Cloning, Expression And Functional Characterization Of Antioxidants From The Jellyfish Cyanea Capillata

Jellyfish is one group of numerous marine planktons. Scientists have discovered thatthere are many kinds of highly bioactive substances in the body of jellyfish, including avariety of toxins and new functional proteins, which may have good prospects for thedevelopment of new marine drugs. As a representative of macroplankton, jellyfish livesin pelagic oceanic waters, which are continuously exposed to harsh environmental factors,such as strong sunlight and ultraviolet (UV) radiation, which may lead to an increase inthe production of reactive oxygen species (ROS). At low concentrations, ROS mayfacilitate processes, such as intracellular signaling and defense against microorganisms.However, oxidative stress may occur in the case of excessive production andaccumulation of ROS, which would result in a disturbance of metabolic balance, causingdamage to cellular lipids, proteins and DNA. It is already established that manyorganisms have both enzymatic and non-enzymatic antioxidant defense mechanisms tominimize such injuries. Scientists have isolated some proteins from the jellyfish,Rhopilema esculentum and Stomolophus meleagris, which have strong free radicalscavenging abilities and can protect mouse skin lipid and collagen from UV radiationdamage. However, the composition of the antioxidant system in jellyfish species and thesequences, expression levels and bioactivities of some important antioxidant enzymeshave not yet been reported until now.Research objectives:To define the activity of three important antioxidants from the jellyfish Cyaneacapillata, to understand the material basis of the antioxidant system of jellyfish, and toprovide scientific foundation for developing new types of marine drugs as a result.Methods:1. Screening of the antioxidants: Large-scale sequencing on the cDNA library of thetentacle of the jellyfish C. capillata we made before, then the EST sequences werecompared with those in the GenBank database using the BLASTx program to identify thegenes encoding possible antioxidants. The BLASTx algorithm revealed that three of theEST sequences from the cDNA library showed a significant similarity to proteinsequences of the thioredoxin1, peroxiredoxin4or Cu/Zn superoxide dismutase family, respectively. Thus, these two EST sequences were chosen for further analysis anddesignated as CcTrx1, CcPrx4and CcCuZnSOD, respectively. Subsequently, completesequencing of both strands of CcTrx1, CcPrx4and CcCuZnSOD cDNA were carried outto confirm that they were full-length cDNA.2. Bioinformatics analysis of the full-length cDNA: The open reading frames (ORF),amino acid composition, conserved domains, signal peptide, phylogenetic trees,molecular mass, isoelectric points, secondary structures and three-dimensional structuresof CcTrx1, CcPrx4and CcCuZnSOD protein were determined or predicted through theuse of a variety of bioinformatic softwares and online tools.3. Construction of the recombinant plasmids, and expression and purification ofrecombinant proteins in Escherichia coli: Primers designed with restriction enzyme wereused to amplify the coding region of CcTrx1, CcPrx4and CcCuZnSOD cDNA, and PCRfragments were ligated with the pET-24a vector to construct the recombinant plasmids.The recombinant plasmid was then transformed into the E. coli for protein expression.After the protein induction, the bacteria were subjected to a centrifugation, and thepellets were collected and resuspended in binding buffer. Subsequently, the resuspendedbacterial pellets were lysed by sonication in an ice bath, and the lysate was centrifuged at12,000×g for30min at4°C. The supernatant was collected and applied to an KTAprotein purification system using a HisTrap High Performance (HP) chelating column.4. Antioxidant activities of rCcTrx1, rCcPrx4and rCcCuZnSOD protein:(1) Disulfide reductase activity of the rCcTrx1protein: The reaction mixturecontained a final volume of1mL with100mM phosphate buffered saline (PBS, pH7.0),1.25mg/mL bovine insulin (Sigma, USA),2mM ethylenediamine tetraacetic acid(EDTA) and8μg/mL purified rCcTrx1protein or the heat-inactivated purified rCcTrx1protein as negative control. The reaction was initiated by adding2μL of1M DTT andmonitored by measuring absorbance at650nm, and a group of8μg/mL purified rCcTrx1protein without adding DTT was used as another negative control.