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- Matrix gla protein in turbot (Scophthalmus maximus): gene expression analysis and identification of sites of protein accumulationPublication . Roberto, Vania Palma; Cavaco, S.; S B Viegas, Carla; Simes, D; Ortiz-Delgado, J. B.; Sarasquete, C.; Gavaia, Paulo J.; Cancela, LeonorMatrix Gla protein (Mgp) is a secreted vitamin K-dependent extracellular matrix protein and a physiological inhibitor of calcification whose gene structure, amino acid sequence and tissue distribution have been conserved throughout evolution. In the present work, the turbot (Scophthalmus maximus) mgp cDNA was cloned and the sequence of the deduced protein compared to that of other vertebrates. As expected, it was closer to teleosts than to other vertebrate groups but there was a strict conservation of amino-acids thought to be important for protein function. Analysis of mgp gene expression indicated branchial arches as the site with higher levels of expression, followed by heart, vertebra and kidney. These results were confirmed by in situ hybridization with a strong mgp expression in branchial arch chondrocytes. Mgp was found to accumulate in gills where it appeared to be restricted to chondrocytes from branchial filaments, while in vertebrae it was localized in vertebral end plates, in growth zones, in vertebral arches and spines and in notochord cells. In the soft tissues analysed, Mgp was mainly detected in kidney and heart, consistent with previous data and providing further evidence for a role of Mgp as a calcification inhibitor and a modulator of the mineralization process. Our studies provide evidence that turbot, an important new species for aquaculture, is also a useful model to study function and expression of Mgp.
- Gla-rich protein (GRP), a new vitamin K-dependent protein identified from sturgeon cartilage and highly conserved in vertebratesPublication . S B Viegas, Carla; Simes, D; Laizé, Vincent; Williamson, M. K.; Price, P. A.; Cancela, LeonorWe report the isolation of a novel vitamin K-dependent protein from the calcified cartilage of Adriatic sturgeon (Acipenser nacarii). This 10.2-kDa secreted protein contains 16 -carboxyglutamic acid (Gla) residues in its 74-residue sequence, the highest Gla percent of any known protein, and we have therefore termed it Gla-rich protein (GRP). GRP has a high charge density (36 negative 16 positive 20 net negative) yet is insoluble at neutral pH. GRP has orthologs in all taxonomic groups of vertebrates, and a paralog (GRP2) in bony fish; no GRP homolog was found in invertebrates. There is no significant sequence homology between GRP and the Gla-containing region of any presently known vitamin K-dependent protein. Forty-seven GRP sequences were obtained by a combination of cDNA cloning and comparative genomics: all 47 have a propeptide that contains a -carboxylase recognition site and a mature protein with 14 highly conserved Glu residues, each of them being carboxylated in sturgeon. The protein sequence of GRP is also highly conserved, with 78% identity between sturgeon and human GRP. Analysis of the corresponding gene structures suggests a highly constrained organization, particularly for exon 4, which encodes the core Gla domain. GRP mRNA is found in virtually all rat and sturgeon tissues examined, with the highest expression in cartilage. Cells expressing GRP include chondrocytes, chondroblasts, osteoblasts, and osteocytes. Because of its potential to bind calcium through Gla residues, we suggest that GRP may regulate calcium in the extracellular environment.
- Whole-genome sequence analysis: Evidences for new osteocalcin isoforms in fish and tetrapodsPublication . Laizé, Vincent; Gavaia, Paulo J.; S B Viegas, Carla; Cancela, LeonorThe evolution of calcified tissues is a defining feature in vertebrate evolution. Investigating evolution of proteins involved in tissue calcification should help elucidate how calcified tissues have evolved. Osteocalcin (OC) is a small calcium-binding protein accumulated exclusively in bone and teeth, whose function, although essential for tissue calcification, remains unclear. Until recently, only a single osteocalcin isoform had been described (OC I).
