Structural and functional divergence of two fish aquaporin-1 water channels following teleost-specific gene duplication
© Tingaud-Sequeira et al; licensee BioMed Central Ltd. 2008
Received: 23 June 2008
Accepted: 23 September 2008
Published: 23 September 2008
Teleost radiation in the oceans required specific physiological adaptations in eggs and early embryos to survive in the hyper-osmotic seawater. Investigating the evolution of aquaporins (AQPs) in these vertebrates should help to elucidate how mechanisms for water homeostasis evolved. The marine teleost gilthead sea bream (Sparus aurata) has a mammalian aquaporin-1 (AQP1)-related channel, termed AQP1o, with a specialized physiological role in mediating egg hydration. However, teleosts have an additional AQP isoform structurally more similar to AQP1, though its relationship with AQP1o is unclear.
By using phylogenetic and genomic analyses we show here that teleosts, unlike tetrapods, have two closely linked AQP1 paralogous genes, termed aqp1a and aqp1b (formerly AQP1o). In marine teleosts that produce hydrated eggs, aqp1b is highly expressed in the ovary, whereas in freshwater species that produce non-hydrated eggs, aqp1b has a completely different expression pattern or is not found in the genome. Both Aqp1a and Aqp1b are functional water-selective channels when expressed in Xenopus laevis oocytes. However, expression of chimeric and mutated proteins in oocytes revealed that the sea bream Aqp1b C-terminus, unlike that of Aqp1a, contains specific residues involved in the control of Aqp1b intracellular trafficking through phosphorylation-independent and -dependent mechanisms.
We propose that 1) Aqp1a and Aqp1b are encoded by distinct genes that probably originated specifically in the teleost lineage by duplication of a common ancestor soon after divergence from tetrapods, 2) Aqp1b possibly represents a neofunctionalized AQP adapted to oocytes of marine and catadromous teleosts, thereby contributing to a water reservoir in eggs and early embryos that increases their survival in the ocean, and 3) Aqp1b independently acquired regulatory domains in the cytoplasmatic C-terminal tail for the specific control of Aqp1b expression in the plasma membrane.
Membrane intrinsic proteins (MIP) such as aquaporins (AQP) are molecular channels present from bacteria to humans that transport water and small molecular weight solutes across biological membranes . These membrane proteins are classified into two groups: those that are water-selective, and those that also transport small neutral molecules, such as glycerol and urea, termed aqua(glycero)porins. All known AQPs have six transmembrane domains connected by five loops (A-E), in which both the N- and C-termini are cytoplasmic. Their primary structure can be divided into two similar halves each of which bear the highly conserved Asn-Pro-Ala (NPA) motif in loops B and E that are involved in the formation of the water pore, which is the hallmark of the MIP family [1, 2]. In higher vertebrates, 13 different AQPs have been described, which differ in tissue expression, regulation and selectivity [1, 3].
Recent studies have shown that, in mammals, AQP1 and AQP2 are essential for water resorption in the kidney , AQP4 is involved in cerebral water balance, astrocyte migration and neural signal transduction , and AQP3 and AQP7 seem to play important roles during skin hydration and metabolism of adipocytes, respectively . However, there is little information on the functional properties and physiological functions of AQPs in teleosts, vertebrates which form a highly diverse group of organisms adapted to living both in freshwater and seawater.
Marine teleosts are thought to have colonized the oceans after a long freshwater ancestry, which is supported by the fossil record and by the hypo-osmotic condition and the presence of a glomerular kidney in extant marine species (see  for review). The re-entry of teleosts into seawater, however, most likely required new molecular adaptations to maintain water homeostasis, which is especially important for gametes and early embryos that do not have osmoregulatory systems. The spawning of pelagic eggs by many marine teleosts (termed pelagophils), where water content may reach up to 95% in weight, has been proposed as a mechanism to provide a water reservoir in the embryo to compensate for the passive water efflux due to the hyper-osmotic effect of the seawater until osmoregulatory organs develop [7, 8]. In addition, hydration of the egg contributes to buoyancy, thereby improving oxygen exchange and dispersal in the ocean.
