Tingaud-Sequeira A et al. (2010), The zebrafish genome encodes the largest verteb...

XB-ART-41123

BMC Evol Biol 2010 Feb 11;10:38. doi: 10.1186/1471-2148-10-38.

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The zebrafish genome encodes the largest vertebrate repertoire of functional aquaporins with dual paralogy and substrate specificities similar to mammals.

Tingaud-Sequeira A , Calusinska M , Finn RN , Chauvigné F , Lozano J , Cerdà J .

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BACKGROUND: Aquaporins are integral membrane proteins that facilitate the transport of water and small solutes across cell membranes. These proteins are vital for maintaining water homeostasis in living organisms. In mammals, thirteen aquaporins (AQP0-12) have been characterized, but in lower vertebrates, such as fish, the diversity, structure and substrate specificity of these membrane channel proteins are largely unknown. RESULTS: The screening and isolation of transcripts from the zebrafish (Danio rerio) genome revealed eighteen sequences structurally related to the four subfamilies of tetrapod aquaporins, i.e., aquaporins (AQP0, -1 and -4), water and glycerol transporters or aquaglyceroporins (Glps; AQP3 and AQP7-10), a water and urea transporter (AQP8), and two unorthodox aquaporins (AQP11 and -12). Phylogenetic analyses of nucleotide and deduced amino acid sequences demonstrated dual paralogy between teleost and human aquaporins. Three of the duplicated zebrafish isoforms have unlinked loci, two have linked loci, while DrAqp8 was found in triplicate across two chromosomes. Genomic sequencing, structural analysis, and maximum likelihood reconstruction, further revealed the presence of a putative pseudogene that displays hybrid exons similar to tetrapod AQP5 and -1. Ectopic expression of the cloned transcripts in Xenopus laevis oocytes demonstrated that zebrafish aquaporins and Glps transport water or water, glycerol and urea, respectively, whereas DrAqp11b and -12 were not functional in oocytes. Contrary to humans and some rodents, intrachromosomal duplicates of zebrafish AQP8 were water and urea permeable, while the genomic duplicate only transported water. All aquaporin transcripts were expressed in adult tissues and found to have divergent expression patterns. In some tissues, however, redundant expression of transcripts encoding two duplicated paralogs seems to occur. CONCLUSION: The zebrafish genome encodes the largest repertoire of functional vertebrate aquaporins with dual paralogy to human isoforms. Our data reveal an early and specific diversification of these integral membrane proteins at the root of the crown-clade of Teleostei. Despite the increase in gene copy number, zebrafish aquaporins mostly retain the substrate specificity characteristic of the tetrapod counterparts. Based upon the integration of phylogenetic, genomic and functional data we propose a new classification for the piscine aquaporin superfamily.

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Species referenced: Xenopus laevis
Genes referenced: aqp1 aqp11 aqp3 aqp5 aqp7 aqp8 aqp9 h1-8 mip

