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J Biol Chem, Vol. 273, Issue 41, 26765-26771, October 9, 1998
From the The enzyme acyl coenzyme A:cholesterol
acyltransferase 1 (ACAT1) mediates sterol esterification, a crucial
component of intracellular lipid homeostasis. Two enzymes catalyze this
activity in Saccharomyces cerevisiae (yeast), and several
lines of evidence suggest multigene families may also exist in mammals.
Using the human ACAT1 sequence to screen data bases of expressed
sequence tags, we identified two novel and distinct partial human
cDNAs. Full-length cDNA clones for these ACAT related gene
products (ARGP) 1 and 2 were isolated from a hepatocyte (HepG2)
cDNA library. ARGP1 was expressed in numerous human adult tissues
and tissue culture cell lines, whereas expression of ARGP2 was more
restricted. In vitro microsomal assays in a yeast strain
deleted for both esterification genes and completely deficient in
sterol esterification indicated that ARGP2 esterified cholesterol while
ARGP1 did not. In contrast to ACAT1 and similar to liver
esterification, the activity of ARGP2 was relatively resistant to a
histidine active site modifier. ARGP2 is therefore a tissue-specific
sterol esterification enzyme which we thus designated ACAT2. We
speculate that ARGP1 participates in the coenzyme
A-dependent acylation of substrate(s) other than
cholesterol. Consistent with this hypothesis, ARGP1, unlike any other
member of this multigene family, possesses a predicted diacylglycerol
binding motif suggesting that it may perform the last acylation in
triglyceride biosynthesis.
The intracellular formation of sterol esters from fatty acid and
sterol is mediated by acyl-CoA:cholesterol acyltransferase (ACAT).1 The pathological
accumulation of cholesterol esters in atherosclerotic lesions has lead
to intense pursuit of ACAT inhibitors as pharmacological agents.
Microsomal ACAT preparations from various tissues display differential
sensitivities to some of these agents (1) including histidine modifiers
(2). This suggests that more than one protein mediates the
esterification reaction, such as occurs in yeast (reviewed in Ref. 3).
Saccharomyces cerevisiae (budding yeast) has two ACAT
related enzymes, Are1 and Are2, which are derived from separate genes
and have been shown to independently esterify sterols (4, 5). In terms
of contribution to the sterol ester mass of the cell, Are1 is the minor
isoform relative to Are2. These genes were identified based on sequence
conservation to a human gene, ACAT1, which encodes an ACAT enzyme with
homologs in many mammalian species (6, 7). The human ACAT1 gene encodes a 550-amino acid polypeptide and is expressed in most tissues, predominantly placenta, lung, kidney, and pancreas (6). ACAT1 has been
predicted to have two transmembrane domains (6) and has been
immunolocalized to the endoplasmic reticulum (8, 9). When murine ACAT1
was disrupted in induced mutant mice, homozygotes for the deletion were
found to essentially lack ACAT activity in embryonic fibroblasts and
have negligible amounts of cholesterol ester in the adrenal cortex and
peritoneal macrophages (10). However, cholesterol ester accumulation
was normal in hepatocytes while dietary cholesterol absorption, an
indirect marker for intestinal cholesterol esterification, was
indistinguishable from control littermates. This is consistent with the
concept of a multigene family for this activity.
ACAT isoenzymes may be required to perform the variety of physiological
roles mediated by cholesterol esterification. Increases in cellular
free cholesterol above certain levels are cytotoxic and are ameliorated
by cholesterol ester formation (11). In hepatocytes, the bulk of
cholesterol secreted in very low density lipoprotein is esterified
intracellularly and determines apolipoprotein B secretion rates
(12-14). Cholesterol esterification in the enterocyte may be necessary
for cholesterol absorption from the lumen and secretion in chylomicrons
into the lymph (15). The formation of cholesterol ester stores could
also provide a readily available substrate for steroid hormone
synthesis in steroidogenic tissues (16, 17). It is likely that
different ACAT isozymes mediate each of these processes, and the data
presented here support that hypothesis.
We reasoned that additional human ACAT proteins would have sequence
similarity to regions conserved between human ACAT1 and yeast Are1 and
Are2. (4). Accordingly, an ACAT consensus sequence was used to screen
the data base of expressed sequence tags (dbEST). Several cDNA
entries were identified which were transcribed from two independent
human genes. This study is a description of the isolation of
full-length cDNA clones for two
ACAT-related gene products (ARGP1 and ARGP2), examination of their pattern
of tissue expression, and assays of enzymatic activity. We show that
ARGP2 can catalyze the formation of sterol ester from cholesterol and oleoyl-CoA, leading us to rename this gene, ACAT2. By contrast, ARGP1
did not detectably esterify cholesterol and we propose that it performs
acyl-CoA-dependent acylation of other molecules, such as
diacylglycerol.
General--
Molecular biology techniques were performed by
conventional protocols (18, 19) and DNA modifying reagents were
purchased from Life Technologies, Inc., New England Biolabs, or Promega as indicated. The Prime It random priming probe synthesis kit was
obtained from Stratagene. The DIG Genius probe synthesis kit and CSPD
were supplied by Boerhinger Mannheim. Radioactive reagents ([14C]oleoyl-CoA and [32P]dCTP) were
purchased from NEN Life Science Products Inc. Ethidium bromide-stained
agarose gels were visualized by the Kodak Digital Science 1D system.
