Molecular characterization and expression of rat acyl-CoA synthetase 3.

Isolation and characterization of a rat brain cDNA identified a third acyl-CoA synthetase (ACS) designated ACS3. The deduced amino acid sequence of the cDNA revealed that ACS3 consists of 720 amino acids and exhibits a structural architecture common to ACSs from various origins. ACS3 expressed in COS cells was purified to near homogeneity. The purified ACS3 resolved by SDS-polyacrylamide gel electrophoresis into two major proteins of 79 and 80 kDa. Cell-free translation of a synthetic mRNA encoding the entire region of ACS3 revealed that the two isoforms were derived from the same mRNA. The purified ACS3 utilizes laurate and myristate most efficiently among C8-C22 saturated fatty acids and arachidonate and eicosapentaenoate among C16-C20 unsaturated fatty acids. Northern blot analysis revealed that ACS3 mRNA is most abundant in brain and, to a much lesser extent, in lung, adrenal gland, kidney, and small intestine. During the development of the rat brain, expression of ACS3 mRNA reached a maximum level at 15 days after birth and then declined gradually to 10% of the maximum in the adult brain.

Activation of fatty acids catalyzed by acyl-CoA synthetase (ACS, 1 EC 6.2.1.3) is an initial reaction of fatty acid metabolism in eukaryotic cells. This reaction is indispensable in fatty acid utilization; thereby ACS plays a key role in lipid metabolism. Acyl-CoA produced by this enzyme is a key intermediate in two major metabolic pathways: degradation of fatty acids via the ␤-oxidation system; and synthesis of cellular lipids that include triglycerides, phospholipids, and cholesterol esters. In addition, acyl-CoA is utilized in numerous reactions, including protein modification (1) and intracellular protein transport (2). Acyl-CoA also functions as a modulator of protein kinase C (3) and nuclear thyroid hormone receptor (4).
ACS is a member of a growing family of enzymes that resemble firefly luciferase in amino acid sequence (5,6). This ACS/ luciferase family includes acetyl-CoA synthetases from Neurospora crassa and Aspergillus nidulans (7), 4-coumarate:CoA ligase 1 from parsley (8), Bacillus brevis gramicidin S synthetase 1 (9), and tyrocidine synthetase 1 (10), Penicillium chrysogenum ␦-(L-␣-aminoadipyl)-L-cysteinyl-D-valine synthetase (11,12), luciferases from click beetle (13) and firefly (14), and ACS itself. All of the members of this family require ATP as a cofactor and catalyze a two-step reaction: adenylation of substrates with subsequent thioester formation (5,6,15). The brain is one of the most lipid-enriched organs in the body. Most brain lipids are actively synthesized in the brain itself and deposited in large amounts during the early phase of development of the nervous system (16,17). Despite the importance of lipids in the brain, relatively little is known about lipid metabolism in the brain.
In a previous study, we have shown the presence of a brainspecific isozyme designated brain ACS (hereafter referred to as ACS2) by cDNA cloning (18). ACS2 resembles the well characterized ACS (hereafter referred to as ACS1) in amino acid sequence (5,6); approximately 65% of the amino acids in the two enzymes are identical. Although ACS1 and ACS2 are structurally similar and exhibit similar fatty acid specificity, the patterns of tissue expression of the two enzymes are completely different. ACS1 mRNA is most abundant in liver, heart, and adipose tissue and, to a much lesser extent, in brain (5). In contrast, ACS2 is predominantly expressed in the brain (18). Although the brain contains two types of ACS, the presence of other brain types of ACS specific for arachidonate (19), docosahexaenoate (20,21), and lignocerate (22)(23)(24) have been shown by enzymatic characterizations. However, none of these enzymes have been fully characterized by purification or cDNA cloning.
To further extend our studies on ACS in brain, we have isolated a cDNA that encodes a new brain-type ACS distinct from ACS1 and ACS2. We describe here the primary structure, expression, and regulation of this brain-type ACS designated ACS3. We also describe the enzymatic properties of the purified enzyme from COS cells transfected with ACS3 cDNA.
