(Received for publication, February 6, 1996)
From the
The cDNA encoding rat brain cytosolic acyl-CoA thioester hydrolase (ACT) has been cloned and sequenced, and the primary structure of the enzyme has been deduced. A partial amino acid sequence (38 amino acids) of the enzyme was determined using the peptides generated after CNBr digestion of the purified enzyme. Primers synthesized on the basis of this information were used to isolate two cDNA clones, each encoding the full length of the enzyme. The nucleotide sequences of these clones contained an open reading frame encoding a 358-amino acid polypeptide with a calculated molecular mass of 39.7 kDa, similar to that determined for the purified enzyme (40.9 kDa). The deduced ACT sequence showed no homology to the known sequences of any other thioesterases nor to any other known protein sequence. However, there was a strong homology to a number of expressed sequence tag human brain cDNA clones. The identity of the ACT cDNA was confirmed by the expression of ACT activity in Escherichia coli. There was a 10-15-fold increase in ACT-specific activity in the bacterial extracts after induction with isopropyl thiogalactoside, and the properties of the expressed enzyme (fusion protein) were the same as those of the purified rat brain ACT. Northern blot analysis showed that a 1.65-kilobase ACT transcript was present in rat brain and testis but not in any other rat tissues examined. However, the ACT mRNA was induced in the liver of rats that were fed Wy-14,643, a peroxisome proliferator and inducer of rodent liver cytosolic acyl-CoA thioesterase. These results indicate that the induced rat liver ACT is homologous to the constitutive rat brain ACT.
Long chain acyl coenzyme A thioester hydrolase (E.C. 3.1.2.2) is
present in all living organisms (Waku, 1992). Isoforms of this acyl-CoA
thioesterase (ACT) ()are present in a membrane-bound form in
various subcellular organelles (Berge and Farstad, 1979; Berge et
al., 1984) and in soluble form inside mitochondria (Svensson et al., 1995a), peroxisomes (Svensson et al., 1995b),
and cytosol (Srere et al., 1959). The best characterized of
these enzymes are those that are associated with the fatty
acid-synthesizing enzyme, where ACT specificity determines the chain
length of the synthesized fatty acids (Naggert et al., 1991a;
Tai et al., 1993; Witkowski et al., 1991). Likewise,
the estrogen-induced thioesterase in duck uropygial gland has also been
shown to regulate the chain lengths of fatty acid end products (Hwang
and Kolattukudy, 1993). The mitochondrial and microsomal membrane-bound
enzymes may have a similar function in controlling the chain length of
the fatty acid products during the enzymatic chain elongation process
(Berge, 1979). Some of these enzymes may also act as acyltransferases
(Lehner and Kuksis, 1993); however, their physiological function in
many systems is not known (Anderson and Erwin, 1971; Cho and Cronan,
1993; Naggert et al., 1991a). These enzymes have been purified
to homogeneity from plants, animals, and bacteria, and the cDNAs
encoding several of them have been cloned and sequenced (Cho and
Cronan, 1993; Dormann et al., 1994; Hwang and Kolattukudy,
1993; Loader et al., 1993; Naggert et al., 1991a,
1991b). The properties, physiological regulation, and catalytic
mechanisms of the purified and cloned enzymes have been extensively
studied (Naggert et al., 1991a, 1991b; Cho and Cronan, 1993;
Tai et al., 1993; Witkowski et al., 1991, Hwang and
Kolattukudy, 1993).
Mammalian tissues contain a cytosolic acyl-CoA thioesterase not associated with fatty acid synthase that has high activity in brain and testis (Anderson and Erwin, 1971; Broustas and Hajra, 1995; Kurooka et al., 1972, Smith and Sun, 1981, Srere et al., 1959). Although the activity of this cytosolic thioesterase is normally very low in mammalian liver, the enzyme is highly induced in rodent livers when peroxisome-proliferating agents are administered to the animals (Kawashima et al., 1981; Miyazawa et al., 1981). Homologous soluble enzymes have also been reported to be induced inside liver mitochondria and peroxisomes (Svensson et al., 1995a; Wilcke and Alexson, 1994).
The mammalian soluble ACT has been purified to homogeneity from heart cytosol (Gross, 1983), and from liver cytosol (Miyazawa et al., 1981; Yamada et al., 1994), mitochondrial matrix (Svensson et al., 1995a), and peroxisomal matrix (Wilcke and Alexson, 1994) after induction of the enzyme by peroxisome-proliferating agents. Miyazawa et al.(1981) reported that the antibodies raised against the induced rat liver cytosolic thioesterase precipitated rat brain cytosolic thioesterase. Recently, Yamada et al.(1994) reported that two ACTs are induced in liver by peroxisome-proliferators, and the antibodies raised against the higher specific activity liver enzyme (ACH1) cross-reacted on a Western blot with a 36-kDa brain protein.