Egg hydration in marine fish occurs during the later stages of oogenesis, prior to ovulation (i.e., oocyte meiotic maturation). It is driven by the osmotic gradient created by the generation of a large pool of free amino acids (FAAs) in the oocyte, produced from the hydrolysis of vitellogenin (Vtg)-derived yolk proteins, and the accumulation of inorganic ions (see  for review]. In the pelagophil teleost gilthead sea bream (Sparus aurata), we recently showed that water influx into the oocyte is facilitated by a novel water-selective AQP, predominantly expressed in the ovary, which, being structurally and functionally similar to mammalian AQP1, was named the S. aurata aquaporin-1 of the ovary (SaAQP1o) [10, 11]. This finding illustrates how marine teleosts have evolved novel molecular mechanisms to face life in the ocean. However, sea bream also expresses another water-selective, AQP1-related channel, termed SaAQP1, which is more similar to mammalian AQP1 and is ubiquitiously distributed in tissues, including osmoregulatory organs such as the kidney, gills and intestine [10, 12]. Water channels related to SaAQP1o and SaAQP1 have also been found in other teleosts [13–15], but the phylogenetic and functional relationships between these two vertebrate AQPs remain unclear.
Now that the genome of several teleosts has been completely or partially sequenced, and there is an increasing number of expressed sequence tags (ESTs) and cDNAs available, the phylogeny and functional properties of teleost AQP1-like proteins can be investigated. Using phylogenetic reconstruction and genomic analysis, we show here that the AQP1o gene (aqp1b) is teleost specific and probably originated by local duplication of a vertebrate AQP1 ancestor, while tetrapods have only a single AQP1 gene structurally more similar to teleost AQP1 (aqp1a). Expression analyses and functional characterization in Xenopus laevis oocytes suggest that teleost Aqp1b represents a neofunctionalized water channel in the ovary and some osmoregulatory organs of marine species, which has evolved unique regulatory domains at the C-terminal cytoplasmic tail for the control of intracellular trafficking.
Duplication of AQP1 in teleosts
Genomic organization of teleost aqp1 and aqp1band primary structure of the encoded polypeptides
Functional Aqp1b is predominantly expressed in the ovary of marine and catadromous teleosts
The highly conserved amino acid sequence of loops B and E of teleost Aqp1a and Aqp1b with respect to those of human AQP1 suggest that fish Aqp1b paralogs might encode functional water channels. To test this, X. laevis oocytes injected with cRNAs encoding sea bream Aqp1a or Aqp1b, sole Aqp1b, eel Aqp1b or zebrafish Aqp1b were compared with oocytes injected with 50 nl of water (Figure 4E). Coefficients of water osmotic permeability (Pf) were determined from rates of oocyte swelling after transfer to hypoosmotic MBS. Water-injected oocytes exhibited low water permeability, whereas the Pf of sea bream Aqp1a oocytes was increased by 10-fold, sea bream Aqp1b and eel Aqp1b oocytes by 8-fold, sole Aqp1b oocytes by 6-fold, and zebrafish Aqp1b oocytes by 4-fold. The presence of 0.7 mM HgCl2 reduced the Pf of both Aqp1a- and Aqp1b-injected oocytes (82.6 ± 2.1% and 50.2 ± 3.1%, respectively). The inhibition was partially recovered (52.2 ± 8.3% and 27.5 ± 10.3%, for Aqp1a and Aqp1b, respectively) by incubation of oocytes with 5 mM β-mercaptoethanol.
