From the Department of Bioorganic Chemistry,
Institute of Chemistry, Tallinn Technical University, Akadeemia tee 15, Tallinn 12618, Estonia, the § Estonian Biocentre, 23 Riia
Street, Tartu 51010, Estonia, and the ¶ Department of
Pharmacology, Vanderbilt University School of Medicine,
Nashville, Tennessee-37232-6602
Received for publication, October 26, 2000, and in revised form, November 14, 2000
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ABSTRACT |
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In vertebrates, the synthesis of prostaglandin
hormones is catalyzed by cyclooxygenase (COX)-1, a constitutively
expressed enzyme with physiological functions, and COX-2, induced in
inflammation and cancer. Prostaglandins have been detected in high
concentrations in certain corals, and previous evidence suggested their
biosynthesis through a lipoxygenase-allene oxide pathway. Here we
describe the discovery of an ancestor of cyclooxygenases that is
responsible for prostaglandin biosynthesis in coral. Using a
homology-based polymerase chain reaction cloning strategy, the
cDNA encoding a polypeptide with ~50% amino acid identity to
both mammalian COX-1 and COX-2 was cloned and sequenced from the Arctic
soft coral Gersemia fruticosa. Nearly all the amino acids
essential for substrate binding and catalysis as determined in the
mammalian enzymes are represented in coral COX: the
arachidonate-binding Arg120 and Tyr355 are
present, as are the heme-coordinating His207 and
His388; the catalytic Tyr385; and the target of
aspirin attack, Ser530. A key amino acid that determines
the sensitivity to selective COX-2 inhibitors (Ile523 in
COX-1 and Val523 in COX-2) is present in coral COX
as isoleucine. The conserved Glu524, implicated in the
binding of certain COX inhibitors, is represented as alanine.
Expression of the G. fruticosa cDNA afforded a
functional cyclooxygenase that converted exogenous arachidonic acid to
prostaglandins. The biosynthesis was inhibited by indomethacin, whereas
the selective COX-2 inhibitor nimesulide was ineffective. We conclude
that the cyclooxygenase occurs widely in the animal kingdom and that
vertebrate COX-1 and COX-2 are evolutionary derivatives of the
invertebrate precursor.
Prostaglandins have been found in a diverse range of vertebrates
and invertebrates (1, 2). In vertebrates, they are synthesized by
prostaglandin-H2 synthase, known also as cyclooxygenase (COX)1 (3-5). COX is a
hemoprotein with two distinct catalytic activities: the cyclooxygenase
activity involved in the formation of PGG2 from arachidonic
acid and the peroxidase activity that catalyzes the reduction of
PGG2 to PGH2 (3). There are two COX isozymes called COX-1 and COX-2 (6, 7). COX-1 is expressed constitutively in
nearly all mammalian tissues and forms prostaglandins with housekeeping
functions. COX-2, although absent from most cells, can be rapidly
induced in many cell types upon treatment with inflammatory cytokines,
growth factors, and tumor promoters (8). These two isoforms share
~60% amino acid sequence identity. They have similar structural
topology and an identical catalytic mechanism (4, 9). The
three-dimensional x-ray crystal structures of COX-1 and COX-2 are
virtually superimposable. The residues that form the substrate-binding
channel, catalytic sites, and residues immediately adjacent are all
identical except for some small variations (9-12). These small
differences in sequence lead to clear biochemical differences in
substrate selectivity and sensitivity to various nonsteroidal
anti-inflammatory drugs (4, 6).
The mechanism of prostaglandin biosynthesis in invertebrates,
particularly in the prostaglandin-containing corals (13, 14), has been
the object of intense studies and speculations over the years. A
proposal that coral uses a fundamentally different mechanism from the
mammalian pathway, i.e. a lipoxygenase-allene oxide synthase route similar to the jasmonic acid pathway in plants (15-17), has not
found experimental support more recently. The highly active peroxidase-lipoxygenase fusion protein identified in
Plexaura homomalla that catalyzes conversion of
arachidonic acid into allene oxide is not involved in prostaglandin
synthesis (17). Our previous biochemical studies showed that (i) crude
enzyme preparations of the Arctic soft coral Gersemia
fruticosa convert exogenous arachidonic acid into a mixture of
prostaglandins with typical mammalian stereochemistry; (ii) the
biosynthetic pathway involves a common hydroperoxyendoperoxide
intermediate, PGG2; and (iii) the synthesis is inhibited by
nonsteroidal anti-inflammatory drugs (18-20). These findings provide
strong evidence that a relative of mammalian cyclooxygenases is
responsible for prostaglandin synthesis in coral. However, no enzyme
has been isolated, cloned, or otherwise characterized from coral or any
other invertebrate.
Here we report investigations aimed at characterization of this enzyme.
Using a homology-based PCR strategy, we cloned and sequenced the
cDNA encoding the functional cyclooxygenase that catalyzes
transformation of arachidonic acid into prostaglandins in the
prostaglandin-containing Arctic coral G. fruticosa. In expression studies, the coral enzyme was located in the endoplasmic reticulum and nuclear envelope of COS-7 cells, and the putative N-terminal signal peptide of the enzyme was cleaved. Our results indicate that the first cloned COX from non-vertebrates is an ancestor
of vertebrate cyclooxygenase isozymes.
