Cellular and plasma levels of human glutaredoxin 1 and 2 detected by sensitive ELISA systems

doi:10.1016/j.bbrc.2004.04.199

Biochemical and Biophysical Research Communications

Volume 319, Issue 3, 2 July 2004, Pages 801-809

https://doi.org/10.1016/j.bbrc.2004.04.199 Get rights and content

Abstract

Glutaredoxins (Grx) catalyze glutathione-dependent thiol-disulfide oxidoreduction reactions. Mammalian cells contain at least two dithiol glutaredoxins, the well-characterized cytoplasmic (12 kDa) Grx1 and the recently identified (18 kDa) Grx2 with mitochondrial and nuclear isoforms. We have developed two sensitive and specific sandwich ELISAs to study the levels of human Grx1 and Grx2. Both Grx1 and Grx2 were present in placenta extracts and in cell lysates prepared from various tumor cell lines. However, the levels of Grx1 were at least 20 times higher than those of Grx2. Plasma from healthy blood donors contained 13.4 ± 7.9 ng/ml of Grx1, while Grx2 was not detected. Unstimulated peripheral blood mononuclear cells were shown to secrete Grx1, but upon 12-O-tetradecanoylphorbol-13-acetate activation, the secretion of Grx1 was strongly suppressed. This effect was shown to occur at the transcriptional level. The secretion of Grx1 and its presence in plasma suggests extracellular functions as found for mammalian thioredoxin 1.

Section snippets

Materials and methods

Reagents. 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and dithiothreitol (DTT) were obtained from Sigma Chemical, St. Louis, MO/USA. All cell mediums and supplements were purchased from Gibco-BRL if not stated otherwise. Recombinant human Grx1 and Grx2 (6× His-Grx2Δ41–164) were purified to homogeneity as previously described by Padilla et al. [9] and Lundberg et al. [10], respectively. Human thioredoxin 1 (Trx1) was purified as previously described by Ren et al. [22]. All other chemicals and

Sandwich ELISA for human Grx1 and Grx2

The standard curves for the Grx1 and Grx2 ELISAs showed good titrations in the range of 0.1–25 ng/ml (Fig. 1). The intra-assay coefficient of variation for Grx1 and Grx2 ELISAs was 7.3% (n=27) and 4.5% (n=7), respectively [25]. The inter-assay coefficient of variation for the Grx1 and Grx2 ELISAs was 7.3% (n=10) and 10.5% (n=10), respectively [25]. Moreover, the detection limit, determined as three times the standard deviation above the blank, is approximately 0.2 ng/ml for Grx1 and 0.1 ng/ml for

Discussion

Mammalian cells contain at least two dithiol glutaredoxins, the well-characterized Grx1 [31], [32] with a cytoplasmic localization pattern, and the recently identified Grx2 [10] with the potential to localize both in the mitochondria and in the nucleus or at the nuclear membrane due to differential splicing. Here we present the development of two novel sandwich ELISAs for human Grx1 and Grx2 based upon mono-specific polyclonal antibodies. The assays were shown to be highly sensitive, specific,

Acknowledgements

This research was supported by grants from the Swedish Cancer Society (961), the Knut and Alice Wallenberg Foundation, the Inga-Britt and Arne Lundberg Foundation, and the Karolinska Institute.

