|Identification of Sparus aurata bone morphogenetic protein 2: molecular cloning, gene expression and in silico analysis of protein conserved features in vertebrates.
|Rafael, MS, Laizé, V, M. Cancela, L
|Year of Publication
|Amino Acid Sequence, Animals, Base Sequence, Bone Morphogenetic Proteins, Cell Line, Chondrocytes, Cloning, Molecular, Conserved Sequence, DNA, Complementary, Evolution, Molecular, Gene Expression Regulation, Developmental, Molecular Sequence Data, Osteoblasts, Phylogeny, Protein Structure, Tertiary, RNA, Messenger, Sea Bream, Tissue Distribution
Bone morphogenetic protein 2 (BMP-2) is a secreted signaling molecule that acts as an inducer of bone formation and a regulator of embryonic development. The objectives of this work were as follows: (1) to clone the full-length cDNA of BMP-2 in a marine fish model, (2) analyze its gene expression during development, in adult tissues and in cell lines, and (3) identify protein conserved features of vertebrate BMP-2. Using a combination of RT- and 5'-RACE-PCR, a 1653-bp fragment corresponding to Sparus aurata BMP-2 cDNA (SaBMP-2) was amplified. Levels of SaBMP-2 gene expression were estimated using quantitative real-time PCR and shown to be strongly increased (150-fold induction) at gastrulation, thus suggesting a key role for BMP-2 in fish development. Tissue distribution of SaBMP-2 mRNA revealed highest levels in the calcified tissues bone, caudal fin and scales and in liver. BMP-2 was also found to be highly expressed in S. aurata bone-derived cell lines VSa13 and VSa16 and to be up-regulated (more than 10-fold induction) in mineralized VSa13 chondrocyte-like cells. Using bioinformatic tools and all vertebrate protein sequences available, conserved features of BMP-2 were characterized. The mature protein was shown to be highly conserved across 20 species indicating that BMP-2 function has been conserved throughout evolution, a finding that is in agreement with the widely accepted view of the important role played by BMPs in vertebrate development.