Preparation of Total RNA
For preparation of total RNA, the method of Chomczynski
and Sacchi (21) gave the crude product with a remarkable amount of low
molecular mass decomposition products as determined by denaturing RNA
gel electrophoresis. The best results were obtained with the method
used by Su and Gibor (22) for RNA isolation from marine red or green
algae. Stage A gave an almost colorless pellet of RNA that was
successfully used for cDNA synthesis. Approximately 3 g of
G. fruticosa stored at mRNA purification for 5'-RACE was carried out with an
oligo(dT)-cellulose column and purification kit (Amersham Pharmacia Biotech) and yielded 10 µg of poly(A)-rich RNA from 1.45 mg of G. fruticosa total RNA. For Northern blot analysis, the
mRNA was purified using an Oligotex spin column (Oligotex mRNA
midi kit, QIAGEN Inc.). Approximately 20 µg of mRNA was recovered
from 330 µg of total RNA using this protocol.
cDNA Synthesis and PCR Cloning
cDNA reactions were run as described previously (23) using
total RNA and an oligo(dT) sequence linked at the 5'-end to an adaptor
sequence (5'-ATG-AAT-TCG-GTA-CCC-GGG-ATC-C(T)17-3').
Initial PCR Clone--
Upstream degenerate primers were based on
the conserved sequence of mammalian COX-1 and COX-2,
FAFFAQHFTHQFFKT, 5'-TTT-GCN-TTY-TTY-GCN-CAR-CAY-TT-3' (see Fig. 1,
primer F1) for the first round and 5'-TTC-ACN-CAY-CAR-TTY-TTY-AAR-AC-3' (primer F2) (where R is A or G; Y is C or T; and N is A, G, C, or T)
for the second round of nested PCR. Downstream primers were based on
the conserved sequence TFGGDVG, 5'-CC-CAC-NTC-NCC-NCC-RAA-NGT-3' (see
Fig. 1, primer R1), and for the second round of nested PCR, the
conserved sequence in the region of the active-site tyrosine, WHPLLPD,
5'-TC-AGG-CAK-NAR-NGG-RTG-CCA-3' (primer R2) (where K is G or T).
The first round of PCR was primed with G. fruticosa cDNA
prepared from 0.4 µg of total RNA using 10 mM Tris-HCl
(pH 8.3), 50 mM KCl, and 3 mM MgCl2
with 0.2 mM each dNTP, 0.4 µM primers, and
0.25 µl (1.25 units) of AmpliTaq DNA polymerase (PerkinElmer Life
Sciences) in a PerkinElmer Life Sciences 480 thermal cycler. After a hot start at 80 °C, the PCR was programmed as follows: 94 °C for 45 s for one cycle; 47 °C for 1 min, 72 °C for
1.5 min, and 94 °C for 45 s for 30 cycles; and 72 °C for 10 min. The second round reaction was primed with the equivalent of 0.1 µl of the first round reaction products (added as a 10-fold
dilution). The protocol used was as follows: 94 °C for 45 s for
one cycle; 50 °C for 1 min, 72 °C for 1 min, and 94 °C 45 s for 30 cycles; and 72 °C for 10 min. The PCR products visualized
on 2.0% agarose gels containing ethidium bromide were purified on an
agarose gel (GENO-BIND, CLONTECH) and
subcloned into the TA cloning vector pCR2.1 (Invitrogen).
3'-RACE--
This was accomplished using first strand cDNA
prepared using the adaptor-linked oligo(dT) primer. The upstream
primers for the first and second rounds of PCR were gene-specific:
5'-CA-GAG-CTA-GTG-TTT-GAC-CAT-GGC-3' and
5'-C-TAC-GAC-AAT-CGT-ATC-CAC-GTC-G-3'. The downstream primer was based
on the adaptor sequence of the cDNA synthesis primer 5'-ATG-AAT-TCG-GTA-CCC-GGG-3'. A PCR program of one cycle at 94 °C
for 45 s; 30 cycles at 47 °C for 1 min, 72 °C for 1.5 min, and 94 °C for 45 s; and one cycle at 72 °C for 10 min was
used for the first round of PCR. A PCR program of 30 cycles at 58 °C for 30 s, 72 °C for 1.5 min, and 96 °C for 15 s
followed by one cycle at 72 °C for 10 min was used for the second
round of half-nested PCR.
5'-RACE--
This was accomplished using a Marathon cDNA
amplification kit (CLONTECH). The first strand
cDNA was synthesized using 1.6 µg of mRNA according to the
manufacturer's instructions. The adaptor-ligated double-stranded
cDNA was diluted 1:50, and 1 µl of the dilution was used per
50-µl PCR. For the 5'-RACE reaction, the upstream primer for the
first and second rounds of PCR was specific for the ligated adaptor
sequence, 5'-CCATCCTAATACGACTCACTATAGGGC-3'. The downstream primer for
the first round reaction was 5'-TT-TTG-TCG-CTC-CAT-ATC-CTG-ACC-3'. The
PCR program was one cycle at 94 °C for 45 s; 30 cycles at 55 °C for 30 s, 72 °C for 2.5 min, and 96 °C for 15 s; and one cycle at 72 °C for 10 min. For the second round reaction
(primed with 0.1 µl of first round reaction products), the downstream primer was 5'-GA-GAC-ATC-CAC-TCC-ATG-ATT-CCC-3', and a similar PCR
program was used except that the extension time was shortened to 2 min.
