Integrated proteomic analysis of normal and diseased red cells. Role of physiological stresses in erythrocyte signaling pathways

Peroxiredoxin 2 (Prx2) is the third most abundant cytoplasmic protein in red blood cells (RBCs) and is involved at least in part in defence against oxidative stress. Here, we examined the expression and localization of Prx2 in RBCs from two murine models of β thalassemia (Hbbth3/+, Hbbth/th) and in normal RBCs treated with phenylhydrazine (PHZ) as β thalassemic-like RBCs. Prx2 expression was higher in β thalassemic mouse models than in normal RBCs without effects of cell density/age. Dimeric Prx2 was only detectable in β thalassemic RBC lysates and related with the severity of the hematological phenotype. Although β thalassemic RBC membrane is characterized by a severe oxidative damage, we found Prx2 translocated from the membrane to the cytosol, without detectable dimers as also observed in β thalassemic-like RBCs. We generated 2D maps of the phosphoenriched RBC membrane proteins, and in the area where Prx2 was expected to migrate, anti-phosphotyrosine staining was observed, which was further identified as Prx2 by both mass-spectrometric and immunoblot-analysis. These data demonstrate that a population of Prx2 associated with the membrane is tyrosine-phosphorylated and that tyrosine phosphorylation might regulate both the oligomeric state and membrane association of Prx2, regardless of the oxidation state of the cell.