Comparative study of two thioredoxin peroxidases from disk abalone (Haliotis discus discus): cloning, recombinant protein purification, characterization of antioxidant activities and expression analysis.
Thioredoxin peroxidase (TPx), also named peroxiredoxin (Prx), is an important peroxidase, which can protect organisms against various oxidative stresses. Two TPxs were isolated from a disk abalone (Haliotis discus discus) cDNA library, named as AbTPx1 and AbTPx2, respectively. AbTPx1 and AbTPx2 consist of 1315 and 1045 bp full-length cDNA with 753 and 597 bp open reading frames encoding 251 and 199 amino acids, respectively. The TPx signature motif 1 (FYPLDFTFVCPTEI) and motif 2 (GEVCPA) were conserved in both AbTPx1 and AbTPx2 amino acid sequences. Purified recombinant abalone TPx fusion proteins catalyzed the reduction of H2O2 and butyl hydroperoxide in peroxidase assays. Furthermore, both AbTPx fusion proteins were shown to protect super-coiled DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Escherichia coli cells transformed with AbTPx1 and AbTPx2 coding sequences in pMAL-c2x showed resistance to H2O2 at 0.8 mM concentration by in vivo H2O2 tolerance assay. AbTPx1 and AbTPx2 mRNA were constitutively expressed in gill, mantle, abductor muscle and digestive tract in a tissue specific manner. Additionally, both TPxs mRNA were up-regulated in gill and digestive tract tissues against H2O2 at 3h post injection. The results indicate that AbTPx1 and AbTPx2 gene expressions are induced by oxidative stress and their respective proteins function in the detoxification of different ROS molecules to maintain efficient antioxidant defense in disk abalone.
Pushpamali
Wickramaarachchilage Anoja
WA
Department of Marine Biotechnology, College of Ocean Science, Cheju National University, 66 Jejudaehakno, Ara-Dong, Jeju 690-756, Republic of Korea.
De Zoysa
Mahanama
M
Kang
Hyun-Sil
HS
Oh
Cheol Hong
CH
Whang
Ilson
I
Kim
Se Jae
SJ
Lee
Jehee
J
D000595:Amino Acid Sequence; D000818:Animals; D000975:Antioxidants / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004278:DNA, Superhelical; D004926:Escherichia coli / Q000235:genetics; D049851:Gastropoda / Q000187:drug effects / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008969:Molecular Sequence Data; D054464:Peroxiredoxins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D015854:Up-Regulation / Q000187:drug effects
D000975:Antioxidants; D004278:DNA, Superhelical; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D054464:Peroxiredoxins