Full-length cDNA Clones Obtained by PCR--
The upstream
primer encoded the N terminus of the polypeptide and the natural Kozak
consensus sequence from the coral (AAG-ATG-G) for
translation initiation (24); the BamHI site was added at the
5'-end to facilitate subcloning:
5'-T-CAC-GGA-TCC-AAG-ATG-GTG-GCC-AAG-TTT-GTC-G-3'. The downstream
primer encoded the C terminus of the polypeptide with an added
5'-EcoRV site to facilitate subcloning:
5'-TC-TAG-GCC-TGA-TAT-CTA-AAG-TTC-ATC-TCT-TGC-TTC-3'. PCRs were run
using 1 µl of the first strand cDNA and the cloned Pfu
DNA polymerase (Promega) with the buffer supplied (10× buffer: 100 mM KCl, 100 mM
(NH4)2SO4, 200 mM
Tris-HCl (pH 8.75), 20 mM MgSO4, 1% Triton
X-100, and 1 mg/ml bovine serum albumin), 200 µM each
dNTP, and 0.4 µM each primer using the hot start
protocol: one cycle at 95 °C for 5 min; cooling to 4 °C and
addition of DNA polymerase (2.5 units/50-µl reaction); one cycle at
94 °C for 1 min; three cycles at 94 °C for 45 s, 50 °C
for 45 s, and 72 °C for 5 min; 30 cycles at 94 °C for
45 s, 55 °C for 45 s, and 72 °C for 5 min; and one
cycle at 72 °C for 10 min. The 3'-A overhang was added by a 10-min
incubation at 72 °C with AmpliTaq DNA polymerase. The PCR product
was purified on agarose gel and subcloned into the TA cloning vector
pGEM-T Easy (Promega).
DNA Sequencing and Sequence Analyses
Plasmid DNA was isolated using the QIAGEN plasmid purification
system. The clones were sequenced using an ABI Prism dye terminator cycle sequencing kit (PerkinElmer Life Sciences) and an ABI Prism 310 genetic analyzer. The amino acid sequence data were compared with
entries in the GenBankTM/EBI Data Bank using BLAST (25).
The signal peptide cleavage site was predicted using SignalP Version
1.1 (26). Multiple sequence alignments were obtained with the Clustal
method using the Lasergene program (DNASTAR, Inc.).
Northern Blot Analysis
Poly(A)-rich RNA (1 and 10 µg) from G. fruticosa
was fractionated on a 1.0% denatured formaldehyde-agarose gel and
immobilized on a Hybond-N+ 0.45-µm nylon membrane
(Amersham Pharmacia Biotech RPN119B). Immobilized RNA was hybridized
with radiolabeled DNA by incubating the nylon membranes at 65 °C for
2 h with the initial 566-bp PCR product labeled with
[ Plasmids
The ORF of the coral COX cDNA was subcloned into the
eukaryotic expression vector pCR3.1 (Invitrogen) for expression in the vaccinia/HeLa expression system and into pCR3.1, pcDNA3
(Invitrogen), and pCG-E2Tag (27) vectors for transient expression in
COS-7 cells. The 1.8-kilobase BamHI/EcoRV
fragment was isolated from the coral COX-pGEM-T Easy construct and
cloned into a suitable expression vector. This fragment contains the
translational start and stop codons, and the cohesive ends allow the
fragment to be ligated into expression vectors in the proper
orientation. The XbaI/BamHI digestion removed the
E2Tag from the N terminus of the coral COX sequence in the
pCG-E2Tag construct (see Fig. 4A). For insertion of the
E2Tag epitope in the middle of the protein, the Eco91I
(BstEII) site, 23 amino acids from the C terminus, was used
(see Fig. 4A). The pcDNA3.1 expression vector containing rabbit COX-2 cDNA was a kind gift from Dr. Matthew Breyer
(Vanderbilt University).
Cells and Transfections
The COS-7 cells were maintained in Iscove's modified
Dulbecco's medium with 10% fetal bovine serum. For transfection,
COS-7 cells were trypsinized, harvested by centrifugation, and
resuspended in Iscove's modified Dulbecco's medium containing 10%
fetal calf serum at a density of 1 × 107 cells/ml.
250 µl of cell suspension was mixed with 800 ng of plasmid DNA and 50 µg of salmon sperm DNA in a disposable electroporation cuvette and
was subjected to an electric discharge of 180 V using a Bio-Rad Gene
Pulser at 970-microfarad capacity. After the discharge, the cell/DNA
mixture was left at room temperature for 15 min, and then cells were
washed and plated. Cells were grown either at 37 °C for 48 h or
at 28 °C for 72 h. Cells were collected using a rubber
policeman, washed with ice-cold PBS, and collected by centrifugation at
1000 × g for 10 min. Transfected cells from 2-12
tissue culture plates (100 mm, 2-2.5 × 106
cells/plate) were resuspended in 1 ml of ice-cold 50 mM
Tris-HCl (pH 8.0) containing 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol and
disrupted by sonication. The sonicated cells were centrifuged at
200,000 × g for 1 h in a Beckman Ti-70.1 rotor to
yield the microsomal fraction. The membranes were resuspended by
homogenization in a volume of 50 mM Tris-HCl (pH 8.0)
convenient for cyclooxygenase assay.