Standard Procedures-Standard molecular biology techniques were carried out essentially as described by Sambrook et al. (26). cDNA fragments were subcloned into pUC vectors in both orientations and sequenced by the dideoxy chain termination method (27) performed with T7 DNA polymerase (Sequenase, version 2.0; U. S. Biochemical Corp.) and [␣-35 S]dCTP. To sequence the entire region of the cDNA, the cDNA fragments were shortened successively by exonuclease III (28) and subcloned into pUC vectors. For Northern blotting, total RNA prepared by the guanidinium thiocyanate/CsCl method (29) was denatured with 1 M glyoxal and 50% dimethyl sulfoxide, fractionated in a 1.5% agarose gel, and transferred to a Zeta Probe nylon membrane (Bio-Rad).
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)-Poly(A) RNA was prepared from adult rat brain total RNA using oligo(dT)-Latex (Takara Shuzo) as described (30). A mixture of oligo(dT)primed cDNAs was synthesized from 3 g of brain poly(A) RNA using oligo(dT) 15 (1.5 g), 25 units of RNasin, and 400 units of Superscript (Life Technologies, Inc.) in 30 l of standard reverse transcription buffer (50 mM Tris-HCl, pH 8.3, 15 mM MgCl 2 , 75 mM KCl, 1 mM dithiothreitol, and 1 mM of each deoxynucleotide triphosphate) at 37°C for 1 h. The reaction products were then subjected to replacement synthesis of the second strand cDNA (31). The resulting doublestranded cDNA was precipitated in ethanol and dissolved in 20 l of 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA. Five l of the cDNA were amplified with PCR (32) using two highly degenerate primers derived from the most highly conserved amino acid sequences among ACS1, ACS2, and click beetle luciferase (18). The sequences of the sense primer and the antisense primer were 5Ј-GG 1% Triton X-100, and 0.2 mM of each deoxynucleotide triphopshate). The thermal profile used was 94°C for 30 s, 54°C for 1 min, and then 72°C for 2 min. After 30 cycles, the PCR products were separated by electrophoresis on a 5% polyacrylamide gel. Major reaction products (0.5-1.0 kb) were eluted from the gel, subcloned into T-vectors (33), and sequenced. By sequencing 50 clones, we obtained a cDNA encoding a new type of ACS (pOCT5-5).
Isolation of cDNAs-Rat brain cDNA library (18) constructed in Okayama-Berg vector (34) was screened using a 290-base pair cDNA fragment from pOCT5-5 as a probe. By screening of 3 ϫ 10 5 clones, we obtained eight positive clones; one representative clone containing the largest cDNA insert (pACS3) was further characterized.
DNA Transfection-COS-7 cells were grown in monolayer culture in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin. Cells were transfected with 10 g of plasmid DNA by the DEAE-dextran procedure (35). Three days after DNA transfection, the cells were harvested for measurement of ACS activity.
Assay of ACS Activity-ACS activity was determined at 37°C either by the isotopic method (19) or by the spectrophotometric method (25); the latter was used only for the purified enzyme. The ratio of the activity measured by the spectrophotometric method (with the standard mixture) to that measured by the isotopic method (with the standard mixture) was 0.75. All assays were carried out within the range where the reaction proceeded linearly with time and the initial rate of reaction was proportional to the amount of enzyme added. The protein content of cell extract was measured by the Lowry method (36) with bovine serum albumin as the standard. The reaction product of the enzyme reaction was determined as described (25).