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	Figure 1. Amino acid sequence alignment of zebrafish aquaporins. (A) The consensus sequence logo is scaled according to amino acid conservation. Highest residue similarity (blue: 100%, green: 80-100% or sand; 60-80%) is found within the Î±-helical regions (H1-8). The transmembrane domains (TMD1-6) are annotated for DrAqp0a based upon a molecular sequence wrap to the crystallographically resolved structure of Bos taurus AQP0 (B). The structure wrap consists of the complete peptides (263 amino acids) with a gapless identity/similarity of 70.3/85.9%. The render shows identical residues in red, non-identical in blue. The hemi-helices H3 and H7 (yellow) on loops B and E, respectively, fold such that the opposing NPA motifs (pink in the alignment) interact to present the arginine constriction (DrAqp0a R187 green ball and stick, and arrow in alignment). The C-terminal domain is shown with a grey Î±-helix.
	Figure 2. Phylogenetic relationships among zebrafish aquaporins. The unrooted phylogenetic tree was constructed using the NJ method. The Escherichia coli homologs (EcAqpZ and EcGlpF) cluster as aquaporins and aquaglyceroporins, respectively. The bar indicates the mean distance of 0.2 changes per amino acid residue.
	Figure 3. Genomic organization of zebrafish aquaporins. Schematic representation of zebrafish aquaporin gene structures and chromosomal loci. The boxes indicate exons with coding regions only. Distances are in kb or in Mb when indicated. In the case of draqp9a, the quality of the genomic sequence available was insufficient to establish the size of the introns.
	Figure 4. Bayesian majority rule consensus tree for the codon alignment of piscine and human aquaporins. The upper right panel shows the summarized topology of the complete tree rooted with archaean aqpm. The left panel shows the topology of the classical aquaporins (aqp0, -1, and -4), unorthodox aquaporins (aqp11 and -12) and Aqp8. Accession numbers are annotated with the taxa. Bayesian posterior probabilities are shown at each node. Scale bar indicates the rate of expected nucleotide substitutions per site.
	Figure 5. Bayesian majority rule consensus tree for the codon alignment of piscine and human Glps. The tree illustrates the expanded topology of the classical Glps (Aqp9, -3, -7 and -10) from the summarized complete tree shown in Figure 4. Accession numbers and other annotations are as described for Figure 4.
	Figure 6. Structural features of zebrafish aquaporins. (A-C) Water-selective aquaporins and aquaglyceroporins (Glps; D-F). (A and D) Three-dimensional reconstruction of DrAqp4 wrapped to the crystallographically resolved structure of Escherichia coli AqpZ (1RC2 chain B), and DrAqp3a wrapped to the crystallographically resolved structure of E. coli GlpF (1LDI chain A). Molecules are mirror tube-worm renders rotated to show identical (red) and non-identical (blue) residues and the annotated features including the blue space-filled conserved sites (P1-P5) and the opposing yellow ball and stick Asn-Pro-Ala (NPA) motifs between hemi-helices H3 and H7. Despite low primary identity/similarity (21.1/36.6% for DrAqp4; 34.1/53.0% for DrAqp3a) the secondary and tertiary structures appear conserved. (B and E) Schematic diagram of aquaporin monomers showing the 6 transmembrane helices (H), the two NPA motifs, the amino acids forming the aromatic/arginine (ar/R) constriction, and the five residues (P1-P5) conserved in water-selective (B) and Glps (E). In each position the conserved residues are indicated. (C) Amino acid sequence alignment of human AQP1 and AQP8 (HsAQP1 and HsAQP8), mouse AQP8 (MmAQP8), DrAqp0a, -0b, -1a, -1b, -4, and zebrafish AQP8-related sequences (DrAqp8aa, -8ab and -8b). The arrowheads point to the positions of the ar/R constriction. The P1-P5 conserved amino acids are shaded in blue. The asterisks indicate identical residues, whereas conserved amino acid substitutions and substitutions with similar amino acids are indicated by a double or single dot, respectively. The potential mercury-sensitive Cys site before the second NPA motif is underlined. (F) Amino acid sequence alignment of EcGlpF, human AQP3 (HsAQP3) and zebrafish Glps. Symbols and notes as in C.
	Figure 7. Functional characterization of zebrafish aquaporins. Osmotic water permeability (Pf; left), and glycerol and urea uptake (right), of Xenopus laevis oocytes expressing zebrafish aquaporins. The Pf was assayed in the presence or absence of HgCl2 and Î²-mercaptoethanol (ME). Values (mean Â± SEM; n = 8-10 oocytes) with an asterisk are significantly (p < 0.01) different from water-injected oocytes in a representative experiment.
	Figure 8. Aquaporin gene expression in adult tissues of zebrafish. Representative RT-PCR analysis of aquaporin and b-actin1 (drbactin1) transcripts. PCR on genomic DNA was used as control. Minus indicates absence of RT during cDNA synthesis. The size (kb) of PCR products and molecular markers are indicated on the left; from top to bottom: 21.23, 5.15, 4.27, 3.53, 2.03, 1.91, 1.58, 1.37, 0.95, 0.83 and 0.56. A summary of the presence or absence of the aquaporin transcripts in the different tissues is shown to the lower right.

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