Automated DNA sequencing was performed at the Columbia University
Cancer Center sequencing facility, and oligonucleotides were
synthesized by Genset. DNA and amino acid sequence analysis and
comparisons were performed using DNAStrider (20), PILEUP, and GAP
programs (GCG Inc. (21)), Prosite (22), and Identify (Ref. 23, website
http://dna.stanford.edu/identify/). Yeast media components were
prepared as described (18).
Screening the dbEST--
A 30-amino acid ACAT concensus peptide
sequence (FAEMLRFGDRMFYKDWWNSTSYSNYYRTWN) was used as the query in a
tblastn (which compares a protein sequence against a nucleotide
sequence data base translated in all reading frames (24, 25)) search of the data base of expressed sequence tags at NCBLI (dbEST). Three clones, H24971, R07932, and R99213, derived from a common gene (named
ACAT related gene product 1, ARGP1), were identified (p < 10 5' Rapid Amplification of cDNA Ends (RACE) of
ARGP1--
Oligo(dT) primed, double stranded cDNA was reverse
transcribed from human, ileal, poly(A)+ mRNA, kindly
provided by Dr. Paul Dawson, and ligated to adapters using a
commercially available kit (CLONTECH, Palo Alto,
CA). Touchdown PCR (26) was performed for 35 cycles with a forward primer complementary to the adapter (AP1,
5'-CCATCCTAATACGACTCACTATAGGGC) and a reverse primer (End4A,
5'-CCACCTGGAGCTGGGTGAAGAAC) complementary to the ARGP1 dbEST clone
Z43867. The PCR mixture included 200 nM each oligo, 200 µM dNTPs, 1.75 mM MgCl2, 2.5 units of Taq, and the cDNA diluted 1:500. The 700-bp
reaction product was gel isolated, ligated into YEp352 with a T
overhang generated by Taq polymerase, and sequenced.
5' RACE of ARGP2--
A human, fetal (20 weeks post-conception)
liver/spleen, oligo(dT)-primed, cDNA library in the vector pT7T3D
was kindly provided by Dr. Bento Soares. PCR was performed with the
cDNA, a forward primer (M13 reverse, 5'-TGAGCGGATAACAATTTCACACAGG)
complementary to the vector and a reverse primer (203, 5'-CCCCATGCTGAGGTCTGTGATCAG), complementary to the ARGP2 dbEST clone
R10272, using the above conditions. The 800-bp reaction product was gel
isolated, ligated into pBS:SK (Stratagene) with a T overhang generated
by Taq polymerase, and sequenced.
Hybridization Screening of a HepG2 cDNA Library--
A yeast
expression library of HepG2 cDNA (size selected for inserts greater
than 2.0 kb in pAB23BXN, commercially available from Austral
Biologicals, San Ramon, CA), was propagated in the E. coli
strain MC1061 and plated onto 135-mm LB + ampicillin (50 µg/ml)
plates at an approximate density of 5000 colonies per plate. Membrane
(Hybond-N, Amersham) replicas of the plates were probed by
hybridization with a digoxigenin-labeled probe specific for ARGP1
(synthesized using a 420-bp NotI, PstI digestion
product of the 5' RACE product) or ARGP2 (synthesized using the 5' RACE product) in 5 × SSC, 0.05% SDS, 0.1%
N-laurolysarcosine, 0.1 mg/ml salmon sperm DNA, and 2%
(w/v) blocking reagent (Boerhinger Mannheim) at 65 °C for 14-18 h.
The membranes were washed in 0.2 × SSC, 0.1% SDS at 60 °C for
80 min, incubated with an anti-digoxigenin antibody (1:10,000), washed
in Tris-buffered saline, incubated with the peroxidase substrate CSPD
(Boerhinger Mannheim), and detected by enhanced chemiluminescence
(ECL). For ARGP1, 4 single positive clones were isolated after
screening ~20,000 clones. For ARGP2, 4 single positive clones were
isolated after screening ~30,000 clones. The longest clones for each
were sequenced multiple times on both strands using vector and
gene-specific oligonucleotides.
Tissue Culture--
Cultured human Caco2, HeLa, HepG2, and THP1
cell lines were donated by Dr. R. J. Deckelbaum and originally
obtained from the ATCC. HepG2, HeLa, and Caco2 cells were maintained as
cell monolayers in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) + 10% fetal bovine serum (HyClone) in 5%
CO2. THP1 monocyte cells were maintained in suspension in
RPMI (Life Technologies, Inc.) + 10% fetal bovine serum in 5%
CO2. Differentiation of THP1 cells was stimulated with 150 ng/ml tetramyristate phorbol ester and 140 µM
Human Adult and Fetal Multi-tissue Northern Blot
Analysis--
Commercially obtained multi-tissue Northern blot
(CLONTECH) contained 2 µg of poly(A)+
RNA from human adult or fetal (18-24 weeks postconception) tissues originally resolved on a 1.2% agarose, formaldehyde gel. The adult tissue membrane was hybridized with a random-hexamer primed,
[32P]dCTP-labeled probe, generated using the insert of
the ARGP1 dbEST clone R99213, in ExpressHyb buffer
(CLONTECH) for 1 h at 68 °C. The membrane
was washed in 0.1 × SSC, 0.5% SDS at 50 °C. After stripping
the membrane was probed with ARGP2 (dbEST clone 10272 insert and the
ARGP2 5' RACE product) using the conditions above. The fetal tissue
Northern blot was hybridized with the same ARGP2 probe.
