A New Coral Carbonic Anhydrase in Stylophora pistillata - Springer Link

Mar Biotechnol (2011) 13:992–1002 DOI 10.1007/s10126-011-9363-x

ORIGINAL ARTICLE

A New Coral Carbonic Anhydrase in Stylophora pistillata Anthony Bertucci & Sylvie Tambutté & Claudiu T. Supuran & Denis Allemand & Didier Zoccola

Received: 13 July 2010 / Accepted: 10 January 2011 / Published online: 12 February 2011 # Springer Science+Business Media, LLC 2011

Abstract Scleractinian corals are of particular interest due to their ability to establish an intracellular mutualistic symbiosis with phototrophic dinoflagellates and to deposit high rates of calcium carbonate in their skeleton. Carbonic anhydrases have been shown to play a crucial role in both processes. In this study, we report the molecular cloning and characterization of a novel α-CA in the coral Stylophora pistillata. This enzyme shares homologies with the human isoform CA II and is referred to as STPCA-2. STPCA-2 is 35.2 kDa and possesses all key amino acids for catalytic activity. With a ratio between catalytic and Michaelis constants (kcat/Km) of 8.3.107 M−1 s−1 is considered as highly active. Owing to its intracellular localisation in the oral endoderm and in the aboral tissue, we propose that STPCA-2 is involved in pH regulation and/or inorganic carbon delivery to symbiont and calcification. Keywords Scleractinian corals . Carbonic anhydrase . Symbiosis . Calcification . Intracelullar pH

Introduction Carbonic anhydrases (CA, EC 4.2.1.1) are ubiquitous metalloenzymes that catalyse the reversible hydration of carbon dioxide into bicarbonate (CO2 +H2O⇆HCO3− +H+). A. Bertucci : S. Tambutté : D. Allemand : D. Zoccola (*) Centre Scientifique de Monaco, Avenue Saint Martin, 98000 Monaco, Monaco e-mail: [email protected] C. T. Supuran Dipartimento di Chimica, University of Florence, via della Lastruccia, 3, Rm. 188, Polo Scientifico, 50019 Sesto Fiorentino (Florence), Italy

The CA family is polyphyletic, with at least five classes: α, β, γ, ∂ and ζ. They are involved in numerous physiological processes from invertebrates to vertebrates like, for example, respiration with CO2/HCO3− transport and exchange between tissues and lungs, pH regulation, electrolyte secretion, calcification and also pathologic processes (for a review, see Supuran 2008 and Xu et al. 2008). Scleractinian corals have been of particular interest for research over the past two decades owing to their fundamental role in reef formation and maintenance of this highly diverse ecosystem in oligotrophic tropical regions. The ecological success of corals comes from two major features: the symbiotic association that many of them establish with photosynthetic dinoflagellates from the genus Symbiodinium (commonly named zooxanthellae), and the precipitation of an aragonitic calcium carbonate (CaCO3) skeleton. Both these processes involve carbonic anhydrases. In seawater, where pH is about 8.2, most dissolved inorganic carbon (DIC) is under the form of bicarbonate (HCO3−). Moreover, zooxanthellae are enclosed in a hostderived vacuole inside the endodermal cell layer. Thus Symbiodinium, as a broad array of marine algae, has developed CO2-concentrating mechanisms (CCM) in which CAs play a crucial role (for review see Giordano et al. 2005) to increase the CO2 availability for DIC transport and/or photosynthesis. Thus, CCM allow to maintain high rate of carbon fixation. Studies on this role of CAs in DIC uptake for photosynthesis in symbiotic Cnidarians have mainly been performed in non-calcifying organisms such as the sea anemones Aiptasia pulchella (Weis 1993), Anthopleura elegantissima (Weis and Reynolds 1999) or Anemonia viridis (Furla et al. 2000a). In reef-building corals, CCM also exist (Al-Moghrabi et al. 1996; Leggat et al. 1999; Furla et al. 2000a; Leggat et al. 2002), but CAs are particularly studied for their involvement in biomineraliza-

