Cloning and expression of a novel human glutaredoxin (Grx2) with mitochondrial and nuclear isoforms

Glutaredoxin (Grx) is a glutathione-dependent hydrogen donor for ribonucleo tide reductase, Today glutaredoxins are known as a multifunctional family o f GSH-disulfide-oxidoreductases belonging to the thioredoxin fold superfami ly, In contrast to Escherichia coli and yeast, a single human glutaredoxin is known. We have identified and cloned a novel 18-kDa human dithiol glutar edoxin, named glutaredoxin-2 (Grx2), which is 34% identical to the previous ly known cytosolic 12-kDa human Grx1. The human Grx2 sequence contains thre e characteristic regions of the glutaredoxin family: the dithiol/disulfide active site, CSYC, the GSH binding site, and a hydrophobic surface area. Th e human Grx2 gene, located at chromosome 1q31.2-31.3, consisted of five exo ns that were transcribed to a 0.9-kilobase human Grx2 mRNA ubiquitously exp ressed in several tissues. Two alternatively spliced Grx2 mRNA isoforms tha t differed in their 5 ' region were identified. These corresponded to alter native proteins with a common 125-residue C-terminal Grx domain but with di fferent N-terminal extensions of 39 and 40 residues, respectively. The 125- residue Grx domain and the two full-length variants were expressed in E. co li and exhibited GSH-dependent hydroxyethyl disulfide and dehydroascorbate reducing activities. Western blot analysis of subcellular fractions from Ju rkat cells with a specific anti-Grx2 antibody showed that human Grx2 was pr edominantly located in the nucleus but also present in the mitochondria, We further showed that one of the mRNA isoforms corresponding to Grx2a encode d a functional N-terminal mitochondrial translocation signal.