A novel carbonic anhydrase from the giant clam Tridacna gigas contains two carbonic anhydrase domains William Leggat 1,2 , Ross Dixon 1 , Said Saleh 1 and David Yellowlees 1 1 Biochemistry and Molecular Biology, James Cook University, Townsville, Queensland, Australia 2 Centre for Marine Studies, University of Queensland, Queensland, Australia Carbonic anhydrase (CA; EC 4.2.1.1) catalyses the hydration of CO 2 to HCO 3 – , is ubiquitous amongst all living organisms and fulfils a variety of metabolic roles [1]. Currently there are five evolutionarily distinct CA gene families (a, b, c, d and e) [1,2] and it is generally believed that all animal CAs belong to the a-CA fam- ily. a-CAs are characterized by 36 amino acids found around the active site [3]. Of these, 15 are conserved in all active CAs, suggesting that they are required for CA activity [3]. To date 15 a-CA or a-CA-like proteins have been identified in mammals. These can be divided into five broad subgroups, the cytosolic CAs (CA I, CA II, CA III, CA VII and CA XIII), mitochondrial CAs (CA VA and CA VB), secreted CAs (CA VI), mem- brane-associated CAs (CA IV, CA IX, CA XII and CA XIV) and those without CA activity, the CA-related proteins (CA-RP VIII, X and XI). The cytosolic and mitochondrial CAs and the secreted and membrane-associated CAs are often further Keywords carbonic anhydrase; clam; symbiosis Correspondence W. Leggat, Centre for Marine Studies, University of Queensland, Queensland 4072, Australia Fax: +61 7 33654755 Tel: +61 7 33469576 E-mail: b.leggat@marine.uq.edu.au Notes The nucleotide sequences for carbonic anhydrase from T. gigas have been deposited in the GenBank database under GenBank accession numbers AY790884 and AY799986-AY799998. The alignment for the genomic sequence of tgCA between positions 101 and 1810 of the cDNA has been submitted to EMBL-ALIGN database under the accession number ALIGN_000833. (Received 14 February 2005, revised 11 April 2005, accepted 28 April 2005) doi:10.1111/j.1742-4658.2005.04742.x This report describes the presence of a unique dual domain carbonic anhydrase (CA) in the giant clam, Tridacna gigas. CA plays an important role in the movement of inorganic carbon (C i ) from the surrounding sea- water to the symbiotic algae that are found within the clam’s tissue. One of these isoforms is a glycoprotein which is significantly larger (70 kDa) than any previously reported from animals (generally between 28 and 52 kDa). This a-family CA contains two complete carbonic anhydrase domains within the one protein, accounting for its large size; dual domain CAs have previously only been reported from two algal species. The protein contains a leader sequence, an N-terminal CA domain and a C-terminal CA domain. The two CA domains have relatively little identity at the amino acid level (29%). The genomic sequence spans in excess of 17 kb and con- tains at least 12 introns and 13 exons. A number of these introns are in positions that are only found in the membrane attached ⁄ secreted CAs. This fact, along with phylogenetic analysis, suggests that this protein represents the second example of a membrane attached invertebrate CA and it con- tains a dual domain structure unique amongst all animal CAs characterized to date. Abbreviations CA, carbonic anhydrase; C i , inorganic carbon; GPI, glycosylphosphatidylinositol. FEBS Journal 272 (2005) 3297–3305 ª 2005 FEBS 3297 consolidated into two separate groups based upon sequence similarity [3]. Of the membrane-associated CAs, CA IX, XII and XIV contain integral trans- membrane domains while CA IV is membrane atta- ched through a glycosylphosphatidylinositol (GPI) anchor. Mammalian CAs function in a variety of roles inclu- ding pH balance, H + secretion, HCO 3 – secretion, CO 2 exchange, bone resorption and ion transport [1]. The diversity in mammalian genes and the presence of homologs in other animals suggests that a large num- ber of CAs are yet to be characterized from the animal kingdom. Complete cDNA sequences from inverte- brate sources have only been obtained from the tube- worm Riftia pachyptila [4], cnidarians [5] and mosquitoes [6,7]. In addition a cDNA sequence enco- ding a protein involved in calcification from the pearl oyster Pinctada fucata contains an a-CA-like domain [8]. All animal CAs purified to date have subunit molecular weights less than 58 kDa. The only excep- tion is a report of the purification of a protein display- ing CA activity from the giant clam Tridacna gigas, where the purified protein has a molecular weight of 70 