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Molecular and Functional Characterization of Thioredoxin 1 from Korean Rose Bitterling (Rhodeus uyekii).

Kim J, Moon JY, Kim WJ, Kim DG, Nam BH, Kim YO, Park JY, An CM, Kong HJ - Int J Mol Sci (2015)

Bottom Line: Thioredoxin is a multifunctional antioxidant enzyme that belongs to the reductase family.In Raw264.7 cells, Dihydroethidium (DHE) staining for ROS production indicated the antioxidant activity of rRuTrx.Together, these findings suggest that RuTrx may play a role in maintaining the redox state balance in Korean rose bitterling R. uyekii.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Research Division, National Fisheries Research and Development Institute, Busan 619-705, Korea. tks1010@hanmail.net.

ABSTRACT
Thioredoxin is a multifunctional antioxidant enzyme that belongs to the reductase family. In this study, we cloned and characterized thioredoxin 1 cDNA from the Korean rose bitterling Rhodeus uyekii (RuTrx). The full-length RuTrx cDNA consists of 674 bp with a 324 nt open reading frame (ORF) encoding a 107 aa protein. The deduced RuTrx amino acid sequence indicated a characteristic redox active site, (31)WCGPC(35). Pairwise alignment revealed RuTrx amino acid identity (55.1%-83.2%) with orthologs from various species of mammalia, amphibia, fish and bird. Phylogenetic analysis was conducted to determine the evolutionary position of RuTrx. Expression analysis showed that RuTrx transcripts were present in all of the tissues examined, and was high in the hepatopancreas of R. uyekii. During early development, the expression of RuTrx transcripts was increased. Recombinant RuTrx protein (rRuTrx) was tested for its capacity to serve as an antioxidant enzyme using a metal-catalyzed oxidation (MCO) system. The ability of rRuTrx to protect against supercoiled DNA cleavage due to oxidative nicking increased in a dose-dependent manner. In Raw264.7 cells, Dihydroethidium (DHE) staining for ROS production indicated the antioxidant activity of rRuTrx. Together, these findings suggest that RuTrx may play a role in maintaining the redox state balance in Korean rose bitterling R. uyekii.

No MeSH data available.


Nucleotide and deduced amino acid sequences of the R. uyekii thioredoxin (RuTrx). The start codon and stop codon are shown in bold. The active cysteine and two other cysteine residues are in bold and underlined, and the active site WCGPC is italicized.
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ijms-16-19433-f001: Nucleotide and deduced amino acid sequences of the R. uyekii thioredoxin (RuTrx). The start codon and stop codon are shown in bold. The active cysteine and two other cysteine residues are in bold and underlined, and the active site WCGPC is italicized.

Mentions: The RuTrx full-length cDNA sequence was identified by expressed sequence tag (EST) analysis from a Korean rose bitterling R. uyekii cDNA library (GenBank accession no. KT279874). The nucleotide and deduced amino acid sequences are presented in Figure 1. The 674 bp RuTrx cDNA contains a 324 nt open reading frame (ORF) encoding a 107 aa protein preceded by a 40 nt 5′ UTR and followed by a 310 nt 3′ UTR with a poly (A) tail. The putative isoelectric point (pI) and molecular weight (Mw) of the deduced protein of 107 aa were calculated as 5.28 and 12 kDa, respectively. There was no evidence of a signal sequence or an N-linked glycosylation site, indicating that the RuTrx protein might be a cytosolic form. In the deduced protein sequence, there was a characteristic redox active site, 31WCGPC35, and two other cysteine residues (Cys71 and Cys75). Two predicted disulfide bonds were identified (Cys32–Cys35, Cys71–Cys75). The cysteine residues found in the catalytic CGPC motif are essential for the activity of Trx. The CGPC motif is located on the surface of the protein in a short segment at the amino end of α-helix 2 [24].


Molecular and Functional Characterization of Thioredoxin 1 from Korean Rose Bitterling (Rhodeus uyekii).

Kim J, Moon JY, Kim WJ, Kim DG, Nam BH, Kim YO, Park JY, An CM, Kong HJ - Int J Mol Sci (2015)

Nucleotide and deduced amino acid sequences of the R. uyekii thioredoxin (RuTrx). The start codon and stop codon are shown in bold. The active cysteine and two other cysteine residues are in bold and underlined, and the active site WCGPC is italicized.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581305&req=5

ijms-16-19433-f001: Nucleotide and deduced amino acid sequences of the R. uyekii thioredoxin (RuTrx). The start codon and stop codon are shown in bold. The active cysteine and two other cysteine residues are in bold and underlined, and the active site WCGPC is italicized.
Mentions: The RuTrx full-length cDNA sequence was identified by expressed sequence tag (EST) analysis from a Korean rose bitterling R. uyekii cDNA library (GenBank accession no. KT279874). The nucleotide and deduced amino acid sequences are presented in Figure 1. The 674 bp RuTrx cDNA contains a 324 nt open reading frame (ORF) encoding a 107 aa protein preceded by a 40 nt 5′ UTR and followed by a 310 nt 3′ UTR with a poly (A) tail. The putative isoelectric point (pI) and molecular weight (Mw) of the deduced protein of 107 aa were calculated as 5.28 and 12 kDa, respectively. There was no evidence of a signal sequence or an N-linked glycosylation site, indicating that the RuTrx protein might be a cytosolic form. In the deduced protein sequence, there was a characteristic redox active site, 31WCGPC35, and two other cysteine residues (Cys71 and Cys75). Two predicted disulfide bonds were identified (Cys32–Cys35, Cys71–Cys75). The cysteine residues found in the catalytic CGPC motif are essential for the activity of Trx. The CGPC motif is located on the surface of the protein in a short segment at the amino end of α-helix 2 [24].

Bottom Line: Thioredoxin is a multifunctional antioxidant enzyme that belongs to the reductase family.In Raw264.7 cells, Dihydroethidium (DHE) staining for ROS production indicated the antioxidant activity of rRuTrx.Together, these findings suggest that RuTrx may play a role in maintaining the redox state balance in Korean rose bitterling R. uyekii.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Research Division, National Fisheries Research and Development Institute, Busan 619-705, Korea. tks1010@hanmail.net.

ABSTRACT
Thioredoxin is a multifunctional antioxidant enzyme that belongs to the reductase family. In this study, we cloned and characterized thioredoxin 1 cDNA from the Korean rose bitterling Rhodeus uyekii (RuTrx). The full-length RuTrx cDNA consists of 674 bp with a 324 nt open reading frame (ORF) encoding a 107 aa protein. The deduced RuTrx amino acid sequence indicated a characteristic redox active site, (31)WCGPC(35). Pairwise alignment revealed RuTrx amino acid identity (55.1%-83.2%) with orthologs from various species of mammalia, amphibia, fish and bird. Phylogenetic analysis was conducted to determine the evolutionary position of RuTrx. Expression analysis showed that RuTrx transcripts were present in all of the tissues examined, and was high in the hepatopancreas of R. uyekii. During early development, the expression of RuTrx transcripts was increased. Recombinant RuTrx protein (rRuTrx) was tested for its capacity to serve as an antioxidant enzyme using a metal-catalyzed oxidation (MCO) system. The ability of rRuTrx to protect against supercoiled DNA cleavage due to oxidative nicking increased in a dose-dependent manner. In Raw264.7 cells, Dihydroethidium (DHE) staining for ROS production indicated the antioxidant activity of rRuTrx. Together, these findings suggest that RuTrx may play a role in maintaining the redox state balance in Korean rose bitterling R. uyekii.

No MeSH data available.