Identification, expression pattern and characterization of mouse glutaredoxin 2 isoforms.

HUDEMANN, C.; LÖNN, M.; REY FUNES, M.; COIRINI, H.; CAPANI, F.; HOLMGREN, A.; LILLIG, C.H.

Mosbach, Alemania

Congreso; Annual Spring Meeting, German Society for Biochemistry and Molecular Biology (GBM); 2007

German Society for Biochemistry and Molecular Biology

The reducing power of NADPH in cells is used by both the thioredoxin (Trx) and glutaredoxin (Grx) system to maintain cellular thiol homeostasis. Both Trxs and Grxs utilize the characteristic Cys-X-X-Cys active site motif for the reversible reduction of disulfide bonds. Grxs, as Trxs, catalyze the reduction of protein disulfides via a dithiol mechanism and the reduction of protein disulfides with GSH via a unique monothiol mechanism. The recently discovered human Grx2 is mainly localized in mitochondria (hGrx2a), but an alternative variant (hGrx2b) has been proposed to be targeted to the nucleus[1]. Here, we present the identification and characterization of new Grx2 isoforms in mouse. We have systematically screened for new transcript variants of mouse Grx2 and identified a total of six exons:. three constitutive exons (II, III, and IV), encoding the Grx core structure, two alternative first exons (exons Ia and Ic) and one single cassette exon (exon IIIb) located between exons III and IV. These exons give rise to five different transcript variants. First, mGLRX2_v1, encoding mitochondrial Grx2a. Secondly, mGLRX2_v2, mGLRX2_v4 and mGLRX2_v5 encoding a cytosolic variant named Grx2c and mGLRX2_v3 encoding the testis-specific cytosolic Grx2d. We found both Grx2a and Grx2c expressed globally, with the remarkable exception of testis, where only transcript variants encoding Grx2c and Grx2d were present. Next, we have expressed and characterized the newly identified protein isoforms Grx2c and Grx2d. Cytosolic Grx2c complexes a [2Fe,2S]-cluster at the interface of two Grx2 molecules as demonstrated for human Grx2 before[2]. The monomeric, cofactorless protein showed a specific activity of 11.3 ±1.3 µmol mg-1 min-1 in the HED-assay compared to 27.4 ±2.4 for human Grx2. Mouse Grx2d, on the other hand, lacks part of the GSH binding site and one cysteinyl residue necessary to complex the cluster. Grx2d was not expressed in soluble form in E. coli. The in vitro refolded protein was enzymatically inactive, free of any chromophore and, unlike Grx2a and Grx2c, did not react with the human anti-Grx2 antibody. Highest concentrations of immunoreactive mouse Grx2 where measured in heart (7.02 ng mg-1), kidney (5.96 ng mg-1) and brain (5.66 ng mg-1). Our results suggest additional functions of Grx2 in mice during oxidative stress and in cellular differentiation.