Molecular cloning and functional characterization of a Cu/Zn superoxide dismutase from jellyfish Cyanea capillata.

We identified and characterized a novel superoxide dismutase (SOD), designated as CcSOD1, from the cDNA library from the tentacle tissue of the jellyfish Cyanea capillata. The full-length cDNA sequence of CcSOD1 consists of 745 nucleotides with an open reading frame encoding a mature protein of 154 amino acids, sharing a predicted structure similar to the typical Cu/Zn-SODs. The CcSOD1 coding sequence was cloned into the expression vector pET-24a and successfully expressed in Escherichia coli Rosetta (DE3) pLysS. The recombinant protein rCcSOD1 was purified by HisTrap High Performance chelating column chromatography and analyzed for its biological function. Our results showed that the purified rCcSOD1 could inhibit superoxide anion and keep active in a pH interval of 4.5-9 and a temperature interval of 10-70°C. Even when heated at 70°C for 60 min, rCcSOD1 retained 100% activity, indicating a relatively high thermostability. These results suggest that CcSOD1 protein may play an important role in protecting jellyfish from oxidative damage and can serve as a new resource for antioxidant products.

Global Transcriptome Analysis of the Tentacle of the Jellyfish Cyanea capillata Using Deep Sequencing and Expressed Sequence Tags: Insight into the Toxin- and Degenerative Disease-Related Transcripts.

BACKGROUND: Jellyfish contain diverse toxins and other bioactive components. However, large-scale identification of novel toxins and bioactive components from jellyfish has been hampered by the low efficiency of traditional isolation and purification methods. RESULTS: We performed de novo transcriptome sequencing of the tentacle tissue of the jellyfish Cyanea capillata. A total of 51,304,108 reads were obtained and assembled into 50,536 unigenes. Of these, 21,357 unigenes had homologues in public databases, but the remaining unigenes had no significant matches due to the limited sequence information available and species-specific novel sequences. Functional annotation of the unigenes also revealed general gene expression profile characteristics in the tentacle of C. capillata. A primary goal of this study was to identify putative toxin transcripts. As expected, we screened many transcripts encoding proteins similar to several well-known toxin families including phospholipases, metalloproteases, serine proteases and serine protease inhibitors. In addition, some transcripts also resembled molecules with potential toxic activities, including cnidarian CfTX-like toxins with hemolytic activity, plancitoxin-1, venom toxin-like peptide-6, histamine-releasing factor, neprilysin, dipeptidyl peptidase 4, vascular endothelial growth factor A, angiotensin-converting enzyme-like and endothelin-converting enzyme 1-like proteins. Most of these molecules have not been previously reported in jellyfish. Interestingly, we also characterized a number of transcripts with similarities to proteins relevant to several degenerative diseases, including Huntington's, Alzheimer's and Parkinson's diseases. This is the first description of degenerative disease-associated genes in jellyfish. CONCLUSION: We obtained a well-categorized and annotated transcriptome of C. capillata tentacle that will be an important and valuable resource for further understanding of jellyfish at the molecular level and information on the underlying molecular mechanisms of jellyfish stinging. The findings of this study may also be used in comparative studies of gene expression profiling among different jellyfish species.

First report of a thioredoxin homologue in jellyfish: molecular cloning, expression and antioxidant activity of CcTrx1 from Cyanea capillata.

Thioredoxins (Trx proteins) are a family of small, highly-conserved and ubiquitous proteins that play significant roles in the resistance of oxidative damage. In this study, a homologue of Trx was identified from the cDNA library of tentacle of the jellyfish Cyanea capillata and named CcTrx1. The full-length cDNA of CcTrx1 was 479 bp with a 312 bp open reading frame encoding 104 amino acids. Bioinformatics analysis revealed that the putative CcTrx1 protein harbored the evolutionarily-conserved Trx active site 31CGPC34 and shared a high similarity with Trx1 proteins from other organisms analyzed, indicating that CcTrx1 is a new member of Trx1 sub-family. CcTrx1 mRNA was found to be constitutively expressed in tentacle, umbrella, oral arm and gonad, indicating a general role of CcTrx1 protein in various physiological processes. The recombinant CcTrx1 (rCcTrx1) protein was expressed in Escherichia coli BL21 (DE3), and then purified by affinity chromatography. The rCcTrx1 protein was demonstrated to possess the expected redox activity in enzymatic analysis and protection against oxidative damage of supercoiled DNA. These results indicate that CcTrx1 may function as an important antioxidant in C. capillata. To our knowledge, this is the first Trx protein characterized from jellyfish species.

First report of a peroxiredoxin homologue in jellyfish: molecular cloning, expression and functional characterization of CcPrx4 from Cyanea capillata.

We first identified and characterized a novel peroxiredoxin (Prx), designated as CcPrx4, from the cDNA library of the tentacle of the jellyfish Cyanea capillata. The full-length cDNA sequence of CcPrx4 consisted of 884 nucleotides with an open reading frame encoding a mature protein of 247 amino acids. It showed a significant homology to peroxiredoxin 4 (Prx4) with the highly conserved F-motif (93FTFVCPTEI101), hydrophobic region (217VCPAGW222), 140GGLG143 and 239YF240, indicating that it should be a new member of the Prx4 family. The deduced CcPrx4 protein had a calculated molecular mass of 27.2 kDa and an estimated isoelectric point of 6.3. Quantitative real-time PCR analysis showed that CcPrx4 mRNA could be detected in all the jellyfish tissues analyzed. CcPrx4 protein was cloned into the expression vector, pET-24a, and expressed in Escherichia coli Rosetta (DE3) pLysS. Recombinant CcPrx4 protein was purified by HisTrap High Performance chelating column chromatography and analyzed for its biological function. The results showed that the purified recombinant CcPrx4 protein manifested the ability to reduce hydrogen peroxide and protect supercoiled DNA from oxidative damage, suggesting that CcPrx4 protein may play an important role in protecting jellyfish from oxidative damage.