kDa [9]. This protein is glycosylated through both N- and O-links and is thought to be involved in the transport of inorganic carbon (C i ) from seawater to symbiotic photosynthetic dinoflagellates that are found intercellularly within the clam tissue [9,10]. It has been demonstrated that these alga can supply up to 100% of the clam’s energy requirements [11]. In addition T. gigas contains two other CA isoforms, one of 32 kDa and one of approximately 200 kDa [9,10]. Here we present further characterization of the 70 kDa CA isoform from T. gigas, including cDNA sequence indicating that it codes for a unique animal CA containing two putative CA catalytic domains within the one protein. Each domain contains those residues thought to be essential for CA activity. Results tgCA cDNA sequence, deduced amino acid sequence and domains Only cDNA sequence for the first 1438 bp of the 70 kDa CA could be obtained from both the gill and mantle cDNA libraries; both sequences were identical. In both cases the sequence terminated in an identical position (1438 bp after the start codon), suggesting that the mRNA secondary structure prevented complete sec- ond strand synthesis. For this reason cDNA sequence was also obtained using RT-PCR using a higher than normal temperature for second strand synthesis. Using RT-PCR the 3¢ end of the cDNA was obtained yielding an open reading frame of 1803 bp encoding 600 amino acids and a protein of 66.7 kDa (Fig. 1). This is flanked by a 58 bp 5¢-UTR and an 89 bp 3¢-UTR. Although no polyadenylation signal was found a poly A tail was sequenced. The translated protein sequence (tgCA) was found to contain three domains, based upon database searches, a signal sequence (Met1–Ala17) and two domains with homology to the a-CA family, n-tgCA (Ala18–Thr289) and c-tgCA (Ala290–Ser600) (Fig. 2). The predicted cleavage point of the signal sequence, between Ala17 and Ala18 (Centre for Biological Sequence Analysis Database), produces a mature N-terminal amino acid sequence almost identical (21 out of 22 residues) to an N-terminal peptide sequence previously obtained [9], suggesting that this is the cor- rect cleavage point for the signal sequence. A potential GPI-modification site was identified at Gly577 by the DPGI database. Five consensus sites for N-glycosylation (NXS or NXT) were found in the deduced amino acid sequence at positions Asn66, Asn97, Asn177, Asn421 and Asn452. Phylogenetic comparison of both CA domains with a number of characterized human CA isoforms and representative invertebrate CAs shows the clear group- ing of the three recognized a-CA groups, the cytosolic, Fig. 1. Translated protein sequence of tgCA from T. gigas. The first CA domain (n-tgCA) begins at Ala18, the second CA domain (c-tgCA) begins at Ala290 (bold). The signal sequence is highlighted and the five poten- tial glycosylation sites (three in n-tgCA and 2 in c-tgCA) are underlined. Gly577 (double underline) is the predicted GPI-anchor site. A dual domain carbonic anhydrase W. Leggat et al. 3298 FEBS Journal 272 (2005) 3297–3305 ª 2005 FEBS secreted ⁄ membrane-associated and the CA-related pro- teins (Fig. 3). Both domains of the clam CA group with the membrane-associated CAs. Intron ⁄ exon mapping There are a number of intron ⁄ exon locations that are specific for the various CA classes [3,12]. With this in mind the introns for tgCA were mapped to further characterize the two CA domains. The genomic sequence of tgCA, between positions 101 and 1810 of the cDNA, was amplified in a number of PCR reac- tions spanning in excess of 17 kb. These sequences included the complete coding sequence for the gene between positions 101 and 1810. Twelve introns and 13 exons were found in this region, five in n-tgCA, six Fig. 2. Alignment of n-tgCA and c-tgCA with other CAs showing intron position and conserved motifs. The 15 amino acids thought to be required for CA activity are indicated (#), while cysteine residues involved in disulfide bonding in CA IV (two disulfide bonds) and CA VI, XII, XIV (one disulfide bond) are indicated (Ù) above the alignment. Numbers below the alignment indicate the intron number while intron posi- tions are represented by: ( ⁄ ) intron between amino acids, (\) intron located after the first codon position of the following amino acid, (+) intron located after the second codon position of the following amino acid, and (*) represents no intron present. Residues shared by more than 50% of the CAs examined are shaded. The alignment was performed using CLUSTALW. Note that hCA1 contains