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Originally published In Press as doi:10.1074/jbc.M313258200 on January 19, 2004

J. Biol. Chem., Vol. 279, Issue 14, 13624-13633, April 2, 2004
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On the Evolutionary Origin of Cyclooxygenase (COX) Isozymes

CHARACTERIZATION OF MARINE INVERTEBRATE COX GENES POINTS TO INDEPENDENT DUPLICATION EVENTS IN VERTEBRATE AND INVERTEBRATE LINEAGES*

Reet Järving{ddagger}, Ivar Järving{ddagger}, Reet Kurg§, Alan R. Brash¶, and Nigulas Samel{ddagger}||

From the {ddagger}Department of Chemistry, Tallinn Technical University, Akadeemia tee 15, Tallinn 12618, §Institute of Technology, Tartu University, 23 Riia Street, Tartu 51010, Estonia, and the Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6602

Received for publication, December 4, 2003 , and in revised form, January 14, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In vertebrates, COX-1 and COX-2, two cyclooxygenase isozymes with different physiological functions and gene regulation, catalyze identical reactions in prostaglandin synthesis. It is still not understood why there are multiple forms of COX enzyme in the same cell type and when the evolutionary duplication of the COX gene occurred. Here we report the structure of two genes encoding for COX isozymes in the coral Gersemia fruticosa, the first non-vertebrate organism from which a cyclooxygenase was characterized. Both genes are about 20 kb in size and consist of nine exons. Intron/exon boundaries are well conserved between coral and mammalian COX genes. mRNAs of the previously reported G. fruticosa COX-A (GenBankTM accession number AY004222 [GenBank] ) and the novel COX-B share 94% sequence identity in the coding regions and less than 30% in the 5'- and 3'-untranslated region. Transcripts of both COX genes are detectable in coral cells, although the transcriptional level of COX-A is 2 orders of magnitude higher than COX-B. Expression of both coral genes in mammalian cells gave functional proteins with similar catalytic properties. By data base analyses we also detected and constructed different pairs of COX genes from the primitive chordates, Ciona savignyi and Ciona intestinalis. These two gene pairs encode proteins with 50% intra-species and only 70% cross-species sequence identity. Our results suggest that invertebrate COX gene pairs do not correspond to vertebrate COX-1 and COX-2 and are consistent with duplication of the COX gene having occurred independently in corals, ascidians, and vertebrates. It is evident that due to the importance and complexity of its regulatory role, COX has multiple isoforms in all organisms known to express it, and the genes encoding for the isozymes may to be regulated differently.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostaglandins are important signaling molecules that are involved in inflammation, ovulation, modulation of immune responses, and mitogenesis. The key enzyme in prostaglandin biosynthesis is prostaglandin-endoperoxide G/H synthase (EC 1.14.99.1 [EC] ), commonly known also as cyclooxygenase (COX)1 (1, 2). Vertebrates from fish to human beings have two different COX isozymes, COX-1 and COX-2 (3–6). COX-1 is constitutively expressed in most mammalian tissues, and expression levels of this enzyme do not vary greatly in adult animals. COX-2, although absent in most cells, can be rapidly induced in many cell types by inflammatory cytokines, growth factors, and tumor promoters (2, 7). Mammalian COX isozymes are encoded by separate single copy genes that map to distinct chromosomes (1, 8). The human gene for COX-1 is ~22 kb in length with 11 exons and is transcribed as 2.8- and 5.2-kb mRNA (9, 10). The gene for COX-2 is about 8.3 kb long with 10 exons, and it is transcribed as 2.8- and 4.6-kb mRNA variants (11–15). The gene structures of COX-1 and COX-2 demonstrate remarkable conservation of exon/intron junctions (16–19). The main differences between the COX-1 and COX-2 genes consist of different intron lengths and one missing intron in COX-2. Although encoded by different genes, the two COX isozymes share a relatively conserved primary structure (~60% of amino acid sequence identity), similar structural topology, and an identical catalytic mechanism (20–24); but the patterns of gene expression and regulation differ greatly, pointing to distinct biological roles (7, 15, 25).

The origin of prostaglandins, as well the mechanism of their biosynthesis in invertebrates, has been an object of intensive studies and speculations over the years (26–29). The first nonvertebrate COX has been cloned and characterized from the prostaglandin-containing corals Gersemia fruticosa (30, 31) and Plexaura homomalla (32), indicating the evolutionary conservation of the COX pathway of prostaglandin formation from marine invertebrates to mammals.

Here we report on cloning and characterization of two functional cyclooxygenase-encoding genes in the coral G. fruticosa. The exon/intron structure is highly conserved between two coral genes and with all vertebrate COX genes characterized. However, significant differences in the transcriptional level of the coral COX genes point to differences in the gene regulation and/or mRNA stability in cells. In addition, by using data base searches, we constructed and analyzed the sequences and exon/intron structures of COX genes from two ascidians species, Ciona intestinalis and Ciona savignyi.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of RNA and cDNA Synthesis
Total RNA was prepared as described previously (31) using the method for RNA isolation from marine red or green algae (33). mRNA was prepared from total RNA by using an oligo(dT)-cellulose column and purification kit (Amersham Biosciences). First strand cDNA was prepared using an oligo(dT)-adapter primer (34).

