Molecular characterization and antioxidant function of glutaredoxin 3 in Sepiella japonica

Glutaredoxins (Grxs) are essential antioxidant proteins that protect cells from oxidative damage and participate in multiple biological processes. However, their roles in the common Chinese cuttlefish (Sepiella japonica) remain uncharacterized. Here, we identified a glutaredoxin 3 homolog from S. japonica (SjGrx3). The SjGrx3 gene encodes a 981 bp open reading frame producing a 326-amino-acid protein (36.47 kDa) that contains an N-terminal thioredoxin domain and two C-terminal Grx domains, each containing the conserved CGFS active-site motif. Phylogenetic analysis clustered SjGrx3 within the cephalopod Grx3 clade, showing high sequence identity to other molluscan homologs. The SjGrx3 mRNA was detected in all examined tissues, with the highest levels in skin and kidney and moderate levels in gill, gut, and liver. In naturally Vibrio harveyi-infected individuals, SjGrx3 transcripts increased significantly in skin, gill, liver, white body, and brain, implicating it in innate immune responses. Confocal microscopy showed that SjGrx3 localized to both the cytoplasm and nucleus. Functional assays demonstrated that recombinant SjGrx3 was soluble and enhanced Escherichia coli resistance to H₂O₂-induced oxidative damage. Moreover, overexpression of SjGrx3 in HEK293T cells significantly reduced H₂O₂-induced intracellular reactive oxygen species (ROS) accumulation and inhibited H₂O₂-mediated cell death, confirming its role in oxidative stress protection. Collectively, these findings indicate that SjGrx3 contributes to redox regulation and immune defense against bacterial infection in S. japonica.