From the Departments of Molecular Genetics and
§ Internal Medicine, University of Texas Southwestern
Medical Center, Dallas, Texas 75390-9046
Received for publication, November 15, 2002, and in revised form, December 12, 2002
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ABSTRACT |
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The de novo synthesis of fatty acids
occurs in two distinct cellular compartments. Palmitate (16:0) is
synthesized from acetyl-CoA and malonyl-CoA in the cytoplasm by the
enzymes acetyl-CoA carboxylase 1 and fatty acid synthase. The synthesis
of fatty acids longer than 16 carbons takes place in microsomes and
utilizes malonyl-CoA as the carbon source. Each two-carbon addition
requires four sequential reactions: condensation, reduction,
dehydration, and a final reduction to form the elongated fatty
acyl-CoA. The initial condensation reaction is the regulated and
rate-controlling step in microsomal fatty acyl elongation. We
previously reported the cDNA cloning and characterization of a
murine long chain fatty acyl elongase (LCE) (1). Overexpression of LCE
in cells resulted in the enhanced addition of two-carbon units to
C12-C16 fatty acids, and evidence was provided that LCE catalyzed the
initial condensation reaction of long chain fatty acid elongation. The
remaining three enzymes in the elongation reaction have not been
identified in mammals. Here, we report the identification and
characterization of two mammalian enzymes that catalyze the
3-ketoacyl-CoA and trans-2,3-enoyl-CoA reduction reactions
in long and very long chain fatty acid elongation, respectively.
Ninety percent of all fatty acids present in mammalian cells are
derived from de novo synthesis. The predominant fatty acids synthesized in mammals are long chain fatty acids 16-18 carbons in
length. Long chain fatty acids are important components of phospholipids, represent the largest energy storage reservoir in the
form of triglycerides, and are the preferred fatty acids used for the
esterification of cholesterol. The highest rate of de novo
fatty acid synthesis occurs in the liver, which converts excess glucose
into fatty acids for storage and transport. During times of caloric
excess, glucose is converted to pyruvate, which is converted to citrate
in the mitochondria and transported to the cytosol where ATP citrate
lyase uses citrate to produce acetyl-CoA. Acetyl-CoA is carboxylated by
acetyl-CoA carboxylase 1 to form malonyl-CoA. Fatty acid
synthase (FAS)1 then uses
malonyl-CoA, acetyl-CoA, and NADPH to elongate fatty acids in
two-carbon increments in the cytosol (2). The principal fatty acid
produced by FAS in rodents is palmitic acid, which contains 16 carbons
and is designated 16:0 (3).
The mammalian enzymes that elongate palmitic acid (16:0) and very long
chain fatty acids (>C18) have been localized to the endoplasmic
reticulum (ER) and are shown schematically in Fig. 1 (4). Microsomal
fatty acid elongation uses malonyl-CoA as the two-carbon donor and
consists of four sequential and independent reactions: 1) a
condensation between a fatty acyl-CoA and malonyl-CoA to form
3-ketoacyl-CoA; 2) a reduction of the 3-ketoacyl-CoA using NADPH to
form 3-hydroxyacyl-CoA; 3) a dehydration of 3-hydroxyacyl-CoA to
trans-2,3-enoyl-CoA; and 4) a reduction of
trans-2,3-enoyl-CoA to saturated acyl-CoA (5). Unlike the
multifunctional FAS enzyme, the enzymes that carry out microsomal fatty
acid elongation are encoded by separate genes.
Enzymes involved in microsomal fatty acid elongation have been
characterized most extensively by genetic deletion studies in
Saccharomyces cerevisiae. Three proteins, designated Elo1p, Elo2p, and Elo3p, participate in the initial condensation reaction of
microsomal fatty acyl elongation. Elo1p is required for the elongation
of C14 to C16 fatty acids (6), and Elo2p and Elo3p are required for the
synthesis of very long chain fatty acids (7).
Six mammalian homologues of the yeast ELO genes have been described
(Fig. 1). Like their yeast counterparts,
these enzymes exhibit some fatty acid chain length substrate
specificity. The first mammalian elongase identified was Cig30
(cold-induced glycoprotein of
30 kDa), which is the functional equivalent of yeast Elo2p (8). Ssc1 and Ssc2 (sequence
similarity to Cig30 1 and
2) subsequently were identified based on homology to
Cig30 (9). Ssc1 is the functional equivalent of Elo3p in
yeast (9). Definitive fatty acid substrate specificity has not been
assigned to Ssc2, although two of its substrates are arachidonic (20:4)
and eicosapentaenoic (20:5) acids (1). ELOVL4
(elongation of very long chain
fatty acids-like 4) was identified by linkage and haplotype
analysis in families with two forms of autosomal dominant macular
dystrophy and is expressed only in tissues with high contents of very
long chain fatty acids; therefore, it is likely that ELOVL4 is involved in the elongation of very long chain fatty acids (10). HELO1 was
identified based on sequence homology with yeast Elo2p and has a broad
range of very long chain fatty acid substrate specificity (11).
