THE JOURNAL OF BIOLOGICAL CHEMISTRY
7033–7040, 2001
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
The Basis of Prostaglandin Synthesis in Coral
MOLECULAR CLONING AND EXPRESSION OF A CYCLOOXYGENASE FROM THE ARCTIC SOFT CORAL
GERSEMIA FRUTICOSA*
Received for publication, October 26, 2000, and in revised form, November 14, 2000
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M009803200
Reet Koljak‡, Ivar Järving‡, Reet Kurg§, William E. Boeglin¶, Külliki Varvas‡, Karin Valmsen‡,
Mart Ustav§, Alan R. Brash¶, and Nigulas Samel‡储
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
In vertebrates, the synthesis of prostaglandin hor-
mones is catalyzed by cyclooxygenase (COX)-1, a consti-
tutively expressed enzyme with physiological functions,
and COX-2, induced in inflammation and cancer. Pros-
taglandins have been detected in high concentrations in
certain corals, and previous evidence suggested their
biosynthesis through a lipoxygenase-allene oxide path-
way. Here we describe the discovery of an ancestor of
cyclooxygenases that is responsible for prostaglandin
biosynthesis in coral. Using a homology-based polymer-
ase 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 se-
quenced from the Arctic soft coral Gersemia fruticosa.
Nearly all the amino acids essential for substrate bind-
ing and catalysis as determined in the mammalian en-
zymes 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 con-
clude that the cyclooxygenase occurs widely in the ani-
mal 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 verte-
brates and invertebrates (1, 2). In vertebrates, they are syn-
thesized by prostaglandin-H2 synthase, known also as cy-
clooxygenase (COX)1 (3–5). COX is a hemoprotein with two
* 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. This
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/EBI Data Bank with accession number(s)AY004222.
储 To whom correspondence should be addressed. Tel.: 372-6204-376;
Fax: 372-6703-683; E-mail: samel@chemnet.ee.
1
The abbreviations used are: COX, cyclooxygenase; PG, prosta-
This paper is available on line at http://www.jbc.org
Vol. 276, No. 10, Issue of March 9, pp.
Printed in U.S.A.
distinct catalytic activities: the cyclooxygenase activity in-
volved 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 housekeep-
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ing functions. COX-2, although absent from most cells, can be
rapidly induced in many cell types upon treatment with inflam-
matory cytokines, growth factors, and tumor promoters (8).
These two isoforms share ⬃60% amino acid sequence identity.
They have similar structural topology and an identical cata-
lytic 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 ex-
cept 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-inflam-
matory drugs (4, 6).
The mechanism of prostaglandin biosynthesis in inverte-
brates, 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 path-
way in plants (15–17), has not found experimental support
more recently. The highly active peroxidase-lipoxygenase fu-
sion protein identified in Plexaura homomalla that catalyzes
conversion of arachidonic acid into allene oxide is not involved
in prostaglandin synthesis (17). Our previous biochemical stud-
ies 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 ste-
reochemistry; (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 cyclooxygen-
ase that catalyzes transformation of arachidonic acid into pros-
glandin; PCR, polymerase chain reaction; RACE, rapid amplification of
cDNA ends; bp, base pair(s); ORF, open reading frame; PBS, phosphate-
buffered saline.
7033
7034 Cloning of a Non-vertebrate Cyclooxygenase
taglandins in the prostaglandin-containing Arctic coral G. fru-
ticosa. 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.
MATERIALS AND METHODS
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
⫺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 chlo-
roform/isoamyl alcohol (49:1, v/v), and the phases were separated by
centrifugation at 10,000 ⫻ g for 20 min. The aqueous phase was col-
lected 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.
mRNA purification for 5⬘-RACE was carried out with an oligo(dT)-
cellulose column and purification kit (Amersham Pharmacia Biotech)
and yielded 10 ␮g 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.). Approx-
imately 20 ␮g of mRNA was recovered from 330 ␮g 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, FAFFAQH-
FTHQFFKT, 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⬘ (prim-
er R2) (where K is G or T).
The first round of PCR was primed with G. fruticosa cDNA prepared
from 0.4 ␮g 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 ␮M primers, and 0.25 ␮l
(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 ␮l 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 amplifi-
cation kit (CLONTECH). The first strand cDNA was synthesized using
1.6 ␮g of mRNA according to the manufacturer’s instructions. The
adaptor-ligated double-stranded cDNA was diluted 1:50, and 1 ␮l of the
dilution was used per 50-␮l PCR. For the 5⬘-RACE reaction, the up-
stream primer for the first and second rounds of PCR was specific for
the ligated adaptor sequence, 5⬘-CCATCCTAATACGACTCACTAT-
AGGGC-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 ␮l 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.
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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 initi-
ation (24); the BamHI site was added at the 5⬘-end to facilitate sub-
cloning: 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 us-
ing 1 ␮l 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 ␮M each dNTP, and 0.4
␮M 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-␮l reac-
tion); 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 ␮g) from G. fruticosa was fractionated on