Sea bream Aqp1a and Aqp1b are differentially translocated into the oocyte plasma membrane
Role of sea bream Aqp1b C-terminus for Aqp1b cell surface expression
In silico analysis of teleost Aqp1b C-terminal amino acid sequence
Involvement of specific residues in sea bream Aqp1b C-terminus in intracellular trafficking
C-Terminal amino acid sequences of sea bream wild-type (WT) and mutated Aqp1b
Aqp1b C-terminus sequence
PRAQNFR A RRNVLLNGSEDEDAGFDAPREGNSSPGPSQGPSQWPKH
PRAQNFR D RRNVLLNGSEDEDAGFDAPREGNSSPGPSQGPSQWPKH
PRAQNFRTRRNV AA NGSEDEDAGFDAPREGNSSPGPSQGPSQWPKH
PRAQNFRTRRNVLLNG A EDEDAGFDAPREGNSSPGPSQGPSQWPKH
PRAQNFRTRRNVLLNGSEDEDAGFDAPREGN A SPGPSQGPSQWPKH
PRAQNFRTRRNVLLNGSEDEDAGFDAPREGNS A PGPSQGPSQWPKH
PRAQNFRTRRNVLLNGSEDEDAGFDAPREGNS D PGPSQGPSQWPKH
PRAQNFRTRRNVLLNGSEDEDAGFDAPREGNSSPGP A QGPSQWPKH
PRAQNFRTRRNVLLNGSEDEDAGFDAPREGNSSPGPSQGP A QWPKH
We present here strong evidence confirming the presence of two AQP isoforms in vertebrates that are structurally and functionally related to mammalian AQP1. These isoforms, Aqp1a and Aqp1b, seem to coexist exclusively in teleost fish since Aqp1b was not found in mammals, amphibians or birds. The main difference between Aqp1a and Aqp1b is in the C-terminal tail, which contains specific residues for regulation of intracellular trafficking in Aqp1b.
Previous sequence analyses of MIPs suggest that substrate selective modes (AQPs and aquaglyceroporins) were acquired early in the history of the family by gene duplication and functional shift, with the highest level of diversification occurring in vertebrates and higher plants . Analysis of Aqp1a and Aqp1b distribution by searching currently available genome sequence information and by cDNA cloning suggest that both isoforms are present exclusively in the teleost genome. Phylogenetic reconstruction of vertebrate AQP1-like proteins indicates that Aqp1a and Aqp1b share a common origin and are likely to have evolved from duplication of a common ancestor. Because both isoforms are present in fish species belonging to distant taxonomic groups, from basal (e.g., Anguilliformes) to more modern (e.g., Gasterosteiformes, Perciformes, Pleuronectiformes and Tetraodontiformes) groups [26, 27], this duplication must be ancient and is likely to have affected most teleosts. As suggested for many duplicated genes in teleosts, the origin of Aqp1b might be the whole-genome duplication (WGD) event that occurred specifically in the ray-finned (Actinopterygian) lineage after splitting from the tetrapod lineage about 350 million years ago . However, as it has not been possible to identify Aqp1b in most basal actinopterygians (e.g., paddlefish and sturgeon) with the genomic information available, it is not known whether the AQP1 duplication also affected these groups. In all teleosts examined, the aqp1a and aqp1b loci were found to be closely linked, indicating that Aqp1b possibly arose by a gene duplication event at a local level rather than at the chromosome or genome level. Local gene duplication has also been proposed, for instance, to explain the repertoire of teleost opsins [29, 30] and the generation of the Xiphophorus Xmrk oncogene .
The radiation of teleosts in the ocean most likely required the evolution of new osmoregulatory mechanisms in eggs and early embryos to alleviate the passive water loss imposed by the hyper-osmotic environment . In this scenario, it is plausible to hypothesize that duplication of aqp1 genes in teleosts allowed for one duplicate to encode a product with a new function through innovating mutations in regulatory and/or structural sequences ('neofunctionalization'). This seems to be the case for sea bream Aqp1b which plays a specialized physiological role in the oocyte mediating water uptake during meiotic maturation [10, 11]. In other marine and catadromous teleosts, such as sole and eel, respectively, that like sea bream also spawn highly hydrated eggs, we show here that aqp1b also encodes a functional water channel whose RNA is predominantly accumulated in the ovary, suggesting a similar role of Aqp1b during oocyte hydration. In other pelagophil teleosts [32, 33], as well as in some species of catfish, in which oocyte hydration may also occur despite having a freshwater life cycle (e.g., ), Aqp1b-encoding ESTs have also been found in the ovary. In contrast, in zebrafish, a freshwater species where almost no oocyte hydration is observed , we found a completely different aqp1b expression pattern, together with a higher mutation rate in the amino acid sequence of the encoded protein. Based on these data, we argue that, in marine teleosts producing highly hydrated eggs, Aqp1b possibly represents a neofunctionalized water channel adapted to oocytes to facilitate water transport. Finn and Kristoffersen  recently proposed that neofunctionalization of duplicated Vtg genes, which allowed one paralog to be proteolyzed into FAAs driving hydration of the maturing oocytes, was a key event in the evolution and success of the teleosts in the oceanic environment. The duplication and neofunctionalization of aqp1b may have occurred in parallel to this mechanism to facilitate oocyte water uptake in marine teleosts.