Reverse Transcription PCR--
Human cDNA obtained as part
of a Quick Screen cDNA Panel of Human tissues
(CLONTECH) or reverse transcribed (Life
Technologies, Inc. kit) from human ileal poly(A)+ mRNA
was used as the template in a PCR reaction with primers specific for
ARGP1 (106, GGCATCCTGAACTGGTGTGTGGTG; 110, AGCTGGCATCAGACTGTGTCTGG), ARGP2 (202, GAGTTCCCCCACATTCATCAAATCC; 206, CATGCTGCTGCTCATCTTCTTTGCA), or In Vitro Assay of ACAT Activity in Yeast Microsomes--
The
cDNA inserts of the longest ARGP1 and ARGP2 HepG2 library clones
were removed by NotI, EcoRI digestion and ligated
into the yeast expression vector pRS426GP which utilizes the galactose inducible GAL1/GAL10 promoter. A cDNA corresponding to the coding region of human ACAT1 flanked by 5 bp of 5'-untranslated region and 1 bp of 3'-untranslated region, in pRS426GP was described previously
(27). Yeast strain, SCY059 (MAT Isolation of Full-length cDNA Clones for Two ACAT Related Human
Genes--
A comparison of the human ACAT1 protein and the two yeast
ACAT orthologs (Are1, Are2) identified a highly conserved (70%
identical) region of 30 amino acids (ACAT1 amino acids 391-420) near
the carboxyl terminus. This peptide was used to screen the data base of
expressed sequence tags (dbEST). The search identified several human
cDNAs, the longest being 890 bp (GenBank accession number H45923),
derived from a common gene we call the ARGP1. To date, 26 clones for
human ARGP1 are present in the dbEST from fetal liver/spleen, infant
brain, breast, cerebellum, hippocampus, kidney, placenta, testis, ovary
tumor, colon tumor, and lung tumor libraries, suggesting ubiquitous and
abundant expression. In addition, ARGP1 is also represented as several
murine entries (e.g. GenBank accession number C75990). The
dbEST was then searched using the entire ACAT1 protein sequence. Four
human cDNAs, distinct from ARGP1 cDNA clones, were identified
in fetal liver/spleen and fetal heart libraries and are derived from a
common gene we call ARGP2. The longest entry was 600 bp (GenBank
accession number R10272). To date these are the only dbEST entries for
human ARGP2, although several murine entries have been identified
(e.g. GenBank accession number AA410072).
Characterization of Two Human Genes Encoding Acyl Coenzyme
A:Cholesterol Acyltransferase-related Enzymes*
§,,§
Institute of Human Nutrition,
Departments of Pediatrics and Physiology and Molecular
Biophysics, Columbia University College of Physicians and Surgeons, New
York, New York 10032 and ¶ DuPont Pharmaceutical Company,
Experimental Station, Wilmington, Delaware 19880-0400
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
4). The entire human ACAT1 protein was then used in
an identical search. In addition to clones of ACAT1 and ARGP1, two
entries, R10272 and W76421, with significant similarity were identified (p < 105). They were derived from a gene
we named ARGP2. Rescreening the dbEST with these clones identified two
more ARGP2 entries. Escherichia coli clones with the largest
inserts corresponding to these sequences were obtained from the
I.M.A.G.E. consortium and resequenced with T3, T7, or gene specific
primers.
-mercaptoethanol. Whole cell RNA was isolated from confluent monolayer cultures or pelleted THP1 cells using TRIzol (Life
Technologies, Inc.) extraction. The Caco2 cells had been confluent for
approximately 21 days.
-actin (Act1, GAGCTGCCTGACGGCCAGGTC; Act2,
CACATCTGCTGGAAGGTGGACAG). The PCR mixture included 1.5 mM
MgCl2, 200 µM dNTPs, 400 nM of each primer, and 2 units of Taq (Life Technologies, Inc.).
Following 35 cycles (94 °C, 45 s; 60 °C, 45 s;
72 °C, 2 min), the products were resolved on a 1% agarose, 0.5 µg/ml ethidium bromide gel. For RNA prepared from human cultured
cells, first strand cDNA synthesis was performed, with and without
reverse transcriptase (SuperscriptII, Life Technologies, Inc.) using 4 µg of whole cell RNA. A fraction of each reaction (10%) was the
template in a PCR reaction (30 cycles of 94 °C, 30 s; 68 °C,
2 min) with primers specific for ARGP1 (103, GCTTCATGGAGTTCTGGATGGTGG;
106, GGCATCCTGAACTGGTGTGTGGTG), ARGP2 (201, GACACCTCGATCTTGGTCCTGCC;
202, as above) or human ACAT1 (ACATa, CGGAATATCAAACAGGAGCCCTTC; ACATb,
CATTCCAAAGAACATGAAGATGCACG).