a 1.0% denatured formaldehyde-agarose gel and immobilized on a Hy-
bond-N⫹ 0.45-␮m 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 [␣-32P]dCTP using ExpressHyb hy-
bridization 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.
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.
 Cloning of a Non-vertebrate Cyclooxygenase
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 ␮l of cell suspension was mixed with
800 ng of plasmid DNA and 50 ␮g 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 phenylmethyl-
sulfonyl 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 mem-
branes 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 ␮l 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 concen-
tration of 0.1 ␮g/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 us-
ing 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 (⫺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/␮l (in bovine serum albumin/PBS) and anti-
COX-2 monoclonal antibody in a dilution of 1:100, and both were incu-
bated for 1 h at room temperature and washed with PBS. As a second-
ary 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.
Enzyme Assay
Incubations of the microsomal fraction of transfected cells with
[14C]arachidonic acid (final concentration of 50 ␮M) were performed in 1
ml of 50 mM Tris-HCl (pH 8.0) containing 1 mM adrenaline and 1 ␮M
hemin for 10 min at room temperature. The reactions were terminated
by addition of 100 ␮l 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 recon-
stituted with hemin (1 ␮M) in 50 mM Tris-HCl (pH 8.0) containing 1 mM
adrenaline and preincubated at room temperature for 5 min with var-
ious 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 ␮M). The
7035
FIG. 1. Cloning strategy. Shown are the positions of degenerate
primers based on conserved regions of the known mammalian cyclooxy-
genases and three overlapping PCR products.
incubation was carried out for 10 min, followed by treatment with
SnCl2. The products were extracted and analyzed as described above.
RESULTS
Cloning of COX by Degenerate Oligonucleotide-primed
PCR—Vertebrate cyclooxygenases have highly conserved resi-
dues in their functionally important regions of the catalytic
domain. In an effort to isolate the gene responsible for prosta-
glandin synthesis in the soft coral G. fruticosa, a homology-
based reverse-transcription-PCR cloning strategy and degen-
erate primers were used to amplify COX sequences from coral
cDNA (Fig. 1). Upstream degenerate primers were based on the
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conserved amino acid sequence of mammalian COX-1 and
COX-2, FAFFAQHFTHQFFKT (primers F1 and F2). Down-
stream primers were based on the conserved sequence TFGG-
DVG (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. Sec-
ond 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 mamma-
lian COX genes. Extension of the 5⬘- and 3⬘-ends was achieved
using RACE-PCR methodology. Overall, the nucleotide se-
quence obtained from the three overlapping products trans-
lated in one reading frame to give the full-length cDNA se-
quence. 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 induc-
ible enzymes are ⬃2.8 and 4.5 kilobases, respectively (8). Sub-
sequently, 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 acces-
sion 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 deter-
mined 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).
7036 Cloning of a Non-vertebrate Cyclooxygenase
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
␮g of mRNA; lane 2, 10 ␮g of mRNA. Molecular mass markers and
ribosomal (R) bands of coral total RNA are indicated.
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 oligo-
nucleotide 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 re-
striction site for the 5⬘-end and an EcoRV site for the 3⬘-end of
the ORF. This initial construct lacked detectable enzyme ac-
tivity 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-termi-
nal 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 an-
alyzed 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 mamma-
lian 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 pep-
tide, 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]arachi-
donic 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 signifi-
cantly during the year. Even in the summer months, the water
temperature is ⬃5 °C. To survive under such extreme condi-
tions, enzymes must catalyze efficiently at low temperatures.
The molecular basis of cold adaptation of psychrophilic en-
zymes is relatively poorly understood (31, 32). Some recombi-
nant 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 trans-
fected COS-7 cells expressing coral COX at 28 °C. To follow the
expression level of the COX enzyme under such extreme con-
ditions, 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
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coral COX protein, although at a reduced level compared with
the normal temperature (37 °C). To obtain sufficient recombi-
nant enzyme for cyclooxygenase assay, the COS-7 cells trans-
fected with pCG-E2Tag-COX were grown at 28 °C for 72 h. The
microsomal fraction was prepared and incubated with [14C]ar-
achidonic 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 prosta-
glandins (Fig. 5). The prostaglandins PGD2, PGE2, PGF2␣, 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 measure-
ments 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).
The effect of typical mammalian cyclooxygenase inhibitors
(indomethacin and nimesulide) was tested on product forma-
tion by recombinant coral COX. As shown in Table I, indo-
methacin 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 ␮M.
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).
DISCUSSION
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
 Cloning of a Non-vertebrate Cyclooxygenase 7037