Recently, we identified a long chain fatty acyl elongase (LCE) that is
homologous to the very long chain fatty acyl elongases; however, unlike
other family members, the activity of LCE is restricted to long chain
fatty acids (C12-C16) (1).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Microsomal fatty acyl elongation. The
diagram shows the enzymes and fatty acyl-CoA intermediates that
comprise the two-carbon microsomal elongation of fatty acyl-CoAs. The
fatty acyl-CoA first undergoes a condensation, which is catalyzed by
one of six condensing enzymes discussed in the Introduction. The
condensing enzyme utilized is determined by the fatty acyl chain length
and degree of desaturation. All of the products of the fatty acyl-CoA
condensation reaction then undergo a reduction mediated by the KAR, a
dehydration mediated by an unidentified enzyme, and a final reduction
mediated by the TER that results in the final elongated fatty acyl-CoA
product. The identification and characterization of KAR and TER are
described in the current report.
Proteins that participate in the post-condensation reactions of
microsomal fatty acid elongation recently were characterized in yeast.
Two proteins were identified that participated in the 3-ketoacyl-CoA
reductase reaction. The YBR159w gene encoded the protein
that was responsible for the majority of the 3-ketoacyl-CoA reductase
activity in yeast microsomes (12). Studies using Ybr159p mutants
revealed that a small amount of residual 3-ketoacyl-CoA reductase
activity was still present, which was subsequently ascribed to
1-acyldihydroxyacetone-phosphate reductase (13). No proteins have been
identified that carry out the third dehydratase reaction. The final
reduction of the trans-2,3-enoyl-CoA is carried out by
Tsc13p in yeast (14). In mammals, the enzymes that carry out reactions
distal to the condensation reaction have not been identified. In the
current studies, we identify and characterize human and mouse
reductases that catalyze the second and fourth reactions in microsomal
fatty acid elongation.
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EXPERIMENTAL PROCEDURES |
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Materials--
[2-14C]Malonyl-CoA (40-60
mCi/mmol) was obtained from PerkinElmer Life Sciences. Redivue
[-32P]dCTP (3000 Ci/mmol) was obtained from Amersham
Biosciences. Palmitoyl-CoA, palmitic acid (16:0),
-linolenic acid
(18:3n-6), arachidonic acid (20:4n-6),
eicosapentaenoic acid (20:5n-3), fatty acid-free BSA,
coenzyme A sodium salt, ATP, NADPH, malonyl-CoA, and monoclonal anti-HA
(clone number HA-7) were obtained from Sigma.
Cloning of Mammalian 3-Ketoacyl-CoA and trans-2,3-Enoyl-CoA Reductase cDNAs and Construction of Expression Plasmids-- cDNAs encoding the putative human and mouse microsomal 3-ketoacyl-CoA reductase (KAR) were identified by a BlastP search of the NCBI data bases using the S. cerevisiae protein Ybr159p. Human and mouse cDNAs (GenBankTM accession numbers NM_016142 and AF064635, respectively) were identified that encode proteins that are ~31% identical to the yeast 3-ketoacyl-CoA reductase protein Ybr159p. The trans-2,3-enoyl-CoA reductase (TER) proteins were identified by a BlastP search using the S. cerevisiae protein Tsc13p, which encodes the yeast trans-2,3-enoyl-CoA reductase. Human and mouse cDNAs (GenBankTM accession numbers AAF32373 and AK010984, respectively) were identified that encode proteins that are ~34% identical to Tsc13p.
The expression plasmids for human KAR and TER were constructed as follows. cDNAs encoding the full-length protein were obtained by PCR amplification using human adipose tissue first strand cDNA (Clontech, catalogue number 7128-1) as the template and the following primers: KAR, 5' primer, 5'-GCCACCATGGGCGGCCGCGAGAGCGCTCTCCCCGCCGCC-3', and 3' primer, 5'-TTAGTTCTTCTTGGTTTTCTTCAG-3'; and TER, 5' primer, 5'-GCCACCATGGGCGGCCGCAAGCATTACGAGGTGGAGATT-3', and 3' primer, 5'-TCAGAGCAGGAAGGGGATGATGGG-3'. The 5' primers used to amplify each cDNA contained a Kozak sequence followed by an ATG codon and a NotI restriction enzyme site. The resulting PCR products were ligated into pCMV-Script (Stratagene, La Jolla, CA) and digested with NotI, and three copies of the HA epitope (YPYDVPDYA) were inserted at the 5' end of each cDNA. The resulting plasmids were designated pCMV-HA-KAR and pCMV-HA-TER, respectively. The integrity of all PCR products and ligations was confirmed by DNA sequencing. The LCE expression plasmid (pCMV-HA-LCE) was constructed as described previously (1).
