Elsevier

Fish & Shellfish Immunology

Volume 100, May 2020, Pages 368-377
Fish & Shellfish Immunology

Full length article
Identification and molecular characterization of peroxiredoxin 6 from noble scallop Chlamys nobilis revealing its potent immune response and antioxidant property

https://doi.org/10.1016/j.fsi.2020.03.021Get rights and content

Highlights

  • Prx6 gene (designed as CnPrx6) was first time identified in the noble scallop Chlamys nobilis.

  • CnPrx6 was involved in the immune response against pathogen infection.

  • Different responses were existed between golden and brown scallop under immunity stress.

  • Purified recombinant CnPrx6 protein can protect supercoiled plasmid DNA from damages.

Abstract

The 1-cyseine peroxiredoxin (Prx6) is an importantly antioxidant enzyme that protects cells from oxidative damage caused by excessive production of reactive oxygen species (ROS). In this study, we described the molecular characteristics of the noble scallop Chlamys nobilis peroxiredoxin 6 (designed as CnPrx6), immune responses and DNA protection activity of the recombinant protein. The complete ORF (696 bp) of CnPrx6 encoded a polypeptide (25.5 kDa) of 231 amino acids, harboring a conserved peroxidase catalytic center (41PVCTTE46) and the catalytic triads putatively involved in peroxidase and phospholipase A2 activities. The deduced amino acid sequence of CnPrx6 shared a relatively high amino acid sequence similarity (more than 50%). The qRT-PCR revealed that the CnPrx6 mRNA was constitutively expressed in all examined tissues, with the highest expression observed in adductor. Upon immunological challenge with Vibrio parahaemolyticus, lipopolysaccharides (LPS) and polyinosinic-polycytidylic acid (Poly I:C), the expression level of CnPrx6 mRNA was significantly up-regulated (P < 0.05). Furthermore, there was a significant difference (P < 0.05) in the expression level of CnPrx6 between golden and brown scallops. The purified recombinant CnPrx6 protein protected the supercoiled plasmid DNA from metal-catalyzed ROS damage. Taken together, these results indicated that the CnPrx6 may play an important role in modulating immune responses and minimizing DNA damage in noble scallop Chlamys nobilis.

Introduction

Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide anion and singlet oxygen, are thought to be vital for a variety cellular functions, such as cell proliferation, differentiation, immune response, and signal transduction [[1], [2], [3], [4]]. However, excessive accumulation of ROS could cause serious oxidative damage to host cells, including DNA base modifications, DNA strand breaks, protein oxidation, lipid peroxidation and cell death [5]. The antioxidant system is one of the most important immune systems in shellfish [6], and its most important function is to maintain and control ROS at the intracellular level using non-enzymatic natural antioxidants including vitamins E, A, C and intracellular antioxidant enzymes like peroxiredoxins [7].

Peroxiredoxin (Prx) is a ubiquitous family of peroxidases that plays a protective antioxidant role by scavenging H2O2 and alkyl hydroperoxides [8]. Six isoforms of Prx (Prx1-6) have been detected in mammals, all of which are divided into 2-Cys (Prx1-4), atypical 2-Cys (Prx5) and 1-Cys (Prx6) based on the number and position of cysteine (Cys) residues that participated in catalysis [9,10]. As an indispensable member of the Prx family, Prx6, is referred as a bi-functional enzyme due to its glutathione-peroxidase activity and phospholipase A2 (PLA2) activity [11,12], which has been involved in immune responses against different oxidative stresses including live bacteria [13,14], virus [15,16], pathogen-associated molecular patterns (PAMPs) [14,[16], [17], [18], [19]] etc. However, study and sequence information for the bivalves Prx6 has been limited to few species such as Crassostrea gigas [13], Laternula elliptica [7], and Saccostrea glomerate [20]. Hence, further studies on other bivalve species such as scallops would broaden the understanding of Prx6 and its associated functions.

The noble scallop Chlamys nobilis, a warm-water edible marine bivalve, is one of the most important and popular cultured species in southern China with huge annual production and enormous economic benefits. Nevertheless, over the last few decades, diseases and infections have seriously threatened and hampered the sustainable development of the scallops' aquaculture industry, leading to scallop mass mortality thereby resulting in severe economic losses [21,22]. In order to solve this problem, our laboratory has carried out a genetic breeding project since 2008, and in 2015, a new variety named “Nan'ao Golden Scallop” with abundant carotenoids in the soft tissues was successfully bred [23]. Its resistance and immunity are stronger than other individuals with less carotenoids in its soft tissues [[24], [25], [26]]. Carotenoids are lipid-soluble bioactive substances that widely present in the tissues of marine aquatic animals, which refer to tetraterpenes with long hydrocarbon chains containing multiple conjugated double bonds and ionone rings [27,28]. Thank to such structure, these compounds not only act as powerful antioxidants that provide protection against singlet oxygen and free radicals [29], but also play a role in biological processes such as immune enhancement, reproduction, and growth [30,31].

In the present study, in order to investigate whether carotenoids and Prx6 in noble scallop Chlamys nobilis are involved in immunity, Vibrio parahaemolyticus, lipopolysaccharides (LPS) and polyinosinic-polycytidylic acid (Poly I:C) challenge tests were separately conducted by using golden and brown scallops from the same stock with different carotenoids content. The antioxidant activity of recombinant CnPrx6 protein was evaluated by mix-function oxidase assay. The results of present study will provide further insight for better understanding the function of CnPrx6 and innate immune system of aquatic animals.

Section snippets

Experimental animals

Healthy noble scallop Chlamys nobilis with shell length of 51.26 ± 0.84 mm were obtained from Nan'ao Marine Biology Experimental Station of Shantou University (24.41°E, 117.88°N). All scallops were cleaned and acclimatized in three 500 L-tanks with aerated seawater at a temperature of 24 ± 0.5 °C for seven days prior to the experiment.

Immune challenge experiment

Two hundred golden and 200 brown scallops were randomly divided into three experiment groups (V. parahaemolyticus, LPS and Poly I:C challenge) and one control

Identification and characterization of CnPrx6 cDNA sequence

The full-length coding sequence of CnPrx6 was 2319 bp in length (GeneBank accession number: MN594763), which encodes a polypeptide of 231 amino acids with a molecular weight of 25.5 kDa and theoretical isoelectric point of 6.90. The cDNA ends with a poly-A tail which was preceded at 16 bp upstream by a polyadenylation signal, AATAAA. Predicated CnPrx6 protein has four cysteine residues at Cys43, Cys69, Cys80 and Cys139 (Fig. 1). Moreover, no signal peptide was found at the N-terminus by SignalP.

Discussion and conclusion

In the present study, we identified the full-length cDNA of Prx6 from the noble scallop Chlamys nobilis. The deduced amino acid sequence of CnPrx6 has common structural features with Prx6 from vertebrates and invertebrates. The phylogenetic tree showed that the CnPrx6 was fall into the 1-Cys subgroup. CnPrx6 contained only a single conserved cysteine residue (Cys43), which was the representative characteristic of Prx6 [39] that distinguished from 2-Cys Prxs [9]. Although, three non-conserved

CRediT authorship contribution statement

Dewei Cheng: Writing - original draft. Hongxing Liu: Investigation. Xinxu Zhang: Writing - original draft. Karsoon Tan: Writing - review & editing. Shengkang Li: Writing - original draft. Hongyu Ma: Writing - original draft.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (31872563), China Modern Agro-industry Technology Research System (CARS-49), and Department of Education of Guangdong Province (2017KCXTD014), China, Shenzhen Fundamental Research (JCYJ20180305163524811), China.

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