Loading...
8 results
Search Results
Now showing 1 - 8 of 8
- Isolation and characterization of piscine osteonectin and downregulation of Its expression by PTH-related proteinPublication . Redruello, Begoña; Estêvão, M. Dulce; Rotllant, J.; Guerreiro, P. M.; Anjos, Liliana; Canario, Adelino V. M.; Power, DeborahThe skeleton is the main source of osteonectin mRNA in adults of the seawater teleost sea bream Sparus auratus. It is expressed by cells forming the basement membrane of calcifying tissue indicating that, as in mammals, it may play a role in osteoblast differentiation. PTHrP induced downregulation of osteonectin mRNA in vitro in scales, a mineralizing tissue with bone-like metabolism. This indicates a means to redirect calcium to activities such as vitellogenesis when this ion is in high demand.
- Ligand binding and signalling pathways of PTH receptors in sea bream (Sparus auratus) enterocytesPublication . Rotllant, J.; Guerreiro, P. M.; Redruello, Begoña; Fernandes, H.; Apolonia, L.; Anjos, Liliana; Canario, Adelino V. M.; Power, DeborahWhole animal studies have indicated that Ca2+ uptake by the gastrointestinal tract is regulated by the action of parathyroid hormone-related peptide (PTHrP) in teleost fish. We have characterised PTH receptors (PTHR) in piscine enterocytes and established, by using aminoterminal PTHrP peptides, the amino acid residues important for receptor activation and for stabilising the ligand/receptor complex. Ligand binding of 125I-(1–35tyr) PTHrP to the membrane fraction of isolated sea bream enterocytes revealed the existence of a single saturable high-affinity receptor (KD=2.59 nM; Bmax=71 fmol/mg protein). Reverse transcription/polymerase chain reaction with specific primers for sea bream PTH1R and PTH3R confirmed the mRNA expression of only the later receptor. Fugu (1–34) PTHrP increased cAMP levels in enterocytes but had no effect on total inositol phosphate accumulation. The aminoterminal peptides (2–34)PTHrP, (3–34)PTHrP and (7–34) PTHrP bound efficiently to the receptor but were severely defective in stimulating cAMP in enterocyte cells indicating that the first six residues of piscine (1–34)PTHrP, although not important for receptor binding, are essential for activation of the adenylate cyclase/phosphokinase A (AC-PKA)-receptor-coupled intracellular signalling pathway. Therefore, PTHrP in teleosts acts on the gastrointestinal tract through PTH3R and the AC-PKA intracellular signalling pathway and might regulate Ca2+ uptake at this site. Ligand-receptor binding and activity throughout the vertebrates appears to be allocated to the same amino acid residues of the amino-terminal domain of the PTHrP molecule.
- Production and characterisation of gilthead sea bream (Sparus auratus) recombinant parathyroid hormone related proteinPublication . Anjos, Liliana; Rotllant, J.; Guerreiro, P. M.; Hang, X. M.; Canario, Adelino V. M.; Balment, R.; Power, DeborahThe production and puriWcation of gilthead sea bream recombinant parathyroid hormone related protein [sbPTHrP(1–125)] using an Escherichia coli system and one step puriWcation process with continuous elution gel electrophoresis is reported. The cDNA encoding sbPTHrP(1–125) was cloned into a prokaryotic expression vector pET-11a. The recombinant plasmid was used to transfect E. coli BL21(DE3) pLysS and sbPTHrP(1–125) synthesis was induced by addition of 1mM isopropyl- -D-thiogalactopyranoside. The rapid one step isolation method gave pure sbPTHrP(1–125) as judged by SDS–PAGE and yielded up to 40mg/L of culture medium (3.3mg protein/g of bacteria). The bioactivity of recombinant sbPTHrP(1–125) assessed using an in vitro scale bioassay was found to be equipotent to PTHrP(1–34) in stimulating cAMP accumulation. Assessment of the immunological reactivity of the isolated protein by Western blot revealed it cross-reacts with antisera speciWc for the N-terminal and C-terminal region of PTHrP. In a radioimmunoassay speciWc for piscine N-terminal (1–34 aa) PTHrP, the recombinant sbPTHrP(1–125) was equipotent with PTHrP(1–34) in displacing labelled 125I-PTHrP(1–36) PTHrP from the antisera. The availability of recombinant sbPTHrP will allow the development of region speciWc assays and studies aimed at deWning post-secretory processing of this protein and its biological activity in fish.
- Stimulation of cortisol release by the N terminus of teleost parathyroid hormone-related protein in interrenal cells in vitroPublication . Rotllant, J.; Guerreiro, P. M.; Anjos, Liliana; Redruello, Begoña; Canario, Adelino V. M.; Power, DeborahThe mode of action of PTHrP in the regulation of sea bream (Sparus auratus) interrenal cortisol production was studied in vitro using a dynamic superfusion system. Piscine (1–34)PTHrP (10−6–10−11 m) stimulated cortisol production in a dose-dependent manner. The ED50 of (1–34)PTHrP was 2.8 times higher than that of (1–39)ACTH, and maximum increase in cortisol production in response to 10−8 m of (1–34)PTHrP was approximately 7-fold lower than for 10−8 m of (1–39)ACTH. In contrast to (1–34)PTHrP, piscine (10–20)PTHrP, (79–93)PTHrP, and (100–125)PTHrP (10−9–10−7 m) did not stimulate cortisol production. The effect of piscine (1–34)PTHrP on cortisol production was abolished by N-terminal peptides in which the first amino acid (Ser) was absent and by simultaneous addition of inhibitors of the adenylyl cyclase-protein kinase A and phospholipase C-protein kinase C intracellular pathways but not by each separately. The PTHrP-induced signal transduction was further investigated by measurements of cAMP production and [H3]myo-inositol incorporation in an interrenal cell suspension. Piscine (1–34)PTHrP increased cAMP and total inositol phosphate accumulation, which is indicative that the mechanism of action of PTHrP in interrenal tissue involves the activation of both the adenylyl cyclase-cAMP and phospholipase C-inositol phosphate signaling pathways. These results, together with the expression of mRNA for PTHrP and for PTH receptor (PTHR) type 1 and PTHR type 3 receptors in sea bream interrenal tissue, suggest a specific paracrine or autocrine steroidogenic action of PTHrP mediated by the PTHRs.
