Peroxiredoxin-2 (Prx-2) Shows a Dual Role as Antioxidant and Chaperone in Red Cells from a β Thalassemic Mouse Model.

Peroxiredoxins are a ubiquitous family of anti-oxidant enzymes, which have been recently involved in different cellular functions such as cell proliferation or intracellular signaling. In red blood cells (RBCs) Prx-2 is present as scavenger of the reactive oxygen species generated by hemoglobin auto-oxidation as supported also by the evidences in mouse RBCs genetically lacking Prx-2. β thalassemia (thal) is an inherited RBC disorder due to defective β-globin chain synthesis, leading to ineffective erythropoiesis and chronic hemolytic anemia related to severe RBCs membrane oxidative damage. Here, we evaluated Prx-2 expression and localization in RBCs from a mouse model of β thal, which shows biological and clinical features similar to human β thal intermedia. We studied βthal (Hbbth/th) and wild-type mice (WT) at different ages (2- 6 months old), divided into groups of 10 mice each. Since β thal is characterized by high reticulocyte count due to the chronic hemolytic anemia, we divided RBCs into reticulocyte enriched fraction (F1) and dense RBCs fraction (F2), corresponding to the oldest cells. Then, RBCs from F1 and F2 were studied as total RBCs lysate and as RBCs membrane fraction. No significant differences in Prx-2 expression related to animals aging were present within mouse strains. βthal total RBCs lysates showed increase expression of Prx-2 as monomers and dimers in both F1 and F2 fractions compared to WT ones; increase dimerization of Prx-2 in F1 compared to F2, while no differences were present between F1 and F2 in WT RBCs, suggesting an upregulation of Prx-2 synthesis in β thal reticulocytes, since it is already present, and a Prx-2 hyperoxidation in circulating β thal RBCs. Since a chaperone activity for Prx-2 has been recently shown in yeasts and bacteria and being the assistance to denaturated proteins one of the major activity of chaperones, we evaluated Prx-2 membrane localization in β thal RBCs, characterized by severe membrane oxidative damage. Prx-2, as monomers and dimers, was recruited to the membrane in both mouse strains, suggesting a possible dual role of Prx-2 as antioxidant and chaperone. We also observed that the amount of Prx-2 bound to the RBCs membrane was higher in WT than in β thal mice, most likely related to either removal of the more severely damaged β thal RBCs from circulation or to perturbation of Prx-2 function in β thal RBCs. In order to evaluate Prx-2 functions we exposed WT RBCs to either Phenylhydrazine (PHZ 10–20 μM), mimicking β thal RBCs membrane damage or H2O2 at low concentration (5 μM). PHZ treatment significantly reduced the amount of Prx-2 monomers and dimers bound to the membrane in a dose dependent manner, whereas H2O2 did not significantly modify them, indicating that PHZ and H2O2 differently affect Prx-2 membrane recruitment. Since Prx-2 functions seem to be related to phosphorylation events, we analysed Prx-2 localization in RBCs lacking protein tyrosin phosphatase- ε (PTP-ε). In the absence of PTP-ε we observed a lower amount of Prx-2 bound to the membrane as monomers, with undetectable Prx-2 dimers compared to WT ones. When PTP-ε RBCs were treated with PHZ, Prx-2 was almost undetectable on RBCs membrane, suggesting a possible role of PTP-ε in changes of Prx-2 oligomeric state. In conclusion, in β thal mouse RBCs Prx-2 expression is up-regulated, is present in both monomeric and dimeric state, and is modulated by oxidative damage and phosphorylation events supporting a dual role of Prx-2 as antioxidant and chaperone.