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Sci Rep
2016 Feb 26;6:21506. doi: 10.1038/srep21506.
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Shedding light on ovothiol biosynthesis in marine metazoans.
Castellano I
,
Migliaccio O
,
D'Aniello S
,
Merlino A
,
Napolitano A
,
Palumbo A
.
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Ovothiol, isolated from marine invertebrate eggs, is considered one of the most powerful antioxidant with potential for drug development. However, its biological functions in marine organisms still represent a matter of debate. In sea urchins, the most accepted view is that ovothiol protects the eggs by the high oxidative burst at fertilization. In this work we address the role of ovothiol during sea urchin development to give new insights on ovothiol biosynthesis in metazoans. The gene involved in ovothiol biosynthesis OvoA was identified in Paracentrotus lividus genome (PlOvoA). PlOvoA embryo expression significantly increased at the pluteus stage and was up-regulated by metals at concentrations mimicking polluted sea-water and by cyclic toxic algal blooms, leading to ovothiol biosynthesis. In silico analyses of the PlOvoA upstream region revealed metal and stress responsive elements. Structural protein models highlighted conserved active site residues likely responsible for ovothiol biosynthesis. Phylogenetic analyses indicated that OvoA evolved in most marine metazoans and was lost in bony vertebrates during the transition from the aquatic to terrestrial environment. These results highlight the crucial role of OvoA in protecting embryos released in seawater from environmental cues, thus allowing the survival under different conditions.
Figure 1. Schematic representation of SpOvoA and PlOvoA gene loci.The exons are illustrated as green boxes. The promoter responsive elements are reported in different colors as indicated in the figure legend.
Figure 2. PlOvoA expression profile and response to environmental stress conditions.(A) PlOvoA expression during embryo development. Eggs and embryos collected at different development stages (virgin eggs VE, fertilized eggs FE, early blastula EB, swimming blastula SB, prisma Pr and pluteus Pl) were examined for gene expression by Real Time qPCR. Fold differences in the expression levels of PlOvoA with respect to the reference gene Pl-Z12-1 were expressed in percentage with respect to highest levels of mRNA in VE (100%). *represents the significance respect to VE, **P < 0.01, ***P < 0.001. #represents the significance respect to Pr, ##P < 0.01. (B) Pl OvoA expression analysis in developing embryos after metal treatment. FE were treated with 1, 5, 10 μM Cd2+ and 36 μM Mn2+ and different developmental stages were examined for the transcriptional expression of PlOvoA. Data are reported as a fold difference in PlOvoA expression levels, compared to control (mean ± SEM), embryos developed in seawater without metals. Fold differences greater than ± 2 (see dotted horizontal guidelines at values of 2 and −2) were considered significant. (C) Ovothiol levels in developing embryos after metal treatment. FE were treated with 5 μM Cd2+ and SB and Pr were examined for ovothiol production. Data are reported as μmol ovothiol/mg of embryos. Results are representative of 3 independent experiments and expressed as means ± SEM, and analyzed by unpaired t-test. *represents the significance respect to the control, **P < 0.01. D. PlOvoA expression analysis in developing embryos after maternal exposure to O. cf ovata bloom. Sea urchin females, exposed to Ostreopsis cf. ovata bloom in July at Gaiola site, were collected in October and fertilized. Different developmental stages (EB, SB, Pr and Pl) were examined for PlOvoA expression. Data (mean ± SEM) are reported as a fold difference in PlOvoA expression levels, compared to control embryos derived from sea urchins collected at control site. Fold differences greater than ± 2 were considered significant.
Figure 3. Characterization of OvoA protein in sea urchins.(A) Sequence alignment of PlOvoA and SpOvoA. DinB superfamily domain (36–176 aa, PlOvoA) in the N-terminal region is boxed in blue. The putative iron binding motif (HX3HXE) is indicated in red. The FGE-sulfatase domain (211–491 aa) is boxed in magenta and the SAM-transferase domain (572–743) in the C-terminal region is boxed in green. The residues (581–587, 602–603, 661–663, 680) belonging to the SAM-binding site are indicated in blue. The putative residues accounting for binding to cysteine and histidine are highlighted in yellow. (B) Schematic representation of OvoA and EgtB primary structure. DinB_2 superfamily domain is boxed in blue and the putative iron-binding site is indicated by an arrow, FGE-sulfatase domain is boxed in magenta. SAM-transferase domain is boxed in green. SAM binding sites are highlighted by arrows.
Figure 4. Structural model of PlOvoA.(A,B) Ribbon representation of the two domains shared by MtEgtB (panel A, in cyan) and PlOvoA (panel B, in green). The iron-binding site and the conserved His residues are also shown as ball and stick. (C,D) Comparison between the active sites of MtEgtB and PlOvoA. MtEgtB (cyan) and PlOvoA (green) catalyze C-S bond formation and sulfoxidation between gamma-glutamyl cysteine and N-alpha-trimethyl histidine or between cysteine and histidine as the central steps in the synthesis of ergothioneine and ovothiol, respectively. Residues involved in the recognition of iron are conserved, whereas those involved in the recognition of N-alpha-trimethyl histidine or important for the binding of gamma-glutamyl cysteine in MtEgtB are not conserved in PlOvoA, according to the different substrates (N-alpha-trimethyl histidine versus His and gamma-glutamyl cysteine versus cysteine) of these enzymes.
Figure 5. Schematic phylogenetic tree of OvoA in metazoans.The diagram is representative of the different metazoan phyla, in which OvoA is present (see Table 1 for protein sequence ID).
Figure 6. Proposed model for ovothiol biosynthesis regulation.Broadcast spawners, i.e. sea urchins, release eggs and sperm in the column seawater, where fertilization occurs. Toxins and heavy metals, dispersed in seawater, induce oxidative stress in the embryos. ROS production can affect transcription factors, which bind to MRE and/or SRE and activate OvoA transcription. Finally, OvoA enzyme catalyzes the synthesis of ovothiol to counteract redox unbalance.
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