Western Blotting
COS-7 cells transfected with expression plasmid in 100-mm
diameter dishes were lysed 48 h after electroporation in 200 µl of Laemmli sample buffer (28). Proteins were separated by SDS-8% polyacrylamide gel electrophoresis. After transfer of proteins by a
semidry blotting method, a polyvinylidene difluoride membrane (Millipore Corp.) was incubated with anti-E2Tag mouse monoclonal antibody (final concentration of 0.1 µg/ml) (30), ovine
COX-1-specific polyclonal or monoclonal antibodies (Cayman Chemical
Co., Inc.), or rat COX-2-specific mouse monoclonal antibody
(Pharmingen) and with a secondary horseradish peroxidase-conjugated
antibody (LabAs Ltd., Tartu, Estonia) according to the manufacturers'
recommendations. Detection was performed using an ECL detection kit
(Amersham Pharmacia Biotech) following the manufacturer's manual.
Immunofluorescence Analysis
For immunofluorescence analysis, COS-7 cells transfected with
the appropriate expression vector were grown on coverslips for 48 h and fixed in cold ( Enzyme Assay
Incubations of the microsomal fraction of transfected cells with
[14C]arachidonic acid (final concentration of 50 µM) were performed in 1 ml of 50 mM Tris-HCl
(pH 8.0) containing 1 mM adrenaline and 1 µM
hemin for 10 min at room temperature. The reactions were terminated by
addition of 100 µl of 100 mM SnCl2 as an
aqueous suspension. After acidification to pH 3, the products were
recovered by extraction with ethyl acetate and subjected to TLC
analysis with unlabeled authentic standards (a generous gift from
Kevelt Ltd., Tallinn, Estonia) (20).
For inhibition studies, the enzyme (microsomal fraction) was
reconstituted with hemin (1 µM) in 50 mM
Tris-HCl (pH 8.0) containing 1 mM adrenaline and
preincubated at room temperature for 5 min with various amounts of
inhibitors added in a few microliters of ethanol. An equal amount of
ethanol was added to the control. The reaction was initiated with
[14C]arachidonic acid (final concentration of 30 µM). The incubation was carried out for 10 min, followed
by treatment with SnCl2. The products were extracted
and analyzed as described above.
Cloning of COX by Degenerate Oligonucleotide-primed
PCR--
Vertebrate cyclooxygenases have highly conserved residues in
their functionally important regions of the catalytic domain. In an
effort to isolate the gene responsible for prostaglandin synthesis
in the soft coral G. fruticosa, a homology-based
reverse-transcription-PCR cloning strategy and degenerate primers were
used to amplify COX sequences from coral cDNA (Fig.
1). Upstream degenerate primers were
based on the conserved amino acid sequence of mammalian COX-1 and COX-2, FAFFAQHFTHQFFKT (primers F1 and F2). Downstream primers were
based on the conserved sequence TFGGDVG (primer R1) and, for the second
round of nested PCR, on the conserved sequence in the region of the
active-site tyrosine, WHPLLPD (primer R2). First round PCR experiments
gave no visible bands on an ethidium bromide-stained agarose gel.
Second round half-nested or nested PCR with primers F2 and R2 gave a
product of the expected size (566 bp) that, when cloned and sequenced,
was found to have high homology to mammalian COX genes. Extension of
the 5'- and 3'-ends was achieved using RACE-PCR methodology. Overall,
the nucleotide sequence obtained from the three overlapping products
translated in one reading frame to give the full-length cDNA
sequence. The resulting clone contained, in addition to 1767 bp of
coral COX ORF, also 161 bp of 5'-untranslated region and 197 bp of
3'-untranslated region, including the polyadenylation signal AATAAA.
Northern blot analysis of G. fruticosa poly(A)-rich RNA with
the 32P-labeled initial PCR product showed that the coral
COX mRNA is ~2.1 kilobases in size, substantially smaller than
the mRNAs for mammalian COX gene products (Fig.
2). The mRNAs of the mammalian constitutive and inducible enzymes are ~2.8 and 4.5 kilobases, respectively (8). Subsequently, the ORF of the full-length clone was
amplified from the first strand cDNA by PCR using gene-specific primers and a proofreading polymerase. Five full-length clones were
sequenced.
Sequence Analysis--
The coral cDNA
(GenBankTM/EBI accession number AY004222) contains an ORF
encoding a protein of 589 amino acid residues with a calculated
molecular mass of 67,934 Da. Multiple alignment of the predicted
sequence of the coral enzyme with other known COX sequences revealed a
high level of conservation within these molecules (Fig.
3). The amino acid sequence identity to
vertebrate COX-1 and COX-2 was approximately equal, 45-49% to each.
There are 21 amino acids in the coral sequence that can be considered
COX-1-specific (i.e. conserved in all COX-1 sequences and
not in COX-2, e.g. Ile523), and 23 coral
residues are COX-2-specific (e.g. Leu503). (The
numbering of residues used here corresponds to the amino acids in sheep
COX-1.) The key residues determined to be essential for substrate
binding, catalysis, and inhibition are well conserved in the coral
enzyme. Also, five pairs of cysteines that form disulfide bonds
(Cys36-Cys47,
Cys37-Cys159,
Cys41-Cys57,
Cys59-Cys69, and
Cys569-Cys575) are conserved in coral COX
(10).