Immunofluorescence Microscopy--
Chinese hamster ovary K-1
cells (ATCC CCL-61) were set up on glass coverslips in 6-well plates at
a density of 1.0×105/well in Dulbecco's modified Eagle's
medium/Ham's F-12 medium supplemented with 5% fetal calf serum, 100 units/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate (day
0). On day 1, 0.5 µg of indicated plasmids was transfected using 3 µl of FuGENE 6 (Roche Molecular Biochemicals) in serum-free
Dulbecco's modified Eagle's medium/Ham's F-12 medium. On day 3, the
cells were washed with PBS and then fixed and permeabilized by
incubating in 2 ml of methanol at 20 °C for 10 min. After three
washes with PBS, the cells were incubated for 1 h at 4 °C in
1% BSA in PBS (buffer A). The cells were then incubated in buffer A at
4 °C with a mouse monoclonal HA antibody (HA probe F-7; Santa Cruz
Biotechnology, Inc., Santa Cruz, CA) (20 µg/ml) and a rabbit
anti-calnexin polyclonal antibody (StressGen Biotechnologies Corp.,
Victoria, Canada) (1:200 dilution) for 1 h. The cells were washed
three times with buffer B (0.1% BSA in PBS), and primary antibodies
were localized by incubating the cells for 1 h at room temperature
in buffer A containing 2 µg/ml goat anti-rabbit IgG conjugated to
Alexa Fluor 568 and goat anti-mouse IgG conjugated to Alexa Fluor 488 (Molecular Probes, Inc., Eugene, OR). After incubation with the
secondary antibody, the cells were washed three times with buffer B,
quickly rinsed with PBS and distilled water, and analyzed with a Leica
TCS SP confocal microscope (Leica Microsystems Inc., Heidelberg, Germany).
Co-expression of KAR or TER with LCE in HEK-293
Cells--
HEK-293 cells (ATCC CRL-1573) were plated at a density of
4 × 105 cells/60-mm dish in Dulbecco's modified
Eagle's medium supplemented with 10% (v/v) fetal calf serum, 100 units/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate (day
0). On days 1 and 2, the cells were transfected with expression
plasmids using 6 µl of FuGENE 6 according to the manufacturer's
instructions. Transfection studies with KAR contained 0.6 µg of
pCMV-HA-LCE, 0.6 µg of pCMV-HA-KAR, 0.8 µg of pVAI (15), and 0.02 µg of pCMV -galactosidase plasmids/dish. Transfection studies with
TER contained 1.0 µg of pCMV-HA-LCE, 0.2 µg of pCMV-HA-TER, 0.8 µg of pVAI (15), and 0.02 µg of pCMV
-galactosidase
plasmids/dish. The total amount of plasmid DNA in each transfection was
adjusted to 2 µg/dish by adding pCMV-Script. On day 3, the cells were
harvested, and cytosolic and membrane proteins were prepared as
described previously (1). The microsomal protein was stored at
80 °C after snap freezing in liquid nitrogen. Transfection
efficiencies were determined by measuring the
-galactosidase activity in the supernatant from the 1.3 × 105 × g spin using a
-galactosidase assay kit (Stratagene, La
Jolla, CA), and the expression of HA-tagged proteins in membranes was confirmed by immunoblot analysis as described (1). The protein concentrations were determined using the method of Lowry et
al. (16).
In Vitro Fatty Acid Elongation Assay-- Palmitoyl-CoA or BSA-bound fatty acids were used as substrates for all reactions. BSA-bound fatty acids were prepared as 5 or 10 mM solutions as described (1). The elongation assays contained 0.05 mg of microsomal protein in 50 mM potassium phosphate, pH 6.5, 5 µM rotenone, 20 µM palmitoyl-CoA, 150 µM [2-14C]malonyl-CoA (6.5 dpm/pmol), 1 mM NADPH, 20 µM BSA in a final reaction volume of 0.2 ml. For assays using BSA-bound fatty acids, the reaction mixtures contained 0.05 mg of microsomal protein in 50 mM potassium phosphate, pH 6.5, 5 µM rotenone, 20 µM BSA-bound fatty acid, 100 µM coenzyme A, 1 mM ATP, 1 mM MgCl2, 150 µM [2-14C]malonyl-CoA (6.5 dpm/pmol), and 1 mM NADPH in a final volume of 0.2 ml.
To initiate the elongation reaction, 0.05 mg of microsomal protein from transfected cells was added, and the incubation was continued for the indicated times. The reactions were stopped by adding 0.1 ml of 75% KOH (w/v) and 0.2 ml of ethanol, saponified at 70 °C for 1 h, and then acidified by adding 0.4 ml of 5 N HCl with 0.2 ml of ethanol. Fatty acids were collected in three independent extractions using 1 ml of hexane. The extractions were pooled, dried under nitrogen, and separated by TLC using hexane/diethyl ether/acetic acid (30:70:1) as described (1). The TLC plates were exposed to a PhosphorImager screen, the resulting image was analyzed, and the lipids were quantified using a Bio-Imaging Analyzer with BAS1000 MacBAS 2.1 software (Fuji Medical Systems, Stamford, CT).