, ade2-1, can1-1, trp1-1,
ura3-1, his3-11, 15, leu2-3, 112, met14
14HpaI-SalI,
are1NA::HIS3, are2::LEU2) with deletions in ARE1
and ARE2, the yeast homologs of human ACAT1 (4), was
transformed with the above constructs or pRS426GP using lithium acetate
and nucleic acid prototrophy selection (28). Expression of the
constructs was verified by RT-PCR analysis of RNA isolated from the
transformed cells. Culturing of the transformed yeast, induction of
expression, microsome isolation, and sterol esterification assays were
as described previously (27). In those experiments involving
diethylpyrocarbonate (DEPC) to modify histidine residues, a
preincubation with 100 µM DEPC was performed as described
(2).
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
ARGP1 Predicted Peptide-- The longest open reading frame of ARGP1, flanked by a 244 nucleotide 5'-untranslated region and a 265-nucleotide 3'-untranslated region, encodes a 488-amino acid protein (Fig. 1) with a calculated molecular mass of 55,216 daltons. The predicted initiator methionine lies within a consensus for initiation of translation (29) and downstream of an in-frame termination codon. Comparison to ACAT1 revealed 22% amino acid sequence identity (29% similarity) over the entire molecule. The conservation of these molecules is greatest toward the COOH terminus, such that ACAT1 and ARGP1 are 28% identical over the last 250 residues. This pattern of sequence similarity is strikingly similar to that observed from comparison of ACAT1 with the yeast Are1 and Are2 proteins. ARGP1 is predicted to be a membrane bound protein with nine putative transmembrane domains and one N-linked glycosylation site. Uniquely, ARGP1 contains a diacylglycerol/phorbol ester binding signature sequence (H.[FWY]..[KR].F..P) at amino acids 382-392 which was originally identified by comparison of protein kinase C isoforms and diacylglycerol kinases (Fig. 7) (36, 37)). This motif is also conserved in the murine homolog of ARGP1 residing at the dbEST (GenBank accession number AA764382).
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ARGP2 Predicted Peptide-- The longest ARGP2 open reading frame, flanked by a 51-nucleotide 5'-untranslated region and a 420-nucleotide 3'-untranslated region, predicts a 522-amino acid protein with a calculated molecular mass of 59,942 daltons (Fig. 2). The predicted initiator methionine lies within a consensus for initiation of translation (29). Over the entire molecule, the predicted protein is 47% identical (54% similar) to human ACAT1. This conservation is even more pronounced at the COOH-terminal end of the molecules, raising to 63% identity over the last 250 residues. ARGP2 is predicted to be a membrane bound protein with seven putative transmembrane domains and two N-linked glycosylation sites. ARGP2 is similar to ACAT1 in that it contains a leucine zipper (338-359) which may mediate multimerization or interaction with other proteins. ARGP2 does not possess a predicted diacylglycerol/phorbol ester-binding site. A sequenced tag entry (number WI-11660) for ARGP2 localizes to human chromosome 12, further distinguishing it from ACAT1, which is located on chromosome 1 (30).
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ARGP1 and ARGP2 Expression in Human Tissues and Tissue Culture Cell
Lines--
Expression of a second ACAT would be expected in tissues
(e.g. liver and intestine) which exhibit normal ACAT
activity in the induced mutant ACAT1
(acact) mice (10). Expression of
ARGP1 and ARGP2 was thus examined by Northern blot of human RNA (Figs.
3 and 4). Hybridization of an ARGP1
cDNA probe to a panel of adult tissue mRNAs detected a
transcript in all tissues examined (Fig. 3). However, ARGP1 expression
levels varied qualitatively among tissues with moderate expression in
thyroid, stomach, heart, skeletal muscle, and liver and high expression
in adrenal cortex, adrenal medulla, testes, and small intestine. The
presence of a 2.0-kb transcript was invariable among the tissues while
a 2.4-kb transcript was observed in about half the tissues, most
notably the tissues with high expression. The origin of these two
transcripts has not been identified, however, their heterogeneity is
unlikely to lie at the 3' end of the message since all dbEST entries
for ARGP1 cDNAs terminate at a similar position. Hybridization of
the same membrane, under identical conditions, with an ARGP2 cDNA
probe failed to detect a transcript in any tissue (data not shown).
Since the four ARGP2 dbEST clones were in human fetal libraries, ARGP2
expression was examined in human fetal tissues by Northern blot (Fig.
4). A 2.2-kb transcript was detected in
fetal liver but not in fetal brain, lung, or kidney.
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Assay of ACAT Activity in ACAT Negative Yeast Transformed with ARGP1 and ARGP2-- The ability of ARGP1 and ARGP2 to esterify sterols was assayed in a sterol esterification deficient yeast strain (SCY059) in which the endogenous ARE genes were deleted (27). Microsomes from these yeast, transformed with an expression vector harboring no insert or cDNA inserts for ARGP1, ARGP2, or human ACAT1 were assayed in vitro for the incorporation of [14C]oleate into sterol ester. Since we previously demonstrated that cholesterol is the preferred substrate for mammalian ACAT enzymes (27, 32), assays were performed with exogenous cholesterol supplied in Triton WR-1339. As shown in Table I, ARGP2 forms cholesterol ester at a rate of 49 pmol/min/mg of microsomal protein. This is 24-fold over background and about 15% of the activity detected in microsomes from ACAT1 transformants. We therefore renamed ARGP2 as ACAT2. ARGP1 did not display significant ACAT activity. None of the enzymes showed the ability to use ergosterol, the major sterol in yeast microsomes, as a substrate (data not shown). While the ACAT1 and ACAT2 mediated activities were equally sensitive (75% inhibition) to the ACAT inhibitor Dup128 (0.5 µM; not shown), they showed significantly different sensitivity to the histidine/tyrosine modifying agent diethylpyrocarbonate (DEPC, Table I). This reagent was previously demonstrated to distinguish liver and adrenal ACAT activities, the latter being significantly more sensitive. Since adrenal ACAT would primarily represent ACAT1, our data are consistent with ACAT2 representing the DEPC-resistant isoform identified by Kinnunen et al. (2).