Expression of Coral COX cDNA--
The ORF of the coral COX
cDNA was subcloned into the eukaryotic expression vector pCR3.1 for
transient expression in COS-7 cells and vaccinia/HeLa cell expression
systems. For deletion of the 5'- and 3'-untranslated regions, it was
necessary to synthesize two oligonucleotide linkers to rebuild the
initiator methionine with a Kozak translational start sequence and a
termination codon at the 3'-end of the ORF. These linkers contained a
BamHI restriction site for the 5'-end and an
EcoRV site for the 3'-end of the ORF. This initial construct
lacked detectable enzyme activity in transfection experiments at
37 °C in COS-7 cells as well as in vaccinia/HeLa cell expression
systems. We were also unable to detect the expression level of the
protein at this stage due to the lack of a suitable antibody.
To follow the expression of the protein, the ORF of the coral COX
cDNA was subcloned into the epitope-tagged eukaryotic expression vector pCG-E2Tag (27, 29). In this way, we fused the bovine papilloma
virus type 1 E2 protein-derived epitope (E2Tag, GVSSTSSDFRDR) (30) in
frame into the N terminus of coral COX. As both extreme ends of
cyclooxygenases have been shown to be important for processing the
enzyme within a mammalian cell (6), we also inserted the tag into the
C-terminal part of the protein, 23 amino acids from the C terminus
(Fig. 4A). The resulting
plasmids, pCG-E2Tag-COX and pCG-COX(E2Tag), were expressed in
COS-7 cells; and 48 h after transfection, the expression of the
coral COX protein was analyzed by immunoblot analysis. At the same
time, we found that a mouse monoclonal antibody raised against a
236-amino acid C-terminal part of the rat COX-2 peptide, but not
anti-ovine COX-1 polyclonal or monoclonal antibodies (data not shown),
reacted specifically with both native (Fig. 4B, lane
8) and recombinant (lanes 6 and 7) coral
enzymes upon Western blotting. Rabbit COX-2 in pcDNA3.1 was used as
a positive control (lane 5). As shown in Fig. 4B, anti-E2Tag monoclonal antibody recognized the COX(E2Tag) protein (lane 3), where the tag was fused in the middle of the
enzyme, but was not able to recognize N-terminally fused E2Tag
(lane 2). As the protein E2Tag-COX was readily detectable by
COX-specific antibody (lane 7), we can conclude that coral
COX, similar to mammalian COX enzymes, contains a signal peptide in its
N terminus and that this signal peptide is cleaved during the
processing of the protein within the cell. In conclusion, Western
blotting of recombinant coral COX revealed that (i) recombinant coral
COX expressed in a mammalian cell expression system gives one distinct band of ~72 kDa, similar to that of the native coral enzyme; and (ii)
coral COX contains an N-terminal signal peptide, which is cleaved to
yield the mature protein.
Expression at 37 °C in either COS-7 cells or vaccinia-infected HeLa
cells produced COX protein as determined by Western analysis, but there
was no detectable conversion of [14C]arachidonic acid
with the recombinant enzyme. The soft coral G. fruticosa
grows in the Arctic White Sea at depths below 20 m, where
temperature and light conditions do not change significantly during the
year. Even in the summer months, the water temperature is ~5 °C.
To survive under such extreme conditions, enzymes must catalyze
efficiently at low temperatures. The molecular basis of cold adaptation
of psychrophilic enzymes is relatively poorly understood (31, 32). Some
recombinant psychrophilic enzymes that have been used to measure
specific activity may not fold properly in mesophilic hosts or may be
partially inactivated as a result of the high temperature (>30 °C)
used for their expression (32).
Based on these considerations, we decided to grow the transfected COS-7
cells expressing coral COX at 28 °C. To follow the expression level
of the COX enzyme under such extreme conditions, the pCG-COX(E2Tag)
plasmid was transfected into the COS-7 cells; the cells growing at two
different temperatures were harvested at different time points; and the
expressed protein level was determined by Western blot analysis. As
shown in Fig. 4C, COS-7 cells grown at 28 °C did express
the coral COX protein, although at a reduced level compared with the
normal temperature (37 °C). To obtain sufficient recombinant enzyme
for cyclooxygenase assay, the COS-7 cells transfected with
pCG-E2Tag-COX were grown at 28 °C for 72 h. The microsomal
fraction was prepared and incubated with [14C]arachidonic
acid. The products were separated by TLC, followed by monitoring of the
radioactivity. Four well separated polar bands comigrated with natural
mammalian prostaglandin standards and gave color reactions with
anisaldehyde spray reagent characteristic of the corresponding
authentic prostaglandins (Fig. 5).
The prostaglandins PGD2, PGE2,
PGF2
The effect of typical mammalian cyclooxygenase inhibitors (indomethacin
and nimesulide) was tested on product formation by recombinant
coral COX. As shown in Table I, indomethacin inhibited the COX activity
in microsomal fractions of COS-7 cells transfected with coral COX
cDNA. The selective COX-2 inhibitor nimesulide was found to be
ineffective up to concentrations of 40 µM.
Intracellular Localization of Coral COX--
To determine the
subcellular localization of recombinant coral COX, pCG-E2Tag-COX,
pCG-COX(E2Tag), and pcDNA3.1 rabbit COX-2 were transfected into
COS-7 cells. The cells were grown and fixed on coverslips, and
immunofluorescence staining was performed. As shown in Fig.