an alanine at position 122 rather then the conserved Val122, however, the consensus for the CA-1 isoform from vertebrates is valine [3]. hCA, human CA; NCBI accession numbers in brackets, hCA1 (NM_001738), hCA2 (NM_000067), hCA3 (NM_005181), hCA4 (NM_000717), hCA5 (NM_001739), hCA6 (NM_001215), hCA7 (NM_005182), hCA8 (NM_004056), hCA12 (NP_001209), hCA14 (NP_036245). W. Leggat et al. A dual domain carbonic anhydrase FEBS Journal 272 (2005) 3297–3305 ª 2005 FEBS 3299 in c-tgCA and one between the two domains (Fig. 2). Unfortunately, despite repeated attempts it was not possible to obtain genomic sequence corresponding to the cDNA sequence prior to position 101 of the cDNA sequence, this may have been due to the presence of an extremely large intron. Alignment of the intron positions with those of the human CAs shows that both n-tgCA and c-tgCA share the majority of intron locations with the secreted ⁄ membrane-associated CAs (Fig. 2). All introns conformed to the gt-ag rule [13] (Table 1). In three cases (introns 2, 7 and 11) multiple sequences were obtained for introns, suggesting that tgCA is a multiple copy gene, this was confirmed by the Southern analysis (data not shown). Only one base pair of the genomic sequence differed to that previ- ously obtained for the cDNA sequence (1586CfiT). Of the  15.7 kb of intron in the gene, sequence data was obtained for  9.8 kb. The GC content of the coding sequence (44.5%) was significantly higher than that of the introns (36.0%). In addition microsatellite repeat sequences were found in exon 5 (CAAA, 21 repeats) and exon 7 (GTTT, 13 repeats) (data not shown). tgCA subunit size In addition to the 70 kDa CA, another protein of 200 kDa with characteristics of CA was also identified [9]. Although a minor component of the purified CA fraction this protein had an identical N-terminal amino acid sequence to the 70 kDa isoform [9]. When gel purified and separated on an 8% SDS ⁄ PAGE gel it was found that the apparent molecular mass of this Fig. 3. Phylogeny of the CA domains of tgCA (n-tgCA and c-tgCA) with representa- tives of other CA classes using maximum likelihood. Alignments were performed using CLUSTALW, bootstrapped 1000 times and the trees constructed using maximum likelihood. hCA, human CA; ce, Caenorhabditis elegans;Dr,Drosophila melanogaster;ae, Anthopleura elegantissma; CAH1 from the alga Clamydomonas reinhardtii was used as an outgroup. Table 1. Exon ⁄ intron junctions of tgCA. No genomic sequence was obtained before the position corresponding to base pair 101 in the cDNA sequence. Numbering of the cDNA sequence begins at the first codon position of the first in-frame methionine. The exact intron size is given where known; estimates were made from the size of PCR products. Uppercase letters indicate coding sequence, while lowercase indicate intron sequence. Intron cDNA codon position preceding intron (bp) Intron size (bp) 5¢ Splice donor 3¢ Splice acceptor 1265  1040 CACGG gtaaac ttccag TGGTA 2417  956 a TTGAG gtaggt ttgtag GTACA 3510  1200 TTGAG gtgggt ttctag ATCGA 4583  1830 TAAAA gttagt ttctag ATGGA 5744  2280 CTCAG gtatat tttcag CTTGC 6868  2000 TACAG gttggg ttacag CTCAA 7910  1380 a TCAAG gtatgt ttacag GAGTG 8 1093  950 TACAA gtactt ctacag TCCAA 9 1242  1100 TCGAG gtactg tttcag CTACA 10 1335  1370 TTGAA gtaagt tttcag ATCGG 11 1399  1300,  1200 a TAAAG gtatgt tttcag ATGCC 12 1581 347 GTCAG gtaagt ttccag TTGGT a Two intron sequences were found for these introns. A dual domain carbonic anhydrase W. Leggat et al. 3300 FEBS Journal 272 (2005) 3297–3305 ª 2005 FEBS protein is approximately 145 kDa, as opposed to 200 kDa suggested in [9]. In addition a protein of 70 kDa was also observed (Fig. 4A). When purified CA containing the 32, 70 and 145 kDa isoform was separated in the presence of increasing concentrations of the reducing agent 2-mercaptoethanol, it was found that the 145 kDa isoform disappeared (Fig. 4B). These two observations, the presence of the 70 kDa isoform in gel purified 145 kDa extract, and the disappearance of the 145 kDa isoform under reducing conditions, in addition to the identical N-terminal acid sequences [9] suggests that the 70 kDa forms a homodimer of  145 kDa. Separation of affinity purified CA by 2D-PAGE shows that both the 32 and 70 kDa CAs have multiple isoforms with identical masses but differing pI values between 4 and 4.5 for the 32 kDa isoform and between 5.2 and 6.0 for the 70 kDa isoform (Fig. 5). The pre- dicted pI point of the mature protein derived from the cDNA sequence is 5.84. Discussion The