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- The serendipitous origin of chordate secretin peptide family membersPublication . Cardoso, João CR; Vieira, Florbela A.; Gomes, Ana S.; Power, DeborahBackground: The secretin family is a pleotropic group of brain-gut peptides with affinity for class 2 G-protein coupled receptors (secretin family GPCRs) proposed to have emerged early in the metazoan radiation via gene or genome duplications. In human, 10 members exist and sequence and functional homologues and ligand-receptor pairs have been characterised in representatives of most vertebrate classes. Secretin-like family GPCR homologues have also been isolated in non-vertebrate genomes however their corresponding ligands have not been convincingly identified and their evolution remains enigmatic. Results: In silico sequence comparisons failed to retrieve a non-vertebrate (porifera, cnidaria, protostome and early deuterostome) secretin family homologue. In contrast, secretin family members were identified in lamprey, several teleosts and tetrapods and comparative studies revealed that sequence and structure is in general maintained. Sequence comparisons and phylogenetic analysis revealed that PACAP, VIP and GCG are the most highly conserved members and two major peptide subfamilies exist; i) PACAP-like which includes PACAP, PRP, VIP, PH, GHRH, SCT and ii) GCG-like which includes GCG, GLP1, GLP2 and GIP. Conserved regions flanking secretin family members were established by comparative analysis of the Takifugu, Xenopus, chicken and human genomes and gene homologues were identified in nematode, Drosophila and Ciona genomes but no gene linkage occurred. However, in Drosophila and nematode genes which flank vertebrate secretin family members were identified in the same chromosome. Conclusions: Receptors of the secretin-like family GPCRs are present in protostomes but no sequence homologues of the vertebrate cognate ligands have been identified. It has not been possible to determine when the ligands evolved but it seems likely that it was after the protostome-deuterostome divergence from an exon that was part of an existing gene or gene fragment by rounds of gene/genome duplication. The duplicate exon under different evolutionary pressures originated the chordate PACAP-like and GCG-like subfamily groups. This event occurred after the emergence of the metazoan secretin GPCRs and led to the establishment of novel peptide-receptor interactions that contributed to the generation of novel physiological functions in the chordate lineage.
- Cloning, tissue and ontogenetic expression of the taurine transporter in the flatfish Senegalese sole (Solea senegalensis)Publication . Pinto, W.; Rønnestad, I.; Jordal, A. E. O.; Gomes, Ana S.; Dinis, Maria Teresa; Aragão, C.Flatfish species seem to require dietary taurine for normal growth and development. Although dietary taurine supplementation has been recommended for flatfish, little is known about the mechanisms of taurine absorption in the digestive tract of flatfish throughout ontogeny. This study described the cloning and ontogenetic expression of the taurine transporter (TauT) in the flatfish Senegalese sole (Solea senegalensis). Results showed a high similarity between TauT in Senegalese sole and other vertebrates, but a change in TauT amino acid sequences indicates that taurine transport may differ between mammals and fish, reptiles or birds. Moreover, results showed that Senegalese sole metamorphosis is an important developmental trigger to promote taurine transport in larvae, especially in muscle tissues, which may be important for larval growth. Results also indicated that the capacity to uptake dietary taurine in the digestive tract is already established in larvae at the onset of metamorphosis. In Senegalese sole juveniles, TauT expression was highest in brain, heart and eye. These are organs where taurine is usually found in high concentrations and is believed to play important biological roles. In the digestive tract of juveniles, TauT was more expressed in stomach and hindgut, indicating that dietary taurine is quickly absorbed when digestion begins and taurine endogenously used for bile salt conjugation may be recycled at the posterior end of the digestive tract. Therefore, these results suggest an enterohepatic recycling pathway for taurine in Senegalese sole, a process that may be important for maintenance of the taurine body levels in flatfish species.
- The transcriptome of metamorphosing flatfishPublication . Alves, Ricardo N.; Gomes, Ana S.; Stueber, Kurt; Tine, Mbaye; Thorne, M. A. S.; Smáradóttir, H.; Reinhard, Richard; Clark, M. S.; Rønnestad, Ivar; Power, DeborahBackground Flatfish metamorphosis denotes the extraordinary transformation of a symmetric pelagic larva into an asymmetric benthic juvenile. Metamorphosis in vertebrates is driven by thyroid hormones (THs), but how they orchestrate the cellular, morphological and functional modifications associated with maturation to juvenile/adult states in flatfish is an enigma. Since THs act via thyroid receptors that are ligand activated transcription factors, we hypothesized that the maturation of tissues during metamorphosis should be preceded by significant modifications in the transcriptome. Targeting the unique metamorphosis of flatfish and taking advantage of the large size of Atlantic halibut (Hippoglossus hippoglossus) larvae, we determined the molecular basis of TH action using RNA sequencing. Results De novo assembly of sequences for larval head, skin and gastrointestinal tract (GI-tract) yielded 90,676, 65,530 and 38,426 contigs, respectively. More than 57 % of the assembled sequences were successfully annotated using a multi-step Blast approach. A unique set of biological processes and candidate genes were identified specifically associated with changes in morphology and function of the head, skin and GI-tract. Transcriptome dynamics during metamorphosis were mapped with SOLiD sequencing of whole larvae and revealed greater than 8,000 differentially expressed (DE) genes significantly (p < 0.05) up- or down-regulated in comparison with the juvenile stage. Candidate transcripts quantified by SOLiD and qPCR analysis were significantly (r = 0.843; p < 0.05) correlated. The majority (98 %) of DE genes during metamorphosis were not TH-responsive. TH-responsive transcripts clustered into 6 groups based on their expression pattern during metamorphosis and the majority of the 145 DE TH-responsive genes were down-regulated. Conclusions A transcriptome resource has been generated for metamorphosing Atlantic halibut and over 8,000 DE transcripts per stage were identified. Unique sets of biological processes and candidate genes were associated with changes in the head, skin and GI-tract during metamorphosis. A small proportion of DE transcripts were TH-responsive, suggesting that they trigger gene networks, signalling cascades and transcription factors, leading to the overt changes in tissue occurring during metamorphosis.
