PDI family thioredoxin from disk abalone (Haliotis discus discus): Responses to stimulants (PAMPs, bacteria, and viral) and functional characterization

doi:10.1016/j.fsi.2021.11.037

Fish & Shellfish Immunology

Volume 120, January 2022, Pages 261-270

https://doi.org/10.1016/j.fsi.2021.11.037 Get rights and content

Highlights

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AbTXNDC5 is an important gene in redox regulation and innate immunity.
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AbTXNDC5 upregulated in hemocytes and gills in response to PAMPs, bacteria, and virus.
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RAbTXNDC5 has actively precipitated the insulin by breaking A and B chain bonds.
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AbTXNDC5 transfected FHM cells exhibited cell protection against apoptosis and hypoxia.

Abstract

Thioredoxin, a highly conserved class of proteins involved in redox signaling, is found in a range of organisms from bacteria to higher-level eukaryotes. Thioredoxin acts as an active regulatory enzyme to eliminate excessive reactive oxygen species, thereby preventing cellular damage. In this study, the cDNA sequence of thioredoxin domain-containing 5 (AbTXNDC5) from the disk abalone transcriptomic database was characterized. An in silico analysis of AbTXNDC5 was performed, and its spatial and temporal expression patterns in hemocytes and gills in response to bacteria (Vibrio parahaemolyticus, Listeria monocytogenes), viral hemorrhagic septicemia virus, and pathogen-associated molecular pattern molecules were observed. Furthermore, AbTXNDC5 expression was examined in different developmental stages. Functional assays to explore insulin disulfide reduction, anti-apoptotic activity, and protection against hypoxic cell death of AbTXNDC5 were conducted through recombinant proteins or overexpression in cells. AbTXNDC5 contains a 1179-bp open reading frame coding for 392 amino acids. Conserved thiol-disulfide cysteine residues within two Cys-X-X-Cys motifs were found in AbTXNDC5. Quantitative real-time polymerase chain reaction indicated that healthy digestive tract and hemocyte tissues expressed high levels of AbTXNDC5 mRNA, which may protect the host from invading pathogens. Immune-challenged abalone hemocytes and gills exhibited upregulated expression of AbTXNDC5 at different time points. rAbTXNDC5 also exhibited a functional insulin disulfide reductase activity. AbTXNDC5 conferred protection to cultured cells from apoptosis and hypoxia-induced stress_, compared to the pcDNA3.1⁽⁺⁾ transfected control cells. Therefore, AbTXNDC5 can be considered an important gene in abalones in relation to the primary immune system and regulation of redox homeostasis and confers protection from stress.

Introduction

Reactive oxygen species (ROS) are highly reactive molecules that readily react with macromolecules such as DNA and RNA. ROS comprise free radicals, like O₂•⁻, •OH, and non-radicals, such as H₂O₂ and O₂ [1] and are involved in several signaling pathways related to cell proliferation, metabolism, cell survival, antioxidants, anti-inflammatory reactions, ion homeostasis, and DNA damage responses [2,3]. They are generated naturally in cells during oxidative metabolism and cellular responses to xenobiotics, cytokines, and bacterial invasion. They may also be generated in response to external stimuli, such as ultraviolet rays and heat [2]. Excessive ROS can be highly toxic to cells and can lead to carcinogenesis, neurodegeneration, and aging [4]. Thioredoxin can convert oxidized ROS to its reduced form via thiol-disulfide reduction. Cellular proteins may be oxidized and malfunction as a direct response to ROS or after reacting with their derivatives generated under stress conditions [5].

The regulation of oxidative and reductive stress is essential to defend cells from ROS. Eukaryotes and prokaryotes have integrated antioxidant systems, which contain enzymatic and non-enzymatic antioxidants that are effective in impeding the injurious effects of ROS [6]. Furthermore, numerous proteins such as thioredoxin, thioredoxin domain-containing proteins, catalase, superoxide dismutase, peroxiredoxin, glutathione peroxidase, glutathione transferase, and glutaredoxin act as antioxidants [6]. Herein, we studied thioredoxin domain-containing 5 (TXNDC5), which is involved in redox homeostasis, inhibition of apoptosis, hypoxia, cell proliferation, migration, and invasion [7].