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tion since Goreau (1959) showed the reduction in calcification rates by CA-specific inhibitors. In these last organisms, CAs convert metabolic CO2 produced by calcifying cells into HCO3− that will be used for CaCO3 deposition (Isa and Yamazato 1984; Kingsley and Watabe 1987; Tambutté et al. 1996; Furla et al. 2000b; Al-Horani et al. 2003b; Marshall and Clode 2003; Tambutté et al. 2007b; Moya et al. 2008b). CAs are also found in several other calcifying cnidarians such as gorgonians (Lucas and Knapp 1997) and alcyonarians (Rahman et al. 2008). In the literature, only five α-CAs have been described in Scleractinian corals. Two atypical membrane-bound isozymes in Fungia scutaria, FCA-a and -b, that could play a role in the onset of calcification in larvae (deBoer et al. 2006), a secreted one in Stylophora pistillata, STPCA, which plays a key role in biomineralization (Moya et al. 2008b; Bertucci et al. 2009a, b; Bertucci et al. 2010a; Bertucci et al. b) and two genes found in Acropora millepora: A030-E11, expressed in the septa where calcification is occurring to form adult structures, and C007-E7, involved in the onset of calcification (Grasso et al. 2008). Here, we report the cloning, sequencing and characterization of a new α-CA in the Scleractinian coral Stylophora pistillata. This protein shares similarities with the human cytoplasmic isoform II, which is known as the most efficient CA isozyme with a kcat =1,4.106 s−1 and one of the most active enzyme with a kcat/Km =1,5.108 M−1 s−1. This isoform is usually involved in intracellular pH regulation in different human cell types (for a review, see Supuran 2008). As this enzyme is localised intracellularly, we propose that this new coral CA, called STPCA-2, is likely to have the same role in coral cells.

Materials and Methods Biological Material Experiments were conducted in the laboratory on the branching symbiotic Scleractinian coral Stylophora pistillata (Esper 1797). Colonies were stored in a 300-L aquarium, supplied with seawater from the Mediterranean Sea (exchange rate 2% per hour) under controlled conditions: semi-open circuit, temperature of 26.0±0.2°C, salinity of 38.2 psu, light of 175 μmol photons per square metre per second (using fluorescent tubes Custom Sea Life®) on a 12:12 photoperiod. Corals were fed three times weekly with a mix of Artemia salina nauplii, frozen adults of Artemia salina, and frozen krill. RNA Extraction, cDNA Synthesis, PCR Amplification, Cloning and Sequencing Total RNAs extraction and cDNA synthesis were performed as described previously (Moya et al. 2008a). Polymerase chain reaction (PCR) amplification

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was carried out using Platinum Taq DNA polymerase (Invitrogen) and degenerate primers: forward (5′CAGTTNCAYTTYCAYTGGGG-3′) designed on the conserved Q(L/F)HFHWG region, and reverse (5′-ACCRAS CACWGCCARGCC-3′) designed on the conserved PDGLAVLG region. PCR amplification product was ligated into a plasmid vector (pGEM-T easy vector system II, Promega). DNA sequencing of three independent clones were carried out on both strands with SP6 and T7 primer sequences by Macrogen Inc. To obtain the 5′ and 3′ ends of the sequence, rapid amplification of cDNA ends (RACE) experiments were performed using the 5′/3′ RACE Kit by Roche. Both ends were cloned in pGEM-T easy vector and sequenced as mentioned above. To know which partner of the symbiosis does this CA belong to, we performed a present/absent PCR assay on both cDNA and gDNA from either holobiont (coral+ zooxanthellae) or cultured zooxanthellae using the following primers: 5′-GGCCACTTGCCCAGGAAACAGGG-3′ and 5′-CGTCTACGGCTGTCACATTGGC-3′, respectively forward and reverse. Control genes are 36B4 (Moya et al. 2008a) and RuBisCO, respectively for the animal and algal gene expression (for primers sequences, see Bertucci et al. 2010a). Sequence and Phylogenetic Analyses BLASTX analyses were conducted on the NCBI server (Altschul et al. 1997) on GenBank database. Signal peptide presence was determined using the server SignalP-3 (Emanuelsson et al. 2007). Phosphorylation and glycosylation prediction analyses were performed on the Center for Biological Sequence Analysis prediction server (Blom et al. 1999). Glycosylphosphatidylinositol (GPI) modification site prediction was performed using both the big-PI predictor (Eisenhaber et al. 1999) and DGPI servers. Phylogenetic trees were constructed with both the maximum likelihood (ML) and Bayesian methods in order to check for concordance of the results. The alignments of all amino acid sequences were performed with the Multalin server (Corpet 1988). Based upon the amino acid alignment, ML estimates of the topology and branch length were obtained using PhyML v3.0 (Guindon and Gascuel 2003) with the WAG+I+G model of substitution as recommended by alignment analysis with ProtTest v2.0 (Abascal et al. 2005). Support for individual branches was inferred by bootstrap analyses (500 replicates). Further phylogenetic relationships were investigated using Bayesian techniques as implemented in the computer programme MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001) starting from a random tree, using the WAG+I+G model generating 850,000 generations with sampling every 100 generations, and with four chains in order to obtain the final (consensus) tree and to determine the posterior probabilities at the different nodes.

