Peroxiredoxin-2 (Prx2) Membrane Displacement is A New Additional Factor to the Severity of β-Thalassemic Mouse Red Cell Membrane Damage.
Authors: Mattè Alessandro , Low Philip S. , Turrini Franco , Bertoldi Mariarita , Campanella Maria Estela , Pantaleo Antonella , Siciliano Angela , De Franceschi Lucia 
Published: October 2019

Abstract 2009

Poster Board I-1031

Peroxiredoxin 2 (Prx2) is the third most abundant cytoplasmic protein in red blood cells (RBCs) and is involved in defence against oxidative stress. Although much is known regarding the structure and function of Prx2 in healthy RBCs, its role in protecting pathological RBCs remains largely unexplored. Here, we studied Prx2 dimerization and membrane translocation in mouse RBCs (wild-type mice, n=20) in response to in vitro oxidative stress by either phenylhydrazine-PHZ (50 μM) or diamide (2mM) or H2O2 (75-100 μM) and in vivo in β thalassemic (β thal) mouse RBCs (Hbbth/th and Hbbth3/+ mouse strains; n=16 from each group).

In RBCs lysates we observed (i) slightly increased Prx2 dimerization in PHZ treated RBCs; (ii) complete Prx2 dimerization in presence of diamide; (iii) not apparent Prx2 dimerization in H2O2, suggesting a different sensitivity of Prx2 to the various oxidant agents. In RBCs membrane, Prx2 association was (i) reduced in PHZ treated RBCs; (ii) increased in diamide treated RBCs as both monomers and dimers and (iii) reduced to almost undetectable amount in H2O2 treated RBCs. In order to evaluate whether Prx2 native or from RBCs exposed to oxidative stress possess different degrees of affinity to native or oxidized membrane, we compared Prx2 membrane association in control, diamide, PHZ treated membranes with the cytoplasmic RBC fractions obtained from control, diamide and PHZ treated RBCs. We observed a remarkable loss of interactions only between Prx2 and the membranes obtained from PHZ treated red cells (n=3), suggesting that PHZ may inhibit Prx2 binding to the membrane through the masking of its docking sites. Using multiple techniques (immunofluorescence confocal microscopy, immunoblot and flow cytometric analysis), we showed that Prx2 was displaced from PHZ treated RBC membrane in a dose dependent manner. We then investigated the connection between Prx2 membrane displacement and hemicrome binding in time course experiments with PHZ treated RBCs. Prx2 was displaced from the membrane concomitantly to hemichrome membrane binding, indicating that PHZ blocks the Prx2 binding to the membrane through the masking of its docking sites by hemichromes.

Since PHZ membrane oxidative damage mimics that of b thal RBCs we have examined Prx2 in RBCs from two mouse models of b thalassemia. We showed that Prx2 content was higher in b thal mouse RBCs than in controls and correlated with the clinical severity of b thalassemia, but the amount of Prx2 associated to the membrane was markedly reduced as observed in PHZ-treated RBCs. These data suggest that in b thal RBCs the masking of Prx2 membrane binding sites by hemichromes and the accumulation of its oxidized/dimerized state in the cytoplasm may affect Prx2 catalytic efficiency on b thal membrane proteins, therefore contributing to their lack of repair from oxidative insults. Thus, the abnormally low membrane association of Prx2 in b thal mouse RBCs represents a new additional factor amplifying the oxidative damage characterizing b thal RBCs and responsible for their reduced lifespan in the periferal circulation.

Disclosures:

No relevant conflicts of interest to declare.