Cloning and characterization of human glutaredoxins

Reactive oxygen species, ROS, are generated in cells during aerobic metabolism. High and sustained levels of ROS pose a serious threat to all major cellular components, DNA, proteins mid lipids whereas controlled and physiological concentrations play a central role as a second messenger signal molecule involved in cell functions including, differentiation, proliferation, and apoptosis. In the antioxidant system shown to balance ROS to physiologically relevant levels the tripeptide glutathione (GSH, gamma-glutamylcysteinylglycine) and the GSH-dependent enzyme systems, have central positions.

The aim of this study was to further explore glutathione and the glutathione-dependent enzyme glutaredoxin. Glutaredoxin (Grx) catalyzes glutathione-dependent thioldisulfide oxidoreduction reactions and was originally discovered as a hydrogen donor for ribonucleotide reductase in DNA synthesis. GSH and glutaredoxin, studied in a broad range of organism are implicated in numerous processes as cellular differentiation, regulation of transcription factors, and control of apoptosis.

When testing the role of GSH and the GSH prodrug N-acetyl-L-cysteine (NAC) as immunomodulators of T-lymphocyte function it was found that both NAC and GSH decrease the production of cytokines (IL-4, IL-5 and IFN-gamma) in activated T-lymphocytes, as determined by ELISA. The most marked effect was observed for the IL-4 production. Both NAC arid GSH also affected the expression of the cell surface molecule CD30, while CD28 and CD40L were unaffected. Previously NAC was shown to exert its effect by regulating transcription of genes. Using a novel plate assay to determine free thiols we showed that both NAC and GSH could exert their effect also by directly reducing biologically important protein disulfides.

A novel human glutaredoxin, Grx2, localized at chromosome 1 was identified and cloned. Recombinant human Grx2 exhibited GSH-dependent hydroxyethyl disulfide reductase activity (BED-assay), typical for glutaredoxins. Secondary structure prediction of Grx2 showed high structural homology to the previously known cytosolic human Grx 1, despite relatively low identity (34%) at the primary structure. Two mRNAs encoding Grx2 were identified encoding two identical proteins only differing by a short stretch at the Nterminus. Western blot analysis and transfection analysis indicated that one of the tonus might be a mitochondrial protein. The other form might be nuclear.

We developed two sandwich ELISAs specific for human Grx1 and Grx2, respectively. Both Grx1 and Grx2 were expressed in placenta and present in cell lysates prepared from various tumor cell lines, while only Grx1 was detected in plasma. The concentration of Grx1 determined by BED assay and Grx1 sandwich ELISA respectively correlated, indicating that Grx1 is the major intracellular reductase of low-molecular-weight mixed disulfides with GSH. Moreover, unstimulated peripheral blood mononuclear cells (PBMC) were shown to secrete Grx1 which was strongly suppressed upon 12-0tetradecanoylphorbol-13-acetate (TPA) activation. TPA also lead to reduction of Grx 1 mRNA levels. The results indicate that Grx1 is a protein also with unknown extracellular functions.

Analyzing the expression of human Grx1 in the cervix dining pregnancy we found that the level of Grx1 increased at the mRNA level in term pregnancy and immediately after delivery. The levels were increased in particular after prostaglandin-induced delivery. This may suggests that Grx 1 is involved in the process of cervical ripening, a process also known to involve an inflammatory response with release of inflammatory mediators and influx of leucocytes.

We like others failed, by DNA transfection, to produce cells over-expressing human Grx1 for studies of its biological role suggesting that the level is controlled. We explored VP22, a protein reported to translocate across cell membranes as a carrier for protein Grx delivery. However, we found that previously observed translocation of VP22 is due to a fixation artifact. VP22 due to it's positively charge adheres to cell membranes of living cells and fixation by methanol releases membrane bound VP22 which then redistributes within the cell, preferentially to the cell nucleus. The results in this study give substantial evidence to the notion that several glutaredoxins play important biological roles.

List of scientific papers

I. Bengtsson A, Lundberg M, Avila-Carino J, Jacobsson G, Holmgren A, Scheynius A (2001). Thiols decrease cytokine levels and down-regulate the expression of CD30 on human allergen-specific T helper (Th) 0 and Th2 cells. Clin Exp Immunol. 123(3): 350-60.
https://pubmed.ncbi.nlm.nih.gov/11298119

II. Sahlin L, Wang H, Stjernholm Y, Lundberg M, Ekman G, Holmgren A, Eriksson H (2000). The expression of glutaredoxin is increased in the human cervix in term pregnancy and immediately post-partum, particularly after prostaglandin-induced delivery. Mol Hum Reprod. 6(12): 1147-53.
https://pubmed.ncbi.nlm.nih.gov/11101698

III. Lundberg M, Johansson C, Chandra J, Enoksson M, Jacobsson G, Ljung J, Johansson M, Holmgren A (2001). Cloning and expression of a novel human glutaredoxin (Grx2) with mitochondrial and nuclear isoforms. J Biol Chem. 276(28): 26269-75.
https://pubmed.ncbi.nlm.nih.gov/11297543

IV. Lundberg M, Potamiou A, Kumar S, Holmgren A (2002). Expression of human glutaredoxin 1 (Grx1) and glutaredoxin 2 (Grx2) in cells: Grx1 is secreted by peripheral blood mononuclear cells while phorbol-ester activation down regulated secretion. [Manuscript]

V. Lundberg M, Johansson M (2001). Is VP22 nuclear homing an artifact? Nat Biotechnol. 19(8): 713-4.
https://pubmed.ncbi.nlm.nih.gov/11479552

VI. Lundberg M, Johansson M (2002). Positively charged DNA-binding proteins cause apparent cell membrane translocation. Biochem Biophys Res Commun. 291: 367-71.