Irreversible AE1 Tyrosine Phosphorylation Leads to Membrane Vesiculation in G6PD Deficient Red Cells

et al. (2011) Irreversible AE1 Tyrosine Phosphorylation Leads to Membrane Vesiculation in G6PD Deficient Red Cells. PLoS ONE 6(1): e15847. doi:10.1371/journal.pone.0015847 Irreversible AE1 Tyrosine Phosphorylation Leads to Membrane Vesiculation in G6PD Deficient Red Cells Antonella Pantaleo 0 Emanuela Ferru 0 Franco Carta 0 Franca Mannu 0 Luigi F. Simula 0 Amina Khadjavi 0 Proto Pippia 0 Francesco Turrini 0 Hendrik W. van Veen, University of Cambridge, United Kingdom 0 1 Department of Genetics, Biology and Biochemistry, University of Turin , Turin , Italy , 2 Section of Internal Medicine, Department of Clinical and Experimental Medicine, University of Verona , Verona, Italy, 3 Nurex S.r.l., Sassari , Italy , 4 Hospital of Alghero, ASL 1- Sassari , Sassari , Italy , 5 Department of Physiological, Biochemical and Cell Sciences, University of Sassari , Sassari , Italy Background: While G6PD deficiency is one of the major causes of acute hemolytic anemia, the membrane changes leading to red cell lysis have not been extensively studied. New findings concerning the mechanisms of G6PD deficient red cell destruction may facilitate our understanding of the large individual variations in susceptibility to pro-oxidant compounds and aid the prediction of the hemolytic activity of new drugs. Methodology/Principal Findings: Our results show that treatment of G6PD deficient red cells with diamide (0.25 mM) or divicine (0.5 mM) causes: (1) an increase in the oxidation and tyrosine phosphorylation of AE1; (2) progressive recruitment of phosphorylated AE1 in large membrane complexes which also contain hemichromes; (3) parallel red cell lysis and a massive release of vesicles containing hemichromes. We have observed that inhibition of AE1 phosphorylation by Syk kinase inhibitors prevented its clustering and the membrane vesiculation while increases in AE1 phosphorylation by tyrosine phosphatase inhibitors increased both red cell lysis and vesiculation rates. In control RBCs we observed only transient AE1 phosphorylation. Conclusions/Significance: Collectively, our findings indicate that persistent tyrosine phosphorylation produces extensive membrane destabilization leading to the loss of vesicles which contain hemichromes. The proposed mechanism of hemolysis may be applied to other hemolytic diseases characterized by the accumulation of hemoglobin denaturation products. - G6PD deficiency affects more than 400 million people worldwide, with a prevalence varying from 10 to 25% in most areas where malaria is endemic. This genetic defect provides partial protection against malaria, but may lead to severe hemolytic episodes after the administration of some drugs (antimalarials, anti-inflammatories, vitamin K, etc.), the ingestion of fava beans (favism) or infection [13]. Typically the appearance of the first symptoms occurs 2448 hours after the intake of prooxidant drugs or fava beans. While the molecular biology of G6PD deficiency has been extensively studied [2], the molecular mechanisms leading to the hemolytic crisis are still unclear. G6PD deficient red cells (G2 RBCs) display a failure of the protective response to oxidant stress, which leads to irreversible oxidation of glutathione [1,2,46]. The accumulation of large hemichrome aggregates (Heinz bodies) is an additional hallmark of the hemolytic crisis in G2 individuals [7]. Some membrane alterations have been described in G2 RBCs, such as the oxidation and clustering of membrane proteins, the binding of hemichromes to the internal face of the membrane, the destabilization of the membrane and the release of micro-vesicles [810]. Interestingly, increased hemichrome formation has been observed in G2 RBCs infected by malaria parasites [11]. The data available on membrane modifications are in any case insufficient to formulate a clear hypothesis as to the mechanisms of membrane destabilization and G2 RBC destruction. The dearth of information concerning the mechanisms of red cell lysis represents a practical drawback which impedes both any prediction about the hemolytic activity of drugs and the understanding of the large individual susceptibility even in presence of the same G6PD mutation [1]. The authors, as well as others have shown that band 3 red cell membrane protein (AE1) displays a marked tendency to become tyrosine phosphorylated in G- RBCs after SH group oxidation or GSH depletion by 1-chloro-2,4-dinitrobenzene (CDNB) or diamide [12,13]. We have also demonstrated that Syk tyrosine kinase strongly increases its affinity to oxidized AE1 and induces its selective phosphorylation [13]. Hyper-phosphorylated AE1 showed a manifest tendency to cluster, indicating a change in its interactions with the cytoskeletal network. Furthermore, abnormal AE1 tyrosine phosphorylation has been observed in a number of red cell disorders [14]. In the present study we have demonstrated that following SH group oxidation induced by diamide (SH group oxidant) and divicine, an oxygen reactive compound held responsible for favism [15], AE1 becomes increasingly and irreversibly phosphorylated in G2 RBCs. Syk kinase inhibition largely prevents red cell membrane lysis and vesiculation, strongly suggesting a functional role of AE1 tyrosine phosphorylation in the red cell membrane destabilization. Short and long term effects of oxidants in G6PD deficient red cells Previous work has described how oxidant treatments induce more intense AE1 tyrosine phosphorylation in G2 RBCs than in control RBCs [13,14,16]. In the present study, we analyzed AE1 phosphorylation and a series of additional parameters for longer time exposure with diamide, an -SH group oxidant reagent, or with divicine [15], a compound extracted from fava beans considered responsible for severe hemolytic crises in G2 deficient subjects [5,15]. The long term effects of oxidants in the G2 RBCs were not easily predictable as, although the G2 RBC samples used in our experiments had low G6PD levels (Mediterranean variant 563 C . T with approximately 23% of normal red cell G6PD activity level), the hexose monophosphate shunt activity in these cells presented normal activity and could be 23 fold further activated following oxidant treatments [17]. Following 0.25 mM treatment with diamide, approximately 80% of reduced glutathione (GSH) was oxidized within 5 minutes and the pre-treatment levels were restored within 45 minutes in control RBCs. Conversely, in G2 RBCs reduced GSH further declined and reached un-measurable levels within 2 hours of incubation (Fig. 1A). Indistinguishable GSH response was elicited by divicine 0.5 mM. On the basis of these results we decided to use these concentrations both for diamide and divicine. Figure 1C shows that following oxidant treatment, in G2 RBCs, AE1 phosphorylation progressively increased during the course of incubation (10 hours). In control RBCs AE1 phosphorylation was completely reverted in approximately 1 hour. Syk kinase inhibitors markedly reduced the (...truncated)