Isolation of Genomic DNA
For isolation of genomic DNA, the CTAB procedure (35) was used with slight modifications. Three pieces of G. fruticosa (~1 g) stored at -70 °C were transferred into 2 ml of prewarmed (65 °C) isolation buffer (2% w/v CTAB, 1.4 M NaCl, 50 mM EDTA, 100 mM Tris-HCl (pH 8.0), 0.2% {beta}-mercaptoethanol). Proteinase K (100 µg/ml final concentration) was added, and the tube was incubated at 65 °C for 2 h. The pellet was separated by centrifugation at 2000 x g for 5 min. The supernatant was gently extracted with 2 ml of chloroform/isoamyl alcohol (49:1, v/v), and the phases were separated by centrifugation at 2000 x g for 5 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). For precipitation, 2.5 volumes of ice-cold ethanol were added. DNA precipitated as a whitish network, which was transferred into the washing solution (1 ml of ice-cold 70% ethanol) using a glass hook. The pellet was collected by centrifugation (10 min, 5000 x g), dissolved in 200 µl of TE buffer, and quantified by UV spectroscopy. Approximately 1.2 mg of genomic DNA was recovered by using this protocol.

PCR Cloning
Amplification and Cloning of Genomic DNA Sequences—Fragments of genomic DNA were amplified by PCR using 500 ng of G. fruticosa genomic DNA and 0.4 µM gene-specific primers (see Table I) in 50 µl of 75 mM Tris-HCl (pH 8.8) containing 20 mM (NH4)2SO4, 0.01% Tween 20, 2.5 mM MgCl2, and 0.2 mM each dNTP. After a hot start at 95 °C for 5 min, 1.25 units of TaqDNA polymerase (Naxo Ltd., Estonia) was added, and the PCR was programmed as follows: 95 °C for 45 s, 50–58 °C for 45 s, and 72 °C for 5 min for 35 cycles; and 72 °C for 10 min. For amplification of longer genomic DNA fragments (3–9 kb), Expand Long Template PCR System with buffer 3 (Roche Diagnostics), 0.5 mM each dNTP, and 0.3 µM primers were used. The PCR program was 1 cycle at 94 °C for 2 min; 10 cycles at 93 °C for 30 s, 53–62 °C for 30 s, 68 °C for 8–15 min; 20 cycles at 93 °C for 30 s, 53–62 °C for 30 s, 68 °C for 8–15 min + 20 s for each cycle; and 68 °C for 15 min. The PCR products visualized on 0.7% agarose gels containing ethidium bromide were purified (Agarose Gel DNA Extraction Kit, Roche Diagnostics; QIAEX II Gel Extraction Kit, Qiagen Inc.) and subcloned into the TA cloning vector pGEM-T Easy (Promega). The exon/intron junctions were determined by comparing the cDNA sequence and genomic sequence. All exons and intron five were sequenced full length. In other cases, the sequences at the borders of the introns were determined by sequencing across the exon/intron borders, and the sizes of introns were estimated by PCR.


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  		TABLE I
Oligonucleotide primers used for RT-PCR Positions are given relative to the ATG start codon of G. fruticosa COX-A (or COX-B) cDNA. The melting temperatures of primers, Tm, for COX-A and COX-B are calculated using the Lasergene program (DNAstar, Inc.).

 
cDNA Cloning of G. fruticosa COX-B, 5'-RACE—5'-RACE was accomplished using a 5'/3'-RACE kit (Roche Diagnostics). The first strand cDNA was synthesized using 2 µg of G. fruticosa poly(A)+ RNA and COX-B-specific primer TNTS-down (Table I) according to the manufacturer's instructions. The first strand cDNA was purified by the QIA-quick PCR purification kit (Qiagen Inc.). Terminal transferase was used to add a homopolymeric A-tail to the 3'-end of the cDNA. 5 µl (1:5) of tailed cDNA was amplified by PCR using a nested gene-specific primer DCTR-down, the oligo(dT)-anchor primer (0.3 µM), and the Expand Long Template PCR System with buffer 1 (Roche Diagnostics). The PCR program was 1 cycle at 94 °C for 2 min; 10 cycles at 93 °C for 30 s, 55 °C for 45 s, 68 °C for 5 min; 20 cycles at 93 °C for 30 s, 55 °C for 45 s, 68 °C for 5 min + 20 s for each cycle; and 68 °C for 10 min. 0.01 µlofthe first round reaction products (added as a 100-fold dilution) was further amplified by a second PCR using a nested gene-specific primer TCDC-down and the PCR anchor primer.

3'-RACE—3'-RACE of COX-B was accomplished by using first strand cDNA prepared using G. fruticosa mRNA and the adaptor-linked oligo(dT) primer (31). The upstream primers for the first and second rounds of PCR were COX-B-specific; FKG-up and QET-up (Table I). The PCR program was 1 cycle at 94 °C for 2 min; 10 cycles at 93 °C for 30 s, 55 °C for 45 s, 68 °C for 5 min; 20 cycles at 93 °C for 30 s, 55 °C for 45 s, 68 °C for 5 min + 20 s for each cycle; and 68 °C for 10 min.

