A newly discovered teleost disulfide isomerase, thioredoxin domain containing 5 (TXNDC5), from big-belly seahorse (Hippocampus abdominalis): Insights into its molecular and functional properties and immune regulatory functions

다국어 초록 (Multilingual Abstract)

The thioredoxin domain containing 5 (TXNDC5) is a recently discovered member of the protein disulfide isomerase family (PDI), which is mainly involved in the proper folding of and the correct formation of disulfide bonds in newly synthesized proteins via its disulfide isomerase and chaperone activities. Although the structural and functional features of mammalian TXNDC5 have been explored in previous studies, no studies have reported the functional characteristics of TXNDC5 in teleost fish. In this study, we report the identification and characterization of TXNDC5 from big-belly seahorse (Hippocampus abdominalis) (ShTXNDC5) accompanied by functional studies. The in-silico analysis revealed that the gene encodes a 433 amino acid (aa) long polypeptide chain with a predicted molecular weight of 49.3 kDa. According to homology analysis, ShTXNDC5 shares more than 55% sequence similarity with other teleost TXNDC5 proteins, and the alignment of the gene sequence convincingly reflects the accepted phylogeny of teleost. Analysis of the spatial distribution of ShTXNDC5 expression showed that its highest expression was observed in the ovary, gill, and pouch of seahorses. Moreover, significant upregulation of ShTXNDC5 transcription was noted in seahorse blood and kidney tissues in a timedependent manner upon viral and bacterial immune challenges. Furthermore, considerable NADPH turnover, insulin reduction ability and significant cell survival effects of ShTXNDC5 were determined by the functional assay, revealing its capability to overcome cellular oxidative stress. Altogether, these findings expand our understanding of TXNDC5 at the molecular and functional levels, and its putative role in seahorse immunity.

목차 (Table of Contents)

• ABSTRACT
• 1. Introduction
• 2. Materials & methods
• 2.1. Identification and sequence characterization of ShTXNDC5
• 2.2. Experimental fish rearing and tissue collection
• 2.3. Immune challenge experiment
• 2.4. RNA extraction and cDNA synthesis
• 2.5. ShTXNDC5 expression analysis – quantitative real time PCR (qPCR)
• 2.6. Cloning, overexpression, and purification of recombinant ShTXNDC5 (rShTXNDC5)
• 2.7. Functional assays
• 2.8. Statistical analysis
• 3. Results & discussion
• 3.1. Sequence characterization and bioinformatics analysis of ShTXNDC5
• 3.2. Homology and phylogenetic analysis of ShTXNDC5
• 3.3. Spatial mRNA expression of ShTXNDC5
• 3.4. Temporal expression of ShTXNDC5 mRNA upon immune stimulation
• 3.5. Expression and purification of rShTXNDC5
• 3.6. Functional assays
• 4. Conclusion
• References