(Received for publication, August 31, 1995; and in revised form, September 29, 1995)
From the
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.
Acyl-coenzyme A:cholesterol acyltransferase (ACAT) ()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.
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 10
cells/well in 6-well dishes for 48 h. To harvest, cells were
rinsed in 4
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
10
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 (1) or 200
µg (2
) 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.''
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.
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.
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.
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 flask, were split by trypsin and plated evenly into
nine 25-cm
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
), 150 µg (1.5
), or 300
µg (3
) 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).
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 (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.
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)
(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
, 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.