Full-length cDNA Clones—Full-length open reading frame of the G. fruticosa COX-B cDNA was obtained by PCR. The BamHI site was added at the 5'-end of the upstream and downstream primer to facilitate subcloning (primers GGGN-up and LSAK-down in Table I). PCR was run using 1 µl of the first strand cDNA and the Expand Long Template PCR System with buffer 3 (Roche Diagnostics), 0.5 mM each dNTP, and 0.3 µM primers. The PCR program was 1 cycle at 94 °C for 2 min; 10 cycles at 93 °C for 30 s, 52 °C for 45 s, 68 °C for 3 min; 20 cycles at 93 °C for 30 s, 55 °C for 45 s, 68 °C for 3 min + 20 s for each cycle; and 68 °C for 10 min. The ends of the PCR product were digested with BamHI. Digested product was purified on agarose gel and cloned into pCG and pCG-E2Tag (36) vector for transient expression in COS-7 cells. Plasmid DNA was isolated using the Qiagen plasmid purification system. Three clones were full-length sequenced and expressed.

The rabbit COX-2 cDNA was isolated from the RabCOX-2-pcDNA3.1 construct (kind gift from Dr. Matthew Breyer, Vanderbilt University), cloned into pCG expression vector, and used as a positive control in transfections for tunicamycin treatments.

DNA Sequencing and Sequence Analysis
The clones were sequenced using a DYEnamic ET terminator cycle sequencing kit (Amersham Biosciences) and an ABI Prism 310 genetic analyzer. Sequence alignments were obtained with the Clustal method using the Lasergene program (DNAstar, Inc.). The signal peptide cleavage site was predicted using SignalP version 1.1 (37). Untranslated regions of COX-A and COX-B mRNA were analyzed using UTRdb (38).

Relative Quantification of G. fruticosa COX-A and COX-B mRNAs by Reverse Transcriptase-PCR
For determination of relative concentration of COX-A and COX-B transcripts in the G. fruticosa mRNA, three different primer pairs were used: VAV-up versus DMV-down, ESGA-up versus DYA-down, and FKG-up versus TNTS-down (see Table I). The primer pairs were tested for specificity in PCR with different dilutions (1 fg to 10 ng per µl) of cloned COX-A and COX-B in the pCG-E2Tag vector (total size about 6100 bp). The cloned DNA was isolated using the Qiagen plasmid purification system. The concentrations of the stock solutions (20 ng per µl) were estimated by UV spectrum and agarose gel electrophoresis. The working solutions were prepared by serial 4–5-fold dilutions of the original stock solution. PCRs were run at three different hybridization temperatures: 50, 55, and 60 °C.

cDNA for quantification was synthesized from 2.9 µg of mRNA in a 50-µl reaction volume. 1 µl of 5–10,000 times diluted cDNA synthesis reaction (equal to 5.8 pg to 11 ng of mRNA) was used in a 25-µl PCR volume. For each run, a master mix, containing all components except DNA, was prepared and aliquoted into separate tubes to ensure that all reactions had the same starting conditions. cDNA dilutions and control dilutions of cloned COX genes were run and analyzed at the same time using the same master mix. Quantitative PCR was performed in 25 µl of 75 mM Tris-HCl (pH 8.8) containing 20 mM (NH4)2SO4, 0.01% Tween 20, 2.5 mM MgCl2, 0.2 mM each dNTP, and 0.4 µM of primers. After the hot start at 95 °C for 5 min, 0.6 units of TaqDNA polymerase (Naxo Ltd.) in 3 µl of buffer was added, and the PCR was programmed as follows: 95 °C for 45 s, 50 °C for 30 s, and 72 °C for 1 min for 30 cycles; and 72 °C for 10 min. The amplification products were visualized on 2% agarose gel using ethidium bromide. Five sets of quantitative PCR were performed independently.

For determination of ratio of COX-A and COX-B in the G. fruticosa genomic DNA, 1–500 ng of genomic DNA and the primer pair FFAQ-up versus FMY-down were used (Table I). PCR was performed in 50 µl of 75 mM Tris-HCl (pH 8.8) containing 20 mM (NH4)2SO4, 0.01% Tween 20, 2.5 mM MgCl2, 0.2 mM each dNTP, and 0.4 µM primers. After the hot start at 95 °C for 5 min, 1.25 units of TaqDNA polymerase (Naxo Ltd.) in 3 µl of buffer was added, and the PCR was run as described for cDNA quantification. The amplification products were visualized on 1.2% agarose gel using ethidium bromide.

Cells and Transfections
The COS-7 cells were maintained in Iscove's modified Dulbecco's medium with 10% fetal bovine serum. Transfections were performed as described previously (31) with slight modifications. 250 µl of cell suspension (1 x 107 cells/ml) was mixed with 400 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. For activity assay, transfected cells were grown at 28 °C for 72 h.

Tunicamycin treatments of COS-7 cells transfected with pCG-COX-A, pCG-COX-B, or pCG-RabCOX-2 constructs were performed by adding tunicamycin (Sigma) to the media at final concentration of 0.3 or 1.0 µg/ml 4 h post-transfection. The cells were grown at 37 °C and harvested at 28 h post-transfection.