data presented here represents the first example of an animal protein containing two CA catalytic domains within the one coding sequence. Only two other pro- teins have been found to contain transcripts containing duplicate CA domains which are translated into the one peptide, one from the green alga Dunaliella salina contains two a-CA domains while the second from the red alga Porphyridium purpureum contains two b-CA repeats. tgCA contains a 17 amino acid leader sequence, an N-terminal CA domain of 272 amino acids and a C-terminal domain of 311 amino acids. The cDNA encodes a protein of 66.7 kDa, when the leader sequence is removed the mature molecular mass is reduced to 64.8 kDa, this is similar to the 62 kDa molecular weight obtained for the deglycosylated pro- tein determined by SDS ⁄ PAGE [9]. Both CA domains of tgCA contain all residues thought to be required for CA activity [3] suggesting that both domains are cata- lytically active. In addition, both domains contain a histidine residue (His87 in n-tgCA, His363 in c-tgCA) conserved with His64 in human CA2 (Fig. 2). This his- tidine residue has been found to act as a proton shuttle in CO 2 hydration in high activity CAs (reviewed in [14,15]) supporting the notion that both CA domains within this protein are catalytically functional. How- ever this will have to be confirmed experimentally through either mutational studies or the use of select- ive inhibitors. The predicted cleavage position of the leader sequence between Ala17 and Ala18 produces a mature N-terminal protein sequence which is identical for the first 21 amino acids to that obtained from N-terminal sequencing of the purified protein. The putative signal sequence for tgCA contains two potential in-frame start codons (Met1 and Met3), similar to that found in the membrane-associated human CA4 (Fig. 2). The presence of this signal sequence is consistent with pre- vious localization studies that have indicated extracel- lular localization of this protein in both the mantle and gills of giant clams [9,10]. Previous studies on the purified tgCA protein [9] and immunolocalization results [9,10] suggest that tgCA is a membrane CA, although its mode of attach- ment was not clear. It was also found [9] that AB Fig. 4. SDS ⁄ PAGE separation of the various 70 kDa CA isoforms. (A) An 8% SDS ⁄ PAGE gel showing the presence of both the 145 kDa and 70 kDa CA isoforms in a purified extract of the 145 kDa isoform. Lane 1, purified 145 kDa CA isoform; lane 2, puri- fied 70 kDa isoform. (B) Purified gill CA separated by SDS ⁄ PAGE in the presence of increasing concentrations of 2-mercaptoethanol. Lane 1, 0.25 M 2-mercaptoethanol; lane 2, 1.3 M 2-mercaptoetha- nol; lane 3. 5.3 M 2-mercaptoethanol. Fig. 5. Two-dimensional electrophoresis separation of affinity puri- fied T. gigas CA. pI values are shown across the top while molecular masses are shown on the right. Box 1 surrounds the 70 kDa isoform while box 2 surrounds the 32 kDa isoform. W. Leggat et al. A dual domain carbonic anhydrase FEBS Journal 272 (2005) 3297–3305 ª 2005 FEBS 3301 phosphoinositol phospholipase C digestion did not result in the release of tgCA from crude gill homogen- ate, suggesting that it is not GPI-anchored and is instead an integral membrane protein. However analy- sis of the predicted protein sequence presented here does not seem to support this hypothesis, as evidence of a putative transmembrane domain region is ambigu- ous (data not shown). In addition the DPGI database predicts that a GPI anchor may be present at Gly577 (it must be stated that the big-PI Predictor did not identify a GPI-anchor for this protein). How tgCA is associated with the membrane is still unknown and requires further research, although the lack of a hydro- phobic domain suggests that, as with human CA4, it is attached through a GPI-anchor. Perhaps surprisingly there is very little identity between the two CA domains of this protein at either the coding (48%) or amino acid level (29%). This level of identity is similar to that seen when comparing the different domains of tgCA to human CAs. Of the ver- tebrate and invertebrate CAs used for the tree con- struction (Fig. 3), n-tgCA had the greatest identity at the amino acid level with human CA2 and human CA7 (32% for both) while c-tgCA was most similar to human CA1 and human CA7 (28% identity for both). The lowest identity was with the alga C. reinhardtii CAH1 gene (13% n-tgCA and 16% c-tgCA). Despite this greater identity with human cytosolic CAs, phylogenetic analysis suggests that