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Expression in CHO Cells and Protein Purification PCR products were prepared from oligo-dT reverse transcription coral cDNAs and cloned into pIRES-DSRED mammalian expression vector (Clontech, Inc.). PCR reaction was carried out with the following primers: forward: (5′C TA G C TA G C TA G A C C AT G TAT T T T C T G T G G C TAATTTC-3′) and reverse (5′-TCCCCCGGGGGATCAGT GATGGTGATGGTGATGCGATCCTCTTTCCCGGCTA GAAAGGACAGAG-3′). The recombinant vector STPCA2ORFHIS contains a chimeric sequence encoding the ORF of the STPCA-2 fused to a polyhistidine region at the Cterminus. After sequencing, the plasmid clone was introduced into Chinese hamster ovary (CHO) cells with the Lipofectamine transfection reagent (Invitrogen). A positive clone, obtained by limiting dilution, was used to produce STPCA-2. When the CHO-STPCA-2 cells were 70–80% confluent, the culture medium was harvested 7 days after and deposited onto Ni-NTA agarose beads (Qiagen) to which the 6×His-Tag of the recombinant protein would bind. The beads were washed with washing buffer (20 mM imidazole, 300 mM NaCI, 50 mM NaH2PO4 and 0,05% Tween 20, pH 8.0), and the 6×His-Tagged protein was eluted with the elution buffer (200 mM imidazole, 300 mM NaCI, 50 mM NaH2P04 and 0,05% Tween 20, pH 8.0). Imidazole was removed by concentration of eluted protein using a Centricon Plus centrifugal filter unit (5,000 MW cutoff) before a washing step with phosphate-buffered saline (PBS). At the end, the retentate was collected, and protein concentration was measured using a Qubit kit assay (Invitrogen). Western Blotting Purified proteins were subjected to 12% SDS-PAGE and electroblotted onto a PVDF membrane. Blots were probed with the monoclonal mouse antiHistidine Tag (Serotec; 1:1,000 dilution) and the STPCA2 polyclonal antibody raised against the whole protein in rabbit (Eurogentec; 1:1,000 dilution). STPCA-2 was also treated overnight by N-glycosidase F (Roche Applied Science) in 250 mM Tris, pH 8.0, 50 mM 2mercaptoethanol at 37°C. Immunolocalisation with Anti-STPCA-2 Apexes of colonies were prepared for immunolocalisation as described previously by Puverel et al. 2005. Briefly, apexes of S. pistillata were fixed in 3% paraformaldehyde in S22 buffer (450 mmol L −1 NaCl, 10 mmol L −1 KCl, 58 mmol L−1 MgCl2, 10 mmol L−1 CaCl2, 100 mmol L−1 Hepes, pH 7.8) at 4°C overnight and then decalcified using 0.5 mol L−1 ethylenediaminetetraacetic acid in Ca-free S22 at 4°C. They were then dehydrated in an ethanol series and embedded in Paraplast. Crosssections (6-μm thick) were cut and mounted on silanecoated glass slides.