Purification of ACS3-A typical purification is described. All purification procedures were carried out at 4°C. One hundred fifty dishes (100-mm diameter) of COS-7 cells transfected with pACS3 were suspended in 30 ml of buffer A (50 mM potassium phosphate, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, and 10% (w/v) glycerol) containing 1 g/ml aprotinin, 1 g/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride, disrupted by sonication (three pulses of 15 s in an ice bath, 100 watts), and centrifuged at 10,000 ϫ g for 10 min. The resulting supernatant was further centrifuged at 230,000 ϫ g for 1 h to obtain microsomes. The microsomal fraction was suspended in 30 ml of buffer A containing 0.5% (w/v) Triton X-100 and 200 mM KCl using a Dounce homogenizer. The suspension was gently stirred for 1 h and then centrifuged at 230,000 ϫ g for 1 h. The resulting supernatant was dialyzed against buffer B (25 mM potassium phosphate, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Triton X-100, and 10% glycerol) and applied to a Q-Sepharose FF (Pharmacia Biotech Inc.) column (2.5 ϫ 5 cm) equilibrated with buffer B. The column was washed with three column volumes of buffer A. The void and wash fractions exhibiting enzyme activities were collected and applied to a Blue-Sepharose CL-6B (Pharmacia) column (1.5 ϫ 8 cm) equilibrated with buffer B. The column was washed with 5 column volumes of buffer C (50 mM potassium phosphate, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Triton X-100, and 10% glycerol) containing 10 mM ATP and then eluted with a linear gradient between 100 ml of buffer C and the same volume of buffer C containing 2 M NaCl. The pooled fractions exhibiting ACS activities were concentrated by ultrafiltration with Amicon CF-25. The concentrated enzyme solution was dialyzed against buffer C and stored at Ϫ80°C. The results of the purification of ACS3 are summarized in Table I. The overall purification was 11-fold with a yield of 22%. The purified enzyme exhibited a specific activity of 4.62 mol/min/mg protein at 37°C when measured by the isotopic method using palmitate as a substrate. The purified enzyme could be stored at Ϫ80°C for at least 3 months without loss of activity.
In Vitro Translation of ACS3 mRNA-The entire coding region of ACS3 cDNA was introduced into pBluescript SK(ϩ) vector, purified by CsCl banding, and translated in a TnT-coupled reticulocyte lysate system (Promega). Each coupled transcription-translation reaction contained 1 g of plasmid DNA in a final volume of 50 l and was incubated at 30°C for 1 h. For radiolabeling, the plasmid was translated in a methionine-free amino acid mixture supplemented with [ 35 S]methionine, according to the instruction of the manufacturer. An aliquot (5 l) of the reaction mixture was subjected to immunoprecipitation with an antibody against the fusion protein of glutathione Stransferase (GST) and a fragment of ACS3 (see below) and analyzed by SDS-polyacrylamide gel electrophoresis.
Antibodies-A fusion protein of GST and a fragment of ACS3 was used to immunize rabbits. To produce the GST-ACS3 fusion protein, a cDNA fragment encoding ACS3 amino acids 72-223 was ligated inframe to a pGEX bacterial expression vector (Pharmacia). The fusion protein was induced in Escherichia coli JM109 with isopropyl ␤-Dthiogalactopyranoside for 4 h. E. coli cells were sonicated in a lysis buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM EDTA, 1% Triton X-100 (w/v), 0.1% sodium deoxycholate (w/v), and 1 mM phenylmethylsulfonyl fluoride) and centrifuged at 12,000 ϫ g for 10 min at 4°C. The pellet was resuspended with the lysis buffer containing 5 M urea and centrifuged at 12,000 ϫ g for 20 min at 4°C. The fusion protein recovered in the pellet was then dissolved by boiling in the SDS-sample buffer, purified by SDS-polyacrylamide gel electrophoresis, and used as an antigen.

RESULTS AND DISCUSSION
Isolation of ACS3 cDNAs from Rat Brain-A new type of ACS designated ACS3 was isolated through PCR-mediated DNA amplification by using two degenerate oligonucleotide primers corresponding to the conserved amino acid sequences among ACS1, ACS2 and click beetle luciferase (18). Fifty clones isolated by PCR amplification were sequenced, and the sequence of one of them (pOCT5-5) showed 25% amino acid identity with the corresponding regions of ACS1 and ACS2.
To obtain a cDNA encoding a complete coding region, an adult rat brain cDNA library was screened by hybridization with a 32 P-labeled insert of pOCT5-5 under high stringency conditions. Screening 5 ϫ 10 5 clones, we obtained eight positive clones. A representative clone (designated pACS3) containing the largest cDNA insert was sequenced. The insert of the cDNA includes a 2160-base pair open reading frame encoding a protein of 720 amino acids with a calculated molecular weight of M r 80,456 (Fig. 1A). The putative initiator methionine was preceded by an in-frame stop codon present 39 nucleotides upstream.