both the N- and C-terminal domains belong to the secreted ⁄ membrane-associated CA group (Fig. 3). Despite low bootstrap values for the tree in general, the support for the division between the cytosolic and secre- ted ⁄ membrane-associated CAs is high (80%). This tree clearly groups the human cytosolic CAs, the CA-like proteins and secreted ⁄ membrane-associated CAs. Of the invertebrate CAs, both the fly and anemone fall within the cytosolic group while the two putative Caenorhabditis elegans CAs (CAH1 and CAH2) group with the CA-RP vertebrate genes. To our knowledge there is only one published report of a cDNA coding for an invertebrate membrane attached CA [7], indica- ting that tgCA represents the second example of a membrane-associated CA from the invertebrates. The phylogenetic grouping of tgCA with the mem- brane-associated CAs from vertebrates is supported by a range of other properties of this protein including the presence of a signal sequence and the presence of a conserved disulfide bond. All CAs of this group have been found to contain two conserved cysteine residues involved in an intrachain disulfide bond (Fig. 2). The tgCA sequence is no exception with each CA domain containing two conserved cysteine residues (Cys41, Cys229, Cys315, Cys508) that are homologous to those found in all other CAs of this group (Fig. 2). The pres- ence of disulfide bonds in tgCA is supported by chan- ges in electrophoretic mobility when the purified protein is subjected to varying levels of reducing agent. In the presence of higher concentrations of 2-merca- ptoethanol, the 70 kDa isoform migrates more slowly (Fig. 4A). This is consistent with human CA4 which shows a similar pattern in the presence of 5% 2-merca- ptoethanol [16]. While four of the six cysteines in tgCA are implica- ted in intrachain disulfide bonds, evidence suggests that at least one of the remaining two cysteines forms a disulfide bond with another tgCA subunit making a dimer of the 70 kDa protein. Gel filtration experiments had previously suggested that tgCA exists as a dimer, with a native weight of approximately 141 kDa [9]. When a gel purified fraction of similar estimated mole- cular mass (145 kDa) is separated by SDS ⁄ PAGE, the 70 kDa band is found in addition to the original 145 kDa protein. Upon addition of high concentra- tions of the reducing agent 2-mercaptoethanol this band ( 145 kDa) disappears (Fig. 4B) supporting the conclusion that this represents a dimer. The gel filtra- tion results in combination with reduction of the 145 kDa protein to the 70 kDa isoform suggest that there are interchain disulfide bonds between two 70 kDa subunits. Analysis of the genomic sequence data, where differ- ent intron sequences have been obtained (Table 1), Southern blots (data not shown) and protein two- dimensional gels all suggest that tgCA is a multicopy gene. Despite this no sequence differences were observed in the coding sequence or intron position where different copies, evidenced by different intron sequences, were obtained. Given this it seems reason- able then to use the combined genomic data, even if it does not represent one gene, for analysis of intron ⁄ exon structure. Intron ⁄ exon positions are considered diagnostic for animal CAs with characteristic pattern differences being found in cytosolic and secreted ⁄ membrane-asso- ciated CAs [3,12]. For example of the 15 possible intron sites shown in Fig. 2, three introns are shared between these two groups, three are found only in the cytosolic CAs and at least two more are found only in the secreted ⁄ membrane-associated CAs. Genomic sequence of tgCA revealed 12 introns and 13 exons (Fig. 2), all of which conformed to the gt ⁄ ag rule for splice junctions [13] (Table 1). The majority of these introns (11) were found to be homologous to those in the secreted ⁄ membrane-associated CAs (intron posi- tions 5, 9 and 13 of all possible introns, Fig. 2) or A dual domain carbonic anhydrase W. Leggat et al. 3302 FEBS Journal 272 (2005) 3297–3305 ª 2005 FEBS those introns common to the majority of vertebrate CAs (introns 7, 8, 11). This distribution of intron ⁄ exon boundaries supports the phylogeny and protein prop- erties discussed above that groups both CA domains of tgCA with the secreted ⁄ membrane attached CAs. However, surprisingly one intron in the c-tgCA was found to differ from this pattern. Intron 3 in c-tgCA (Fig. 2) is diagnostic for the cytosolic CAs. The pres- ence of a cytosolic specific intron in a CA that would otherwise belong to the secreted ⁄ membrane attached CA grouping suggests that this intron was present before the division of these two groups and has subse- quently been lost from the membrane-associated CAs. The dual domain structure of tgCA could have arisen through one of two mechanisms, either the fusion of two separate CA genes or a duplication of a single gene followed by a fusion event. If this protein arose through duplication and fusion event, and given the poor iden- tity between the two CA domains (29%), the duplication event must be old, thereby allowing time for the two domains to diverge. This low identity between domains is especially striking when compared to other duplicated domain CA proteins, 52 and 72% identity for D. salina and P. purpureum, respectively [17,18]. Furthermore there are similar examples of non-CA duplicated domain proteins from invertebrates. Phosphagen kinases from a number of bivalves [19–21], sea anemones [22] and sea urchins [23] have been shown to contain bi- or tripartite repeat domains. In all of these examples, identity between the domains is in excess of 60%. For each pro- tein it has been concluded that the dual domain struc- ture arose through gene duplication of one gene and subsequent fusion. Where genomic sequence is available [20,22] this is supported by the presence of an intron between the two domains. Similarly in tgCA an intron is found between the two domains. Given the low homol- ogy between the two domains of tgCA in comparison to other duplicated domain proteins it is not possible to exclude the possibility that this protein has arisen through the fusion of two different CA genes rather than a duplication event. Given the unique structure of this CA protein it would be interesting to know if both domains display CA activity. Previous studies [9] have shown that the purified protein is active, however, from this data it is not possible to conclude if this is due to one or both domains. As both domains contain all the required residues it seems likely that they are both active. A fur- ther area of study is the interaction of the two domains, for example are both required for activity and ⁄ or do they function cooperatively and what is their three-dimensional arrangement? These questions are areas of future study. To date the dual domain structure of tgCA is unique amongst animals, whether this gene duplication of CA is present in other symbiotic or nonsymbiotic bivalves, and possibly other invertebrates, remains to be seen. If this CA arrangement is restricted to symbiotic bivalves it may represent a mechanism by which a symbiotic animal can increase the rate of inorganic carbon trans- port to their photosynthetic symbionts, and thereby maximize the benefits of symbiosis. Experimental procedures Purification of carbonic anhydrase from clam gills The 70 kDa CA isoform was purified from the gills of the giant clam T. gigas as previously described [9]. The 145 kDa CA isoform was electroeluted from affinity puri- fied CA after separation by SDS ⁄ PAGE. Separation of CA isoforms by two-dimensional gel electrophoresis Affinity purified CA was analyzed by two-dimensional gel electrophoresis (2D-PAGE). Separation in the first dimen- sion was performed using an Immobiline DryStrip (pH 4–7, Pharmacia, Piscataway, NJ, USA) which was then further separated on an 8–18% SDS ⁄ PAGE gradient gel using the manufacturer’s protocol (Pharmacia, Cat # 18-1038-63). Gels were visualized using Sypro-Ruby (Molecular Probes, Eugene, OR, USA). Purification of RNA and cDNA library construction Total RNA was prepared from T. gigas mantle and gill tissue. Fresh tissue (1.3 g) was snap frozen in liquid nitro- gen and ground in a mortar and pestle. Total RNA was prepared from the tissue using cesium chloride [24]. mRNA was then purified from total RNA using the QuickPrepÒ mRNA Purification Kit (Pharmacia). The gill cDNA was synthesized for rapid amplification of cDNA ends by the polymerase chain reaction (RACE-PCR) using the Clontech TM cDNA Amplification Kit. The mantle lib- rary was initially synthesized as a phage library in k-ZapII (Stratagene, La Jolla, CA, USA). It was used as a template for RACE-PCR using specific primers for the adaptors. Clam CA primers were designed to previously known cDNA sequence of the 70 kDa CA isoform from T. gigas [25] and to N-terminal amino acid sequence [9] (Fig. 1). From the derived sequence further primers were designed to amplify the remaining portion of the cDNA. Products were also amplified using RT-PCR. mRNA was purified as previously described and