Immunoblot Analysis
Transfected cells from 100-mm diameter dishes were lysed in 200 µl of Laemmli sample buffer (39). Proteins were separated by SDS-8% PAGE and transferred to a polyvinylidene difluoride membrane (Millipore Corp.) as described previously (31). Membranes were incubated with anti-E2Tag mouse monoclonal antibody (Quattromed Ltd., Estonia) or rat COX-2-specific mouse monoclonal antibody (Pharmingen) and with secondary horseradish peroxidase-conjugated antibody (LabAs Ltd., Estonia) according to the manufacturer's recommendations. Detection was performed using an ECL detection kit (Amersham Biosciences).

The subcellular localization of recombinant G. fruticosa COX-B in COS-7 cells was determined using rat COX-2-specific monoclonal antibody and immunofluorescence analysis as described previously (31).

Enzyme Assay
Transfected cells from four tissue culture plates (~6 x 106 cells) were treated with 0.3 M EDTA/phosphate-buffered saline, collected by centrifugation at 1000 x g for 10 min, washed with ice-cold phosphate-buffered saline, and centrifuged again. The pellet was resuspended in 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 x g for 1 h to yield the microsomal fraction. The membranes were resuspended by homogenization in a small volume of 50 mM Tris-HCl (pH 8.0) followed by cyclooxygenase assay.

Incubations of the microsomal fraction of transfected cells with [1-14C]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 as described previously (31). 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., Estonia) (31). For the product quantification, the TLC plates were cut into zones and extracted with methanol. The radioactivity was measured with a liquid scintillation counter.

Data Base Search
The deduced protein sequences and genomic structures of the pufferfish Takifugu rubripes and ascidians C. intestinalis and Ciona savignyi COX-related genes were obtained using BLAST searches (tblastn) of Department of Energy Joint Genome Institute (Walnut Creek, CA) and Whitehead Institute (Cambridge, MA) corresponding genome data bases (40–42). Search was based on homology with known COX protein sequences.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of the G. fruticosa COX-B mRNA—The initial information about the G. fruticosa COX-B was obtained from PCR cloning and sequencing of fragments of genomic DNA (see below). COX-B-specific primers were designed, and extension of the 5'- and 3'-ends of COX-B cDNA was achieved using RACE-PCR methodology. The obtained nucleotide sequence consisted of a 147-nucleotide 5'-UTR, a 1788-nucleotide protein coding region, and the 250- or 420-nucleotide 3'-UTR (excluding the poly(A) tail), with a total length of 2358 nucleotides (Table II). The open reading frame encoded a protein of 596 amino acids with a calculated molecular mass of 68.5 kDa.


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  		TABLE II
Comparison of mRNAs of G. fruticosa COX-A and COX-B

 
The deduced amino acid sequence of G. fruticosa COX-B was determined from the coding region of the COX-B transcript, and comparisons were made with G. fruticosa COX-A (Table II and Fig. 1) and other known cyclooxygenases. The enzyme appears to share all the important structural and functional domains implicated in enzyme function. The predicted protein is closely related to the G. fruticosa COX-A (93% identity) and shares 79% identity with P. homomalla COX. Overall comparisons between G. fruticosa COX-B and vertebrate COX-1 and COX-2 revealed about 40–43 and 43–45% of amino acid identity, respectively.



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  		FIG. 1.
Deduced amino acid sequence alignment of G. fruticosa COX-A (GenBankTM accession number AY004222 [GenBank] ) and COX-B (GenBankTM accession number AY480052 [GenBank] ). Asterisks indicate positional differences between two coral isozymes. Putative signal peptide sequences are underlined with dashed lines. Putative membrane-binding helixes are underlined with solid lines. Some of the key residues of substrate binding and catalysis are numbered (ovine COX-1 numbering is used). Putative N-glycosylation sites are boxed.

 
The main difference between G. fruticosa COX-A and COX-B coding regions is an insertion of 21 nucleotides near the 5'-end of COX-B, in the region where the first intron is located in vertebrate COX-1 genes. The 21-nucleotide insert in the COX-B mRNA resulted in a 7-residue insert in the N-terminal signal peptide and affects the predicted cleavage site of the signal peptide (Fig. 1) (37). The proposed signal peptide of COX-B, 35 N-terminal amino acids, includes two conserved cysteines (Cys36 and Cys37 in ovine COX-1 numbering) that are involved in formation of disulfide bonds in the ovine COX-1 and murine COX-2 (20, 21).

The key residues known to be essential for substrate binding and catalysis are well conserved in both G. fruticosa enzymes (Fig. 1). Significant differences can be found, however, in the pattern of potential sites for N-glycosylation (Figs. 1 and 2). G. fruticosa COX-A has four potential N-glycosylation sites. One of them is in a conserved position at Asn144 and two are shifted to positions Asn73 and Asn396. One additional potential N-glycosylation site is found at Asn270a, in the region of the prominent protease-sensitive surface loop containing Arg277, above the peroxidase active site of ovine COX-1 (reviewed in Ref. 24). G. fruticosa COX-B has six potential N-glycosylation sites. Four of them are conserved between COX-A and COX-B, and two additional consensus sites are found at residues Asn193 and Asn278 (Fig. 2).



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  		FIG. 2.
Consensus N-glycosylation sequences of mammalian COX isoforms and coral COX proteins. Sites, experimentally proved to be glycosylated, are in black. Hum, human; G.fru, G. fruticosa; P.hom, P. homomalla.