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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We have isolated two independent human cDNAs, ARGP1 and ACAT2, which encode proteins with significant sequence similarities to human ACAT1. The level of nucleotide sequence conservation between ACAT1 and ACAT2 (55%) suggests their common evolution possibly arising from a gene duplication event, as clearly occurred in the case of the yeast ARE gene family. However, ARGP1 is more distantly related, bearing 39 and 43% nucleotide identity with ACAT1 and ACAT2, respectively, and may have evolved independently. The uniform similarity between the human genes and the two yeast ARE genes precludes any assignment of lineage across species.
The similarity among the three human ACAT-like proteins is most distinct over their COOH-terminal regions just as is the case when comparing the yeast Are proteins to ACAT1. The predicted ARGP1 protein displays 28% identity with ACAT1 over this portion of the molecule and includes a FY.DWWN motif present in all cloned ACATs and shown to be important for enzymatic activity (Fig. 7A).3 However, ARGP1 is the most divergent member of this gene family. For example, a HSF motif (residues 268-270) is invariant in ACAT1 and yeast Are enzymes and was critical to ACAT1 activity in CHO cells. Replacement of Ser by Leu produced an inactive and unstable molecule (33). This motif is not conserved in ARGP1, although several serines are present in the region (e.g. Ser227, Fig. 7B). ARGP1 is also unique in its predicted possession of a diacylglycerol/phorbol ester-binding site (Fig. 7A), leading us to speculate that this enzyme might esterify diacylglycerol to produce triglyceride. Sequence similarity between diacylglycerol acyltransferase and ACAT enzymes might be expected since both have a common substrate, acyl-CoA, but differ in the alcohol (cholesterol or diacylglycerol) used as a second substrate.
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). In ARGP1, the underlined sequence
HKWCIRHFYKP represents a candidate for diacylglycerol binding as found
in protein kinase C and diacylglycerol kinases (motif,
H.[FWY]..[KR].F..P). The asterisks identify those
residues critical to definition of this motif that distinguish ARGP1
from the other members of the family. B, the HSF region. The
central serine residue (indicated 
) was found to be
critical to the activity and stability of Chinese hamster ovary
ACAT1.
Of the two new gene products described here, ACAT2 displays significantly greater sequence similarity to ACAT1, with an overall identity of 47% and 63% invariance over the COOH-terminal half of the molecules. The FY.DWWN motif common to this family of proteins is maintained in ACAT2 to the extent that the flanking residues render the tyrosine a candidate for phosphorylation as observed in ACAT1 and in yeast (Fig. 7A). Tyrosine phosphorylation may be a regulator of ACAT activity, although serine and threonine phosphorylation is unlikely to be involved (34, 35). The HSF motif found in ACAT1, Are1 and Are2 is conservatively replaced in ACAT2 by YSF (residues 244-246; Fig. 7B). Interestingly, histidine modifying agents selectively inactivate adrenal microsomal ACAT activity but display a significantly higher Ki (1500 versus 250 µM) against liver microsomes (2). It is intriguing to speculate that sequence variation in the (H/Y)SF motif may explain this observation. In accordance with this, we showed that ACAT1 was significantly more sensitive to DEPC than ACAT2. In common with ACAT1, Are1 and Are2, the ACAT2 sequence predicts a leucine heptad motif which may play a role in multipeptide complex formation. Radiation inactivation studies in rat liver microsomes have shown that the ACAT enzymatic complex is about 200 kDa (36, 37), much larger than the predicted monomer for ACAT 1 (65 kDa) or ACAT2 (60 kDa). There is also evidence that ACAT1 interacts with itself in a yeast two-hybrid system (38) and ACAT2 may be similar in this regard. ARGP1 and ACAT2 are also similar to ACAT1 in terms of hydrophobicity. While previous studies suggested that ACAT1 contains two transmembrane domains (6), the PredictProtein algorithm (39) indicates eight such domains in ACAT1, similar to the number predicted for ARGP1 (nine) and ACAT2 (seven). Membrane spanning domains are expected characteristics of ACAT and diacylglycerol acyltransferase enzymes since both activities are associated with microsomal membranes (40-42).
In addition to sequence similarity with ACAT1, we expect alternate ACAT
enzymes to be expressed in the tissues which retain ACAT activity in
the induced mutant ACAT1 mouse, namely the liver and intestine. ARGP1
met this criteria, however, it is also highly expressed in human adult
adrenal cortex which was depleted of cholesterol esters in the induced
mutant mouse. Monocytes from acact mice were
also devoid of cholesterol ester and yet ARGP1 mRNA was detected in
the human THP1 monocyte cell line. This evidence is contrary to ARGP1
being an ACAT, barring species-specific differences in expression. By
the sensitive technique of RT-PCR, ACAT2 expression was observed in
human adult liver and intestine and in cell culture models of the
hepatocyte and intestinal enterocyte but was undetectable in THP1
monocytes and macrophages. This profile of expression is consistent
with a role for ACAT2 in the livers and intestine of mammals,
particularly ACAT1 knockout mice.