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Immunolocalisation was performed according to the protocol previously described by Puverel et al. (2005). In summary, deparaffinized sections of tissues were incubated for 1 h in saturating medium [1% BSA, 0.2% teleostean gelatin, 0.05% Tween 20 in 0.05 mol L−1 PBS pH 7.4] at room temperature (RT). The samples were then incubated with the anti-STPCA-2 or the pre-immune serum as primary antibodies (1:100 dilution, 1 h at RT and overnight at 4°C in moist chambers). After rinsing in saturating medium, the samples were incubated with biotinylated antirabbit antibodies (Amersham 1:250 dilution, 1 h at RT) as secondary antibodies. After rinsing with PBS pH 7.4, the samples were finally stained for 15 min with streptavidin Alexa Fluor 568 (Molecular Probes, 1:50 dilution) and 4′,6diamidino-2-phenylindole, DAPI (Sigma, 2 μg mL−1). Samples were embedded in ProLong Antifade medium (Molecular Probes) and observed with a confocal laser scanning microscope (Leica, SP5). CA Activity and Inhibition Assay An Applied Photophysics stopped flow instrument has been used for assaying the CA-catalysed CO2 hydration activity (Khalifah 1971). Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the maximum absorbance of 557 nm, with 10 mM Hepes (pH 7.5) or Tris (pH 8.3) as buffer, and 20 mM Na 2SO 4 or 20 mM NaCl (for maintaining constant the ionic strength), following the CA-catalysed CO2 hydration reaction for a period of 10– 100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For the inhibitor at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (1 mM) were prepared in distilled–de-ionised water with 10–20% (v/v) DMSO (which is not inhibitory at these concentrations) and dilutions up to 0.01 nM were done thereafter with distilled–de-ionised water. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the enzyme–inhibitor complex. The inhibition constants were obtained by non-linear least squares methods using PRISM 3, from Lineweaver–Burk plots, as reported earlier, and represent the mean from at least three different determinations.

Results Cloning and Sequencing The complete sequence is 1,417bp long with a 951 nucleotides open reading frame

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beginning 87 nucleotides downstream from the 5′ end. This sequence encodes a protein of 316 amino acids with a calculated molecular mass of about 35.2 kDa. The nucleotide sequence is available on Genbank™ database by accession number EU532164. BLAST searches of GenBank give best matches mainly with vertebrate cytosolic isoforms CA II. Presence/Absence PCR Assays To ensure wether our gene is of host or symbiont origin, we performed PCR amplification of a fragment of our gene both on genomic and complementary DNA using RuBisCO as a vegetal control gene and 36B4 (Moya et al. 2008a) as an animal control gene in holobiont (coral and symbiont) and cultured zooxanthellae (CZ). Figure 1 shows that the holobiont possesses and expresses all the genes cited above. In CZ, only RuBisCO is present in gDNA and cDNA. Our carbonic anhydrase gene, as well as the animal control gene, is not present in either the CZ genome or transcriptome. This assay proves that this CA belongs to the animal partner and was named STPCA-2 for “STylophora Pistillata Carbonic Anhydrase isoform 2”. The gDNA fragments of STPCA-2 and 36B4 contain introns. These sequences are available on GenBank (accession number HM748807 and HM748806). Sequence Analysis A ClustalW alignment of STPCA-2 with human isoforms CA I, CA II, CA VI and coral isoforms FCA-a, FCA-b, A030-E11, C007-E7 and STPCA, respectively from F. scutaria, Acropora millepora and Stylophora pistillata is presented in Fig. 2. It should be noted that STPCA-2 appears very different from STPCA. Indeed they are 35% identical and 48% homologous in nucleotide