 
Unlike the protein-coding regions, the sequence identities between the untranslated regions of COX-A and COX-B do not exceed 30% (Fig. 3), suggesting distinct functions in the regulation of expression at the post-transcriptional level. The 3'-RACE procedure with COX-B-specific primers gave two products of different lengths, indicating that alternative polyadenylation of the gene occurs. The 3'-UTR of COX-B contains four polyadenylation sites in positions 211–216, 222–227, 233–238 (AAUAAU), and 397–402 (AAUAAA) (Fig. 3).



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  		FIG. 3.
Comparison of the 3'-untranslated regions of G. fruticosa COX-A and COX-B. nt, nucleotide.

 
The gene of COX-A has two active polyadenylation signals, AAUAAA and AAUAAU, in positions 141–146 and 316–321, respectively, of the 3'-UTR (Fig. 3). Unlike the COX-B, the 3'-UTR of COX-A is AU-rich (28% A; 45% U) and possesses five copies of the sequence AUUUUA (Fig. 3), similar to the Shaw-Kamen's sequence (AUUUA) found in many immediate-early genes (43). Furthermore, there are seven copies of the sequence RUUUUU (where R is A or G), but the functions of these sequences in the post-transcriptional regulation of coral COX mRNA are unknown at present.

Expression of G. fruticosa COX-B in COS-7 Cells—For expression of G. fruticosa COX-B cDNA in COS-7 cells, the open reading frame of COX-B was subcloned into the eukaryotic expression vector pCG (36). To follow the cleavage of the predicted N-terminal signal peptide, the bovine papilloma virus type 1 E2 protein-derived epitope (E2Tag, GVSSTSSDFRDR) (36, 44) was fused in-frame into the N terminus of COX-B (31). The protein expression was visualized by immunoblot analysis using both anti-E2Tag mouse monoclonal antibody (data not shown) and rat COX-2-specific mouse monoclonal antibody as described previously (31). Anti-E2Tag mouse monoclonal antibody was not able to recognize N-terminally fused E2Tag-COX-B (data not shown), but the protein was readily detectable by COX-specific antibody showing two distinct bands, both bigger than recombinant COX-A. The results of immunoblot analysis clearly indicate that G. fruticosa COX-B, like mammalian cyclooxygenases and G. fruticosa COX-A (31), contains an N-terminal signal peptide, which is cleaved to yield the mature protein.

G. fruticosa COX-A has four and COX-B six potential N-glycosylation sites (Fig. 2). To determine how many of these sites are glycosylated, the coral cyclooxygenases were expressed in COS-7 cells in the presence or absence of the N-glycosylation inhibitor, tunicamycin. For comparison, the rabbit COX-2 was used. Cells were harvested 28 h after transfection, lysed, and analyzed by Western blot analyses. Tunicamycin treatment resulted in the expression of distinct 65-kDa proteins for COX-A (Fig. 4, lanes 2 and 3) and COX-B (Fig. 4, lanes 5 and 6), whereas the untreated cells expressed the proteins with higher molecular masses (Fig. 4, lanes 1 and 4). The glycosylated and unglycosylated proteins were estimated to differ from each other by ~6 (COX-A), 7.5, and 9 kDa (COX-B). These results, and comparison with rabbit COX-2 (Fig. 4, lanes 7–9), suggested that all four potential N-glycosylation sites are glycosylated in COX-A, whereas COX-B is fully N-glycosylated at five sites with the sixth potential N-glycosylation site occupied in about 50% of the molecules. Differences in the glycosylation pattern and in the potential cleavage site of the signal peptide did not affect the subcellular localization of recombinant G. fruticosa COX-B in COS-7 cells. G. fruticosa COX-A and COX-B both gave immunofluorescence signals at the endoplasmic reticulum and the nuclear envelope, similar to the location of the rabbit COX-2 reference protein (data not shown).



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  		FIG. 4.
Immunoblot analysis of G. fruticosa COX-A and COX-B and rabbit (Rab) COX-2 expressed in COS-7 cells with and without the presence of tunicamycin. COS-7 cells were transfected with pCG-COX-A, pCG-COX-B, or pCG-RabCOX-2 expression vectors. 0 µg/ml (lanes 1, 4, and 7), 0.3 µg/ml (lanes 2, 5, and 8), or 1.0 µg/ml (lanes 3, 6, and 9) of tunicamycin was added to the media as described under "Materials and Methods." 28 h after transfection, cell extracts were prepared and analyzed by immunoblotting with anti-COX-2 antibody. G. fru, G. fruticosa.

 
For cyclooxygenase assay, the COS-7 cells transfected with pCG-COX-A or pCG-COX-B were grown at 28 °C for 72 h (31). The microsomal fraction of about 6 million cells was prepared and incubated with [1-14C]arachidonic acid. The products were separated by TLC and compared with authentic standards (30, 31). Results showed that both recombinant G. fruticosa cyclooxygenases were able to convert up to 11 µg of [1-14C]arachidonic acid to prostaglandins and other products with a similar pattern and level of activity (Fig. 5) suggesting that differences in their primary structures and post-translational processing do not substantially influence their catalytic ability.