6 (B and C), coral
COX and coral COX(E2Tag) both exhibited the signal at the endoplasmic
reticulum and the nuclear envelope, similar to the location of the
rabbit COX-2 protein (Fig. 6A). No specific
immunofluorescence signal was detected in control COS-7 cells
transfected with the carrier salmon sperm DNA (Fig. 6D).
G. fruticosa cyclooxygenase cDNA was cloned by PCR
using degenerate primers based on conserved sequences of known
vertebrate COX enzymes. The primary structure of coral COX has ~50%
amino acid identity to both mammalian COX-1 and COX-2. The residues shown to be catalytically important for both peroxidase and
cyclooxygenase activities in mammalian COX isozymes are present in
coral COX. However, significant structural differences can be found
around the catalytic sites as well as in the pattern of glycosylation.
The Peroxidase Active Site--
The hydroperoxide-reducing site of
mammalian COX-1 and COX-2 lies on the surface of the catalytic domain
on the distal side of the liganded heme prosthetic group (10). Amino
acids proposed to be important for heme binding and catalytic activity
(proximal heme ligand His388 and distal heme
His207 and Gln203 (6)) are conserved in the
coral enzyme (Fig. 3). However, there are significant differences in
the amino acid substitutions at residues 289-295 that are proposed to
form a small shield above His207 and Gln203.
The occurrence of the sequence HPFYSML in G. fruticosa
instead of QEVFGLL in COX-1 may reduce the opening to the
hydroperoxide-reducing site and thus sterically restrict access of
bulky fatty acid hydroperoxides to the heme iron. Also, essential
sequence differences between the coral and mammalian COX enzymes can be
found in the loop of residues at positions 211-220 that are supposed
to form binding sites for PGG2 and the reducing substrates
(10). Our earlier studies showed a very low hydroperoxide-reducing
activity of native G. fruticosa preparations as evidenced by
(i) significant amounts of 15-keto-PGs among the endogenous
prostaglandins, (ii) formation of 15-keto-PGs in incubations with
exogenous arachidonic acid, and (iii) accumulation of PGG2
instead of PGH2 in short incubations even in the presence
of different electron donors (18-20). Structural changes around the
peroxidase catalytic site of the coral enzyme that may affect the
hydroperoxide-reducing ability remain to be established by mutagenesis studies.
The Cyclooxygenase Active Site--
The positioning of
Arg120, Tyr355 (important for fatty acid
substrate binding), catalytic Tyr385, and
Ser530 (the residue that is acetylated by aspirin and that
is essential for its inhibitory activity (5, 6, 9, 33, 34)) is well
conserved between mammalian and coral COX proteins (Fig. 3). The volume
of the arachidonate-binding channel of mammalian COX isozymes is
determined by the Ile-to-Val substitution at position 523. Indeed, the V523I replacement in human COX-2 opens access to a side
pocket in the arachidonate-binding channel for specific COX-2
inhibitors (12, 35). The presence of Ile523 in coral COX
resembles the COX-1 active site. However, substitution at another
crucial position (position 503) is different; in G. fruticosa, similar to COX-2 (36), there is a leucine at position 503. Moreover, substitutions with Leu513 (His in COX-1 and
Arg in COX-2), Ala524 (Glu in COX-1 and COX-2), and
Met434 (Ile and Val in COX-1 and COX-2, respectively) in
coral COX indicate additional structural differences in the hydrophobic
tunnel that forms the cyclooxygenase active site (11). These may
reflect different catalytic and inhibition properties of the coral and mammalian enzymes. Our inhibition studies with native and recombinant G. fruticosa COX enzymes showed that both preparations are
inhibited by the nonselective COX inhibitor indomethacin (Ref. 20 and Table I). The relatively high IC50 compared with mammalian
COX isozymes appears to indicate that coral COX is less sensitive to
indomethacin. Also, the selective COX-2 inhibitor nimesulide had no
effect on coral COX at concentrations up to 40 µM,
indicating that the coral enzyme is even less susceptible to this
inhibitor than is mammalian COX-1 (4).
Consensus N-Glycosylation Sequences--
Mutagenesis studies with
vertebrate COX proteins transiently expressed in COS-1 cells indicate
that COX-1 is N-glycosylated at three sites,
Asn68, Asn144, and Asn410 (37).
COX-2 contains one (trout) or two (chicken and mammals) additional
potential N-glycosylation sites (38); in mammals, only one
of them, Asn580, is occupied in ~50% of molecules (37).
The G. fruticosa COX enzyme has three potential
N-glycosylation sites: one is in a conserved position at
Asn144, whereas the first and third are shifted to
Asn73 and Asn396 (Fig. 3).
Signal Sequences--
The major differences in primary structure
between mammalian COX isozymes are a shorter signal peptide in the N
terminus and an 18-amino acid C-terminal insertion in COX-2 (6, 8). The C terminus of coral COX is similar to that of COX-1, but the N terminus
differs from those of both mammalian isozymes. The putative N-terminal
signal peptide of the coral enzyme is cleaved between Ala22
and Val23, at a position corresponding to the cleavage site
of all COX-2 proteins (Fig. 3) (26). However, based on the size of the
cleaved signal peptide, coral COX is more similar to COX-1 (23 cleaved residues) than to COX-2 (17 cleaved residues) (6).
In subcellular localization, the mammalian cyclooxygenases are
associated with the endoplasmic reticulum and nuclear membranes (39,
40). The coral COX primary structure ends with the sequence RDEL (Fig.