The Aqp1b isoform found in freshwater teleosts that spawn non-hydrated eggs, such as zebrafish, might be inactivated by mutations, or be eventually lost in the genome. This hypothesis is supported by the absence of aqp1b in advanced freshwater species, such as medaka, while the synteny between the aqp1a chromosome loci and downstream genes (e.g., thoc1) is conserved. However, in other extant freshwater teleosts that arose later in evolution, such as Tetraodon, aqp1b is retained in the genome. The retention of aqp1b in freshwater pufferfishes is intriguing, given that there appears to have been a massive elimination of DNA after WGD in most modern teleost genomes, resulting in the retention of only a subset of the duplicates [36, 37]. It is possible to speculate, however, that the recent evolution of Tetraodontiformes did not last long enough to allow specific divergence of the genome and hence of aqp1b. The relatively high amino acid sequence identity (77%) between fugu, a marine pufferfish which produces hydrated eggs, and Tetraodon Aqp1b supports this conjecture. In any event, additional studies aiming at the characterization of Aqp1b in more freshwater fish species, as well as the determination of sites of gene expression and protein accumulation, are required to better understand the driving force behind aqp1b isoform evolution.
In addition to the ovary, aqp1b mRNA has been detected in the posterior intestine, kidney, gills and esophagus of marine fish (this work, and [10, 14, 38]). The intestine of marine teleosts has an important osmoregulatory role, as hypo-osmoregulating fish have long been known to drink seawater to replace water lost by diffusion to their environment (see  for review). Accordingly, Aqp1a and Aqp1b proteins have been reported to be localized in the intestinal epithelia of teleosts [12–15]. However, in the sea bream gastrointestinal tract Aqp1a and Aqp1b have a different distribution pattern. Whereas Aqp1a is localized in the apical and basolateral membrane of enterocytes in duodenum and hindgut, Aqp1b is exclusively localized in the apical microvilli of rectal epithelial cells . Moreover, freshwater acclimation reduces the synthesis of Aqp1a in all intestinal segments, and of Aqp1b in rectum. Conversely, seawater acclimation of eels increases Aqp1a expression and protein synthesis in the intestine [13, 19]. Therefore, although the specific physiological functions of Aqp1a and Aqp1b in the teleost gastrointestinal tract remain unknown, these data may point to an additional role of Aqp1b in water movement across the intestinal epithelia.
The primary structure of AQP1-like proteins corresponding to the TM2 and TM5 domains and connecting loops B and E, which are involved in the formation of the water-selective pore, is highly conserved between teleost and mammals. However, Aqp1a and Aqp1b show different permeability efficiencies when expressed in X. laevis oocytes, and teleost Aqp1b isoforms also show a marked structural divergence at the C-terminal cytoplasmic tail with respect to Aqp1a and mammalian AQP1. Functional experiments, using artificial expression in oocytes of sea bream wild-type Aqp1a and Aqp1b, and chimeric proteins in which the C-terminus of Aqp1a was totally exchanged for that of Aqp1b, or the reverse, revealed that the Aqp1a tail drives constitutive targeting to the plasma membrane, unlike that of Aqp1b which produces partial retention of the expressed proteins in intracelluar vesicles. These data strongly suggest that Aqp1b independently acquired specific regulatory domains in the C-terminal region for the control of Aqp1b intracellular trafficking.