Fig. 1 Presence and expression of STPCA-2 gene in holobiont and CZ. PCR products were deposited on 2.5% agarose gel. RuBisCO and 36B4 are respectively used as algal and animal specific genes. Differences between gDNA and cDNA are due to the presence of intron fragments; 1 kb and 25 bp are DNA ladders from Invitrogen

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sequence. All the 36 residues forming the active site (Hewett-Emmett and Tashian 1996) are present in STPCA-2. A conserved domain search allowed us to identify His113, His115 and His138 as the three residues involved in zinc cofactor binding. This analysis also identifies the other 33 amino acid residues that are important for enzymatic activity among which four are of particular relevance. The zinc-bound water molecule establishes hydrogen bond interactions with the hydroxyl moiety of Thr252, which in turn is bridged to the carboxylate moiety of Glu125. These interactions, called gatekeeper, enhance the nucleophilicity of the zinc-bound water molecule and orient the substrate (CO2) in a favourable location for the nucleophilic attack. In order to regenerate the active form of the enzyme, a proton transfer reaction from the active site to the environment takes place, which is assisted by the proton shuttle residue His82. Gln111 is also identified as an important residue in the active site, this residue, corresponding to Gln92 in hCAII is involved in the binding of many sulfonamide/sulfamate inhibitors. This protein contains 16 putative phosphorylation sites (ten serines, one threonine and five tyrosine) and two Nglycosylation sites in positions 31 and 194. Prediction tools indicate that the first 15 amino acids are a signal peptide. This feature seems to be in contradiction with the homologies between STPCA-2 and the cytosolic α-CA II. These tools did not identify any GPI anchor or transmembrane domains. One can also notice the presence of a large insert of 30 amino acids expanding between positions 212 and 243. Biochemical Characterization A Western blotting of the recombinant STPCA-2 labelled with a 6×His-Tag performed either with an anti-Histidine Tag or an antiSTPCA-2 specific antibody shows an apparent weight of about 45 kDa suggesting post-translational modifications. An N-glycosidase F treatment lowers this weight around 35.2 kDa as predicted by the putative amino acids sequence (Fig. 3). Phylogenetic Analyses To further characterise our sequence, we compared it with 60 already published CAs sequences from various animal taxa among which are seven Cnidarian sequences. PhyML and Bayesian analysis are congruent. The resulting consensus tree separates the 61 sequences into four major clusters (Fig. 4). The first cluster represents the bacteria outgroup. The second group contains sponge CAs. This position of Sponges as an ancestral clade is in accordance with the results of Jackson et al. (2007). The third and fourth clusters contain invertebrates and vertebrates sequences. “Extracellular” (i.e. transmembrane, secreted and membrane-bound) CAs are grouped in the third cluster, whereas cytosolic and mitochondrial CAs are

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hCAI hCAII hCAVI FCA-a F. scutaria FCA-b F. scutaria A030-E11 A. millepora C007-E7 A. millepora STPCA S. pistillata STPCA-2 S. pistillata

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261 260 298 201 224 300 260 307 316

261 260 308 201 225 308 260 323 316

Fig. 2 ClustalW alignment of the deduced amino acid sequence of STPCA-2 with the human isoforms I (hCAI), II (hCAII), VI (hCAVI) and the coral enzymes FCA-a and FCA-b from F. scutaria, A030-E11 and C007-E7 from A. millepora and STPCA from S. pistillata. The amino acids are numbered on the right. Identical amino acids are shaded in black. >75%, >65% and 50 % identical amino acids are

shaded in dark, medium and light gray respectively. The three zincbinding histidines are indicated by an asterisk. The active site residues are indicated by inverted black triangles and potential N-glycosylation sites are indicated by black triangles. The signal peptides in Nterminus are indicated in bold

in the fourth group called “intracellular”. We note that in these two last clusters, vertebrates and invertebrates sequences are well separated and Cnidarian sequences are grouped in both clusters. STPCA-2 branches at the base of the intracellular isoforms group. The support value for this branching is higher in Bayesian analysis (100%) than in ML analysis (>50%).

kcat/Km of 8.3.107 M−1 s−1, STPCA-2 is among the most efficient enzymes tested in this assay, similar to hCA II and hCA VB. With a Ki of 74 nM, STPCA-2 is as sensitive to acetazolamide (AZA) as the mitochondrial isoforms hCA VA and hCA VB (63 and 54 nM, respectively). STPCA-2 is less sensitive to AZA than the secreted STPCA (16 nM).