In confirmation of ACAT2 being a candidate for a second ACAT, heterologous expression of ACAT2 in an ACAT-negative yeast strain conferred significant microsomal cholesterol esterification with oleoyl-CoA at a level comparable to the 20-50 pmol/min/mg of protein observed in human liver microsomes supplied with exogenous cholesterol (43). The ACAT2-mediated esterification activity was significantly (85%) less than that mediated by ACAT1 in yeast. This may be due to differences in protein expression (although both mRNAs were produced at high levels as detected by RT-PCR, data not shown), protein stability, or a genuine difference between the two enzymes.
Liver ACAT, predicted to comprise both ACAT1 and ACAT2, utilizes a limited range of sterol substrates but a wide variety (16:0, 18:0, 18:1, 18:2, and 20:4) of fatty acyl-CoAs (27, 44). Determining substrate-specific differences between ACAT1 and ACAT2 may thus explain their redundancy. The redundancy may also be related to substrate affinity such as seen between the hexokinase types I-III and hexokinase type IV (glucokinase) (45). In such a scenario, one ACAT would have a lower affinity for cholesterol and only catalyze esterification at high cholesterol concentrations.
In addition to potential differences in activity, the two enzymes may
have different physiological roles. For storage, cholesterol esters
concentrate as cytoplasmic neutral lipid droplets, whereas for
lipoprotein synthesis, cholesterol esters are incorporated into
lipoprotein particles in the endoplasmic reticulum lumen. Redundant
ACAT enzymes might allow one to be specific for cytoplasmic release of
the cholesterol ester product and another to mediate endoplasmic
reticulum lumenal release. Since lipoprotein synthesis occurs primarily
in the liver and intestine, we speculate that ACAT2 may release
cholesterol ester into the endoplasmic reticulum lumen, leaving ACAT1
to esterify and store sterols in the cytoplasm. The large amount of
cholesterol ester, likely as cytoplasmic droplets, in the livers of
high fat, high cholesterol fed acact mice, is
contrary to this hypothesis. Alternatively, ACAT2's role may be
important in the fetus since it was easily detected by Northern blot in
human fetal liver.
The abundance of ARGP1 entries in the dbEST from a wide variety of cDNA libraries is reflective of the ubiquitous nature of ARGP1 expression in human adult tissues and tissue culture cell lines. This suggests that ARGP1 serves a function important to many cell types. Expression of two independent clones of ARGP1 under the regulation of two yeast promoters, GAL1/10 and GAPDH (not shown), failed to detectably esterify cholesterol or ergosterol. ARGP1-specific mRNA was identified by RT-PCR in each case. We take this as further evidence that unlike ACAT1 and ACAT2, ARGP1 is not involved in cholesterol esterification, at least when expressed in yeast. Based on the conservation of amino acids in ARGP1 that are important for ACAT1 to be active, ARGP1 likely catalyzes a reaction similar to ACAT. Other esterification reactions which use fatty-acyl CoAs as substrates include retinol esterification, methyl ester formation, triterpene esterification, monoacylglycerol transferase, and diacylglycerol transferase. In the latter case our observations of a diacylglycerol-binding site in ARGP1 biases us to the possibility of ARGP1 being diacylglycerol acyltransferase, which to date has not been isolated at the molecular level. We are presently investigating whether ARGP1 can mediate these reactions.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. B. Soares for helpful advice, Fanny Keyserman for technical assistance, and Dr. J. J. Rich for advice regarding this manuscript.
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FOOTNOTES |
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* This work was supported in part by a Grant-in-Aid/Investigatorship from the American Heart Association (NYC Affiliate) and by the Ara Parseghian Medical Research Foundation (to S. L. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF059202 and AF059203.
§ Supported by NHLBI, National Institutes of Health, Training Fellowship HL07343 in Arteriosclerosis.
Established investigator of the American Heart Association. To
whom correspondence should be addressed: Institute of Human Nutrition,
Columbia University College of Physicians and Surgeons, 650 W. 168th
St., New York, NY 10032. Tel.: 212-305-6304; Fax: 212-305-3079; E-mail:
sls37{at}columbia.edu.
The abbreviations used are: ACAT, acyl coenzyme A:cholesterol acyltransferase; ARGP, ACAT related gene product; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s); RT, reverse transcriptase; DEPC, diethylpyrocarbonate.
