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Screening and structural and functional investigation of a novel ferritin from Phascolosoma esculenta.

ABSTRACT: Ferritins are primary iron storage proteins and play a crucial role in iron storage and detoxification. Yeast two-hybrid method was employed to screen the cDNA library of Phascolosoma esculenta. Sequence of positive colony FER147 was analyzed. The higher similarity and conserved motifs for ferritin indicated that it belonged to a new member of ferritin family. The interaction between Ferritin and Fer147 was further confirmed through co-immunoprecipitation. The pET-28a-FER147 prokaryotic expression vector was constructed. The expressed recombinant Fer147 was then isolated, purified, and refolded. When ferritins were treated by different heavy metals, several detection methods, including scanning electron microscopy (SEM), circular dichroism (CD), and inductively coupled plasma-mass spectrometry (ICP-MS) were applied to examine the structures and functions of the new protein Fer147, recombinant P. esculenta ferritin (Rferritin), and natural horse-spleen ferritin (Hferritin). SEM revealed that the three ferritin aggregates changed obviously after different heavy metals treatment, meanwhile, a little different in aggregates were detected when the ferritins were trapped by the same heavy metal. Hence, changes in aggregation structure of the three proteins are related to the nature of the different heavy metals and the interaction between the heavy metals and the three ferritins. CD data suggested that the secondary structure of the three ferritins hardly changed after different heavy metals were trapped. ICP-MS revealed that the ferritins exhibit different enrichment capacities for various heavy metals. In particular, the enrichment capacity of the recombinant Fer147 and Rferritin is much higher than that of hferritin.

SUBMITTER: Ding H

PROVIDER: S-EPMC5606535 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Screening and structural and functional investigation of a novel ferritin from Phascolosoma esculenta.

Ding Hongwei H Zhang Dijun D Chu Shuangshuang S Zhou Jun J Su Xiurong X

Protein science : a publication of the Protein Society 20170904 10

Ferritins are primary iron storage proteins and play a crucial role in iron storage and detoxification. Yeast two-hybrid method was employed to screen the cDNA library of Phascolosoma esculenta. Sequence of positive colony FER147 was analyzed. The higher similarity and conserved motifs for ferritin indicated that it belonged to a new member of ferritin family. The interaction between Ferritin and Fer147 was further confirmed through co-immunoprecipitation. The pET-28a-FER147 prokaryotic expressi ...[more]

PMID: 28726294

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Project description:BACKGROUND:There are many advantages to the application of complete mitochondrial (mt) genomes in the accurate reconstruction of phylogenetic relationships in Metazoa. Although over one thousand metazoan genomes have been sequenced, the taxonomic sampling is highly biased, left with many phyla without a single representative of complete mitochondrial genome. Sipuncula (peanut worms or star worms) is a small taxon of worm-like marine organisms with an uncertain phylogenetic position. In this report, we present the mitochondrial genome sequence of Phascolosoma esculenta, the first complete mitochondrial genome of the phylum. RESULTS:The mitochondrial genome of P.esculenta is 15,494 bp in length. The coding strand consists of 32.1% A, 21.5% C, 13.0% G, and 33.4% T bases (AT = 65.5%; AT skew = -0.019; GC skew = -0.248). It contains thirteen protein-coding genes (PCGs) with 3,709 codons in total, twenty-two transfer RNA genes, two ribosomal RNA genes and a non-coding AT-rich region (AT = 74.2%). All of the 37 identified genes are transcribed from the same DNA strand. Compared with the typical set of metazoan mt genomes, sipunculid lacks trnR but has an additional trnM. Maximum Likelihood and Bayesian analyses of the protein sequences show that Myzostomida, Sipuncula and Annelida (including echiurans and pogonophorans) form a monophyletic group, which supports a closer relationship between Sipuncula and Annelida than with Mollusca, Brachiopoda, and some other lophotrochozoan groups. CONCLUSION:This is the first report of a complete mitochondrial genome as a representative within the phylum Sipuncula. It shares many more similar features with the four known annelid and one echiuran mtDNAs. Firstly, sipunculans and annelids share quite similar gene order in the mitochondrial genome, with all 37 genes located on the same strand; secondly, phylogenetic analyses based on the concatenated protein sequences also strongly support the sipunculan + annelid clade (including echiurans and pogonophorans). Hence annelid "key-characters" including segmentation may be more labile than previously assumed.

Project description:Phascolosoma esculenta, an economically important species inhabiting the high tide areas of the intertidal zone, is particularly sensitive to water pollution. Considering its potential as a bioindicator, studies on the ecotoxicology of P. esculenta are imperative. The toxic effects of cadmium (Cd) were analyzed by exposing P. esculenta to different concentrations of Cd (6, 24, 96 mg/L). In this study, the changes in the antioxidative indexes of total superoxide dismutase (T-SOD), glutathione s-transferase (GST), reduced glutathione (GSH), and microscale malondialdehyde (MDA) were recorded. Copper/zinc superoxide dismutase (Cu/Zn SOD) is one of the most important free radical scavenging members. To reveal the antioxidative function of P. esculenta, an important member of the antioxidative system, designated Pe-Cu/Zn SOD, was cloned and analyzed. Phylogenic analysis revealed that Pe-Cu/Zn SOD was located in the invertebrate evolutionary branch of intracellular Cu/Zn SOD (icCu/Zn SOD). The quantitative real-time polymerase chain reaction results showed that Pe-Cu/Zn SOD messenger ribonucleic acid was widely expressed in all tissues examined. The highest expression levels in coelomic fluid after Cd exposure indicated its function in the stress response. Using a prokaryotic expression system, we obtained a Pe-Cu/Zn SOD recombinant protein, which enhanced the heavy metal tolerance of Escherichia coli. In vivo assays also confirmed that the Pe-Cu/Zn SOD recombinant protein had an antioxidative and free radical scavenging ability. A Cd toxicity experiment, in which purified Pe-Cu/Zn SOD protein was injected into the body cavities of P. esculenta, showed that the reactive oxygen species content in the coelomic fluid of the experimental group was significantly lower compared with the control group. These results suggest that Pe-Cu/Zn SOD played a role in Cd detoxification by chelating heavy metal ions and scavenging reactive oxygen free radicals, and that P. esculenta could be used as a bioindicator to evaluate heavy metal pollution.

Dataset Information

Screening and structural and functional investigation of a novel ferritin from Phascolosoma esculenta.

Publications

Screening and structural and functional investigation of a novel ferritin from Phascolosoma esculenta.

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