To investigate the nature of putative regulatory sites in the Aqp1b C-terminus, we analyzed its amino acid sequence in different teleosts. Based on this analysis, selected residues of sea bream Aqp1b were mutated into Ala or Asp and the resulting proteins were expressed in oocytes to determine their intracellular localization and permeability properties. In the Aqp1b C-terminus, we found typical sorting and internalisation signals that are common in many mammalian transmembrane proteins for targeting from the trans-Golgi network to the lysosomal-endosomal compartment . These motifs, however, were also detected in the Aqp1a C-terminal tail, although their sequence appeared to be different between the Aqp1a and Aqp1b isoforms. Thus, in all six teleost Aqp1b sequences analyzed, but not in Aqp1a, a di-Leu or Leu-Ile signals appear to be conserved. Mutation of sea bream Aqp1b Leu234Leu235 motif into Ala234Ala235 produced the retention of the protein in intracellular compartments and apparently increased its degradation, thereby reducing water permeability of these oocytes. These results are similar to those observed with the mammalian AQP2 mutant which has an altered and extended C-terminal tail, retained in late endosomes/lysosomes triggering degradation . Similarly, mutation of the C-terminal Leu345Leu346 motif in the human vasopressin V3 receptor produces mis-folding of the protein and abolishes receptor export . It is possible therefore, that the di-Leu motif in sea bream Aqp1b also plays a role in conformation, ensuring correct routing to the plasma membrane. However, di-Leu motifs are also involved in basolateral membrane targeting and microvilli anchoring of mammalian cell adhesion proteins, ion channels and receptors [42–45], including AQP4 , in polarized epithelial cells. Further studies are needed to establish whether the di-Leu motif has an additional function in the control of Aqp1b expression on the cell surface.
Most notably, functional analyses revealed that two residues in the sea bream Aqp1b C-terminal sequence, Thr229 and Ser254, were responsible for sea bream Aqp1b translocation from intracellular vesicles to the oocyte plasma membrane. The probability of the Thr229 residue being phosphorylated was low, and accordingly the Aqp1b-T229A mutant did not affect the phosphorylation state of Aqp1b, although it did inhibit Aqp1b cell surface expression and oocyte water permeability. Since Thr229 did not match any kinase phosphorylation consensus site other than protein kinase C (which apparently is not relevant here), the specific function of this residue is unknown and awaits further experimentation. Nevertheless, it was observed that the Aqp1b-S254A mutant prevented phosphorylation and increased Aqp1b translocation into the plasma membrane and subsequent water permeability, whereas the Aqp1b-S254D mutant, which mimicked the constitutively phosphorylated state of Aqp1b, was predominantly located in intracellular vesicles. These results suggest that dephosphorylation of Ser254 triggers Aqp1b shuttling to the cell surface, while its phosphorylation may retain the protein in intracellular vesicles. This mechanism is thus apparently the opposite to that described so far for mammalian and amphibian AQPs (i.e., AQP2 and AQP-h2), where channel insertion in the plasma membrane of collecting duct cells or granular cells of the anuran urinary bladder is triggered by protein kinase A-mediated phosphorylation of specific Ser residues in the C-terminal tail [21, 47]. Interestingly, the Ser254 in sea bream Aqp1b, a consensus site for a Pro-directed kinase, seems to be conserved in modern marine teleosts which produce hydrated eggs (Ser244 in fugu Aqp1b and Ser244 in sole Aqp1b). Pro-directed kinases are a large family of mitogen-activated protein kinases (MAPK) and cyclin-dependent kinase-like kinases, some of which (e.g., p38 MAPK) are involved in transduction pathways leading to the activation of the maturation promoting factor (MPF) during oocyte meiotic maturation [48–50]. In sea bream, Aqp1b translocation into the oocyte plasma membrane is a tightly regulated process thought to occur transiently downstream of MPF activation during meiotic maturation, just before complete hydrolysis of yolk proteins and maximum K+ accumulation is reached in the oocyte . Therefore, it will be of interest to investigate the potential role of cell-cycle related kinases, or other kinases activated during oocyte maturation, in Ser254 phosphorylation and regulation of Aqp1b trafficking.
We provide phylogenetic and functional evidence for the teleost lineage-specific duplication of AQP1 channels and further divergence at the C-terminal tail. The generation and neofunctionalization of the Aqp1b isoform in oocytes of marine teleosts most likely contributed with the production of highly hydrated eggs to ensure survival in seawater. The neofunctionalization of Aqp1b has also been accompanied by the acquisition of regulatory domains in the cytoplasmic C-terminal tail for the specific control of Aqp1b intracellular trafficking, which are currently being investigated. The elucidation of the biological functions of Aqp1a and Aqp1b in teleosts will contribute to our understanding of the evolution of phenotypic complexity, diversity and innovation in vertebrates.