first strand synthesis performed using Omniscript TM Reverse Transcriptase W. Leggat et al. A dual domain carbonic anhydrase FEBS Journal 272 (2005) 3297–3305 ª 2005 FEBS 3303 (Qiagen, Valencia, CA, USA) using the following primer: 5¢-CCAgTgAgCAg AgTgACggAggACTCgAgCTCA AgCTT TTTTTTTTTTTTTT-3¢. PCR products were then amplified using a gene specific primer and Q 0 (5¢-CCAgTgAgCAgAg TgACg-3¢) whose sequence was contained in the poly(T) primer. The second-strand synthesis was conducted at 38 °C rather than 16 °C to overcome problems associated with sec- ondary structure inhibition of second-strand synthesis which had previously been observed. DNA sequencing Gel purified PCR products (High Pure PCR Product Purifi- cation Kit, Roche, Mannheim, Germany) or 1.5 lL of the PCR reaction were ligated into T-Vector Easy (Promega, USA) and transformed into XL-1 Blue cells. After plasmid purification (High Purity Plasmid Isolation Kit, Roche) clones were sequenced using capillary separation on a ABI 3730xl sequencer using the ABI v3.0 sequencing kit (Applied Biosystems, Foster City, CA, USA). Sequence analysis Sequence alignments were performed using the program clustal w [26], bootstrapped 1000 times and trees con- structed using maximum likelihood [27], the C. reinhardtii a-CA gene (CAH1) was used as an outgroup. All analyses were performed using biomanager by ANGIS (http:// www.angis.org.au). Signal sequences were identified using the Centre for Biological Sequence Analysis database [28]. Potential GPI-anchor sites were examined using the DPGI database (http://129.194.185.165/dgpi/DGPI_demo_en.html) and the big-PI Predictor (http://mendel.imp.univie.ac.at/gpi/ gpi_server.html) [29]. Isolation of genomic DNA Genomic DNA was isolated from T. gigas sperm. Spawning was induced by the injection of approximately 5 mL of a 2mm serotonin solution into the gonads. The sperm was collected from the water and centrifuged (1000 g for 5 min). Sperm (1 mL packed cell volume) was diluted with 11 mL proteinase K solution [50 mm Tris ⁄ HCl pH 7.5, 20 mm EDTA, 100 mm NaCl, 1% (w ⁄ v) SDS, 100 lgÆmL )1 proteinase K] and the sample was incubated overnight at 55 °C. The solution was centrifuged (1000 g for 15 min at 4 °C), the supernatant removed, mixed with 10 mL of ultra-pure phenol (Sigma, St Louis, MO, USA) and then equilibrated with 4 mL of TE (10 mm Tris pH 8.0, 1 mm EDTA) buffer. After adding an equal volume of chloro- form, the solution was left overnight. The solution was again centrifuged (1000 g for 15 min at 4 °C) and the aque- ous phase removed. This was re-extracted twice with chlo- roform and the DNA precipitated with 0.1 volume sodium acetate (3 m, pH 5.2) and 2.5 volume 100% (v ⁄ v) ethanol. After precipitation the DNA was spooled and resuspended in TE buffer. Genomic sequencing The intron ⁄ exon structure of the 70 kDa CA was mapped using a series of sequence specific primers obtained from the cDNA sequence that bracketed possible intron positions [3]. Acknowledgements This work was supported by an Australian Research Council grant to David Yellowlees. We would like to thank three anonymous referees for their helpful com- ments. References 1 Pastorekova S, Parkkila S, Pastorek J & Supuran CT (2004) Carbonic anhydrases: current state of the art, therapeutic applications and future prospects. J Enz Inhib Med Chem 19, 199–229. 2 So AKC, Espie GS, Williams EB, Shively JM, Hein- horst S & Cannon GC (2004) A novel evolutionary line- age of carbonic anhydrase e is a component of the carboxysome shell. J Bacteriol 186, 623–630. 3 Hewett-Emmett D & Tashian RE (1996) Functional diversity, conservation, and convergence in the evolu- tion of the a-, b-, and c-carbonic anhydrase gene famil- ies. Mol Phylo Evol 5, 50–77. 4 De Cian M, Bailly X, Morales J, Strub J, van Doressel- aer A & Lallier FH (2003) Characterization of carbonic anhydrases from Riftia pachyptila, a symbiotic inverte- brate from deep-sea hydrothermal vents. Proteins Struct Func Genet 51, 327–339. 5 Weis VM & Reynolds WS (1999) Carbonic anhydrase expression and synthesis in the sea anemone Anthopleura elegantissima are enhanced by the presence of dinoflagel- late symbionts. Physiol Biochem Zool 72, 307–316. 6 Pilar Corena M, Seron TJ, Lehman HK, Ochrietor JD, Kohn A, Tu C & Linser PJ (2002) Carbonic anhydrase in the midgut of larval Aedes aegypti: cloning, localiza- tion and inhibition. J Exp Biol 205, 591–602. 7 Seron TJ, Hill J & Linser PJ (2004) A GPI-linked car- bonic anhydrase expressed in the larval mosquito mid- gut. J Exp Biol 207, 4559–4572. 