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Circulation through different vascular beds continuously exposes blood components to tissue-derived oxidants. To counteract this continuous challenge, blood counts on a fast and effective set of antioxidant systems. Circulating thiol-containing molecules are key players in blood antioxidant systems. In red blood cells, the peroxiredoxin/thioredoxin/thioredoxin reductase system effectively contributes to the removal of hydrogen peroxide and peroxynitrite. These cells also retain a high concentration of reduced glutathione due to the concerted action of the enzymes dedicated to glutathione biosynthesis and homeostasis. The glutathione/glutathione peroxidase/glutathione reductase system contributes mainly to the removal of lipid hydroperoxides, while glutathione transferase and glutaredoxin participate in detoxification and repairing pathways. On the other hand, plasma is relatively poor in antioxidants—the single reduced cysteine of albumin is the most abundant thiol in plasma and a preferential target of oxidants in this milieu.
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Citation Excerpt :
Although there is increasing evidence of the importance of experimental models of CVD, few clinical studies are available on Glrx. Despite being a cytosolic enzyme, Glrx is detectable in human plasma [14]. A few cross-sectional studies with small subjects demonstrated that Glrx is a potential disease maker in clinical settings [15,16].
Reactive oxygen species that increase during cardiovascular disease (CVD) react with protein cysteine residues to form a glutathione adduct by S-glutathionylation, which is selectively removed by glutaredoxin-1 (Glrx). We previously showed that S-glutathionylation and Glrx play important roles in mouse models of CVD, such as heart failure and peripheral artery disease models. However, there are few clinical studies on Glrx in CVD. Although Glrx is a cytosolic protein expressed in various organs, it is detectable in human plasma. Studies have reported that Glrx in plasma is a potential disease maker, such as CVD and chronic kidney disease and diabetes, however, it remains unclear whether Glrx is related to the prognosis of patients with CVD. The purpose of this study was to elucidate whether Glrx levels in plasma are associated with future events in patients with CVD. Plasma levels of Glrx were measured in 555 patients with CVD who underwent cardiac catheterization using enzyme-linked immunosorbent assay. All patients were followed prospectively for ≤36 months or until occurrence of adverse events, including all-cause death, non-fatal myocardial infarction, and worsening heart failure. During a mean follow-up period of 33 months, 54 adverse events occurred. Kaplan-Meier analysis showed that higher levels of Glrx (>0.622 ng/mL, determined by receiver-operating characteristic curve) resulted in a higher probability for adverse events compared with lower levels of Glrx (≤0.622 ng/mL) (P < 0.01, log-rank test). Multivariate Cox proportional hazards analysis showed that Glrx was a significant predictor of adverse events after adjustment for known risk factors. In conclusion, levels of plasma Glrx >0.662 ng/mL can predict future events in patients with CVD.
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Glutaredoxin, Grx, is a small protein containing an active site cysteine pair and was discovered in 1976 by Arne Holmgren. The Grx system, comprised of Grx, glutathione, glutathione reductase, and NADPH, was first described as an electron donor for Ribonucleotide Reductase but, from the first discovery in E.coli, the Grx family has impressively grown, particularly in the last two decades. Several isoforms have been described in different organisms (from bacteria to humans) and with different functions.
The unique characteristic of Grxs is their ability to catalyse glutathione-dependent redox regulation via glutathionylation, the conjugation of glutathione to a substrate, and its reverse reaction, deglutathionylation. Grxs have also recently been enrolled in iron sulphur cluster formation. These functions have been implied in various physiological and pathological conditions, from immune defense to neurodegeneration and cancer development thus making Grx a possible drug target.
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Glutaredoxins are a group of heat stable oxidoreductases ubiquitously found in prokaryotes and eukaryotes. They are widely known for GSH (glutathione)-dependent protein disulfide reduction and cellular redox homeostasis. This study was performed to identify and characterize rockfish (Sebastes schlegelii) glutaredoxin 1 (SsGrx1) at molecular, transcriptional, and functional levels. The coding sequence of SsGrx1 was 318 bp in length and encoded a protein containing 106 amino acids. The molecular weight and theoretical isoelectric point of the putative SsGrx1 protein were 11.6 kDa and 6.71 kDa, respectively. The amino acid sequence of SsGrx1 comprised a CPYC redox active motif surrounded by several conserved GSH binding sites. The modeled protein structure was found to consist of five α-helices and four β-sheets, similar to human Grx1. SsGrx1 showed a tissue specific expression in all the tissues tested, with the highest expression in the kidney. Immune stimulation by lipopolysaccharides (LPS), polyinosinic:polycytidylic acid (polyI:C), and Streptococcus iniae (S. iniae) could significantly modulate the SsGrx1 expression pattern in the blood and gills. Analysis of its subcellular localization disclosed that SsGrx1 was prominently localized in the cytosol. Recombinant SsGrx1 (rSsGrx1) exhibited significant activity in insulin disulfide reduction assay and HED (β-Hydroxyethyl Disulfide) assay. Furthermore, transient overexpression of SsGrx1 in FHM (fathead minnow) cells significantly enhanced cell survival upon H₂O₂-induced apoptosis. Collectively, our findings strongly suggest that SsGrx1 plays a crucial role in providing rockfish immune protection against pathogens and oxidative stress.

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^☆: Abbreviations: Grx, glutaredoxin; Grx1, cytosolic glutaredoxin; Trx, thioredoxin; Trx1, cytosolic thioredoxin; HED, 2-hydroxyethyl disulfide; GSSG, glutathione disulfide; PBS, phosphate buffered saline; PBMC, peripheral blood mononuclear cells; ELISA, enzyme linked immunosorbent assay.

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Cellular and plasma levels of human glutaredoxin 1 and 2 detected by sensitive ELISA systems☆

Abstract

Section snippets

Materials and methods

Sandwich ELISA for human Grx1 and Grx2

Discussion

Acknowledgements

J. Biol. Chem.

Structure

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Immunol. Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biol. Chem.

J. Biol. Chem.

Free Radic. Biol. Med.

Anal. Biochem.

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Proc. Hydrogen donor system for Escherichia coli ribonucleoside-diphosphate reductase dependent upon glutathione

Proc. Natl. Acad. Sci. USA

Reduction of ribonucleotides

Annu. Rev. Biochem.

Role of reversible oxidation–reduction of enzyme thiols-disulfides in metabolic regulation

Annu. Rev. Biochem.

High-level expression of fully active human glutaredoxin (thioltransferase) in E. coli and characterization of Cys7 to Ser mutant protein

FEBS Lett.