3), which closely resembles the classic endoplasmic reticulum targeting
signal for soluble proteins, KDEL. A similar sequence, (P/S)TEL, found
on the carboxyl termini of all mammalian cyclooxygenases, has been
reported to be necessary for retention of these enzymes within the
endoplasmic reticulum (41). Some other studies indicate that the
extreme C-terminal region is not an essential part of the intracellular
targeting mechanism of COX-1 and COX-2 (42, 43). In our
immunofluorescence studies, the COX protein of the Arctic soft coral
exhibited subcellular localization similar to that of the mammalian
cyclooxygenases, on the endoplasmic reticulum and nuclear envelope. The
localization was not affected by the inserted tag within the
protein, 23 amino acids from the C terminus. However, when the
pCG-coral COX(E2Tag) plasmid expressing the protein containing E2Tag
was used for transfection, no cyclooxygenase activity was detected
(Table I). The result was quite unexpected because it has been reported
that a green fluorescent protein-tagged COX-2 with a 27-kDa green
fluorescent protein polypeptide inserted into the C-terminal 18-amino
acid insert region of the COX-2 polypeptide was catalytically
indistinguishable from wild-type COX-2 (44). On the other hand, the
extreme C-terminal region has been shown to be important to the
functional integrity of COX-1 (41, 42). One explanation for the loss of
activity in the case of the coral COX(E2Tag) protein may be structural changes in the functional part of the protein caused by the positioning of the tag within a conserved region of the enzyme.
The relationship between the coral COX amino acid sequence and other
known COX sequences is shown in the phylogenetic tree in Fig.
7. Coral sequences form a separate arm on
the phylogenetic tree, indicating that invertebrate COX might be a
common ancestor of vertebrate COX isozymes and that divergence of
COX-1 and COX-2 genes occurred later in evolution, after divergence of
the animal kingdom to vertebrates and invertebrates.
In summary, the results of this study demonstrate the existence of a
COX isozyme in marine invertebrates. This is the first study to confirm
experimentally the presence of a cyclooxygenase in non-vertebrates.
Our results establish that the COX enzyme is conserved from lower
animals to human beings. They also lay to rest the long-standing issue
of the origin of prostaglandins in coral, showing clearly that
prostaglandins in coral are synthesized from arachidonic acid via an
endoperoxide intermediate and that the conversion is catalyzed by an
enzyme highly homologous to vertebrate COX isozymes. The expressed
coral enzyme has some unusual catalytic activities that will make it
a striking research object from both the evolutionary and mechanistic
standpoints. The interrelationship of the peroxidase and oxygenase
activities in initiating and maintaining PG endoperoxide synthesis
remains a subject of intense investigation. The different structure and
coupling of the peroxidase in coral COX present an opportunity to gain
additional insights.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
70 °C was pulverized to a fine powder in liquid nitrogen. 10 ml of lysis buffer (150 mM
Tris-HCl (pH 7.5), 2% SDS, and 1%
-mercaptoethanol) was added
immediately to the coral powder and vortexed for 2 min. Then 1 ml of 8 M guanidinium-H Cl was added, and the mixture was vortexed
for 1 min. The solution was extracted with 1 volume of
chloroform/isoamyl alcohol (49:1, v/v), and the phases were separated
by centrifugation at 10,000 × g for 20 min. The
aqueous phase was collected and re-extracted with 1 volume of
phenol/chloroform/isoamyl alcohol (50:49:1, by volume), and the sample
was then centrifuged again. To remove the traces of phenol, the aqueous
phase was re-extracted with chloroform/isoamyl alcohol (49:1, v/v).
Precipitation of the total RNA was carried out by addition of
0.33 volume of 12 M LiCl and
-mercaptoethanol
(final concentration of 1% (v/v)) at
20 °C for 24 h. The
total RNA was pelleted by centrifugation at 20,000 × g
for 90 min and washed with 75% ethanol, followed by centrifugation at
20,000 × g for 15 min. All operations were carried out
at 0 to +4 °C. The pellet of RNA was dissolved in 1 mM
EDTA and quantified by UV spectroscopy. Approximately 2 mg of total RNA
was recovered using this protocol.
-32P]dCTP using ExpressHyb hybridization solution
(CLONTECH 8015-2). The membranes were then washed
as follows: 2 × 25 min with 2× SSC + 0.1% SDS at room
temperature; 2 × 15 min with 2× SSC + 0.1% SDS at 65 °C; and
1 × 15 min with 1× SSC + 0.1% SDS at 65 °C. Hybridization
was visualized by autoradiography after overnight exposure at
70 °C using Kodak x-ray film.
20 °C) acetone/methanol (1:1) for 15 min.
Coverslips were washed with PBS and blocked with bovine serum albumin
(1 mg/ml in PBS) for 30 min. Anti-E2Tag monoclonal antibody was added
at a concentration of 5 ng/µl (in bovine serum albumin/PBS) and
anti-COX-2 monoclonal antibody in a dilution of 1:100, and both were
incubated for 1 h at room temperature and washed with PBS. As a
secondary antibody, fluorescein isothiocyanate-conjugated goat
anti-mouse IgG (LabAs Ltd.) was used at the concentration recommended
by the manufacturer. Cells were examined on an Olympus Vanox-S AH2 microscope.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cloning strategy. Shown are the
positions of degenerate primers based on conserved regions of the known
mammalian cyclooxygenases and three overlapping PCR products.