STPCA-2 Activity The kinetics parameters for the CO2 hydration by the purified recombinant STPCA-2 was measured and compared to human isoform hCA I, hCA II, hCA III, hCA VA and VB, hCA VI, hCA IX and hCA XII (Supuran 2008) as well as the previous enzyme cloned in Stylophora pistillata, STPCA (Moya et al. 2008b) (Table 1). STPCA-2 has a higher catalytic constant (5.6.105 s−1) than the previous coral enzyme, STPCA (3.1.105 s−1). With a

Immunolocalisation Coral tissue sections were labelled with anti-STPCA-2 antibody (Fig. 5). Figure 5a, b show a tissue section of Stylophora pistillata incubated with preimmune serum and anti-STPCA-2 antibody, respectively. Figure 5a is not different from natural autofluorescence of coral tissues. Magnifications on aboral (Fig. 5c) and oral (Fig. 5d) tissues indicate that STPCA-2 is mainly located inside aboral tissue and oral endoderm.

Mar Biotechnol (2011) 13:992–1002

Fig. 3 Western blot: recombinant STPCA (see Moya et al. 2008b) and STPCA-2 are subjected to SDS-PAGE, electroblotted and incubated with anti-His Tag (lanes a, b and c) or anti-STPCA-2 (lanes d and e). a, d Recombinant STPCA. b, e Recombinant STPCA-2. c Recombinant STPCA-2 after N-glycosidase F treatment

Discussion In this study, we report the cloning in the Scleractinian coral Stylophora pistillata of a gene encoding a putative protein with high similarities with the α-class of CAs and more particularly with the cytosolic isoform CAII. This protein belongs to the animal partner (Fig. 1) and has then been named STPCA-2 for “STylophora Pistillata Carbonic Anhydrase isoform 2”. The sequence analysis (Fig. 2) revealed that STPCA-2 possesses key amino acids residues such as the three zinc-binding histidines, the proton shuttle, the gatekeeper residues and other amino acids involved in the hydrogen-bound network (HewettEmmett and Tashian 1996; McCall et al. 2000). Yet, Glu136 (Glu117 in human CAII) is substituted by a Gln in STPCA-2. This modification could lower the hydrogenbound network around the active site and alter STPCA-2 activity. However, this enzyme appears to be highly active and efficient (Table 1). With a kcat/Km of 8.3.107, this new coral CA has, with the human mitochondrial isoform VB (kcat/Km =9.8.107), a catalytic efficiency close to hCA II. The lower sensitivity of STPCA-2 to acetazolamide compared to the previous S. pistillata enzyme STPCA is an encouraging clue to renew inhibition assays in order to better study this novel CA in S. pistillata. Indeed this approach on STPCA (see Bertucci et al. 2009a, b) allowed us to find some specific interacting molecules that potentially inhibit or activate calcification. The same work performed on STPCA-2 would probably identify such molecules and will permit specific in vivo experiments in