- PACAP, VIP and their receptors in the metazoa Insights about the origin and evolution of the ligand-receptor pairPublication . Cardoso, J. C. R.; Vieira, Florbela A.; Gomes, Ana S.; Power, DeborahThe evolution, function and interaction of ligand–receptor pairs are of major pharmaceutical interest. Comparative sequence analysis approaches using data from phylogenetically distant organisms can provide insights into their origin and possible physiological roles. The present review focuses on the pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP) and their receptors in the metazoa. A PACAP-like peptide is present in tunicates and chordates while VIP- and PACAP/VIP-specific receptors have only been isolated in the latter phyla. The apparently disparate evolution of the ligands and their specific receptors raises questions about their evolution during the metazoan radiation and also about how the ligands may have acquired new functions.
- Cartilage Acidic Protein 2 a hyperthermostable, high affinity calcium-binding proteinPublication . Anjos, Liliana; Gomes, Ana S.; Melo, Eduardo; Canario, Adelino V. M.; Power, DeborahCartilage Acidic Protein 2 (CRTAC2) is a novel protein present fromprokaryotes to vertebrateswith abundant expression in the teleost fish pituitary gland and an isoformof CRTAC1, a chondrocyte marker in humans. The two proteins are non-integrins containingN-terminal integrin-like Ca2+-bindingmotifs and their structure and function remain to be assigned. Structural studies of recombinant sea bream (sb)CRTAC2 revealed it is composed of 8.8% α-helix, 33.4% β-sheet and 57.8% unordered protein. sbCRTAC2 bound Ca2+ with high affinity (Kd= 1.46 nM) and favourable Gibbs free energy (ΔG=−12.4 kcal/mol). The stoichiometry for Ca2+ bound to sbCRTAC2 at saturation indicated six Ca2+ ligand-binding sites exist per protein molecule. No conformational change in sbCRTAC2 occurred in the presence of Ca2+. Fluorescence emission revealed that the tertiary structure of the protein is hyperthermostable between 25 °C and 95 °C and the fully unfolded state is only induced by chemical denaturing (4 MGndCl). sbCRTAC has awidespread tissue distribution and is present as highmolecular weight aggregates, although strong reducing conditions promote formation of the monomer. sbCRTAC2 promotes epithelial cell outgrowth in vitro suggesting it may share functional homology with mammalian CRTAC1, recently implicated in cell–cell and cell–matrix interactions.
- Gene structure, transcripts and calciotropic effects of the PTH family of peptides in Xenopus and chickenPublication . Pinheiro, Pedro L. C.; Cardoso, João CR; Gomes, Ana S.; Fuentes, J.; Power, Deborah; Canario, Adelino V. M.Background: Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) belong to a family of endocrine factors that share a highly conserved N-terminal region (amino acids 1-34) and play key roles in calcium homeostasis, bone formation and skeletal development. Recently, PTH-like peptide (PTH-L) was identified in teleost fish raising questions about the evolution of these proteins. Although PTH and PTHrP have been intensively studied in mammals their function in other vertebrates is poorly documented. Amphibians and birds occupy unique phylogenetic positions, the former at the transition of aquatic to terrestrial life and the latter at the transition to homeothermy. Moreover, both organisms have characteristics indicative of a complex system in calcium regulation. This study investigated PTH family evolution in vertebrates with special emphasis on Xenopus and chicken. Results: The PTH-L gene is present throughout the vertebrates with the exception of placental mammals. Gene structure of PTH and PTH-L seems to be conserved in vertebrates while PTHrP gene structure is divergent and has acquired new exons and alternative promoters. Splice variants of PTHrP and PTH-L are common in Xenopus and chicken and transcripts of the former have a widespread tissue distribution, although PTH-L is more restricted. PTH is widely expressed in fish tissue but from Xenopus to mammals becomes largely restricted to the parathyroid gland. The N-terminal (1-34) region of PTH, PTHrP and PTH-L in Xenopus and chicken share high sequence conservation and the capacity to modify calcium fluxes across epithelia suggesting a conserved role in calcium metabolism possibly via similar receptors. Conclusions: The parathyroid hormone family contains 3 principal members, PTH, PTHrP and the recently identified PTH-L. In teleosts there are 5 genes which encode PTHrP (2), PTH (2) and PTH-L and in tetrapods there are 3 genes (PTHrP, PTH and PTH-L), the exception is placental mammals which have 2 genes and lack PTH-L. It is hypothesized that genes of the PTH family appeared at approximately the same time during the vertebrate radiation and evolved via gene duplication/deletion events. PTH-L was lost from the genome of eutherian mammals and PTH, which has a paracrine distribution in lower vertebrates, became the product of a specific endocrine tissue in Amphibia, the parathyroid gland. The PTHrP gene organisation diverged and became more complex in vertebrates and retained its widespread tissue distribution which is congruent with its paracrine nature.