8 Miyamoto H, Miyashita T, Okushima M, Nakano S, Morita T & Matsushiro A (1996) A carbonic anhydrase from the nacreous layer in oyster pearls. Proc Natl Acad Sci USA 93, 9657–9660. 9 Baillie B & Yellowlees D (1998) Characterization and function of carbonic anhydrase in the zooxanthellae- giant clam symbiosis. Proc R Soc Lond B 265, 465–473. A dual domain carbonic anhydrase W. Leggat et al. 3304 FEBS Journal 272 (2005) 3297–3305 ª 2005 FEBS 10 Leggat W, Marendy EM, Baillie B, Whitney SM, Ludwig M, Badger MR & Yellowlees D (2002) Dinofla- gellate symbioses: strategies and adaptations for the acquisition and fixation of inorganic carbon. Funct Plant Biol 29, 309–322. 11 Fisher CR, Fitt WK & Trench RK (1985) Photosynth- esis and respiration in Tridacna gigas as a function of irradiance and size. Biol Bull 169, 230–245. 12 Jiang W & Gupta D (1999) Structure of the carbonic anhydrase VI (CA6) gene: evidence for two distinct groups within the a-CA gene family. Biochem J 344, 385–390. 13 Mount SM (1982) A catalog of splice junction sequences. Nucleic Acids Res 10, 459–472. 14 Lindskog S (1997) Structure and mechanism of carbonic anhydrase. Pharmacol Ther 74, 1–20. 15 Supuran CT, Scozzafava A & Casini A (2003) Carbonic anhydrase inhibitors. Med Res Rev 23, 146–189. 16 Zhu XL & Sly WS (1990) Carbonic anhydrase IV from human lung: purification, characterization, and compar- ison with membrane carbonic anhydrase from human kidney. J Biol Chem 265, 8795–8801. 17 Fisher M, Gokhman I, Pick U & Zamir A (1996) A salt-resistant plasma membrane carbonic anhydrase is induced by salt in Dunaliella salina. J Biol Chem 271, 17718–17723. 18 Mitsuhashi S & Miyachi S (1996) Amino acid sequence homology between N- and C-terminal halves of a carbo- nic anhydrase in Porphyridium purpureum, as deduced from a cloned cDNA. J Biol Chem 271, 28703–28709. 19 Compaan DM & Ellington WR (2003) Functional con- sequences of a gene duplication and fusion event in an arginine kinase. J Exp Biol 206, 1545–1556. 20 Suzuki T, Kawasaki Y, Unemi Y, Nishimura Y, Soga T, Kamidochi M, Yazawa Y & Furukohri T (1998) Gene duplication and fusion have occurred frequently in the evolution of phosphagen kinases – a two-domain arginine kinase from the clam Pseudocardium sachalinen- sis. Biochim Biophys Acta 1388, 253–259. 21 Suzuki T, Sugimura N, Taniguchi T, Unemi Y, Murata T, Hayashida M, Yokouchi K, Uda K & Furukohri T (2002) Two-domain arginine kinases from the clams Solen strictus and Corbicula japonica: exceptional amino acid replacement of the functionally important D62 by G. Int J Biochem Cell Biol 34, 1221–1229. 22 Suzuki T, Kawasaki Y & Furukohri T (1997) Evolution of phosphagen kinase: Isolation, characterization and cDNA-derived amino acid sequence of two-domain arginine kinase from the sea anemone Anthopleura japo- nicus. Biochem J 328, 301–306. 23 Wothe DD, Charbonneau H & Shapiro BM (1990) The phosphocreatine shuttle of sea urchin sperm: flagellar creatine kinase resulted from a gene triplication. Proc Natl Acad Sci USA 87, 5203–5207. 24 Sambrook J, Fritsch EF & Manniatis T (1989) Mole- cular Cloning, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbour, NY. 25 Baillie BK, (1995) Inorganic carbon acquisition and utili- sation in the giant clam symbiosis. PhD Thesis, James Cook University of North Queensland, Australia. 26 Thompson JD, Higgins DG & Gibson TJ (1994) CLUS- TAL W: improving the sensitivity of progressive multi- ple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680. 27 Felsenstein J (1989) P HYLIP – Phylogeny Inference Pack- age Version 3.2. Cladistics, 5, 164–166. 28 Bendtsten JD, Nielsen H, von Heijne G & Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340, 783–795. 29 Eisenhaber B, Bork P & Eisenhaber F (1999) Prediction of potential GPI-modification sites in proprotein sequences. J Mol Biol 292, 741–758. W. Leggat et al. A dual domain carbonic anhydrase FEBS Journal 272 (2005) 3297–3305 ª 2005 FEBS 3305 . A novel carbonic anhydrase from the giant clam Tridacna gigas contains two carbonic anhydrase domains William Leggat 1,2 , Ross Dixon 1 , Said Saleh 1 and David Yellowlees 1 1 Biochemistry and. 2000 TACAG gttggg ttacag CTCAA 7910  1380 a TCAAG gtatgt ttacag GAGTG 8 1093  950 TACAA gtactt ctacag TCCAA 9 1242  1100 TCGAG gtactg tttcag CTACA 10 1335  1370 TTGAA gtaagt tttcag ATCGG 11. describes the presence of a unique dual domain carbonic anhydrase (CA) in the giant clam, Tridacna gigas. CA plays an important role in the movement of inorganic carbon (C i ) from the surrounding sea- water