- CRTAC1 homolog proteins are conserved from cyanobacteria to man and secreted by the teleost fish pituitary glandPublication . Redruello, Begoña; Louro, Bruno; Anjos, Liliana; Silva, Nádia; Greenwell, Roger S.; Canario, Adelino V. M.; Power, DeborahCartilage acidic protein 1 (CRTAC1) gene expression is used as a marker for chondrocyte differentiation instem cell-based tissue engineering. It is also transcribed outside the skeleton where at least two different transcripts are expressed in lung and brain. In the pituitary gland of the teleost fish sea bream Sparus auratus, we have found a transcript with a high degree of sequence identity to CRTAC1 family members but lacking the EGF-like calcium-binding domain encoding sequence of CRTAC1 and designated it as CRTAC2. Database searches revealed many previously unidentified members of the CRTAC1 and CRTAC2 in phylogenetically distant organisms, such as cyanobacteria, bryophyta, lancelets, and diverse representatives of vertebrates. Phylogenetic analyses showed that the genes encoding CRTAC1 and CRTAC2 proteins coexist in teleost fish genomes. Structural prediction analysis identified the N-terminal region of the CRTAC1/CRTAC2 family members as a potential seven-bladed β -propeller structure, closely related to those of integrin α chains and glycosylphosphatidylinositol-specific phospholipase D1 protein families. This relationship is con fi rmed by phylogenetic analysis with the N-terminal domain of sea bream CRTAC2 as the most divergent sequence. Because teleost fi shes are the only phylogenetic group where both CRTAC1 and CRTAC2 genes are present, they occupy a pivotal position in studies of the mechanisms governing the speci fi c expression patterns of each gene/protein subfamily. This will be essential to elucidate their respective biological roles.
- Four stanniocalcin genes in teleost fish: Structure, phylogenetic analysis, tissue distribution and expression during hypercalcemic challengePublication . Schein, V; Schein, Vanessa; Cardoso, João CR; Pinto, Patricia IS; Anjos, Liliana; Silva, Nadia; Power, Deborah; Canario, Adelino V. M.Stanniocalcin (STC), first isolated from the corpuscles of Stannius (CS) of teleost fishes and a systemic regulator of mineral metabolism, is present in all vertebrates as two isoforms, STC1 and STC2, encoded by separate genes. Here we show that the genome of Tetraodon nigroviridis, and other teleosts, possess duplicate genes for each STC isoform, designated stc1-a and -b, and stc2-a and -b. Stc1-a was cloned from CS, stc2-a from muscle and the two novel cDNAs, stc1-b and stc2-b, from brain. However, stc2-b was isolated as a conjoined (read-through) transcript with bod1 (bi-orientation defective 1, or FAM44B), and two additional alternative conjoined transcripts were also isolated. The predicted STC products shared the typical vertebrate 10 conserved cysteine residues and N-linked glycosylation motifs, in addition to specific features. Gene structure was generally conserved with four exons and three introns with the exception of stc1-a which gained an extra intron in exon three, originating one extra exon. Gene order and synteny is also maintained across vertebrates and the cpeb4 gene identified in the homologue region of the chordate Ciona was linked to vertebrate stc2 but not stc1. Immunohistochemistry in different species revealed that STC1-A was found only in CS and in a few cells in kidney. STC1-B had a restricted expression and was more prominent in the gills. STC2-A was detected in a variety of tissues, including pituitary, with most abundant immunoreaction in kidney cells and gill rakers and the CS was negative. Expression of stc1-a in CS of Tetraodon was 15-fold (p < 0.05) up-regulated 2 h after transfer from 2.9 mM Ca2+ to 10 mM Ca2+ water and down-regulated after 12 hours to 11-fold lower than 2.9 mM Ca2+ fish (p < 0.05). With the exception of stc1-a in CS, low expression levels and high individual variation were generally found for the expression of stc transcripts in kidney and gills, with no statistically significant changes in response to the hypercalcemic shock. In conclusion, both stc1 and stc2 genes are represented by paralogues in teleosts genomes and the analysis performed suggests that only stc1-a in the CS is involved in extracellular calcium regulation. The widespread distribution of stcs in fish tissues supports pleiotropic roles.
- Cartilage Acidic Protein 2 a hyperthermostable, high affinity calcium-binding proteinPublication . Anjos, Liliana; 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.
- PTHrP-induced modifications of the sea bream (Sparus auratus) vertebral bone proteomePublication . Anjos, Liliana; 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.