2 T. Seo, P. Oelkers, M. Giattina, R. J. Deckelbaum, and S. L. Sturley, manuscript in preparation.
3 Z. Guo, D. Cromley, J. T. Billheimer, and S. L. Sturley, manuscript in preparation.
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Abstract
Introduction
Procedures
Results
Discussion
References
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 M. Buszczak, X. Lu, W. A. Segraves, T. Y. Chang, and L. Cooley Mutations in the midway Gene Disrupt a Drosophila Acyl Coenzyme A: Diacylglycerol Acyltransferase Genetics, April 1, 2002; 160(4): 1511 - 1518. [Abstract] [Full Text] [PDF]
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P. Oelkers, D. Cromley, M. Padamsee, J. T. Billheimer, and S. L. Sturley The DGA1 Gene Determines a Second Triglyceride Synthetic Pathway in Yeast J. Biol. Chem., March 8, 2002; 277(11): 8877 - 8881. [Abstract] [Full Text] [PDF]
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T. Y. Chang, C. C. Y. Chang, X. Lu, and S. Lin Catalysis of ACAT may be completed within the plane of the membrane: a working hypothesis J. Lipid Res., December 1, 2001; 42(12): 1933 - 1938. [Abstract] [Full Text] [PDF]
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S. Cases, S. J. Stone, P. Zhou, E. Yen, B. Tow, K. D. Lardizabal, T. Voelker, and R. V. Farese Jr. Cloning of DGAT2, a Second Mammalian Diacylglycerol Acyltransferase, and Related Family Members J. Biol. Chem., October 12, 2001; 276(42): 38870 - 38876. [Abstract] [Full Text] [PDF]
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 K. Jensen-Pergakes, Z. Guo, M. Giattina, S. L. Sturley, and M. Bard Transcriptional Regulation of the Two Sterol Esterification Genes in the Yeast Saccharomyces cerevisiae J. Bacteriol., September 1, 2001; 183(17): 4950 - 4957. [Abstract] [Full Text] [PDF]
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Z. Guo, D. Cromley, J. T. Billheimer, and S. L. Sturley Identification of potential substrate-binding sites in yeast and human acyl-CoA sterol acyltransferases by mutagenesis of conserved sequences J. Lipid Res., August 1, 2001; 42(8): 1282 - 1291. [Abstract] [Full Text] [PDF]
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C. Jako, A. Kumar, Y. Wei, J. Zou, D. L. Barton, E. M. Giblin, P. S. Covello, and D. C. Taylor Seed-Specific Over-Expression of an Arabidopsis cDNA Encoding a Diacylglycerol Acyltransferase Enhances Seed Oil Content and Seed Weight Plant Physiology, June 1, 2001; 126(2): 861 - 874. [Abstract] [Full Text] [PDF]
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L. J. Wilcox, N. M. Borradaile, L. E. de Dreu, and M. W. Huff Secretion of hepatocyte apoB is inhibited by the flavonoids, naringenin and hesperetin, via reduced activity and expression of ACAT2 and MTP J. Lipid Res., May 1, 2001; 42(5): 725 - 734. [Abstract] [Full Text]
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K. Aragane, K. Fujinami, K. Kojima, and J. Kusunoki ACAT inhibitor F-1394 prevents intimal hyperplasia induced by balloon injury in rabbits J. Lipid Res., April 1, 2001; 42(4): 480 - 488. [Abstract] [Full Text]
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K. K. Maung, A. Miyazaki, H. Nomiyama, C. C. Y. Chang, T.-Y. Chang, and S. Horiuchi Induction of acyl-coenzyme A:cholesterol acyltransferase-1 by 1,25-dihydroxyvitamin D3 or 9-cis-retinoic acid in undifferentiated THP-1 cells J. Lipid Res., February 1, 2001; 42(2): 181 - 187. [Abstract] [Full Text]
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 A. Ruiz, M. H. Kuehn, J. L. Andorf, E. Stone, G. S. Hageman, and D. Bok Genomic Organization and Mutation Analysis of the Gene Encoding Lecithin Retinol Acyltransferase in Human Retinal Pigment Epithelium Invest. Ophthalmol. Vis. Sci., January 1, 2001; 42(1): 31 - 37. [Abstract] [Full Text]
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R. G. Lee, M. C. Willingham, M. A. Davis, K. A. Skinner, and L. L. Rudel Differential expression of ACAT1 and ACAT2 among cells within liver, intestine, kidney, and adrenal of nonhuman primates J. Lipid Res., December 1, 2000; 41(12): 1991 - 2001. [Abstract] [Full Text]
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C. W. Joyce, G. S. Shelness, M. A. Davis, R. G. Lee, K. Skinner, R. A. Anderson, and L. L. Rudel ACAT1 and ACAT2 Membrane Topology Segregates a Serine Residue Essential for Activity to Opposite Sides of the Endoplasmic Reticulum Membrane Mol. Biol. Cell, November 1, 2000; 11(11): 3675 - 3687. [Abstract] [Full Text]
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 N. N. Izzat, M. E. Deshazer, and D. S. Loose-Mitchell New Molecular Targets for Cholesterol-Lowering Therapy J. Pharmacol. Exp. Ther., May 1, 2000; 293(2): 315 - 320. [Abstract] [Full Text]
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J. Kusunoki, K. Aragane, T. Kitamine, H. Kozono, K. Kano, K. Fujinami, K. Kojima, T. Chiwata, and Y. Sekine Postprandial Hyperlipidemia in Streptozotocin-Induced Diabetic Rats Is Due to Abnormal Increase in Intestinal Acyl Coenzyme A:Cholesterol Acyltransferase Activity Arterioscler. Thromb. Vasc. Biol., January 1, 2000; 20(1): 171 - 178. [Abstract] [Full Text] [PDF]