RNAi-mediated Inhibition of KAR and TER-- Double-stranded (ds) RNA oligonucleotides were synthesized by Dharmacon Research (Lafayette, CO) for human KAR, TER, and an irrelevant control gene, vesicular stomatitis virus glycoprotein. The oligonucleotide sequences are listed in Table I. On day 0, HeLa (ATCC CCL-2) or HepG2 (ATTC HB-8065) cells were set up at a density of 4 × 105 cells/60-mm dish. HeLa cells were cultured in minimum essential medium supplemented with 10% fetal calf serum, 1× nonessential amino acid mix (Cellgro, Herndon, VA), 1 mM sodium pyruvate, 100 units/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate. HepG2 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate, respectively. dsRNAs (0.2 µM) were transfected on days 1, 2, and 3 using OligofectAMINE (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. On day 4, the cells were harvested for membrane protein and total RNA as described (1).
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Blot Hybridization of RNA-- The multiple tissue Northern blot (Clontech, catalogue number 7780-1) was used to determine KAR and TER expression in human tissues. The blot was hybridized according to the manufacturer's instructions with randomly 32P-labeled KAR and TER cDNA probes that were made using the full-length human KAR and TER cDNAs as templates (1). For mouse multiple tissue Northern blots, total RNA was extracted from the indicated tissues of C57BL/6J mice using RNA STAT-60 (TEL-TEST, Friendswood, TX), and poly(A+) RNA was isolated using the MessageMaker mRNA Isolation System (Invitrogen). Three µg of poly(A+) RNA from each tissue was subjected to Northern blot hybridization using 32P-labeled mouse cDNA probes for mouse KAR and TER (1). Mouse full-length cDNA templates for KAR and TER were PCR-amplified from mouse liver first strand cDNA using the following primers: KAR, 5' primer, 5'-ATGGAGTGCGCTCCCCCGGCG-3', and 3' primer, 5'-TTAGTTCTTCTTCCTTTTCTTCAG-3'; TER, 5' primer, 5'-ATGAAGCACTACGAGGTGGAG-3', and 3' primer, 5'-TCAGAGCAGGAAGGGAATAAT-3' (17). The cyclophilin probe used as a control was described previously (1).
For HepG2 and HeLa cell Northern blots, cells from two 60-mm dishes were used to isolate total RNA as described above. Twenty µg of the total RNA was subjected to Northern blot analysis using 32P-labeled human KAR and TER cDNA probes as described above. All of the Northern blot filters were exposed to a Fuji PhosphorImager and quantified using a Bio-Imaging Analyzer with BAS1000 MacBAS software.
Quantitative Real Time PCR-- Real time PCR conditions and oligonucleotides used to measure mRNA levels of mouse 3-hydroxy-3-methylglutaryl CoA synthase and FAS were described previously (18). Primers were designed using Primer Express software (PerkinElmer) for the following mouse genes: LCE, 5' primer, 5'-TGTACGCTGCCTTTATCTTTGG-3', and 3' primer, 5'-GCGGCTTCCGAAGTTCAA-3'; KAR, 5' primer, 5'-GGCAACGAGGCCTTGGT-3', and 3' primer, 5'-CCATCAGTGCCACCTGTAACAA-3'; TER, 5' primer, 5'-TTCGTGCACCGATTCTCTCA-3', and 3' primer 5'-GCCCCAATAGTAGGTGCAGTTT-3'; and 36B4, 5' primer, 5'-CACTGGTCTAGGACCCGAGAAG-3', and 3' primer, 5'-GGTGCCTCTGGAGATTTTCG-3'. 36B4 mRNA was used as an invariant control for all of the studies.
Animal Studies--
All of the mice were housed in colony cages
with a 12-h light/12-h dark cycle and fed Teklad mouse/rat diet 7002 from Harlan Teklad Premier Laboratory Diets (Madison, WI). Studies
using SREBP transgenic mice included five wild-type and five
12-14-week-old TgSREBP-1a, TgSREBP-2 male mice fed a high protein/high
carbohydrate diet for 2 weeks prior to sacrifice as described (17, 19). SCAP liver-specific knockout mice
(SCAPf/f;MX1-Cre) were described previously
(20). Four 8-10-week-old male SCAPf/f;MX1-Cre
and corresponding wild-type mice received four intraperitoneal injections of polyinosinic-polycytidylic acid as described (20). The mice were fed the Teklad chow and sacrificed nonfasted 14 days
after the last injection of polyinosinic-polycytidylic acid. Total RNA
was extracted from liver as described above, and equal aliquots of RNA
from all of the mice were pooled for each treatment group for study by
quantitative real time PCR.
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RESULTS |
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To identify and characterize the mammalian reductases involved in
microsomal long chain fatty acid elongation, we used the sequences of
yeast genes that encode a 3-ketoacyl-CoA reductase (YBR159w)
(12) and a trans-2,3-enoyl-CoA reductase (TSC13)
(14) to identify potential mammalian orthologues in the human and mouse data bases. The BlastP search of the NCBI human data base revealed two
cDNAs (GenBankTM accession numbers NM_016142 and
NM_000197), the predicted amino acids of which were 31% identical to
the yeast 3-ketoacyl-CoA reductase protein Ybr159p. Similarly, the
search of the mouse data base revealed two cDNAs
(GenBankTM accession numbers AF064635 and NM_008291), the
predicted proteins of which were 32% identical to Ybr159p. The human
and mouse cDNAs NM_000197 and NM_008291 encode the hydroxysteroid
17- dehydrogenase 3 protein, which is only expressed in testis (21).
