Identification and characterization of multidomain monothiol glutaredoxin 3 from diploblastic Hydra

Abstract

Intracellular antioxidant glutaredoxin controls cell proliferation and survival. Based on the active site, structure, and conserved domain motifs, it is classified into two classes. Class I contains dithiol Grxs with two cysteines in the consensus active site sequence CXXC, while class II has monothiol Grxs with one cysteine residue in the active site. Monothiol Grxs can also have an additional N-terminal thioredoxin (Trx)-like domain. Previously, we reported the characterization of Grx1 from Hydra vulgaris (HvGrx1), which is a dithiol isoform. Here, we report the molecular cloning, expression, analysis, and characterization of another isoform of Grx, which is the multidomain monothiol glutaredoxin-3 from Hydra vulgaris (HvGrx3). It encodes a protein with 303 amino acids and is significantly larger and more divergent than HvGrx1. In-silico analysis revealed that Grx1 and Grx3 have 22.5% and 9.9% identical nucleotide and amino acid sequences, respectively. HvGrx3 has two glutaredoxin domains and a thioredoxin-like domain at its amino terminus, unlike HvGrx1, which has a single glutaredoxin domain. Like other monothiol glutaredoxins, HvGrx3 failed to reduce glutathione-hydroxyethyl disulfide. In the whole Hydra, HvGrx3 was found to be expressed all over the body column, and treatment with H2O2 led to a significant upregulation of HvGrx3. When transfected in HCT116 (human colon cancer cells) cells, HvGrx3 enhanced cell proliferation and migration, indicating that this isoform could be involved in these cellular functions. These transfected cells also tolerate oxidative stress better.