The rat brain
cytosolic ACT has been partially purified, and its properties have been
studied in different laboratories (Anderson and Erwin, 1971; Lin et
al., 1984; Srere et al., 1959). We have recently purified
this enzyme by 3500-fold to homogeneity, which showed a single band on
SDS-polyacrylamide gel electrophoresis with a molecular mass of 40.9
kDa (Broustas and Hajra, 1995). The enzyme had a very broad substrate
specificity with respect to the acyl-CoA chain length and a very high
specific activity (turnover number = 7 10
min
) compared with other purified
thioesterases. A partial amino acid sequence of the enzyme was
determined by gas phase sequencing of peptides generated by CNBr
digestion of the purified enzyme. From this information, we have cloned
the rat brain cDNA encoding the cytosolic ACT. The validity of the
sequence and the utility of the cDNA clone have been demonstrated by
expressing the thioesterase activity in Escherichia coli.
Figure 1: Primers used for the synthesis of probe by PCR. The sequence of various degenerate primers and the corresponding amino acids for the sense and antisense strands are shown. N in the primer sequence indicates that all four nucleotides were used.
The PCR reaction mixture contained 1 µl of
primer (50 µM), 10 µl of 10 Taq polymerase buffer, 10 µl of dNTP mixture (2.5 mM each), 1 µl of 5` rapid amplification of cDNA ends-ready rat
brain cDNA (Clontech), 5 µl of Me
SO, and water to make
100 µl. The first cycle of the PCR was carried out as follows: 94
°C for 1 min, 50 °C for 30 s, raised to 72 °C in the next
30 s followed by 90 s at 72 °C. The subsequent 40 cycles were all
the same except the denaturation (94 °C) was done for 30 s. 10
µl of the reaction mixture was subjected to 3% NuSieve agarose gel
electrophoresis, and a band of the expected size (114 bp) was seen for
both the primer combinations (Broustas, 1995). This PCR product was
purified from the remaining reaction mixture by preparative agarose gel
electrophoresis (Uhler, 1993).
Figure 2:
Sequencing strategies and restriction map
for brain ACT cDNA clone. The map of the consensus sequence is shown,
the black box shows the open reading frame, and the white
boxes represent the untranslated 3` and 5` ends. The sites for the
restriction endonucleases used to generate probes and confirm the
sequence are shown. The start of translation of the lacZ gene
within pBluescript-SK is also indicated, along with
the T7 and T3 promoter regions. The hatched boxes indicate the
positions and sizes of the four clones used for sequencing the cDNA.
The arrows indicate the direction of sequencing using either
sense (5.x) or antisense (3.x) primers. MCS,
multiple cloning site.
The RNA blot was
first incubated in the hybridization buffer (5% SDS, 400 mM sodium phosphate, pH 7.2, 1 mM EDTA, 1 mg/ml bovine serum
albumin, 50% formamide) at 60 °C for 4 h. The radioactive probe was
then added to the solution, and the blot was further incubated with
gentle shaking at 60 °C for 22 h. The blot was washed two times
with 0.5 SET buffer at 60 °C followed by one wash with the
same buffer at room temperature. After air drying, the blot was used
for autoradiography by exposing x-ray film to it at -70°.
To isolate clones containing the missing 5` end, the cDNA library was again screened, this time with a 250-bp probe generated from the 5` end of ACT 4.0. Among the new clones, ACT 1.1 and ACT 5.1 were found to contain the largest inserts: 1.5 kb and 1.4 kb, respectively. The nucleotide sequences of these two clones were determined using the same strategies described under ``Experimental Procedures.'' The ACT 1.1 insert was found to be a 1400-bp-long polynucleotide that completely contained the nucleotide sequence of ACT 4.0, as mentioned above. The sequence of ACT 5.1 was similar to 1.1 but was truncated at both the 5` and 3` ends (1350 bp). Both these clones contained a putative start methionine codon (ATG) at the 5` end. A comparison of the clones with respect to their relative lengths and positions and a restriction map is given in Fig. 2. The sequence of ACT 1.1, which contained the full-length sequence of clones 5.1, 4.0, and 5.0, is shown in Fig. 3. This cDNA contained a 1074-bp open reading frame with an untranslated region of 93 bp at the 5` end, 231 bp at the 3` end, and a consensus polyadenylation signal (AATAAA) 16 bp upstream from the poly(A) tail. Translation of this open reading frame yielded a 358-amino acid protein with a calculated molecular mass of 39.7 kDa, which is in close agreement with the molecular mass of 40.9 kDa deduced from SDS-polyacrylamide gel electrophoresis analysis of the enzyme (Broustas and Hajra, 1995). The deduced amino acid sequence for the cDNA clone ACT 1.1 is given in Fig. 3. No nucleotide sequence differences were found for any of the cDNAs described.