Therefore, the proteins encoded by these cDNAs were eliminated as
candidates for the microsomal 3-ketoacyl-CoA reductase in liver. The
predicted proteins encoded by the human NM_016142 and mouse AF064635 cDNAs were 82% identical and of unknown function.
An alignment of the predicted yeast, mouse, and human 3-ketoacyl-CoA
reductase amino acids is shown in Fig.
2A. An in-frame stop codon is
present 12 nucleotides prior to the putative initiation methionine in
the human KAR cDNA sequence, indicating that the entire coding
sequence was represented in the identified cDNA. The translational
reading frames of the putative human and mouse KAR cDNAs encode
proteins that are 312 amino acids in length. Like in the yeast protein,
a conserved dilysine ER retention motif is present at the C-terminal
end of human and mouse KAR. Hydropathy analysis using the Kyte and
Doolittle algorithm (22) predicts the presence of as many as four
putative transmembrane domains (Fig. 2B).
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To determine the tissue expression pattern of the putative KAR in mammals, human and mouse multiple tissue Northern blots were performed using species-specific 32P-labeled cDNA probes (Fig. 2C). A single ~2.9-kb mRNA was identified by Northern blot analysis in all of the human tissues represented on the blot. In these, the highest level of expression appeared to be in liver, muscle, and kidney. The expression of KAR also was determined in mouse tissues. The mRNA for mouse KAR was ~2.0 kb in size. The difference in mRNA size between the human and mouse transcripts is due to differences in the 3'-untranslated sequences. In mouse, high levels of KAR expression were also found in white adipose tissue and brown adipose tissue (Fig. 2C).
The putative human and mouse TER proteins were identified by a BlastP search of the NCBI data base using the yeast TER protein Tsc13p. The identified human (GenBankTM accession number AF222742) and mouse (GenBankTM accession number AK010984) cDNAs encode proteins that are ~34% identical to that of Tsc13p (14). The putative human trans-2,3-enoyl-CoA reductase cDNA has an in-frame stop codon 21 nucleotides prior to the initiation methionine. The translational reading frames of the human and mouse putative TER cDNAs predict proteins 308 amino acids in length. The overall identity of the human and mouse TER proteins is 95%.
An alignment of the yeast, mouse, and human TER amino acid sequences is
shown in Fig. 3A. Unlike the
KAR proteins, no consensus ER retention motif is present in the mouse
or human TER sequence. Hydropathy analysis using the Kyte and Doolittle
algorithm (22) predicts the presence of as many as five transmembrane
domains (Fig. 3B). No mitochondrial or peroxisomal targeting
sequences were identified in these proteins. The tissue expression
pattern of TER was determined using human and mouse multiple tissue
Northern blots as described for KAR (Fig. 3C). A single
~1.2-kb mRNA was identified by Northern blotting in all of the
tissues tested from human and mouse (Fig. 3C). The tissue
expression of TER essentially mirrored that of KAR.
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To determine whether KAR or TER participated in microsomal fatty acyl
elongation, expression vectors utilizing the CMV promoter were
assembled that encoded the human KAR or TER proteins with HA epitope
tags at their N terminus. HEK-293 cells were transfected with the human
KAR or TER expression plasmids, and cytosolic and microsomal proteins
were separated by SDS-PAGE to determine the subcellular localization of
the proteins. As predicted from the hydropathy profiles, immunoblot
analysis using an anti-HA antibody revealed that the expressed KAR and
TER proteins were present only in the microsomal fraction (data not
shown). To study the subcellular localization of these proteins
directly, we performed double-label immunofluorescence studies of the
HA epitope-tagged KAR, TER, or LCE proteins that were transfected in
Chinese hamster ovary K1 cells (Fig. 4).
Staining with the anti-HA antibody revealed that KAR and TER
co-localized with the ER resident protein calnexin and the condensing
enzyme LCE (Fig. 4, C, F, I, and
L). Additional stains for a mitochondrial protein, Grp75,
showed no significant co-localization with KAR, TER, or LCE, and stains
for a cis-compartment Golgi resident protein, GM130, showed
no co-localization with KAR and LCE (data not shown). A small degree of
co-localization of GM130 and TER was found, the significance of which
is not known.
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Previously, we showed that the elongation of palmitoyl-CoA (16:0) was increased significantly in microsomes from HEK-293 cells transfected with the condensing enzyme, LCE (1). Elongation activity was determined by measuring the amount of 14C incorporated from [2-14C]malonyl-CoA into elongated fatty acid products. LCE overexpression markedly enhanced the initial condensation of palmitoyl-CoA to 3-ketostearoyl-CoA. The LCE-mediated increase in palmitoyl-CoA condensation caused the subsequent reactions to become rate-limiting, leading to the accumulation of elongation intermediates, which could be separated and identified by TLC (1). The accumulation of elongation intermediates provided a tool to study the potential function of the KAR and TER proteins. Working under the hypothesis that KAR functions as a long chain 3-ketoacyl-CoA reductase, the co-expression of KAR with LCE in cells should result in the selective disappearance of 3-ketostearoyl-CoA intermediate in microsomes incubated with palmitoyl-CoA. Similarly, the co-expression of TER with LCE should result in the selective disappearance of the trans-2,3-stearoyl-CoA intermediate if the TER protein functions as a trans-2,3-enoyl-CoA reductase.
