원문정보
초록
영어
Thioredoxin (Trx) is a small ubiquitous multifunctional protein with a characteristic WCGPC thiol-disulfide active site that is conserved through evolution. Trx plays a crucial role in the antioxidant defense system. Further, it is involved in a variety of biological functions including gene expression, apoptosis, and growth regulation. Trx exists in several forms, with the cytosolic (Trx-1) and mitochondrial (Trx-2) forms being the most predominant. In this study, the mitochondrial Trx protein (HaTrx-2), from the big-belly seahorse (Hippocampus abdominalis) was characterized, and its molecular features and functional properties were investigated. The cDNA sequence of HaTrx-2 consists of a 519 bp ORF, and it encodes a polypeptide of 172 amino acids. This protein has a calculated molecular mass of 18.8 kDa and a calculated isoelectric point (pI) of 7.80. The highest values of identity (78.7%) and similarity (86.2%) were observed with Fundulus heteroclitus Trx-2 from the pairwise alignment results. The phylogenetic analysis revealed that HaTrx-2 is closely clustered with teleost fishes. The qPCR results showed that HaTrx-2 was prevalently expressed at various levels in all the tissues examined. The ovary showed the highest expression, followed by the brain and kidney. HaTrx-2 showed varying mRNA expression levels during the immune challenge experiment, depending on the type of tissue and the time interval. Our results confirmed the antioxidant property of HaTrx-2 by performing the MCO assay, DPPH radical scavenging activity, and cell viability assays. Further, an insulin disulfide reduction assay revealed the dithiol remove the enzymatic activity of HaTrx-2. Altogether these results indicate that HaTrx-2 plays indispensable roles in the regulation of oxidative stress and immune response in the seahorse.
목차
1. Introduction
2. Materials and methodology
2.1. Identification and molecular characterization of HaTrx-2
2.2. Seahorse tissue collection
2.3. In vivo challenge with immune stimulants and bacterial pathogens
2.4. Total RNA extraction and cDNA synthesis
2.5. Analysis of spatial and temporal expression of HaTrx-2 by quantitativereal-time PCR (qPCR)
2.6. Preparation of recombinant HaTrx-2 plasmid constructs
2.7. Recombinant HaTrx-2 fusion protein (rHaTrx-2-MBP) expression andpurification
2.8. Insulin disulfide reduction assay
2.9. DPPH radical-scavenging assay
2.10. Metal-catalyzed oxidation (MCO) protection assay
2.11. Construction of pcDNA3.1(+) vector and transfection of HaTrx-2
2.12. Cell viability analysis via MTT assay
2.13. Statistical analysis
3. Results
3.1. Identification and molecular characterization of HaTrx-2
3.2. Phylogenetic and homology analysis of HaTrx-2
3.3. Spatial expression of HaTrx-2
3.4. Immune challenges modulated HaTrx-2 mRNA expression profile
3.5. Expression and purification of HaTrx-2 recombinant fusion protein(rHaTrx-2-MBP)
3.6. Insulin disulfide reduction assay
3.7. DPPH radical-scavenging assay
3.8. Metal-catalyzed oxidation (MCO) protection assay
3.9. Cell protective role of HaTrx-2 against H2O2
4. Discussion
5. Conclusion
CRediT authorship contribution statement
Acknowledgments
References