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  		FIG. 5.
Products, formed upon incubation of recombinant G. fruticosa COX-A or COX-B with [1-14C]arachidonic acid. A microsomal fraction of the pCG-COX-A- or pCG-COX-B transfected-COS-7 cells (~6 x 106 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 plates were cut into sections and extracted with methanol. The radioactivity was determined by liquid scintillation counting. AA, arachidonic acid; HETEs, hydroxyeicosatetraenoic acids; PG, prostaglandin; G.fru, G. fruticosa.

 
Relative Quantification of G. fruticosa COX-A and COX-B mRNAs—In order to compare the relative quantities of COX-A and COX-B transcripts in the isolated G. fruticosa mRNA, three different primer pairs were used (details in "Materials and Methods" and Table I). All three sets of primers were equally efficient at amplification of their corresponding targets (Fig. 6a, lower block). The primer pair VAV-up versus DMV-down amplified both cloned genes at substantially the same rate (data not shown). The quantification reactions with 5–10,000 times diluted G. fruticosa cDNA showed only specific bands of the expected size (309 bp for COX-A and 394 bp for COX-B). Band intensity of COX-A and COX-B decreased unequally with increasing template dilution (Fig. 6a, upper block). The mRNA level of COX-A was found to be about 2 orders of magnitude higher than the mRNA level of COX-B.



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  		FIG. 6.
PCR analysis of the relative levels of G. fruticosa COX-A and COX-B in the cDNA (a) and genomic DNA (b). a, 1 µl of 5x (lane 1), 25x (lane 2), 100x (lane 3), 500x (lane 4), 2,500x (lane 5), and 10,000x (lane 6) diluted cDNA (upper block) or 100 fg (lane I), 10 fg (lane II), and 1 fg (lane III) of cloned genes A and B in the pCG-E2Tag (lower block) were amplified using primers specific for the COX-A (ESGA versus DYA) or COX-B (FKG versus TNTS). b, 1–500 ng of genomic DNA was amplified using the primer set specific for both genes, FFAQ versus FMY. The PCR products were analyzed by the agarose gel electrophoresis, and the DNA bands were visualized by ethidium bromide staining. Lane M: 0.5 µg of 100 bp DNA Ladder (MBI Fermentas, Lithuania).

 
Determination of the ratio of COX-A and COX-B in the genomic DNA used similar methodology (Table I). The PCR products of COX-A and COX-B contained intron five (217 bp in the COX-A and 504 bp in the COX-B, Table III) and, accordingly, differed in size (540 and 827 bp for COX-A and COX-B, respectively). The intensities of both bands decreased uniformly with decreasing template concentration, indicating equal levels of both COX genes in the G. fruticosa genomic DNA (Fig. 6b).


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  		TABLE III
Exon/intron junctions of the G. fruticosa COX-A and COX-B Capital letters in the DNA sequences represent nucleotides present in exons. Lowercase letters represent nucleotides present in introns. The numbers in the splice position correspond to the nucleotide sequences downstream from the ATG start codon in the COX cDNA.

 
Genomic Structures of the COX Genes of G. fruticosa, C. intestinalis, and C. savignyi—The genomic organization of invertebrate COX genes was determined by a combination of PCR cloning and data base searches. Amplification of G. fruticosa genomic DNA with different COX-A-specific primer sets resulted, in some cases, in two products. This pointed to the existence of at least two COX-related genes with different intron lengths. The PCR products were cloned, partially or full-length sequenced, and compared with G. fruticosa COX-A coding mRNA. The locations of the introns in the human COX-1 (GenBankTM accession number AF440204 [GenBank] ) (9, 45) and COX-2 genes (EMBL sequence data bank D28235 [GenBank] ) (12, 13) were used as a guide. The entire G. fruticosa COX-A and COX-B genes containing the coding (exons) and noncoding parts (introns and untranslated regions) were constructed from the overlapping fragments obtained by PCR using gene-specific primers. The characterization of the complete G. fruticosa COX-B gene became possible after cloning and sequencing of the 5'- and 3'-untranslated regions of the COX-B mRNA. The coding regions of both coral COX genes matched exactly with the corresponding mRNA sequences.

Blast searches of the genome data bases of the pufferfish T. rubripes v. 3.0 (41), and the sea squirts C. intestinalis v. 1.0 (42) and C. savignyi (Whitehead Institute) using known COX sequences gave three hits for T. rubripes (scaffold 150:17553–20773; scaffold 355:35314–38314; scaffold 800:55592–58932) and two hits for C. intestinalis (scaffold 118:62737–69719; scaffold 207:153086–158222). Two COX-related sequences from C. savignyi were obtained in fragments and combined manually. The deduced amino acid sequences of the G. fruticosa cyclooxygenases were aligned with published mammalian and fish COX isozyme sequences and the COX-related sequences from T. rubripes, C. intestinalis, and C. savignyi. The exon/exon borders were compared with those of human COX-1 and COX-2. The maps constructed by these procedures are shown in Fig. 7. The exon/intron structure of one (designated as Fugu COX-2 in Fig. 8) of three T. rubripes COX genes is identical to that of the human COX-2 gene, except the intron lengths. The other two COX-related genes in the T. rubripes genome data base (Fugu COX-1a and COX-1b in Fig. 8) seem to have an additional intron like vertebrate COX-1 genes. The open reading frame of C. intestinalis COX genes contains 12 exons extending only over 5.8 kb (COX-a) or 4.4 kb (COX-b) (Fig. 7). Both G. fruticosa COX genes are composed of nine exons and eight introns and are about 20.0 (COX-A) and 20.7 kb (COX-B) in length. Intron splice sites and intron phases (the placement of introns with respect to reading frame) of the G. fruticosa COX-A and COX-B are shown in Table III. The structure of the coral and sea squirt COX genes is very similar to that of the vertebrate COX genes, except the G. fruticosa COX genes lack introns one and six (numeration corresponds to the human COX-1 gene), and introns one and seven are absent in all the sea squirt COX genes. The sea squirt COX genes have three additional introns that are not found in any other COX gene (Fig. 7 and Table IV). The exon/intron junctions follow the GT-AG rule (46), and all introns in the coral, sea squirt (except for the three additional introns), pufferfish, and reported COX genes share the same positions and identical phases. Unlike the intron positions, the lengths of the corresponding introns vary widely (Table IV).