997

which it would be possible to aim at a specific enzyme and to identify its role. So far, the role of CAs in coral physiology has been determined only by using the nonselective inhibitors AZA and/or ethoxzolamide (EZ). The problem is that the two main processes involving CAs, symbiosis and calcification, are strongly linked together (the phenomenon known as light-enhanced calcification is indisputably the best example), and the effects of AZA and EZ on calcification could as much be due to a disruption in carbon supply to photosynthesis as to a direct effect on the biomineralisation process. The phylogenetic analysis (Fig. 4) confirms that the αCA family expanded from a single ancestral gene through several independent gene duplication events in sponges and eumetazoans as shown by Jackson et al. (2007). These authors suggest that a putative signal peptide was present in the ancestral α-CA that was either secreted or membrane-bound. STPCA-2 possesses a signal peptide in the N-terminus and branches at the base of intracellular enzymes. This could indicate that STPCA-2 is an ancestral isoform of α-CA as indicated by its position, whereas other Cnidarian CAs group well together inside the intracellular as well as in the extracellular clusters. Moreover, STPCA-2 possesses another atypical feature, a 30-amino acid-long insert expanding between Lys212 and Lys243 (Fig. 2). This insert does not affect the active site of STPCA-2. Atypical CAs have already been described in invertebrates. For instance, in pearl oysters, nacrein possesses a split CA domain that may confer a regulatory function to this organic matrix protein (Miyamoto et al. 1996). In Scleractinian corals, deBoer et al. (2006) have identified, in F. scutaria larvae, two isoforms of CA homologues that possess the three zinc-binding histidines but lack crucial amino acids for catalytic activity and exhibit a conserved ATP/GTP binding site. In the same manner, the two CAs described in A. millepora (Grasso et al. 2008) possess a signal peptide, one of these (A030E11, accession number ACJ64663) matches with human CA II as well as STPCA-2. This feature seems to be common in Cnidarians CAs. In vertebrates, including Homo sapiens, the physiological functions of CAs have widely been investigated over the last 70 years (Supuran 2008): hCA II is involved in respiration, regulation of the acid/base homeostasy and in the bone development and function, such as the differentiation of osteoclasts or the provision of acid for bone resorption in osteoclasts. In Fig. 5, we observe that STPCA-2 is mainly localised in oral endoderm and aboral tissue. As mentioned previously, the way the host supplies DIC to its algal symbiont remains still poorly characterised (Al-Moghrabi et al. 1996). Furla and co-workers (2000a) proposed a model to explain the mechanism of inorganic carbon uptake by

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Fig. 4

Phylogenetic analysis of STPCA-2 (indicated in bold red) and 60 previously cloned CA sequences inferred from both ML and Bayesian analyses. The results from ML bootstrap analysis are shown above the branches, only values >50% are indicated. The full, dashed and dotted branches represent >95%, 75–95% and 50–75% Bayesian posterior probabilities, respectively. The accession numbers are from the protein sequence database of Entrez from the National Center of Biotechnology Information. The branch lengths are proportional to the number of substitutions per site (see scale bar in the figure). Other Cnidarian sequences are indicated in green

Cnidarian oral tissue. This model includes two CAs. The first is externally bound to the apical membrane of ectodermal cells and is coupled with a proton pump. This CA is used to convert the external HCO3− into CO2, which easily diffuse into the cell. There, a second CA converts CO2 back to HCO3− that will be transferred to the endodermal cells containing symbionts. This conversion also avoids the leak of inorganic carbon by diffusion of CO2 out of the cell and can be considered as an inorganic carbon trapping mechanism. Our results show that STPCA-2 does not correspond to any of this two CA. This means that other isoforms exist and remain to be characterised in symbiotic Cnidarians. In a recent paper, Venn et al. (2009) performed the first intracellular pH (pHi) measurement in Cnidarians. Two important results came out of this study. First, the pHi of S. pistillata endodermal cells containing symbiotic algae is of 7.41±0.22 in the light. This result suggests that the intracellular DIC form is HCO3−. Secondly, a more acidic area (pH 6.0 or lower) surrounds zooxanthellae. It is not clear whether this more acidic area is associated with the membrane or with the medium surrounding the symbiont (called perisymbiotic space), but this acidification could be achieved by a plasma membrane P-type H +-ATPase belonging to the zooxanthellae (Bertucci et al. 2010a). In such a condition, DIC form is predicted to be CO2. STPCA2 is present in these symbiont-containing cells (Fig. 5d) and

Table 1 Kinetic parameters for the CO2 hydration reaction catalysed by the human carbonic anhydrases isoforms I, II, III, VA, VB, VI, IX and XII and the Stylophora pistillata enzymes STPCA and STPCA-2 at 20°C and pH 7.5 in 10 mM Hepes buffer, and their inhibition data with AZA