Figure 3: Nucleotide and deduced amino acid sequence of cDNA encoding brain ACT. The single line underlined region corresponds to the partial amino acid sequence determined directly from the enzyme, which was used to generate the probe for screening. The polyadenylation signal is shown by the double lines.
Figure 4: A, induction of ACT in E. coli. To the growing E. coli clones (ACT 1.1 and ACT 5.1), the inducer (IPTG) was added (see ``Experimental Procedures''), and cultures were incubated for up to 36 h. Aliquots were taken from both the control and experimental cultures at the indicated time intervals, and the ACT activity in these cells was determined as described under ``Experimental Procedures.'' B, substrate specificity of induced ACT expressed in E. coli. Induced ACT activity with various acyl-CoAs was measured by using the optical assay (see ``Experimental Procedures'') in the extracts of E. coli grown in IPTG-containing medium (24 h).
The properties of the induced thioesterase in the E. coli extract were very similar to those of brain cytosolic
acyl-CoA thioesterase, as seen by its inhibition by bovine serum
albumin, detergents, diethylpyrocarbonate, and p-hydroxymercuribenzoate but not by N-ethylmaleimide
or phenylmethanesulfonyl fluoride (data not shown). The K of the induced E. coli enzyme with
palmitoyl CoA was 4.5 µM, similar to that of the brain ACT
(6.0 µM). Substrate specificity of the recombinant enzyme
was broad with respect to the chain length (Fig. 4B),
similar to that of the rat brain ACT (Broustas and Hajra, 1995).
Figure 5: Northern blot analysis of size and tissue distribution of ACT mRNA in normal and Wy-14,643-fed rats. A, the Northern blot of mRNA from different rat tissues was hybridized with the radioactive riboprobe generated from the ACT cDNA clone and then autoradiographed as described in the text. The mobilities of size marker RNAs are indicated. B, the total RNA extracted from brain, testis, and liver of Wy-14,643-fed (Experimental) and control rats were subjected to formaldehyde-agarose gel electrophoresis, followed by blotting onto Nylon membrane and hybridization with the labeled riboprobe as described in the text. The bottom panel shows long exposure (14-day), and the top panel shows short exposure (overnight) of the same blot to x-ray films.
Northern blot analysis was also performed on RNA from liver, brain, and testis of rats fed Wy-14,643, a strong peroxisome proliferator. As seen previously, ACT mRNA was detected at high levels in testis and at lower levels in brain of control animals. However, in the rats fed Wy-14,643, there was a greatly increased level of ACT mRNA seen in liver, a slight increase in testis, and no increase in brain tissue (Fig. 5B).
Three initial findings support the idea that the cloned cDNA
encodes rat brain ACT: 1) the calculated M of the
enzyme from the deduced amino acid sequence matches well with the
observed value; 2) the transcript size as found by Northern blotting is
compatible with the observed size of the enzyme; and 3) the mRNA
hybridized with the radioactive probe made from the cDNA is observed
only in tissues where cytosolic ACT is expressed, such as normal brain
and testis and Wy-14,643-fed rat liver (Fig. 5). The most
convincing evidence is that enzyme activity is highly induced in E.
coli bearing the cloned cDNA-containing plasmid (Fig. 4).
It is, however, surprising that neither the nucleotide nor the amino
acid sequence of brain ACT shows any homology to any of the known cDNAs
encoding other ACTs, such as thioesterase I and II (Naggert et
al., 1991b; Poulose et al., 1985; Tai et al.,
1993), estrogen-induced duck uropygial gland thioesterase (Hwang and
Kolattukudy, 1993), thioesterase of developing seeds (Dormann et
al., 1994; Loader et al., 1993), and E. coli thioesterases (Cho and Cronan, 1993; Naggert et al.,
1991a). Some of these thioesterases have a serine at the active site in
a GXSXG motif, but no such motif is observed in the
sequence of brain ACT, although this is not altogether unexpected,
because brain ACT is not inhibited by active site serine-specific
reagents such as diisopropylphosphorofluoridate or
phenylmethanesulfonyl fluoride. A C-terminal GXH sequence,
which is present in many thioesterases (Cho and Cronan, 1993), is
present in brain ACT as GQH from amino acid residues 346-348.
Brain ACT is strongly inhibited by diethylpyrocarbonate, indicating
that a histidyl moiety may be involved in catalysis. An active site
histidine has been demonstrated in mammary gland thioesterase II, but
the mammary gland enzyme is also inhibited by phenylmethanesulfonyl
fluoride, indicating that serine is also involved in the catalysis. The
brain enzyme is also strongly inhibited by p-hydroxymercuribenzoate, a sulfhydryl agent, indicating that
a thiol group may be involved in catalysis.