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Fig. 2.
Northern blot analysis of poly(A)-rich RNA
isolated from G. fruticosa with an initial PCR clone
as a probe. Lane 1, 1 µg of mRNA; lane
2, 10 µg of mRNA. Molecular mass markers and ribosomal
(R) bands of coral total RNA are indicated.
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Fig. 3.
Deduced amino acid sequence alignment of
coral COX (GenBankTM/EBI accession number
AY004222) and human COX-1 and COX-2. Black boxes
indicate positional identity for at least one of the compared sequences
with the coral enzyme; similar amino acids (G = A = S, S = T, V = L = I, F = W = Y, E = D = R = K, and Q = N) are indicated by gray boxes. Signal
peptide sequences are underlined. The key residues of
substrate binding and catalysis are numbered. The loops near
the peroxidase active site are indicated by asterisks.
Crucial positions, different in the arachidonate-binding channel of
COX-1 and COX-2, are indicated by arrows. Putative
N-glycosylation sites are boxed.
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Fig. 4.
Expression of the coral COX cDNA in COS-7
cells. A, schematic representation of the
epitope-tagged coral COX proteins used in this study. B,
immunoblot analysis of COS-7 cells transfected with pCG-E2Tag-COX,
pCG-COX(E2Tag), and pcDNA3.1 rabbit COX-2 expression vectors or
carrier DNA (negative control (Neg. Contr.)). 48 h
after transfection, cell extracts were prepared and analyzed by
immunoblotting with anti-E2Tag antibody (lanes 1-3) or with
anti-COX-2 antibody (lanes 4-8) as described under
"Materials and Methods." Lane 8, native COX:
microsomes from the Arctic coral G. fruticosa (10 µg of
protein). C, immunoblot analysis of COS-7 cells transfected
with pCG-COX(E2Tag) cultivated at two different temperatures. Cells
were harvested at the time points indicated (in hours) and
counted, and cell extracts were prepared. An extract prepared from
1 × 105 cells is loaded on each lane. The anti-E2Tag
antibody was used as a probe.
, and 15-keto-PGF2
formed from the
exogenous arachidonic acid with the G. fruticosa acetone
powder were characterized earlier by high pressure liquid
chromatography, gas chromatography-mass spectrometry, 13C
NMR, and optical rotation measurements in comparison with authentic standards (18, 20). So, the active recombinant COX enzyme of the Arctic
coral G. fruticosa was expressed at 28 °C, whereas the
protein expressed at 37 °C was not functional and was unable to
catalyze the conversion of arachidonate to prostaglandins (Table I).
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Fig. 5.
Thin layer radiochromatogram of the products
formed upon incubation of recombinant coral COX with
[1-14C]arachidonic acid. A microsomal fraction of
the pCG-E2Tag-COX-transfected COS-7 cells (2.4 × 107
cells/incubation) was used in the incubation. The products were
separated by TLC using a solvent system of hexane/ethyl acetate (5:1,
v/v), followed by benzene/dioxane/acetic acid (10:5:0.5, by volume).
The TLC plate was cut into sections and extracted with methanol, and
the radioactivity was determined by liquid scintillation counting.
Unlabeled authentic standards of PGF2 ,
PGE2, PGD2, and arachidonic acid were
visualized with an anisaldehyde spray reagent and brief heating.
HETEs, hydroxyeicosatetraenoic acids.
Cyclooxygenase activity of coral COX and rabbit COX-2 expressed in
COS-7 cells
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Fig. 6.
Immunofluorescence staining of COS-7 cells
transfected with pcDNA3.1 rabbit COX-2 (A),
pCG-E2Tag-COX (B), pCG-COX(E2Tag)
(C), or carrier DNA alone (D).
The transfected cells were subjected to indirect immunofluorescence
staining with COX-2-specific antibody (A, B, and
D) or with E2Tag-specific antibody (C) as
described under "Materials and Methods."
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (15K):
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Fig. 7.
Phylogenetic tree showing the relationship
between coral COX amino acid sequences and other known COX
sequences. *, K. Valmsen, unpublished data.
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ACKNOWLEDGEMENTS |
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We are grateful to the National Institute of Chemical Physics and Biophysics (Tallinn, Estonia) for access to molecular biology facilities. The Kartesh White Sea Biological Station of the Russian Academy of Sciences contributed by collection of the coral. We thank Anu Aaspõllu for helpful discussions and assistance in sequencing work and Anne Kalling for assistance with the cell culture. We are also indebted to Dr. Richard Kim and Brenda Leake for help with the vaccinia/HeLa cell expression experiments.
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FOOTNOTES |
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* This work was supported by Grant 3783 from the Estonian Science Foundation, Fogarty International Research Collabration Award Grant TW00404 and Parent Grant GM53638 from the Fogarty International Center of the National Institutes of Health, and Grant IBGMR11499 from the Estonian Innovation 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AY004222.
To whom correspondence should be addressed. Tel.:
372-6204-376; Fax: 372-6703-683; E-mail: samel@chemnet.ee.
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M009803200
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ABBREVIATIONS |
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The abbreviations used are: COX, cyclooxygenase; PG, prostaglandin; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; bp, base pair(s); ORF, open reading frame; PBS, phosphate-buffered saline.
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