Isozyme

Activity level

hCA I hCA II hCA III hCA VA hCA VB hCA VI hCA IX hCA XII STPCA STPCA-2

Moderate Very high Very low Low High Moderate High Moderate Moderate High

could play different roles depending on the surrounding pH, i.e. DIC trapping under the form of bicarbonate in the cytosol and formation of CO2 near the photosynthetic algae to supply photosynthesis. Concerning calcification, it has been shown that 25% of DIC used for CaCO3 deposition comes from the seawater HCO3−, whereas the major source (75%) is the metabolic CO2 (Erez 1978; Furla et al. 2000b) produced by the numerous mitochondrion present in aboral ectoderm, also called calicoderm (Furla et al. 2000b; Tambutté et al. 2007a). In this manner, external DIC must cross the aboral tissue where STPCA-2 is likely to ensure the trapping of this DIC under the form HCO3− prior to its transport toward the calcifying cells through a way that is still unknown. Finally, in these calcifying cells, even if CO2 could diffuse toward the site of calcification (where STPCA is present, Moya et al. 2008b) a part of this internal DIC could be converted into HCO3− intracellularly by STPCA-2. The presence of an intracellular CA in the calicoderm was already suggested by Furla et al. (2000b). All these data are summarised in a model diagram showing the movements of the different forms of DIC and the localisation of STPCA and STPCA-2 enzymes in coral tissues (Fig. 6). Our study confirms the importance of carbonic anhydrases in coral physiology. Owing to its intracellular localisation and similarity with hCA II, we propose that the present STPCA-2 is involved in pH regulation by trapping the DIC under the form of bicarbonate and helps its delivery either to symbionts or to the calcification site. Due to the presence of a signal peptide, we cannot exclude the possibility that STPCA-2 is localised inside intracellular vesicles that are not observable with confocal microscopy. Thus, further pharmacological and physiological studies will attempt to better characterise this enzyme and determine its role.

kcat (s−1)

kcat/Km (M−1 s−1)

Ki (acetazolamide) (nM)

2.0.105 1.4.106 1.0.104 2.9.105 9.5.105 3.4.105 3.8.105 4.2.105 3.1.105 5.6.105

5.0.107 1.5.108 3.0.105 2.9.107 9.8.107 4.9.107 5.5.107 3.5.107 4.6.107 8.3.107

250 12 300,000 63 54 11 25 5.7 16 74

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Fig. 5 Immunolocalisation of STPCA-2. Cross section of Stylophora pistillata tissues stained by a pre-immune serum and b anti-STPCA-2 antibody. c Magnification on aboral tissue. d Magnification on oral tissues. Specific labelling of STPCA-2 appears in orange (Alexa Fluor 568). Nuclei are labelled in blue (DAPI). AEnd, aboral endoderm; AT, aboral tissue; CEct, calicoblastic ectoderm; Co, coelenteron; m, mesoglea; OEct, oral ectoderm; OEnd, oral endoderm; OT, oral tissue; Sw, seawater; Sk, skeleton

Fig. 6 Model of DIC movements through the different layers of coral tissue (modified from Furla et al. 2000a, b and Moya et al. 2008b). Oral tissue is on the left and aboral tissue is on the right. Dotted arrows represent CO2 diffusion. CA, uncharacterized carbonic

anhydrase; Zoox, zooxanthella; M., mitochondrion. pH values are from Furla et al. 1998; Al-Horani et al. 2003a and Venn et al. 2009. Unknown mechanisms of DIC transport are indicated with the symbol question mark

Mar Biotechnol (2011) 13:992–1002 Acknowledgments We are grateful to Nathalie Techer for her technical help and to Dominique Desgré for coral maintenance. This study was conducted as part of the Centre Scientifique de Monaco Research Programme, supported by the Government of the Principality of Monaco. Anthony Bertucci was supported by a fellowship from the Scientific Centre of Monaco.

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