Adult zebrafish, gilthead sea bream, Senegalese sole, and European eel were purchased from local pet stores or fish farms and maintained as described [11, 51–53]. Naturally spawning fish, or hormone-stimulated in the case of eel (see  for details), were sedated by immersion for approximately 15 min in 100 ppm phenoxyethanol, sacrificed by decapitation, and samples of mature ovary and other tissues immediately dissected and frozen at -80°C. Procedures relating to the care and use of animals were approved by the Ethics Committee from IRTA in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.
Cloning and sequencing of teleost AQP1-like cDNAs
Partial cDNAs encoding European eel Aqp1b and Senegalese sole Aqp1a were isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) employing degenerate oligonucleotide primers (see Additional file 3). Total RNA was extracted from kidney, intestine and hydrated ovaries using the RNeasy Maxikit (Qiagen), followed by polyA RNA purification with the Oligotex mRNA Minikit (Qiagen). PolyA RNA (500 ng) was reverse transcribed using 0.5 μg oligo (dT)17, 1 mM dNTPs, 40 IU RNAse inhibitor (Roche), and 10 IU MMLuV-RT enzyme (Roche), for 1.5 h at 42°C. The PCR was carried out with 0.5 μl of the RT reaction in a volume of 50 μl containing 1 × PCR buffer plus Mg2+, 0.2 mM dNTPs, 0.2 μM of each forward and reverse oligonucleotide primers, and 1 IU of Taq polymerase (Roche). Reactions were amplified using one cycle of 95°C, 5 min; then 40 cycles of 95°C, 30 sec; 54°C, 30 sec; 72°C, 1 min; and a final 7-min elongation at 72°C. The products were cloned into the pGEM-T Easy Vector (Promega) and sequenced by BigDye Terminator version 3.1 cycle sequencing on ABI PRISM 377 DNA analyzer (Applied Biosystems). Full-length eel Aqp1b cDNA was isolated by rapid amplification of cDNA ends (RACE; Gibco) followed by a final amplification with a high-fidelity polymerase (Pwo; Roche). Full-length zebrafish Aqp1b cDNA was amplified from total RNA extracted from adult brain using specific forward and reverse primers based on a predicted cDNA (see Additional file 3). The nucleotide sequence of Senegalese sole Aqp1a, European eel Aqp1b, and zebrafish Aqp1b have been deposited in the GenBank database under accession numbers DQ889223, EF011738, and EU327345, respectively.
Genomic organization of teleost aqp1a and aqp1bgenes
Genomic sequences covering the complete sea bream aqp1a and aqp1b loci, as well as the flanking cassette, were amplified by PCR on liver-extracted genomic DNA using the Expand Long Template PCR system 3 (Roche) and gene specific primers (see Additional file 3). Products were cloned into the pGEM-T Easy Vector and sequenced as described. The nucleotide sequence of sea bream aqp1a and aqp1b loci have been deposited in the GenBank database under accession numbers EF011739 and EF011740, respectively. Zebrafish, medaka, fugu, pufferfish, and three-spined stickleback aqp1a and aqp1b genomic sequences were retrieved from ENSEMBL . The exon-intron structure was determined from cloned cDNAs and available ESTs.
Phylogenetic and sequence analyses
Vertebrate and teleost MIP sequences were retrieved from the NCBI database  and ENSEMBL and analyzed at the amino acid level. Amino acid sequence alignments were performed using the ClustalW multiple sequence alignment program  employing the sequence from the first NPA motif to the start of the C-terminus (when sequence data was available), and were manually optimized using the Bioedit software . The alignment is shown in the Additional file 1. The phylogenetic tree and branch support values were estimated by using the NJ, ML and BI methodologies of phylogenetic reconstruction. The NJ analysis  of the amino acid alignment was based on mean character distances using Mega3 software ; bootstrap support values were obtained with 1,000 repetitions. For ML and BI analyses, a Bayesian consensus tree for the sequence data set was built and used to estimate the best-fit evolutionary model by using ProtTest v1.4 . Then, ML (including bootstrapping) analysis was performed with PhyML . To confirm the ML tree, a BI (including posterior probabilities) of phylogeny was conducted by using MrBAYES v3.1  with the ProtTest best-fit model of amino acid substitution (CpRev) provided in the package. Four independent runs, each with four simultaneous Markov Chain Monte Carlo chains, were performed for 1,000,000 generations sampled every 100 generations. Potential Ser, Thr and Tyr phosphorylation sites in amino acid sequences were predicted using NetPhos 2.0 . Candidate functional sites were identified using the Eukaryotic Linear Motif (ELM) server .