(2) In vitro peroxidase activity assay of rCcPrx4protein:1mL reaction mixturescontaining50mM HEPES (pH7.0),5mM DTT and the recombinant CcPrx4protein or100μg/mL heat-inactivated recombinant CcPrx protein (control group), were incubatedat room temperature for10min. H2O2was added to a final concentration of100μM toinitiate the reactions, and then, they were incubated for0,2.5,5,7.5and10min at roomtemperature. Subsequently,100μL of100%(w/v) trichloroacetic acid (TCA) was added to stop the reaction. The mixture was centrifuged to remove the precipitate, followed bythe addition of200μL of10mM Fe(NH4)2(SO4)2and100μL of2.5M KSCN, whichcould react with the remaining H2O2and generated the red-colored ferrothiocyanatecomplex. The remaining amount of H2O2in the mixture was estimated by measurementof the red ferrothiocyanate complex. The absorbance was measured at475nm.(3) SOD activity of rCcCuZnSOD was examined using the SOD Assay Kit (WST-1method) based on the manufacturer’s instruction.(4) Metal-catalyzed oxidation (MCO) assay of rCcTrx1and rCcPrx4protein:50mLreaction mixtures containing50mM HEPES (pH7.0),35μM FeCl3,10mM DTT,1μgsupercoiled plasmid DNA of the pET-24a vector and CcPrx4protein ranging from25to200μg/mL, were incubated at37°C for2h. At the end of the incubation, the reactionmixture was subjected to1%(w/v) agarose gel electrophoresis containing GoldenView as the DNA stain to assess the DNA protection effect.Results:The CcTrx1, CcPrx4and CcCuZnSOD cDNA encoding the mature proteins (notincluding the signal peptide) were amplified from the C. capillata tentacle cDNA libraryand cloned into the pET-24a expression vector. The encoded proteins were expressed in E.coli and then successfully purified using column chromatography. The results ofbioactivity assessment of the rCcTrx1, rCcPrx4and rCcCuZnSOD proteins: the rCcTrx1protein distinctly displayed a specific activity to reduce insulin disulfides in atime-dependent manner and insulin reduction was rapidly increased almost from thebeginning of incubation, indicating that CcTrx1protein could act as an effective disulfidereductase in C. capillata. Moreover, the rCcPrx4protein distinctly displayed a time-andconcentration-dependent activity to reduce H2O2. At25μg/mL of rCcPrx4protein, theclearance rate was slightly increased compared with the control group. When the proteinconcentration of rCcPrx4protein reached75and100μg/mL, the clearance rates weremarkedly increased to very high levels. Before2.5min, the clearance rates were rapidlyincreased. However, the reactions have entered a comparatively stationary process2.5min later. Besides, both of rCcTrx1and rCcPrx4protein showed the ability to protect thesupercoiled structure of pET-24a plasmid DNA against Fe3+catalyzed oxidative damage.And rCcCuZnSOD protein displayed strong activity to reduce superoxide anion (O2-). Conclusions:We first identified and characterized a thioredoxin, a peroxiredoxin and asuperoxide dismutase, designated as CcTrx1, CcPrx4and CcCuZnSOD, respectively,from the cDNA library of the tentacle of the jellyfish C. capillata. Quantitative real-timePCR analysis showed that CcTrx1/CcPrx4mRNA could be detected in all the jellyfishtissues analyzed. CcTrx1, CcPrx4and CcCuZnSOD protein were cloned into theexpression vector pET-24a, and expressed in E. coli. Recombinant proteins was purifiedby a HisTrap High Performance chelating column chromatography and analyzed for itsbiological function. The results showed that the purified recombinant CcTrx1/CcPrx4protein manifested the ability to protect supercoiled DNA from oxidative damage. Inaddition, rCcTrx1protein distinctly displayed a specific activity to reduce insulindisulfides, rCcPrx4protein had a strong efficiency and speed to remove H2O2, andrCcCuZnSOD protein displayed strong activity to reduce superoxide anion (O2-). Thisresearch suggested that CcTrx1, CcPrx4and CcCuZnSOD protein are playing importantroles in protecting jellyfish from oxidative damage.