Abalones (Haliotis spp.) are important marine gastropods cultivated worldwide as a food source. The disk abalone (Haliotis discus discus) belongs to the Haliotidae family and is found in Korea, Japan, and Eastern Asia. In nature, they grow to a diameter of 100–225 mm. The abalone aquaculture industry in Korea continues to face several outbreaks of diseases caused by bacteria, viruses, and other parasites. Furthermore, unfavorable environmental factors can cause stress to cultivated abalones. Abalones are invertebrates with a primitive immune system and lack an adaptive immune system present in most vertebrates. Therefore, elucidation of their immune mechanisms is vital for developing new strategies for disease prevention.

In this study, the TXNDC5 cDNA sequence from the disk abalone transcriptome database was characterized. In silico analysis of the cDNA and protein sequence of disk abalone TXNDC5 (AbTXNDC5) was performed, and their temporal expression patterns were observed in hemocytes and gills upon being challenged by bacteria (Vibrio parahaemolyticus, Listeria monocytogenes), viral hemorrhagic septicemia virus (VHSV), and pathogen-associated molecular pattern molecules (PAMPs) (poly I:C and lipopolysaccharides). Furthermore, AbTXNDC5 expression was examined in different developmental stages, and a recombinant rAbTXNDC5 protein was synthesized to validate its functional properties.

Section snippets

Identification of disk abalone TXNDC5 sequence

The coding sequence of AbTXNDC5 was extracted from the disk abalone transcriptomic database established by Jeju National University, Republic of Korea. It was sequenced by a 454-genome Sequencer FLX machine (Roche, USA). The sequence was identified using the BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) on the National Center for Biotechnology Information (NCBI) curated database [8].

In silico analysis of the AbTXNDC5 sequence

The open reading frames (ORFs) and the deduced amino acid sequences of AbTXNDC5 were obtained from the

Molecular characterization

Homology screening of the AbTXNDC5 coding sequence was performed using NCBI databases. The sequence was deposited to the NCBI GenBank under accession number MH197145.1. As per the in silico analysis, the ORF of AbTXNDC5 is 1179 bp in length and encodes for 392 amino acids. The estimated molecular mass and predicted isoelectric point (pI) of AbTXNDC5 are 44.5 kDa and 4.89, respectively. The instability index of AbTXNDC5 is 38.69, which classifies the protein as stable. The AbTXNDC5 sequence

Discussion

PDI proteins are primarily present in the ER and comprise four tandem thioredoxin-like domains (a, b, b′, x, and a′, where a, b, b′, and a′ are thioredoxin domains and x is the linker domain). In addition to TXNDC5, more than 20 PDI proteins have been identified in human cells [13]. PDI proteins are arranged into a U-shaped structure in nature. However, TXNDC5 lacks a thioredoxin domain in the middle and exhibits an a, b, x, and a′ structure. TXNDC5 has three catalytic activities, namely

Conclusions

In conclusion, our study indicates that TXNDC5 may be an essential gene in the primary immune system and regulation of cellular redox homeostasis in abalones and might confer protection from stress. We performed an in silico analysis of Haliotis discus discus TXNDC5 using different bioinformatics tools. The spatial and temporal expressions of AbTXNDC5 were evaluated by qPCR, and the involvement of AbTXNDC5 in the host immune system was determined. We observed that AbTXNDC5 expression varied in

CRediT authorship contribution statement

D.S. Liyanage: Conceptualization, Methodology, Investigation, Writing – original draft. W.K.M. Omeka: Conceptualization, Methodology, Investigation, Writing – original draft. W.M. Gayashani Sandamalika: Validation, Writing – review & editing. H.M.V. Udayantha: Formal analysis, Writing – review & editing. Taehyug Jeong: Resources, Supervision, Data curation. Sukkyoung Lee: Resources, Supervision, Data curation. Jehee Lee: Resources, Writing – review & editing, Supervision, Project

Acknowledgments

This research was supported by the Golden Seed Project; the Ministry of Agriculture, Food and Rural Affairs (MAFRA); the Ministry of Oceans and Fisheries (MOF); the Rural Development Administration (RDA); and the Korea Forest Service (KFS) (213008-05-1-SB730).

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