Gene expression analysis
The abundance of aqp1b transcripts in sea bream, eel, Senegalese sole and zebrafish adult tissues was assessed by conventional RT-PCR followed by Southern blot. Total RNA from liver, intestine, kidney, gills, brain, ovary and testis was extracted, treated with DNase, and first-strand cDNA synthesized as described above. The PCR was carried out as above on 1 μl of the RT reaction using species-specific aqp1b forward and reverse oligonucleotide primers located in exon 3 and 4, respectively (see Additional file 3). For zebrafish, 500 ng of DNA template was also amplified using the corresponding oligos (not shown). In all experiments, β-actin was used as a reference gene; forward and reverse oligonucleotide primers designed in highly conserved regions of zebrafish β-actin1 (bactin1) (Additional file 3) were employed for all species. PCR reactions were performed with an initial cycle of 95°C, 5 min; then variable number of cycles and temperatures for amplification, depending on the species, to generate half-maximal amounts of PCR products (not shown); and a final 7-min elongation at 72°C. For sea bream aqp1b, the cycles were 28 of 95°C, 30 sec; 62°C, 30 sec; 72°C, 30 sec; for eel and Senegal sole aqp1b the cycles were 37 and 36, respectively, of 95°C, 1 min; 60°C, 1 min; 72°C, 1 min; and for zebrafish aqp1b the cycles were 35 of 95°C, 1 min; 65°C, 1 min; 72°C, 1 min. For bactin1 the cycles were 26 for sea bream and eel, 24 for sole, and 22 for zebrafish, of 95°C, 30 sec; 52°C, 30 sec; 72°C, 45 sec. The PCR products were electrophoresed on 1% agarose gels, and the DNA blotted to nylon membranes (Amersham). Membranes were hybridized with species-specific digoxigenin-labelled aqp1b probes using the DIG DNA Labelling Mix (Roche).
Sea bream Aqp1a and Aqp1b, and zebrafish, eel and Senegalese sole Aqp1b were cloned into the EcoRV/SpeI sites of the oocyte expression vector pT7Ts . To obtain the Aqp1a-Ct1b, in which the C-terminus of Aqp1a was replaced by that of Aqp1b, the C-terminus-coding nucleotide sequence of Aqp1b was PCR amplified using a forward primer partially complementary to Aqp1a, 5'-CCCCCAAATTCCAAAACTTCAGGACGCGCAG-3', and a reverse primer bearing a SpeI restriction site, 5'-ACTAGTGCTTGTTTTTTCAGTGCTTTGG-3'. In parallel, a fragment of Aqp1a cDNA lacking the nucleotide sequence encoding the C-terminus was amplified using a forward primer specific to the 5' end of Aqp1a with an EcoRV site, 5'-GATATCGCCACCACCATGAGAGAG-3', and a reverse primer partially complementary to the nucleotide sequence of the C-terminus of Aqp1b, 5'-GAAGTTTTGGAATTTGGGGGACAG-3'. After purification, the two PCR products were used as templates to synthesize Aqp1a-Ct1b employing the forward and reverse primers bearing EcoRV and SpeI, respectively. The Aqp1b-Ct1a chimera, in which the C-terminus of Aqp1b was replaced by that of Aqp1a, was obtained by amplifying the C-terminus-coding nucleotide sequence of Aqp1a with the primers 5'-CACGAGCGGACGACTTCCCCGAGCGC-3' and 5'-ACTAGTCGTCTGTGTGGGACTATTTTGACG-3'. The Aqp1b-Ct1a cDNA was then amplified using the PCR product as reverse primer and a forward primer specific to the 5' end of Aqp1b, with an EcoRV site (5'-GATATCTCGACGCGGAGATGACAGAA-3'), employing full-length Aqp1b cDNA as a template. In all cases, PCR reactions were performed using Pwo or Easy-A high-fidelity polymerases (Stratagene), and the chimera cDNAs were ligated into pT7Ts after digestion with EcoRV and SpeI. Mutations into the sea bream Aqp1b C-terminal amino acid sequence were introduced by using the QuickChange site-directed mutagenesis kit (Stratagene) on pT7Ts-Aqp1b (Additional file 4). Sequence analysis of selected clones was carried out to confirm that only the desired chimeras or mutations were produced.
