Mitochondrial thioredoxin-2 from Manila clam (Ruditapes philippinarum) is a potent antioxidant enzyme involved in antibacterial response

Thioredoxin (TRx) is a ubiquitous protein involved in the regulation of multiple biological processes. The TRx-2 isoform is exclusively expressed in mitochondria, where it contributes to mitochondrial redox state maintenance. In the present study, a novel thioredoxin-2 gene was identified in the Manila clam, Ruditapes philippinarum. The full-length sequence of RpTRx-2 (1561 bp) consists of a 498 bp coding region encoding a 166 amino acid protein. The N-terminal region of RpTRx-2 harbors a mitochondrial localization signal (56 amino acids), while the C-terminal portion contains the characteristic 89WCGPC93 catalytic active site. Phylogenetic analysis revealed that RpTRx-2 is closest to its ortholog from abalone. The broad distribution pattern of RpTRx-2 mRNA in healthy animal tissues implicates a generally significant function in normal clam physiology. The transcription level of RpTRx-2, however, is highest in hemocytes. Lipopolysaccharide and Vibrio tapetis bacterium caused up-regulation of the RpTrx-2 transcript levels in gill and hemocytes. Interestingly, clam manganese superoxide dismutase (MnSOD) mRNA levels in hemocytes elicited a corresponding response to these immune challenges. RpTRx-2 was recombinantly expressed in Escherichia coli BL21 (DE3) and used in insulin disulfide reduction assay as well as metal-catalyzed oxidation assay to elucidate its antioxidant property by reducing substrate and protecting super-coiled DNA from oxidative damage through free radical scavenging, respectively. Collectively, our data indicated that RpTRx-2, a mitochondrial TRx-2 family member, is an antioxidant enzyme that may be involved in antibacterial defense of clams.

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► Molecular characterization of mitochondrial thioredoxin-2 in Manila clam (RpTRx-2).
► Functional assays for insulin reduction activity and protection of supercoiled DNA.
► Tissue specific expression profile of RpTRx-2.
► Response of RpTRx-2 against Vibrio and LPS in gill and hemocyte.
► Evidences for antioxidant property and antibacterial response of RpTRx-2.