Fig. 5 (lanes 4-6 and
10-12) shows that the overexpression of LCE alone resulted in the
accumulation of all of the elongation intermediates in microsomes from
HEK-293 cells incubated with palmitoyl-CoA as the fatty acid substrate.
Co-expression of LCE and human KAR resulted in the selective
disappearance of 3-ketostearoyl-CoA, suggesting that the KAR protein
enhanced the reduction of 3-ketostearoyl-CoA to 3-hydroxystearoyl-CoA
(Fig. 5, lanes 7-9). Similarly, co-expression of LCE and
human TER resulted in the disappearance of the
trans-2,3-stearoyl-CoA intermediate (Fig. 5, lanes
13-15). This result suggested that TER functions to reduce
trans-2,3-stearoyl-CoA to stearoyl-CoA. A duplicate set of
experiments was performed using the mouse orthologues of KAR and TER,
and similar results were obtained (data not shown).
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To determine whether KAR exhibits fatty acid substrate specificity,
RNAi was employed to selectively reduce the expression of KAR in
cultured cells. Inhibiting KAR expression should result in the
accumulation of the 3-ketoacyl-CoA intermediate if the fatty acid
tested is a substrate of the enzyme. HeLa cells were transfected with
the indicated dsRNAs and microsomal protein, and the total RNA was
isolated (Fig. 6). The endogenous
mRNA level of KAR was selectively reduced 4-fold in cells
transfected with dsRNA oligonucleotides corresponding to KAR, whereas
the expression of TER and cyclophilin was unchanged (Fig. 6,
lower panels). Microsomes from transfected cells were
incubated with long and very long chain fatty acid substrates, and the
14C-labeled elongation products from the fatty acid
elongation reaction were separated by TLC (Fig. 6, upper
panel). Microsomes from cells transfected with dsKAR
oligonucleotides accumulated the 3-ketoacyl-CoA intermediates for all
fatty acids tested in the elongation assay (Fig. 6, lanes 3,
6, 9, and 12). The final elongated
fatty acyl-CoA product was reduced by 40-50% in microsomes from cells
transfected with dsKAR oligonucleotides. These results supported the
conclusion that KAR functioned as a 3-ketoacylCoA reductase and
demonstrated that KAR reduced very long chain 3-ketoacyl-CoA substrates
as well as long chain fatty acyl-CoAs.
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A similar set of RNAi experiments was performed using dsRNAs
oligonucleotides corresponding to human TER in HepG2 cells (Fig. 7). HepG2 cells were used for these
experiments because the lower endogenous expression of TER apparently
facilitated the inhibition of TER by RNAi. The transfection of HepG2
cells with dsTER oligonucleotides resulted in a selective 4-fold
reduction in endogenous TER mRNA levels (Fig. 7, lower
panels). trans-2,3-Enoyl-CoA intermediates accumulated
in microsomes from dsTER oligonucleotide transfected cells for all
fatty acid substrates tested in the elongation assay (Fig. 7,
lanes 3, 6, 9, and 12). The
final fatty acyl-CoA product was reduced by 50-60% in the elongation
assay with all of the fatty acids tested. Together, the data from these
overexpression and inhibition studies suggested that TER functioned as
a trans-2,3-enoyl-CoA reductase and that TER reduced long
and very long chain fatty acid trans-2,3-enoyl-CoA
substrates.
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All of the previously identified enzymes required for long chain fatty acid biosynthesis are regulated by the sterol regulatory element-binding protein (SREBP) family of transcription factors (23). The SREBP family members are designated SREBP-1a, SREBP-1c, and SREBP-2. The SREBP-1 isoforms preferentially activate genes encoding fatty acid biosynthetic enzymes, whereas SREBP-2 preferentially activates genes specifying cholesterol biosynthetic enzymes. To be active, SREBPs undergo two sequential cleavages that require three proteins: an escort protein designated SCAP and two proteases designated S1P and S2P (24). All three proteins are required for normal SREBP activation inasmuch as the deletion of any one results in the absence of all transcriptionally active forms of SREBPs (24).
To determine whether KAR and TER mRNA levels were regulated in a
manner similar to other fatty acid biosynthetic genes, the mRNA
levels of FAS, LCE, KAR, and TER were measured in livers from mice that
either overexpress the transcriptionally active forms of SREBPs or that
lack all SREBP isoforms as a result of inactivating SCAP (17, 19, 20).
Consistent with previous studies, the mRNA levels of FAS and LCE
were increased ~20-fold in livers from SREBP-1a transgenic mice
(TgSREBP-1a) (Table II) (1, 17). SREBP-2
overexpression (TgSREBP-2) also increased the expression of FAS and LCE
mRNAs, but to a lesser extent than the overexpression of SREBP-1a.
