Peroxiredoxin 2 functions as a noncatalytic scavenger of low-level hydrogen peroxide in the erythrocyte

Abstract

Peroxiredoxin 2 (Prx2), a thiol-dependent peroxidase, is the third most abundant protein in the erythrocyte, and its absence in knock-out mice gives rise to hemolytic anemia. We have found that in human erythrocytes, Prx2 was extremely sensitive to oxidation by H 2 O 2 , as dimerization was observed after exposure of 5 ؋ 10 6 cells/mL to 0.5 M H 2 O 2 . In contrast to Prx2 in Jurkat T lymphocytes, Prx2 was resistant to overoxidation (oxidation of the cysteine thiol to a sulfinic/ sulfonic acid) in erythrocytes. Reduction of dimerized Prx2 in the erythrocyte occurred very slowly, with reversal occurring gradually over a 20-minute period. Very low thioredoxin reductase activity was detected in hemolysates. We postulate that this limits the rate of Prx2 regeneration, and this inefficiency in recycling prevents the overoxidation of Prx2. We also found that Prx2 was oxidized by endogenously generated H 2 O 2 , which was mainly derived from hemoglobin autoxidation. Our results demonstrate that in the erythrocyte Prx2 is extremely efficient at scavenging H 2 O 2 noncatalytically. Although it does not act as a classical antioxidant enzyme, its high concentration and substrate sensitivity enable it to handle low H 2 O 2 concentrations efficiently. These unique redox properties may account for its nonredundant role in erythrocyte defense against oxidative stress. ( IntroductionThe peroxiredoxins (Prxs) constitute a family of homodimeric peroxidases that reduce H 2 O 2 and alkyl hydroperoxides to water and alcohol, respectively. They rely on a conserved cysteine residue to catalyze peroxide reduction. There are 6 known mammalian isoforms (Prx1-6), classified as typical 2-Cys, atypical 2-Cys, or 1-Cys Prxs based on the mechanism and number of cysteines involved during catalysis. 1 When peroxiredoxin 2 (Prx2; a typical 2-Cys Prx) reacts with peroxide, the peroxidatic cysteine at the active site on one subunit is oxidized to a sulfenic acid. A second conserved cysteine at the C-terminal end of the other subunit (the resolving cysteine) then reacts with the sulfenic acid to form a disulfide bridge. Reduction of the disulfide by thioredoxin (Trx) regenerates Prx2 and completes the cycle. Trx is in turn regenerated by thioredoxin reductase (TrxR), with reducing equivalents derived from NADPH. 2 An intriguing feature of mammalian 2-Cys Prxs is that in the presence of high levels of peroxide, the peroxidatic Cys becomes overoxidized to the sulfinic (SO 2 H) or sulfonic (SO 3 H) acid form. 3 This abolishes the enzyme's peroxidase activity, although overoxidized Prx can be slowly reverted to the reduced state by sulfiredoxin. 4 It has been suggested that overoxidation allows intracellular accumulation of H 2 O 2 , which can then function as a signal transducer for various pathways. 5,6 Compared with other somatic cells, erythrocytes are exposed to oxidative stress from a wide variety of sources. They contain high levels of O 2 and hemoglobin (Hb), which continually autoxidizes to produce O 2 Ϫ and H 2 O 2 . They also have membranes...

Treating gout with pegloticase, a PEGylated urate oxidase, provides insight into the importance of uric acid as an antioxidant in vivo

“…1B), ∼1% of the RBC count in normal blood. Human RBC previously have been shown to protect murine leukemia cells from reactive oxygen species (ROS) generated by the combination of hypoxanthine and xanthine oxidase (26 (28). Thus, exposing 5 × 10 6 /mL RBC to 5 μM H 2 O 2 caused the quantitative, but reversible, conversion of Prx2 to dimer ( Fig.…”

“…Erythrocytes prepared from fresh heparinized blood were washed three times in PBS, then resuspended in PBS containing 5 mM glucose or in plasma, followed by incubation with H 2 O 2 , or urate plus pegloticase, as indicated in figure legends. After incubation, RBC were centrifuged at 4°C and washed three times with ice-cold 100 mM NEM in PBS as described (28). The RBC then were resuspended in 0.1 mL of nonreducing SDS sample buffer (65.8 mM Tris-HCL pH 6.8, 10.5% glycerol, 2.1% SDS) containing 100 mM N-ethylmaleimide (NEM) and stored at −80°C.…”

“…Heparinized blood obtained from patients at various times during treatment with pegloticase (8 mg every 3 wk) was processed within 15 min at 4°C. After plasma was removed, 10 μL of packed RBC were washed three times with freshly prepared ice-cold 100 mM NEM in PBS, essentially as described (28). The RBC pellets then were resuspended in 0.1 mL of nonreducing SDS sample buffer containing 100 mM NEM and were stored at −80°C before immunoblot analysis of Prx2, as described above.…”

Oxidation of proteins: Basic principles and perspectives for blood proteomics

“…This behaviour makes peroxiredoxin 2 ideally suited to H 2 O 2 sensing at low concentration [155]. When H 2 O 2 concentration increases, catalase and glutathione peroxidase are necessary to dispose of excess H 2 O 2 [156]. What is unknown is the role of peroxiredoxin 2 dimer and overoxidised forms in H 2 O 2 signalling.…”

Levels of two oxidative stress indicators of human sickle erythrocytes incubated in aqueous extracts of Anacardium occidentale, Psidium guajava and Terminalia catappa.

“…[9,22] Other antioxidants are alphatocopherol [23,24] , uric acid [25][26][27] , ascorbic acid [28] , carotenoids [29,30] and varieties of plant secondary metabolites such as flavonoid and related polyphenolic compounds. [31,32] Notable erythrocyte enzymatic ROS scavenging systems include glutathione reductase, [17,19] glutathione peroxidase, [33] glucose-6-phosphate dehydrogenase, [18,34] superoxide dismutase, [5,35] catalases, [36][37][38] peroxiredoxins, [39,40] and NADHmethaemoglobin reductase. [21,[41][42][43] Varieties of xenobiotics of plant origin such as fava beans extracts (Vicia faba) have been reported as agents that can modify the redox status of human erythrocytes especially in individuals with impaired glucose-6-phosphate dehydrogenase activity.…”