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Osteocalcin (OC) and matrix Gla protein (MGP) are considered evolutionarily related because they share key structural features, although they have been described to exert different functions. In this work, we report the identification and characterization of both OC and MGP from the Adriatic sturgeon, a ray-finned fish characterized by a slow evolution and the retention of many ancestral features. Sturgeon MGP shows a primary structure, post-translation modifications, and patterns of mRNA/protein distribution and accumulation typical of known MGPs, and it contains seven possible Gla residues that would make the sturgeon protein the most gamma-carboxylated among known MGPs. In contrast, sturgeon OC was found to present a hybrid structure. Indeed, although exhibiting protein domains typical of known OCs, it also contains structural features usually found in MGPs (e. g. a putative phosphorylated propeptide). Moreover, patterns of OC gene expression and protein accumulation overlap with those reported for MGP; OC was detected in bone cells and mineralized structures but also in soft and cartilaginous tissues. We propose that, in a context of a reduced rate of evolution, sturgeon OC has retained structural features of the ancestral protein that emerged millions of years ago from the duplication of an ancient MGP gene and may exhibit intermediate functional features. Portuguese Science and Technology Foundation [POCTI/MAR/57921/2004, SFRH/BD/9077/2002]; Fundo Europeu De Desenvolvimento Regional (FEDER) (Portugal); National Funding; Center of Marine Sciences (CCMAR)

Osteocalcin 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.

Submitted by Paulo Gavaia (pgavaia@ualg.pt) on 2014-06-06T09:52:10Z No. of bitstreams: 1 PepT1 in a coldwater marine teleost larvae atlantic cod cloning and preliminary studies of expression and phylogeny.pdf: 92089 bytes, checksum: 29d7c838f8ab20cd979bf7fa808abca9 (MD5) Approved for entry into archive by Pedro Sousa (a19661@ualg.pt) on 2014-06-25T12:43:54Z (GMT) No. of bitstreams: 1 PepT1 in a coldwater marine teleost larvae atlantic cod cloning and preliminary studies of expression and phylogeny.pdf: 92089 bytes, checksum: 29d7c838f8ab20cd979bf7fa808abca9 (MD5) Made available in DSpace on 2014-06-25T12:43:54Z (GMT). No. of bitstreams: 1 PepT1 in a coldwater marine teleost larvae atlantic cod cloning and preliminary studies of expression and phylogeny.pdf: 92089 bytes, checksum: 29d7c838f8ab20cd979bf7fa808abca9 (MD5) Previous issue date: 2006

The 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).

Introduction: RUNX2 (also known as CBFA1/Osf2/AML3/PEBP2 alpha A) is a transcription factor essential for bone formation in mammals, as well as for osteoblast and chondrocyte differentiation, through regulation of expression of several bone- and cartilage-related genes. Since its discovery, Runx2 has been the subject of intense studies, mainly focused in unveiling regulatory targets of this transcription factor in high vertebrates. However, no single study has been published addressing the role of Runx2 in bone metabolism of low vertebrates. While analyzing the zebrafish (Danio rerio) runx2 gene, we identified the presence of two orthologs of RUNX2, which we named runx2a and runx2b and cloned a pebp2 alpha A-like transcript of the runx2b gene, which we named pebp2 alpha A2. Materials and Methods: Zebrafish runx2b gene and cDNA were isolated by RT-PCR and sequence data mining. The 3D structure of runx2b runt domain was modeled using mouse Runx1 runt as template. The regulatory effect of pebp2 alpha A2 on osteocalcin expression was analyzed by transient co-transfection experiments using a luciferase reporter gene. Phylogenetic analysis of available Runx sequences was performed with TREE-PUZZLE 5.2. and MrBayes. Results and Conclusions: We showed that the runx2b gene structure is highly conserved between mammals and fish. Zebrafish runx2b has two promoter regions separated by a large intron. Sequence analysis suggested that the runx2b gene encodes three distinct isoforms, by a combination of alternative splicing and differential promoter activation, as described for the human gene. We have cloned a pebp2 alpha A-like transcript of the runx2b gene, which we named pebp2 alpha A2, and showed its high degree of sequence similarity with the mammalian pebp2 alpha A. The cloned zebrafish osteocalcin promoter was found to contain three putative runx2-binding elements, and one of them, located at -221 from the ATG, was capable of mediating pebp2 alpha A2 transactivation. In addition, cross-species transactivation was also confirmed because the mouse Cbfa1 was able to induce the zebrafish osteocalcin promoter, whereas the zebrafish pebp2 alpha A2 activated the murine osteocalcin promoter. These results are consistent with the high degree of evolutionary conservation of these proteins. The 3D structure of the runx2b runt domain was modeled based on the runt domain of mouse Runx1. Results show a high degree of similarity in the 3D configuration of the DNA binding regions from both domains, with significant differences only observed in non-DNA binding regions or in DNA-binding regions known to accommodate considerable structure flexibility. Phylogenetic analysis was used to clarify the relationship between the isoforms of each of the two zebrafish Runx2 orthologs and other Runx proteins. Both zebrafish runx2 genes clustered with other Runx2 sequences. The duplication event seemed, however, to be so old that, whereas Runx2b clearly clusters with the other fish sequences, it is unclear whether Runx2a clusters with Runx2 from higher vertebrates or from other fish. info:eu-repo/semantics/publishedVersion

Gla proteins, as the name indicates, undergo a post-translation modification where specific glutamic acid residues are γ-carboxylated through the action of the ubiquitous enzyme γ-carboxylase and using vitamin K as cofactor. Therefore, these proteins are also called vitamin K dependent proteins or VKD [1, 2]. We can assign VKDs to essentially four different groups: 1) those involved in blood coagulation (such as prothrombin and various coagulation factors; the first group to be discovered), 2) those involved in tissue mineralization (bone and matrix Gla proteins), 3) a nerve growth factor (gas6), and 4) those of unknown function (the latest group to be discovered). The carboxylase enzyme is present in fly and worm but not in yeast, indicating that γ-carboxylation is likely a feature appearing in multicellular eukaryotes.

Functional 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.

Evaluation 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. PG and JD supported by a doctoral and postdoctoral fellowship (PRAXIS/BB/19665/1999). This project was funded by the project MCYT/AGL/2003-03558 (SPARUGENES), from the Spanish Ministry for Science and Technology.

Fish 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.