Conversely, removing all transcriptionally active forms of SREBPs by
deleting SCAP in liver (SCAP/
) resulted in a
4-fold decrease in FAS expression and a ~2-fold reduction in LCE
mRNA. The mRNAs for KAR and TER were largely unaffected, either
by SREBP overexpression or by the absence of SREBPs. These data suggest
that unlike other enzymes required for fatty acid biosynthesis, KAR and
TER mRNA levels are not regulated by SREBPs in vivo.
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DISCUSSION |
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In the current studies, we identified two mammalian reductases that participate in the microsomal elongation of long and very long chain fatty acids. BlastP searches of the NCBI data bases identified human and mouse homologues of the S. cerevisiae proteins Ybr159p, a 3-ketoacyl-CoA reductase, and Tsc13p, a trans-2,3-enoyl-CoA reductase. Biochemical studies of the recombinant human and mouse proteins confirmed that they exhibit KAR and trans-2,3-enoyl-CoA reductase activities. The enzymes responsible for microsomal fatty acyl elongation have not been purified previously. Therefore, the genes identified in this study provide an initial molecular characterization of the reductases that carry out the second and fourth steps in microsomal long and very long chain fatty acyl elongation in mammals.
The 3-ketoacyl-CoA reductase, KAR, identified in this study shares
sequence similarity with members of the short chain dehydrogenase superfamily (25), which are characterized by a nucleotide
co-factor-binding region (Rossmann-fold) and an active site that
consists of a triad of catalytically important and highly conserved
Ser-Tyr-Lys residues. KAR is expressed in all tissues, with the highest
levels of expression occurring in tissues that are directly involved in
lipid metabolism. We provide evidence that KAR is a 3-ketoacyl-CoA
reductase and that it represents the second enzyme in the microsomal
fatty acyl two-carbon elongation cascade. Whether KAR is the only
enzyme that can carry out the 3-ketoacyl-CoA reduction in cells could not be addressed in the current studies. In S. cerevisiae,
the majority of very long chain fatty acyl 3-ketoacyl-CoA activity is
due to Ybr159p; however, the genetic disruption of the
YBR159w does not completely abolish all 3-ketoacyl-CoA
reductase activity (12). The ybr159 mutants are viable
but have a slowed rate of growth. The residual 3-ketoacyl-CoA reductase
activity in the ybr159
mutants was attributed to a gene
that encodes 1-acyldihydroxyacetone-phosphate reductase (13). A
mammalian gene has not been identified; therefore it was not possible
to test whether a mammalian orthologue of AYR1 also could
mediate the reduction of 3-ketoacyl-CoAs.
The trans-2,3-enoyl-CoA reductase, TER, is ~32% identical
to the yeast trans-2,3-enoyl-CoA reductase, Tsc13p. Kohlwein
et al. (14) identified and characterized the yeast Tcs13
protein as a trans-2,3-enoyl-CoA reductase and reported that
it belonged to an evolutionarily conserved family of proteins present
in all mammals, yeast, and Arabidopsis thaliana. The human
and mouse trans-2,3-enoyl-CoA reductase proteins are also
~97% identical to the rat SC2 protein that was originally identified
in a screen for cDNAs that encoded synaptic glycoproteins (27). The
trans-2,3-enoyl-CoA reductase family members share sequence
similarity with steroid 5-reductase, an ER enzyme that catalyzes the
reduction of testosterone to dihydrotestosterone (27, 28). Human TER
and steroid 5
-reductase are ~30% identical and 45% similar over
the C-terminal ~130 amino acids. Neither protein contains classic
NADPH-binding sites; however, at least eight amino acid residues at the
C-terminal end of steroid 5
-reductase type 2 are crucial for NADPH
binding (29). Four of these eight residues are conserved in the yeast
and mammalian TER proteins. Therefore, although the identified TER
protein does not contain a classic NAPDH-binding site, the sequence
similarity with steroid 5
-reductase suggests that it utilizes NADPH
as a co-factor.
The overexpression and inhibition of TER in cultured cells demonstrated that the enzyme is capable of mediating the trans-2,3-enoyl-CoA reduction of both long and very long chain fatty acids. Inhibition of TER by RNAi resulted in the marked accumulation of trans-2,3-enoyl-CoA substrate intermediate for all fatty acids tested (Fig. 7). A small amount of the preceding 3-hydroxyacyl-CoA intermediate also accumulated in microsomes from dsTER oligonucleotide transfected cells. This 3-hydroxyacyl-CoA intermediate is a substrate for the dehydratase enzyme. Although the dehydratase protein has not been identified, Knoll et al. (30) have shown that the dehydratase reaction in microsomal fatty acid elongation is reversible. Therefore, the inhibition of TER could result in the accumulation of the 3-hydroxyacyl-CoA intermediate as a consequence of the reverse reaction. These results do not preclude the possibility that TER may participate in the dehydratase reaction in addition to catalyzing the fourth and final step in the microsomal fatty acyl elongation cascade.