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  		FIG. 7.
Structure of human, sea squirt, and coral COX genes. In the upper panel the exons of the Gersemia fruticosa (G. fru) COX-A and COX-B were compared with exons of human COX-1 (GenBankTM accession numbers NM000962 and AF440204 [GenBank] ) and COX-2 (GenBankTM accession numbers NM000963 and D28235 [GenBank] ) and C. intestinalis (C. int.) COX-a and COX-b genes. The numbers in the boxes indicate the number of nucleotides comprising that exon. The filled areas identify the 5'- and 3'-untranslated regions. The lower panel shows the exon/intron structure of human, sea squirt, and coral COX genes, drawn to the same scale. The numbers identify the exons.

 



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  		FIG. 8.
Schematic presentation of intronic variation during the evolution of metazoan COX genes. Loss or gain of introns is marked on the branches of phylogenetic tree that shows the relationship between the deduced amino acid sequences of vertebrate and non-vertebrate cyclooxygenases. Fugu, T. rubripes; C. sav., C. savignyi; C. int., C. intestinalis; G. fru., G. fruticosa.

 


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  		TABLE IV
Intron lengths of eumetazoan COX genes

 
Although the N-terminal and C-terminal portions of the COX-1 and COX-2 proteins and the corresponding regions in the genes (external exons) differ substantially, seven of the eight internal exons of COX-1 and COX-2 are identical in size. Exon three of the vertebrate COX-2 genes (corresponding to exon four of the COX-1 genes) has an additional three nucleotides absent in mammalian COX-1 (17). These three nucleotides are also present in the corresponding exon of the coral and sea squirt COX genes (Fig. 7). Exon six of the coral genes (corresponding to exon eight of the human COX-1 gene) has an additional three nucleotides encoding an asparagine residue (Asn270a) that is absent in other COX proteins. Exons two and eight of the coral genes have three nucleotides less than the corresponding exons in the human COX genes (Fig. 7). Exon five in the coral genes is six nucleotides short, resulting in two missing amino acid residues in the area of the proposed peroxidase active site (amino acid residues in positions 211–226 of ovine COX-1 (20)).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Evolutionary Comparison of COX Protein Sequences—Here we have characterized two COX genes from the coral G. fruticosa, the first non-vertebrate from which a COX cDNA was cloned. Both genes encode functional COX proteins that make prostaglandin endoperoxide as their major product. These two proteins share 93% of deduced amino acid sequence identity. By using a data base search, we detected pairs of COX-related sequences (COX-a and COX-b) from the ascidians C. intestinalis and C. savignyi. These sequences share only 50% intra-species identity; in other words they are slightly more divergent from each other than are vertebrate COX-1 and COX-2. The ascidian COX-a and COX-b show about 70% primary sequence conservation across the two species of Ciona. Other genes typically also have about 70% sequence divergence between the two Ciona species, which are more divergent from each other than are mouse and human.

The phylogenetic tree of COX proteins (Fig. 8) demonstrates that the coral and ascidian cyclooxygenases form distinct arms that diverge from vertebrate ones prior to the divergence of COX-1 and COX-2. This clearly suggests that duplication of an ancestral cyclooxygenase gene to modern vertebrate COX-1 and COX-2 genes occurred within the vertebrate lineage after divergence of the animal kingdom to invertebrates and vertebrates. Cyclooxygenase gene duplication has also occurred independently in the different lower animals we have studied here. The coral genome contains two functional COX genes that are not an equivalent pair to the pair of COX genes in Ciona, the primitive chordates.

As might be expected, the alignment of amino acid sequences of all known COX proteins reveals several residues that are absolutely conserved through evolution. Such residues are Arg120 (substrate binding), Tyr385 (catalytic residue), Ser530, and Val349 (stereo control at C-15) and some other residues located in the fatty acid substrate-binding channel. Also, residues responsible for heme binding and the peroxidase reaction like His207, His388, and Gln203 are conserved in all COX proteins characterized to date. Among multiple N-glycosylation sites, only one, positioned at Asn144, is absolutely conserved. The others are either absent or shifted to other positions.