In previous studies, we have shown that ACS1 and ACS2 consist of two regions, designated luciferase similar regions 1 and 2 (LS regions 1 and 2) that contain amino acids highly conserved among the members of the ACS/luciferase family (5, 6, 18). Between ACS1 and ACS2, the amino acids in the two LS regions and the COOH terminus are highly conserved (18). Similarly, the amino acids in the two LS regions and the COOH terminus are most highly conserved between ACS2 and ACS3 (amino acid identity of 30 -38%; Fig. 1B); the NH 2 -terminal and a linker connecting LS regions 1 and 2 lacked amino acid conservation. Like ACS1 and ACS2, the two LS regions of ACS3 are most similar to the corresponding regions of click beetle luciferase among the members of the ACS/luciferase family (excluding ACSs) from various origins (Fig. 1B).
Expression and Purification of ACS3-To characterize the fatty acid specificity and kinetic properties of the purified ACS3, the cloned ACS3 cDNA was introduced into COS cells.
When measured with palmitate as a substrate, COS cells transfected with the cDNA exhibited 8 -10-fold increased ACS activities as compared with those transfected with vector alone (data not shown). Attempts to overproduce the recombinant enzyme in E. coli cells were unsuccessful. In COS cells expressing ACS3, most of the enzyme activity was found in the microsomal fraction (data not shown). Using the microsomal fraction of the ACS3-transfected cells as a starting material, ACS3 was purified to near homogeneity. The purification procedure involved solubilization of the enzyme with Triton X-100 and chromatography on Q-Sepharose and Blue-Sepharose (Table I).
When measured by the isotopic method using palmitate as a substrate, the purified enzyme exhibited a specific activity of 4.62 mol/min/mg protein at 37°C. This value is 5-fold lower than that of the purified ACS1.
The purified ACS3 resolved by SDS-polyacrylamide gel electrophoresis into two major proteins with 79 and 80 kDa ( Fig.  2A). Consistent with the purification of the 79-and 80-kDa proteins, cell-free translation of a synthetic mRNA encoding the entire region of ACS3 produced 79-and 80-kDa proteins FIG. 1. Structure of rat ACS3 cDNA. A, nucleotide and deduced amino acid sequence of rat ACS3 cDNA. Nucleotide residues are numbered on the right; amino acids are numbered on the left. Nucleotide 1 is the A of the initiator AUG codon. Negative numbers refer to the 5Ј-untranslated region. Two in-frame translation termination codons at Ϫ39 and 2161 are indicated by asterisks. Potential polyadenylation signals are underlined. B, comparison of the amino acids in rat ACS3. The amino acids in rat ACS3 compared schematically with those in rat ACS2 and click beetle luciferase. The amino acid conservation between ACS1 and ACS2 is also represented. The two luciferase similar regions, LS1 and LS2, are indicated by filled and hatched boxes, respectively. The number of identical residues (expressed as a percentage) in a given region is indicated between the two relevant proteins. that were precipitated with the anti-ACS3-GST antibody (Fig.  2B). This indicates that the two isoforms are derived from the same mRNA. Although the synthetic mRNA contains an inframe stop codon 39 nucleotides upstream of the initiator AUG, a minor protein (87 kDa) copurified with the 79-and 80-kDa proteins was also detected in the cell-free translation. The nature of this 87-kDa protein is currently unknown.
Fatty Acid Specificity and Other Kinetic Properties-Fatty acid specificity of the purified enzyme was determined by the spectrophotometric method using various fatty acids. Fig. 3 compares the fatty acid specificity of the purified ACS3 with that of the purified ACS1. The purified ACS3 utilizes laurate and myristate most efficiently among C 8 -C 22 saturated fatty acids and arachidonate and eicosapentaenoate among C 16 -C 20 unsaturated fatty acids. This fatty acid specificity is completely different from that of the purified ACS1, which uses C 10 -C 18 saturated fatty acids and C 16 -C 20 unsaturated fatty acids with approximately equivalent activities. Unlike the purified ACS1, the purified ACS3 prefers C 16 -C 20 unsaturated fatty acids; the relative activities of ACS3 for these fatty acids are ϳ2-fold higher than those of ACS1. Although the purified ACS1 and ACS3 exhibited different fatty acid specificities, the apparent K m values of the two enzymes for myristate and arachidonate were in the same range (10 -15 M).
