as cycloheximide (Goldstein and Brown, 1977; Doolittle and Chang, 1982b). In fact, subsequent experiments showed that protein synthesis inhibitors actually increased ACAT activities in intact cells (Chang et al., 1986; Tabas and Boykow, 1987). A reconstituted vesicle assay for measuring ACAT activity independent of its surrounding lipid environment was developed (Doolittle and Chang, 1982a, 1982b; Cadigan and Chang, 1988a). Cell extracts prepared from human fibroblasts or from CHO cells treated with or without sterol (or serum lipids) had ACAT activities (assayed conventionally) that varied up to 20-fold. In contrast, the differences in activity were essentially abolished in the reconstituted vesicle assay. In the current study, immunoblot analysis showed that the ACAT protein content in human fibroblast cells, HepG2 cells, or CHO cells was not affected by the presence of sterol (or serum lipids) in the growth medium. This result provides the direct proof to the early notion that modulating the ACAT protein content is not an important mechanism for sterol-dependent regulation of ACAT in cultured mammalian cells. Pape et al.(1995) recently showed that in rabbits, the high fat, high-cholesterol diet caused a 2-3-fold increase in hepatic and aortic ACAT mRNA levels in a tissue-specific manner. Similar observation has been found by Uelmen et al.(1995) in mice fed with high fat, high cholesterol diet. Therefore, in animals, additional regulatory mechanisms may exist, perhaps to guard against overloading of free cholesterol within certain tissues.

Recently, using the ACAT expression system in insect cells as the model, we have shown activation of ACAT by cholesterol or by oxysterol in vitro (Cheng et al., 1995). We hypothesized that inside the mammalian cells, cholesterol itself may serve as an ACAT activator in addition to its role as an ACAT substrate. In the current study, using indirect immunofluorescent microscopy, our results indicated that the ACAT protein is mainly located in the ER. Thus in intact cells, it may be the ER cholesterol concentration in the vicinity of ACAT that serves as a major physiological regulator of ACAT activity. Previous studies have shown that ER is where several sterol-regulated proteins, including the hydroxymethylglutaryl-CoA reductase (for a review, see Goldstein and Brown(1990); also see Gil et al.(1985), Roitelman et al.(1992), Meigs and Simoni(1992), and Correll and Edwards(1994)) and the sterol regulatory element binding protein 2 (SREBP(2)) (for a recent paper, see Sheng et al.(1995)) reside. Other studies have suggested that in various cell types, the ``regulatory sterol pool'' for down-regulating cholesterol biosynthesis and the LDL-receptor activity may be in equilibration with the ACAT cholesterol substrate pool (Tabas et al.(1986), Havekes et al.(1987), Cadigan et al.(1988), Salter et al.(1989), Daumerie et al.(1992), and for review, see Spady et al.(1993)). Within a single-cell type, it is possible that the ``regulatory sterol pool'' may be composed of regional concentration of cholesterol within the ER. Cholesterol pools within different regions of the ER membrane may move rapidly and partially equilibrate with one another. Thus, the main role of ACAT in cellular cholesterol homeostasis may be to maintain a low cholesterol concentration in the ER membrane, such that subtle change(s) in this parameter can produce sensitive signal(s) for regulating hydroxymethylglutaryl-CoA reductase, SREBP(2), and other sterol-regulated protein(s) located in the ER membrane. ER is also the organelle from which newly synthesized cholesterol originates (Kaplan and Simoni, 1985; Reinhart et al., 1987). Most (if not all) of the newly synthesized cholesterol is destined to move to the plasma membrane (DeGrella and Simoni(1982) and Lange and Matthies(1984), and for a recent review, see Liscum and Underwood (1995)). To minimize interference by ACAT of the sterol translocation process between the ER and the plasma membrane, it should be that the cholesterol concentration in the ER must exceed a certain threshold value before it is able to cause significant activation in ACAT activity. The model of ACAT activation induced by cholesterol concentration in the ER proposed from this laboratory can provide a simple and sensitive mechanism for controlling the cholesterol homeostatic process within the cell.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant HL 36709 and a grant from The Council for Tobacco Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 603-650-1622; Fax: 603-6501128.

(^1)
The abbreviations used are: ACAT, acyl-coenzyme A:cholesterol acyltransferase; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; ER, endoplasmic reticulum; Hf, human skin fibroblast; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PC, phosphatidylcholine; BSA, bovine serum albumin; GST, glutathione S-transferase; LDL, low density lipoprotein; PBS, phosphate-buffered saline.

(^2)
Biochemical studies suggested that the rat hepatic ACAT enzyme is preferentially located in the rough ER (see Reinhart et al.(1987) and references therein).


ACKNOWLEDGEMENTS

We thank Drs. Gustav E. Lienhard, Duane A. Compton, Peter Pentchev, George M. Langford, Joel S. Tabb, and Tirso A. Gaglio for helpful advice and discussions.


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