- Functional modifications associated with gastrointestinal tract organogenesis during metamorphosis in Atlantic halibut (Hippoglossus hippoglossus)Publication . Gomes, Ana S.; Kamisaka, Y.; Harboe, Torstein; Power, Deborah; Rønnestad, I.Background: Flatfish metamorphosis is a hormone regulated post-embryonic developmental event that transforms a symmetric larva into an asymmetric juvenile. In altricial-gastric teleost fish, differentiation of the stomach takes place after the onset of first feeding, and during metamorphosis dramatic molecular and morphological modifications of the gastrointestinal (GI-) tract occur. Here we present the functional ontogeny of the developing GI-tract from an integrative perspective in the pleuronectiforme Atlantic halibut, and test the hypothesis that the multiple functions of the teleost stomach develop synchronously during metamorphosis. Results: Onset of gastric function was determined with several approaches (anatomical, biochemical, molecular and in vivo observations). In vivo pH analysis in the GI-tract lumen combined with quantitative PCR (qPCR) of α and β subunits of the gastric proton pump (H+/K+-ATPase) and pepsinogen A2 indicated that gastric proteolytic capacity is established during the climax of metamorphosis. Transcript abundance of ghrelin, a putative orexigenic signalling molecule produced in the developing stomach, correlated (p < 0.05) with the emergence of gastric proteolytic activity, suggesting that the stomach’s role in appetite regulation occurs simultaneously with the establishment of proteolytic function. A 3D models series of the GI-tract development indicated a functional pyloric sphincter prior to first feeding. Observations of fed larvae in vivo confirmed that stomach reservoir function was established before metamorphosis, and was thus independent of this event. Mechanical breakdown of food and transportation of chyme through the GI-tract was observed in vivo and resulted from phasic and propagating contractions established well before metamorphosis. The number of contractions in the midgut decreased at metamorphic climax synchronously with establishment of the stomach’s proteolytic capacity and its increased peristaltic activity. Putative osmoregulatory competence of the GI-tract, inferred by abundance of Na+/K+-ATPase α transcripts, was already established at the onset of exogenous feeding and was unmodified by metamorphosis.Conclusions: The functional specialization of the GI-tract was not exclusive to metamorphosis, and its osmoregulatory capacity and reservoir function were established before first feeding. Nonetheless, acid production and the proteolytic capacity of the stomach coincided with metamorphic climax, and also marked the onset of the stomach’s involvement in appetite regulation via ghrelin.
- PTHrP-induced modifications of the sea bream (Sparus auratus) vertebral bone proteomePublication . Anjos, Liliana; Gomes, Ana S.; Redruello, Begoña; Reinhardt, Richard; Canario, Adelino V. M.; Power, DeborahEndocrine factors play an essential role in the formation and turnover of the skeleton in vertebrates. In the present study sea bream vertebral bone transcripts for PTH1R and PTH3R were identified and the action of intermittent administration of parathyroid hormone related protein (PTHrP) on the proteome of vertebral bone was analysed. Treatment of immature sea bream (Sparus auratus, n = 6) for 5 days with homologous recombinant PTHrP(1–125; 150 ng/g body weight) modified bone metabolism and caused a significant (p < 0.05) reduction in both tartrate resistant acid phosphatase (TRACP) and alkaline phosphatase (ALP) in relation to control fish. However, the ratio of TRACP: ALP in PTHrP treated fish (1.3 to 2.2 cf. control) suggested it had an anabolic response. A sea bream vertebral bone proteome of 157 protein spots was generated and putative identity assigned to 118 (75.2%) proteins of which 72% had homology to proteins/transcripts from teleosts many of which have not previously been reported in teleost bone. Classification of bone proteins using gene ontology revealed those with protein or metal/ion (e.g., calcium, magnesium, zinc) binding (∼53%) activities were most abundant. The expression of eight proteins was significantly (p < 0.05) modified in the vertebra of PTHrP treated compared to control fish; three were up-regulated, betainehomocystein S-methyltransferase, glial fibrillary acidic protein, parvalbumin beta and five were down-regulated, annexin A5, apolipoprotein A1, myosin light chain 2, fast skeletal myosin light chain 3, troponin C. In conclusion, intermittent administration of PTHrP to sea bream is associated with an anabolic response in vertebral bone metabolism and modifies calcium binding proteins in the proteome.