Evolutionary Comparison of COX Gene Structure—Conservation of the positions of metazoan introns from sponges to human has been under inspection for many genes (47–49). To follow the evolution of metazoan cyclooxygenases, we compared the protein and genomic structures of cyclooxygenases from coelenterates (the most primitive eumetazoa) and ascidians (the most primitive chordate) to fish and mammalian species (Fig. 8). To date there is no definitive evidence for the presence of COX genes in unicellular species, sponge, or insects. Our proposition regarding intron variations during the evolution is that the early metazoan COX possessed nine introns, the positions of which are well conserved. The vertebrate COX-1 genes have one and ascidian COX genes possess three additional introns with unique positions. These introns must have been generated by separate intron gain, which occurred only within the particular lineages. The absence of the sixth intron in ascidian COX genes and the fifth intron in coral COX genes might be explained by a single intron loss in each lineage. Alternatively, the eumetazoan ancestral COX may have only eight introns, so the modern fourth intron of chordate COX genes would have been acquired within the chordate lineage after nonvertebrate/chordate divergence.

The introns in G. fruticosa COX-A and COX-B are, on average, of the same size as the introns in human COX-1. This does not correspond to the trend found by Deutsch and Long in their investigation of the distribution of exon/intron structures in over 2900 genes of 10 eukaryotic model organisms (50). Their analysis of variations in exon/intron structures revealed an overall (weak) correlation of genome size with total intron length per gene; for example, invertebrate introns are smaller than those of human genes, whereas yeast introns are shorter than invertebrate introns (50). The COX genes in ascidians and in pufferfish are short (about 3–5 kb), and here the intron lengths do correlate with the size of the whole genome.

Non-coding Regions of COX mRNAs—In contrast to the very high similarity of the protein coding regions of the G. fruticosa COX isozymes, the sequence identity between the 3'-UTR of COX-A and COX-B is only 27.9%. In comparison, the sequence identity between the 3'-UTR of human COX-1 and COX-2 is 19.5% and for zebrafish isozymes is 27.7% (6). The untranslated regions of genes often contain key regulatory elements involved in gene expression control. On mRNA, translational control mechanisms result from the interaction of RNA-binding proteins with the 5'- or 3'-untranslated regions (51). The 3'-UTR of mammalian COX-2 contains multiple control elements that regulate message stability and message translation, many of which represent novel control elements that lie outside of the first 100 nucleotides of the 3'-UTR (52–54). The major translational control element of COX-2, the first 60 nucleotides of the 3'-UTR, is highly conserved across species, is AU-rich, and contains multiple repeats of the regulatory sequence AUUUA (55–57). The sequence of the 3'-UTR of the COX-1 transcripts is highly divergent from that of COX-2, suggesting a distinct function in the regulation of expression at the post-transcriptional and/or translational levels (10, 58). The 3'-UTR of the G. fruticosa COX-A is more similar to COX-2, being AU-rich (73.8%) and containing many repeats. The AU content of the 3'-UTR of the COX-B is 60.5%. Analysis of the untranslated regions of COX-A and COX-B mRNA by using UTRdb, a specialized data base of 5'- and 3'-untranslated sequences of eukaryotic mRNAs (38, 59), revealed one match for 5'-UTR of COX-B, the 5'-terminal oligopyrimidine tract found in all vertebrate ribosomal protein and translation elongation factors, that is required for coordinate translational repression during growth arrest, differentiation, and development (reviewed in Ref. 60). In the 3'-UTR of COX-A, one copy of a Brd box (AGCUUUA) and two copies of similar sequence (AACUUUA) were found (Fig. 4). The Brd box is present in one or more copies in many of the 3'-UTRs of Notch pathway target genes in Drosophila and mediates negative post-transcriptional regulation by affecting transcript stability and translational efficiency (61).

Relative quantification of the mRNA levels of the G. fruticosa COX-A and COX-B indicated about 100 times higher concentration of the COX-A transcript. This substantial difference in transcriptional level might be explained by differences in the basal constitutive expression throughout the coral tissues and/or by a more cell type-selective expression of the minor COX-B. It is proposed that one of the biological functions of prostaglandins in coral is chemical defense against predators. Perhaps this function is assigned to the major isozyme, COX-A, whereas COX-B produces more modest levels of prostaglandins in a signaling role, as is characteristic of prostaglandins in higher animals. The significant structural differences in the untranslated regions together with the widely different transcriptional levels of the two coral COX genes point to differences in gene regulation and functions of the COX isozymes in lower organisms, but further investigations are needed to elucidate the biological role of each isoform.


   FOOTNOTES
 
* This work was supported by Estonian Science Foundation Grants 5639 (to N. S.) and 5100 (to I. J.) and National Institutes of Health Grant GM-53638 (to A. R. B.). 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. Back

|| To whom correspondence should be addressed: Dept. of Chemistry, Tallinn Technical University, Akadeemia tee 15, Tallinn 12618, Estonia. Tel.: 372-620-4376; Fax: 372-670-3683; E-mail: samel{at}chemnet.ee.

1 The abbreviations used are: COX, cyclooxygenase; RACE, rapid amplification of cDNA ends; UTR, untranslated region. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Alfred L. George, Dr. Juhan Sedman, Dr. Külliki Varvas, and Karin Valmsen for helpful discussions.



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