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 N. Sakashita, A. Miyazaki, M. Takeya, S. Horiuchi, C. C. Y. Chang, T.-Y. Chang, and K. Takahashi Localization of Human Acyl-Coenzyme A:Cholesterol Acyltransferase-1 (ACAT-1) in Macrophages and in Various Tissues Am. J. Pathol., January 1, 2000; 156(1): 227 - 236. [Abstract] [Full Text] [PDF]
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C. Yu, J. Chen, S. Lin, J. Liu, C. C. Y. Chang, and T.-Y. Chang Human Acyl-CoA:Cholesterol Acyltransferase-1 Is a Homotetrameric Enzyme in Intact Cells and in Vitro J. Biol. Chem., December 17, 1999; 274(51): 36139 - 36145. [Abstract] [Full Text] [PDF]
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K. A. H. Abo-Hashema, M. H. Cake, G. W. Power, and D. Clarke Evidence for Triacylglycerol Synthesis in the Lumen of Microsomes via a Lipolysis-Esterification Pathway Involving Carnitine Acyltransferases J. Biol. Chem., December 10, 1999; 274(50): 35577 - 35582. [Abstract] [Full Text] [PDF]
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S. Lin, D. Cheng, M.-S. Liu, J. Chen, and T.-Y. Chang Human Acyl-CoA:Cholesterol Acyltransferase-1 in the Endoplasmic Reticulum Contains Seven Transmembrane Domains J. Biol. Chem., August 13, 1999; 274(33): 23276 - 23285. [Abstract] [Full Text] [PDF]
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J. R. Burnett, L. J. Wilcox, D. E. Telford, S. J. Kleinstiver, P. H. R. Barrett, R. S. Newton, and M. W. Huff Inhibition of ACAT by avasimibe decreases both VLDL and LDL apolipoprotein B production in miniature pigs J. Lipid Res., July 1, 1999; 40(7): 1317 - 1328. [Abstract] [Full Text]
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R. L. Hamilton, A. Moorehouse, S. R. Lear, J. S. Wong, and S. K. Erickson A rapid calcium precipitation method of recovering large amounts of highly pure hepatocyte rough endoplasmic reticulum J. Lipid Res., June 1, 1999; 40(6): 1140 - 1147. [Abstract] [Full Text]
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D. Warnecke, R. Erdmann, A. Fahl, B. Hube, F. Muller, T. Zank, U. Zahringer, and E. Heinz Cloning and Functional Expression of UGT Genes Encoding Sterol Glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum J. Biol. Chem., May 7, 1999; 274(19): 13048 - 13059. [Abstract] [Full Text] [PDF]
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B.-L. Li, X.-L. Li, Z.-J. Duan, O. Lee, S. Lin, Z.-M. Ma, C. C. Y. Chang, X.-Y. Yang, J. P. Park, T. K. Mohandas, et al. Human Acyl-CoA:Cholesterol Acyltransferase-1 (ACAT-1) Gene Organization and Evidence That the 4.3-Kilobase ACAT-1 mRNA Is Produced from Two Different Chromosomes J. Biol. Chem., April 16, 1999; 274(16): 11060 - 11071. [Abstract] [Full Text] [PDF]
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S. Cases, S. Novak, Y.-W. Zheng, H. M. Myers, S. R. Lear, E. Sande, C. B. Welch, A. J. Lusis, T. A. Spencer, B. R. Krause, et al. ACAT-2, A Second Mammalian Acyl-CoA:Cholesterol Acyltransferase. ITS CLONING, EXPRESSION, AND CHARACTERIZATION J. Biol. Chem., October 9, 1998; 273(41): 26755 - 26764. [Abstract] [Full Text] [PDF]
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P. Oelkers, A. Tinkelenberg, N. Erdeniz, D. Cromley, J. T. Billheimer, and S. L. Sturley A Lecithin Cholesterol Acyltransferase-like Gene Mediates Diacylglycerol Esterification in Yeast J. Biol. Chem., May 19, 2000; 275(21): 15609 - 15612. [Abstract] [Full Text] [PDF]
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H. Yagyu, T. Kitamine, J.-i. Osuga, R.-i. Tozawa, Z. Chen, Y. Kaji, T. Oka, S. Perrey, Y. Tamura, K. Ohashi, et al. Absence of ACAT-1 Attenuates Atherosclerosis but Causes Dry Eye and Cutaneous Xanthomatosis in Mice with Congenital Hyperlipidemia J. Biol. Chem., July 7, 2000; 275(28): 21324 - 21330. [Abstract] [Full Text] [PDF]
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C. C. Y. Chang, N. Sakashita, K. Ornvold, O. Lee, E. T. Chang, R. Dong, S. Lin, C.-Y. G. Lee, S. C. Strom, R. Kashyap, et al. Immunological Quantitation and Localization of ACAT-1 and ACAT-2 in Human Liver and Small Intestine J. Biol. Chem., September 1, 2000; 275(36): 28083 - 28092. [Abstract] [Full Text] [PDF]
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J.-B. Yang, Z.-J. Duan, W. Yao, O. Lee, L. Yang, X.-Y. Yang, X. Sun, C. C. Y. Chang, T.-Y. Chang, and B.-L. Li Synergistic Transcriptional Activation of Human Acyl-coenzyme A: Cholesterol Acyltransterase-1 Gene by Interferon-gamma and All-trans-Retinoic Acid THP-1 Cells J. Biol. Chem., June 8, 2001; 276(24): 20989 - 20998. [Abstract] [Full Text] [PDF]
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K. K. Buhman, H. C. Chen, and R. V. Farese Jr. The Enzymes of Neutral Lipid Synthesis J. Biol. Chem., October 26, 2001; 276(44): 40369 - 40372. [Full Text] [PDF]
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