- Expression of the oligopeptide transporter PepT1 (Solute carrier family 15, member 1), in fed and starved larval Zebra fish (Danio rerio)Publication . Brito, A. B.; Rønnestad, I.; Gavaia, Paulo J.; S B Viegas, Carla; Cancela, LeonorFish embryos develop while utilizing yolk nutrients supplied by the (...). There normally exists a "first feeding window" between when the larvae (...) able to ingest exogenous feed,and the "point of no return" where (...) exhausted its yolk reserves and also irreversibly degraded critical tissues (...) energetic purposes it feeds is not ingested.
- Cloning and ontogenetic expression of the oligopeptide transporter PepT1 (Solute carrier family 15, member 1), in fed and starved larval Atlantic cod, Gadus morhua LPublication . Rønnestad, I.; Gavaia, Paulo J.; S B Viegas, Carla; Cancela, LeonorFunctional characteristics of marine fish larvae digestive system at the onset of exogenous feeding have long been a subject for discussion. Absorption of digested proteins in vertebrates has been shown to include transporter systems for single amino acids and small peptides.
- Identification of a promoter element within the zebrafish Collagen X¿1 gene responsive to Runx2 isoforms Osf2/Cbfa1 and til-1 but not to pebp2aA2Publication . Simões, B.; Conceição, N.; S B Viegas, Carla; Pinto, Jorge; Gavaia, Paulo J.; Kelsh, R. N.; Cancela, LeonorType X collagen is a short chain collagen specifically expressed by hypertrophic chondrocytes during endochondral ossification. We report here the functional analysis of the zebrafish (Danio rerio) collagen Xa1 gene (colXa1) promoter with the identification of a region responsive to two isoforms of the runt domain transcription factor runx2.
- Oligopeptide transporter PepT1 in Atlantic cod (Gadus morhua L.): cloning, tissue expression and comparative aspectsPublication . Rønnestad, I.; Gavaia, Paulo J.; S B Viegas, Carla; Verri, T.; Nilsen, T. O.; Jordal, A. E. O.; Kamisaka, Y.; Cancela, LeonorA novel full-length cDNA that encodes for the Atlantic cod (Gadus morhua L.) PepT1-type oligopeptide transporter has been cloned. This cDNA (named codPepT1) was 2838·bp long, with an open reading frame of 2190·bp encoding a putative protein of 729 amino acids. Comparison of the predicted Atlantic cod PepT1 protein with zebrafish, bird and mammalian orthologs allowed detection of many structural features that are highly conserved among all the vertebrate proteins analysed, including (1) a larger than expected area of hydrophobic amino acids in close proximity to the N terminus; (2) a single highly conserved cAMP/cGMP-dependent protein kinase phosphorylation motif; (3) a large N-glycosylationrich region within the large extracellular loop; and (4) a conserved and previously undescribed stretch of 8–12 amino acid residues within the large extracellular loop. Expression analysis at the mRNA level indicated that Atlantic cod PepT1 is mainly expressed at intestinal level, but that it is also present in kidney and spleen. Analysis of its regional distribution along the intestinal tract of the fish revealed that PepT1 is ubiquitously expressed in all segments beyond the stomach, including the pyloric caeca, and through the whole midgut. Only in the last segment, which included the hindgut, was there a lower expression. Atlantic cod PepT1, the second teleost fish PepT1-type transporter documented to date, will contribute to the elucidation of the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, it can represent a useful tool for the study of gut functional regionalization, as well as a marker for the analysis of temporal and spatial expression during ontogeny.