Functional expression of teleost AQPs in Xenopus laevisoocytes
Complementary RNA (cRNA) synthesis, expression in X. laevis oocytes, and Pf measurements were performed essentially as described . Oocytes were injected with 0.25 to 10 ng cRNA. The swelling assays were carried out in 10-fold diluted modified Barth's solution (MBS: 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 10 mM Hepes, pH 7.5, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, and 25 μg/ml gentamicin) in the presence or absence of 0.7 mM HgCl2 and 5 mM β-mercaptoethanol. The data shown are an average of 3–5 experiments (each with different batches of oocytes), or from a representative experiment out of three different trials producing similar results. The measured Pf values were statistically analyzed in an unpaired Student's t test; p values < 0.05 were considered significantly different.
Western blotting and immunofluorescence microscopy
Total and plasma membranes were isolated from groups of 10 oocytes as described . Protein samples were denatured at 95°C for 5 min in Laemmli buffer, electrophoresed on a 12% polyacrylamide SDS gel and then blotted onto PVDF or nitrocellulose membranes (Bio-Rad Laboratories). Membranes were blocked for 1 h with TBST (20 mM Tris, 140 mM NaCl, 0.1% Tween, pH 7.6) containing 1% nonfat dry milk, and then incubated with 1:300 diluted affinity-purified rabbit antisera against sea bream Aqp1a or Aqp1b in TBST with 1% nonfat milk powder at 4°C overnight. The Aqp1a and Aqp1b antisera were produced against synthetic peptides corresponding to the C-terminus of the corresponding proteins and they have been characterized elsewhere [10–12]. As secondary antibody, a 1:8000 dilution of goat anti-rabbit IgG coupled to horseradish peroxidase (Sigma) was used. Reactive protein bands were detected using enhanced chemiluminescence (Amersham). In some experiments, protein dephosphorylation was carried out before SDS-PAGE by resuspending total membrane extracts of Aqp1b-expressing oocytes in 10 mM MgCl2, 10 mM Tris-HCl, pH 7.5 and treating with calf intestinal alkaline phosphatase (Fermentas) for 6 h at 37°C, following the manufacturer's instructions.
Immunofluorescence microscopy was carried out on Aqp1a- and Aqp1b-expressing oocytes fixed in Bouin's without acetic acid for 4 h, subsequently dehydrated and embedded in paraplast (Sigma). Sections (7 μm) were blocked with 5% goat serum in PBST (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, 1% BSA, 0.01% Tween, pH 7.5), and incubated at 4°C overnight with Aqp1a or Aqp1b antisera (1:100) in PBST with 1% goat serum. Bound antibodies were detected with mouse FITC anti-rabbit secondary antibodies (1:300). Sections were mounted with Vectashield (Vector Labs) or Prolong Gold antifade reagent (Invitrogen) and photos taken using a Leica SP2 confocal laser scanning microscope.
Membrane intrinsic protein
Free amino acids
Sparus aurata aquaporin-1o (now termed Aqp1b)
S. aurata aquaporin-1 (now termed Aqp1a)
Expressed sequence tag
- P f :
Osmotic water permeability
Casein kinase 1
Casein kinase 2
Mitogen-activated protein kinase
Maturation promoting factor.
This work was supported by grants from the Spanish Ministry of Education and Science (MEC, Spain; AGL2004-00316 and AGL2007-60262) and the European Commission (Q5RS-2002-00784-CRYOCYTE) to JC. Participations of FC, MF and DR were supported by the European Commission (Marie Curie Research Training Network Aqua(glycero)porins, MRTN-CT-2006-035995), a predoctoral scholarship from the Departament d'Universitats, Recerca i Societat de la Informació (Catalan Government, Spain), and by a Ramón y Cajal contract (MEC, Spain).
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