Cloning and expression of glutathione peroxidase genes in the chordate invertebrate Ciona intestinalis.

Ascidians represent interesting models from an evolutionary and ecotoxicology point of view, because of their large distribution in temperate sea and their phylogenetic position of invertebrate chordates. Immune responses imply an increase in oxygen consumption with a consequent risk of oxidative stress. With the aim to study the components of the antioxidant defense system in the solitary ascidian Ciona intestinalis, we have characterized the genes codifying for two glutathione peroxidases (GPx), metalloenzymes that catalyze the reduction of hydrogen peroxide or organic hydroperoxides to water or corresponding alcohols, using reduced glutathione (GSH) as an electron donor. In the GeneBank database five GPx-like sequences from C. intestinalis are present, but only two of those genes demonstrated an effective transcription after cloning and sequencing analyses. The respective proteins, named Ci-GPx7 and Ci-GPxb, show a good level of sequence conservation with metazoan orthologs, especially for residues that are important for the catalytic activity. In the 3’-UTR region of Ci-GPxb cDNA we have found a typical SECIS-element, confirming that this protein may be a selenium GPx. Phylogenetic reconstruction, performed with Bayesian methods using metazoan GPxs, indicate that Ci-GPxb emerges in the tree with the tetrameric GPxs, confirming as previously hypothesized. Preliminary data, obtained by homology modeling, confirmed the tetrameric structure and show that the GPxb is similar to GPx3 from Homo sapiens, a selenium protein. Thus, we propose to name this gene ci-gpx3. As expected, Ci-GPx7 clusterized with other GPx7s. The transcription of both these genes, measured by RT-sqPCR, resulted inducible by Cd, Cu and Zn, which have different effects. The peroxidase activity decreases in the cell-free extract from specimens treated with each considered metals, probably in relation to metal-induction of GSH biosynthesis, as indicated by the presence of positive correlation by the time-dependent Cd accumulation an increase of ROS and GSH production. The data presented here improved our knowledge about the evolution of the antioxidant system in metazoans and emphasize the importance of a complex regulation for the antioxidant system, including the transcription of ci-gpx7 and ci-gpx3 genes, which can create an efficient detoxification pathway allowing C. intestinalis to survive in the presence of metals in the environment.