The purified ACS3 has optimal activity at pH 7.0 -8.5 and requires ATP (K m ϭ 0.5 Ϯ 0.04 mM), CoA (K m ϭ 0.53 Ϯ 0.08 M), and fatty acids. The reaction product formed by the enzyme from [ 14 C]palmitate was identified as palmitoyl-CoA by chromatographic analysis of its hydroxamic acid derivative. When adenylate kinase was omitted from the reaction mixture for the spectrophotometric assay, no oxidation of NADH occurred, indicating that AMP was a reaction product.
Tissue Distribution and Developmental Expression of ACS3 mRNA-Northern blot analysis using a ACS3 cDNA probe revealed that ACS3 mRNA is expressed as a 3.3-kb transcript in normal adult rat tissues (Fig. 4A). The ACS3 mRNA is predominantly expressed in the brain and appears, to a much lesser extent, in lung, adrenal gland, kidney, small intestine, and adipose tissue but is not detected in heart or liver. Consistent with its predominant expression in the brain, ACS3 mRNA was detected in rat C 6 glioma, rat glioma KEG1, and rat adrenal pheochromocytoma PC12 cells (Fig. 4B).
During the development of rat brain, ACS3 mRNA was detectable 5 days after birth, increased to a maximum level at 15 days, and then decreased gradually to 10% of its maximum level in the adult (Fig. 5A). In contrast, ACS2 mRNA increased gradually and reached a maximum level in the adult, and no change was observed in ACS1 mRNA levels during brain development (Fig. 5, B and C).
The presence in the brain of multiple forms of ACS with different fatty acid specificity is of considerable biological significance for controlling the synthesis of brain lipids. Although the exact nature of the enzyme remains to be elucidated, the Total RNA was prepared from the indicated rat tissue and cell line, and then an aliquot (15 g) was subjected to electrophoresis on a 1.5% agarose gel and blotted onto a nylon membrane. The blot was hybridized with a 32 P-labeled 2.2-kb BglII/SacI fragment of pACS3 under stringent conditions. The filter was washed in 0.1 ϫ SSC containing 1% (w/v) SDS at 65°C for 30 min and was then left to expose a Kodak XAR-5 film with an intensifying screen at Ϫ80°C for 48 h. RNA loading was consistent among lanes, as judged by ethidium bromide staining (data not shown). The autoradiograph shown is representative of three independent experiments that gave essentially identical results. availability of the cDNA will allow us to establish a mouse strain lacking this enzyme. This may reveal the role of the enzyme in the brain.
FIG . 5. Expression of ACS3 (A), ACS2 (B), and ACS1 (C) mRNAs during the development of rat brain. In A, total RNAs isolated from rat brains at the indicated postnatal age (days) were analyzed by Northern blotting. Each lane represents total RNA (15 g) from the brain of one rat. The blot was hybridized with 32 P-labeled ACS3 cDNA, as shown in Fig. 5, and left to expose a Kodak XAR-5 film with an intensifying screen at Ϫ80°C for 16 h. B, the same RNA samples analyzed by Northern blotting using a 32 P-labeled 2.0-kb EcoRI/EcoRI fragment from ACS2 cDNA (pBACS9) and left to expose a Kodak XAR-5 film with an intensifying screen at Ϫ80°C for 16 h. C, the same RNA samples in A hybridized with a 32 P-labeled 0.96-kb EcoRI/EcoRV fragment from ACS1 cDNA (pRACS15). The blot was left to expose a Kodak XAR-5 film with an intensifying screen at Ϫ80°C for 72 h. Volume of total RNA in each lane was confirmed with ethidium bromide staining (data not shown). The autoradiograph shown is representative of five independent experiments that gave essentially identical results.