The deduced amino acid sequence of brain ACT does not seem to have any consensus functional sequence motifs, and the sequence itself does not yield any clues regarding its catalytic function. Therefore, it seems that the ACT sequence presented here is unique and represents a new class of enzyme utilizing long chain acyl-CoAs as substrates. Considering that brain ACT is a cytosolic (soluble) enzyme, there is a comparatively large proportion (40%) of hydrophobic amino acids present in the deduced amino acid sequence. A hydropathy plot indicates that the hydrophobic amino acids are almost evenly distributed along the polypeptide backbone, except for the region of amino acid residues 140-156, where no hydrophobic amino acids are present.
Though the ACT sequence shows no homology to any known protein sequence, a very strong homology was seen with a number of human brain EST clones as presented in Table 1. The translated amino acid sequences of these clones have a higher homology with the deduced polypeptide sequence of ACT than the nucleotide sequences because of species-specific codon selection. The sequences of these clones overlap to cover the entire deduced length of rat brain ACT cDNA (Table 1). There is greater than 90% homology between the translated region of the combined human EST clones and rat brain ACT, and in the positions where the amino acids do differ, the replacement amino acid is usually homologous, bringing the sequence similarity close to 100%. Such phylogenetic conservation of structure implies that ACT plays an important physiological function in mammalian brain.
The induction of ACT in E. coli indicates that post-translational modifications are
not necessary for the enzyme to show catalytic activity. It is of
interest to note that this high level of induction (>20 total
ACT endogenous activity) of a thioesterase, which catalyzes the
hydrolysis of acyl-CoAs, vital cellular building blocks, did not hamper
the growth of the bacteria. Expression of other thioesterases in E.
coli have also been shown not to be deleterious to the growth of
the bacteria (Cho and Cronan, 1993; Naggert et al., 1991a,
1991b). The expression of brain ACT in E. coli will provide
the active enzyme in large quantities for studies on its mechanism of
catalysis for antibody production and also for detailed studies on its
structure-function relationships.
Northern blot analysis shows that
the mRNA for ACT is expressed only in rat brain and testis, which
corresponds well to the known tissue distribution of the enzyme. In
rats the enzyme activity is highest in brain followed by testis
(50% of brain activity on a per gram basis) (Kurooka et
al., 1972; Yamada et al., 1994). All other organs have
very low enzyme activity, which may in part be due to the presence of
other isoforms of the enzyme. The surprising finding is that although
the specific activity of ACT is lower in testis, the ACT mRNA level is
much higher in this tissue than in brain (Fig. 5). No ACT mRNA
was found in normal rat liver, but ACT mRNA is present in the liver of
Wy-14,643-fed rats (Fig. 5). The cytosolic ACT activity in liver
is also greatly increased in these rats (from 0.01 to 0.2 units/mg
protein). Though the Northern blot analysis qualitatively indicates
that the ACT mRNA level in testis is also increased in the
Wy-14,643-fed rats (Fig. 5B), no change was observed in
the activity of ACT in testis of these animals. (
)Yamada et al. (1994) also observed no increase in ACT activity in
testis of di(2-ethylhexyl)phthalate-fed rats. The induction of ACT mRNA
in liver and the similarity of properties between the brain ACT and the
induced liver thioesterase reported by other laboratories (Kawashima et al., 1981; Yamada et al., 1994) indicate that
either the same isozyme or an enzyme that is structurally very similar
to the brain ACT is induced in liver.
The physiological function of
this cytosolic thioesterase is not clear. The enzyme activity is
associated with the lipid metabolic activity of tissues (Svensson et al., 1995b) with highest activity in brain and testis,
tissues that have high steroidogenic activity. We have postulated that
this enzyme regulates peroxisomal fatty acid oxidation, one of the
functions of which is to produce acetyl CoAs, the building block for
steroids and also for fatty acids (Broustas and Hajra, 1995; Broustas,
1995). The increase in activity of this enzyme in liver after induction
correlates with the increase in peroxisomal -oxidation of fatty
acids and the increase in liver weight of the animals, indicating that
the high activity of ACT is associated with the high rate of membrane
lipid biosynthesis. Our findings suggest that differences exist in the
regulation of this enzyme in brain, where it is constitutive, and
liver, where it is highly induced by hypolipidemic
peroxisome-proliferating agents. The induction of proteins in rodent
liver by these agents has been shown to be effected via cytosolic
receptors (PPAR), which, after binding to the hypolipidemic agents, are
translocated to the nucleus. Within the nucleus, PPARs have been shown
to form heterodimers with retinoid X receptors and act as transcription
factors (Green and Wahli, 1994). It is possible that in brain these
PPARs are constitutively active due to the presence of endogenous
ligands such as free fatty acids. The cDNA clones described here and
the primary structure of the enzyme presented should prove to be very
useful in establishing the mechanism of this tissue-specific
regulation, as well as the physiological function of this enzyme.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U49694[GenBank].