Studies in yeast and mammals have demonstrated that microsomal fatty
acyl-CoA condensing enzymes exhibit fatty acyl chain length specificity
(1, 7, 9, 10). It has been suggested that the post-condensation enzymes
do not exhibit carbon chain length specificity (4). The current studies
provide support for this hypothesis. Although all possible fatty acid
substrates could not be tested, the data of Fig. 6 show that inhibition
of KAR resulted in the accumulation of the 3-ketoacyl-CoA substrate for
palmitic (16:0), -linolenic (18:3n-6), arachidonic
(20:4n-6), and eicosapentaenoic (0:5n-3) fatty
acids. Similar results were obtained using myristic (14:0), palmitoleic
(16:1), docosatetraenoic (22:4n-6), and docosapentaenoic
(22:5n-3) fatty acids as substrates in the microsomal
elongation assay described in Fig. 6 (data not shown). TER inhibition
by RNAi resulted in the accumulation of the
trans-2,3-enoyl-CoA intermediates for the same broad range of fatty acid substrates identified as KAR substrates. Despite this
accumulation, it is possible that other unidentified 3-ketoacyl-CoA or
trans-2,3-enoyl-CoA reductases have greater activities for a
given fatty acyl substrate than those characterized in the current studies. The current data demonstrate that the identified KAR and TER
do not exhibit the strict fatty acyl chain length substrate specificity
displayed by LCE and other characterized condensing enzymes.
All known fatty acid biosynthetic enzymes isolated to date are regulated by the SREBP family of transcription factors (23). The overexpression of SREBPs in liver results in the accumulation of fatty acids that are 18 carbons in length, because of the activation of FAS and LCE (1, 23, 31). Reducing SREBP levels by eliminating the SCAP protein in liver resulted in a 40-70% reduction in the mRNA levels of all fatty acid biosynthetic genes (20). In contrast to other lipogenic genes, the mRNA levels of KAR and TER were largely unaffected by SREBP expression levels in liver (Table II).
Lipogenesis is hormonally regulated by insulin, and the ability of this hormone to stimulate lipogenesis is mediated by SREBP-1c. The results from the transgenic and knockout mice would suggest that KAR and TER are not regulated by insulin in a manner similar to other lipogenic genes (32, 33). The activities of the four microsomal elongation enzymes previously have been measured under conditions of high and low insulin (34). These studies demonstrated that only the initial condensation reaction catalyzed by LCE is regulated by insulin. The mRNA levels of the identified genes responsible for these reactions follow a similar pattern of regulation. The condensing enzyme, LCE, is suppressed in livers of fasted mice (low insulin) and increased more than 20-fold in liver from mice that were fasted and refed a high carbohydrate diet (high insulin) (35). In similar fasting and refeeding studies, the mRNA levels of TER and KAR remain unchanged in mouse liver (data not shown). Together, the in vivo data support the hypothesis that KAR and TER are constitutively expressed and that the initial condensation reaction is the regulated step in microsomal fatty acyl elongation.
In summary, the overexpression and inhibition of the human KAR and TER
in cultured cells demonstrate that they function as 3-ketoacyl-CoA and
trans-2,3-enoyl-CoA reductases, respectively. The lack of
any measurable fatty acid carbon chain length substrate specificity for
either KAR or TER suggests that the six known condensing enzymes
channel the fatty acyl intermediates to a common series of enzymes that
produce the elongated fatty acyl-CoA product (Fig. 1). Whether KAR and
TER are essential for the long and very long chain fatty acyl
elongation in vivo or whether other proteins also possess
3-ketoacyl-CoA and trans-2,3-enoyl-CoA activities will
require analysis in knockout mice.
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ACKNOWLEDGEMENTS |
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We thank Drs. David W. Russell, Jonathan Cohen, and Joseph L. Goldstein for critical reading of the manuscript and Dr. Richard Anderson for use of the confocal microscope. We also thank Gregory A. Graf for invaluable assistance with the immunohistochemistry. Amy Cox, Norma Anderson, Anh Pho, and Judy Sanchez provided excellent technical assistance.
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FOOTNOTES |
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* This work was supported by grants from the Perot Family Foundation, National Institutes of Health Grants HL-20948 and HL-38049, and funds from the Moss Heart Foundation and the W. M. Keck Foundation.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.
¶ Pew Scholar in the Biomedical Sciences. Recipient of the Established Investigator Grant from the American Heart Association. To whom correspondence should be addressed: Depts. of Internal Medicine and Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Rm. L5-238, Dallas, TX 75390-9046. Tel.: 214-648-9677; Fax: 214-648-8804; E-mail: jay.horton@utsouthwestern.edu.
Published, JBC Papers in Press, December 13, 2002, DOI 10.1074/jbc.M211684200
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ABBREVIATIONS |
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The abbreviations used are: FAS, fatty acid synthase; ER, endoplasmic reticulum; HA, hemagglutinin; HEK, human embryo kidney cells; KAR, 3-ketoacyl-CoA reductase; LCE, long chain fatty acyl elongase; SCAP, SREBP cleavage-activating protein; SREBP, sterol regulatory element-binding protein; TER, trans-2,3-enoyl-CoA reductase; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ds, double-stranded; RNAi, RNA interference.
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