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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.

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 pros-
thetic 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 dif-
ferences 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. fruti-
cosa 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) for-
mation of 15-keto-PGs in incubations with exogenous arachi-
donic 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 cat-
alytic site of the coral enzyme that may affect the hydroperox-
ide-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 mam-
malian COX isozymes is determined by the Ile-to-Val substitu-
tion at position 523. Indeed, the V523I replacement in human
COX-2 opens access to a side pocket in the arachidonate-bind-
ing channel for specific COX-2 inhibitors (12, 35). The presence
of Ile523 in coral COX resembles the COX-1 active site. How-
ever, 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, respec-
tively) 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 inhibi-
tion 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 ␮M,
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
7038 Cloning of a Non-vertebrate Cyclooxygenase
TABLE I
Cyclooxygenase activity of coral COX and rabbit COX-2 expressed in
COS-7 cells
COS-7 cells were transfected with pCG-E2Tag-coral COX, pCG-coral
COX (E2Tag), and pcDNA3.1 rabbit COX-2 (control) for 48 h at 37 °C or
72 h at 28 °C. The microsomal fraction of disrupted cells (8 –10 ⫻ 106
cells/incubation) was incubated with or without inhibitors for 10 min
with 30 or 50 ␮M [14C]arachidonic acid at room temperature as de-
scribed under “Materials and Methods.” Lipids were extracted and
analyzed by TLC, followed by liquid scintillation counting. The data
represent three independent studies. ND, not determined.
[14C]PGs/106 cellsa
37 °C 28 °C

␮g
Sham-transfected ⬍0.01 ⬍0.01
pCG-E2Tag-coral COX ⬍0.01 0.61
Indomethacin
10 ␮M ND 0.42 (36% inhibition)
40 ␮M ND 0.07 (89% inhibition)
Nimesulide
10 ␮M ND 0.69 (0% inhibition)
40 ␮M ND 0.69 (0% inhibition)
pCG-coral COX (E2Tag) ⬍0.01 ⬍0.01
pcDNA3.1 rabbit COX-2 0.91 0.46
a
Sum of PGD2, 15-keto-PGF2␣, PGE2, and PGF2␣.

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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 ex-
pression vectors or carrier DNA (negative control (Neg. Contr.)). 48 h
after transfection, cell extracts were prepared and analyzed by immu-
noblotting 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 ␮g of
protein). C, immunoblot analysis of COS-7 cells transfected with pCG-
COX(E2Tag) cultivated at two different temperatures. Cells were har-
vested 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.

FIG. 6. Immunofluorescence staining of COS-7 cells trans-

fected 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.”

tion sites (38); in mammals, only one of them, Asn580, is occu-

FIG. 5. Thin layer radiochromatogram of the products formed
upon incubation of recombinant coral COX with [1-14C]arachi-
donic 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 meth-
anol, and the radioactivity was determined by liquid scintillation count-
ing. Unlabeled authentic standards of PGF2␣, PGE2, PGD2, and arachi-
donic acid were visualized with an anisaldehyde spray reagent and brief
heating. HETEs, hydroxyeicosatetraenoic acids.
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-glycosyla-
pied 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 struc-
ture 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
 Cloning of a Non-vertebrate Cyclooxygenase
FIG. 7. Phylogenetic tree showing the relationship between
coral COX amino acid sequences and other known COX se-
quences. *, K. Valmsen, unpublished data.
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 pro-
teins, KDEL. A similar sequence, (P/S)TEL, found on the car-
boxyl 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 func-
tional 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 evolu-
tion, after divergence of the animal kingdom to vertebrates and
invertebrates.
In summary, the results of this study demonstrate the exist-
ence 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
7039
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.
Acknowledgments—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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ENZYME CATALYSIS AND
REGULATION:
The Basis of Prostaglandin Synthesis in
Coral: MOLECULAR CLONING AND
EXPRESSION OF A
CYCLOOXYGENASE FROM THE
ARCTIC SOFT CORAL GERSEMIA
FRUTICOSA

Reet Koljak, Ivar Järving, Reet Kurg, William

E. Boeglin, Külliki Varvas, Karin Valmsen,

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Mart Ustav, Alan R. Brash and Nigulas Samel
J. Biol. Chem. 2001, 276:7033-7040.
doi: 10.1074/jbc.M009803200 originally published online November 20, 2000

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