- Identification of an osteocalcin isoform in fish with a large acidic prodomainPublication . Laizé, Vincent; S B Viegas, Carla; Price, P. A.; Cancela, LeonorOsteocalcin is a small, secreted bone protein whose gene consists of four exons. In the course of analyzing the structure of fish osteocalcin genes, we recently found that the spotted green pufferfish has two possible exon 2 structures, one of 15 bp and the other of 324 bp. Subsequent analysis of the pufferfish cDNA showed that only the transcript with a large exon 2 exists. Exon 2 codes for the osteocalcin propeptide, and exon 2 of pufferfish osteocalcin is ∼3.4-fold larger than exon 2 previously found in other vertebrate species. We have termed this new pufferfish osteocalcin isoform OC2. Additional studies showed that the OC2 isoform is restricted to a unique fish taxonomic group, the Osteichthyes; OC2 is the only osteocalcin isoform found so far in six Osteichthyes species, whereas both OC1 and OC2 isoforms coexist in zebrafish and rainbow trout. The larger size of the OC2 propeptide is due to an acidic region that is likely to be highly phosphorylated and has no counterpart in the OC1 propeptide. We propose 1) that OC1 and OC2 are encoded by distinct genes that originated from a duplication event that probably occurred in the teleost fish lineage soon after divergence from tetrapods and 2) that the novel OC2 propeptide could be, if secreted, a phosphoprotein that participates in the regulation of biomineralization through its large acidic and phosphorylated propeptide.
- Gla proteins as markers for studies of skeletal development and malformations in new aquaculture fish species (Pagrus auriga, Diplodus sargo and Scophtalmus maximus)Publication . S B Viegas, Carla; Gavaia, Paulo J.; Ortiz-Delgado, J. B.; Sarasquete, C.; Cancela, LeonorEvaluation of the onset and the development of skeletal structures during larval ontogeny of commercially important species in southern Iberia Peninsula and Mediterranean area (...) is essential for the successful production of high quality fish.
- Evolution of matrix and bone gamma-carboxyglutamic acid proteins in vertebratesPublication . Laizé, Vincent; Martel, Paulo; Viegas, Carla; Price, P. A.; Cancela, LeonorThe evolution of calcified tissues is a defining feature in vertebrate evolution. Investigating the evolution of proteins involved in tissue calcification should help elucidate how calcified tissues have evolved. The purpose of this study was to collect and compare sequences of matrix and bone γ-carboxyglutamic acid proteins (MGP and BGP, respectively) to identify common features and determine the evolutionary relationship between MGP and BGP. Thirteen cDNAs and genes were cloned using standard methods or reconstructed through the use of comparative genomics and data mining. These sequences were compared with available annotated sequences (a total of 48 complete or nearly complete sequences, 28 BGPs and 20 MGPs) have been identified across 32 different species (representing most classes of vertebrates), and evolutionarily conserved features in both MGP and BGP were analyzed using bioinformatic tools and the Tree-Puzzle software. We propose that: 1) MGP and BGP genes originated from two genome duplications that occurred around 500 and 400 million years ago before jawless and jawed fish evolved, respectively; 2) MGP appeared first concomitantly with the emergence of cartilaginous structures, and BGP appeared thereafter along with bony structures; and 3) BGP derives from MGP. We also propose a highly specific pattern definition for the Gla domain of BGP and MGP. Previous Section Next Section BGP1 (bone Gla protein or osteocalcin) and MGP (matrix Gla protein) belong to the growing family of vitamin K-dependent (VKD) proteins, the members of which are involved in a broad range of biological functions such as skeletogenesis and bone maintenance (BGP and MGP), hemostasis (prothrombin, clotting factors VII, IX, and X, and proteins C, S, and Z), growth control (gas6), and potentially signal transduction (proline-rich Gla proteins 1 and 2). VKD proteins are characterized by the presence of several Gla residues resulting from the post-translational vitamin K-dependent γ-carboxylation of specific glutamates, through which they can bind to calcium-containing mineral such as hydroxyapatite. To date, VKD proteins have only been clearly identified in vertebrates (1) although the presence of a γ-glutamyl carboxylase has been reported in the fruit fly Drosophila melanogaster (2) and in marine snails belonging to the genus Conus (3). Gla residues have also been found in neuropeptides from Conus venoms (4), suggesting a wider prevalence of γ-carboxylation.