A New method for the capture of surface proteins in <i>Plasmodium falciparum</i> parasitized erythrocyte

Introduction: We propose a new method for the selective labeling, isolation and electrophoretic analysis of the Plasmodium falciparum protein exposed on the erythrocyte cell surface. Historically, membrane surface proteins have been isolated using a surface biotinylation followed by capture of biotin-conjugated protein via an avidin/streptavidin-coated solid support. The major drawback of the standard methods has been the labeling of internal proteins due to fast internalization of biotin. Methodology: To solve this problem, we used a biotin label that does not permeate through the membrane. As a further precaution to avoid the purification of non surface exposed proteins, we directly challenged whole labeled cells with avidin coated beads and then solubilized them using non ionic detergents. Results: A marked enrichment of most of the RBC membrane proteins known to face the external surface of the membrane validated the specificity of the method; furthermore, only small amounts of haemoglobin and cytoskeletal proteins were detected. A wide range of P. falciparum proteins were additionally described to be exposed on the erythrocyte surface. Some of them have been previously observed and used as vaccine candidates while a number of newly described antigens have been presently identified. Those antigens require further characterization and validation with additional methods. Conclusion: Surface proteins preparations were very reproducible and identification of proteins by mass spectrometry has been demonstrated to be feasible and effective.</br

Irreversible AE1 tyrosine phosphorylation leads to membrane vesiculation in G6PD deficient red cells

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

Band 3 Erythrocyte Membrane Protein Acts as Redox Stress Sensor Leading to Its Phosphorylation by p 72

In erythrocytes, the regulation of the redox sensitive Tyr phosphorylation of band 3 and its functions are still partially defined. A role of band 3 oxidation in regulating its own phosphorylation has been previously suggested. The current study provides evidences to support this hypothesis: (i) in intact erythrocytes, at 2 mM concentration of GSH, band 3 oxidation, and phosphorylation, Syk translocation to the membrane and Syk phosphorylation responded to the same micromolar concentrations of oxidants showing identical temporal variations; (ii) the Cys residues located in the band 3 cytoplasmic domain are 20-fold more reactive than GSH; (iii) disulfide linked band 3 cytoplasmic domain docks Syk kinase; (iv) protein Tyr phosphatases are poorly inhibited at oxidant concentrations leading to massive band 3 oxidation and phosphorylation. We also observed that hemichromes binding to band 3 determined its irreversible oxidation and phosphorylation, progressive hemolysis, and serine hyperphosphorylation of different cytoskeleton proteins. Syk inhibitor suppressed the phosphorylation of band 3 also preventing serine phosphorylation changes and hemolysis. Our data suggest that band 3 acts as redox sensor regulating its own phosphorylation and that hemichromes leading to the protracted phosphorylation of band 3 may trigger a cascade of events finally leading to hemolysis

Band 3 Erythrocyte Membrane Protein Acts as Redox Stress Sensor Leading to Its Phosphorylation by p (72) Syk

In erythrocytes, the regulation of the redox sensitive Tyr phosphorylation of band 3 and its functions are still partially defined. A role of band 3 oxidation in regulating its own phosphorylation has been previously suggested. The current study provides evidences to support this hypothesis: (i) in intact erythrocytes, at 2\u2009mM concentration of GSH, band 3 oxidation, and phosphorylation, Syk translocation to the membrane and Syk phosphorylation responded to the same micromolar concentrations of oxidants showing identical temporal variations; (ii) the Cys residues located in the band 3 cytoplasmic domain are 20-fold more reactive than GSH; (iii) disulfide linked band 3 cytoplasmic domain docks Syk kinase; (iv) protein Tyr phosphatases are poorly inhibited at oxidant concentrations leading to massive band 3 oxidation and phosphorylation. We also observed that hemichromes binding to band 3 determined its irreversible oxidation and phosphorylation, progressive hemolysis, and serine hyperphosphorylation of different cytoskeleton proteins. Syk inhibitor suppressed the phosphorylation of band 3 also preventing serine phosphorylation changes and hemolysis. Our data suggest that band 3 acts as redox sensor regulating its own phosphorylation and that hemichromes leading to the protracted phosphorylation of band 3 may trigger a cascade of events finally leading to hemolysis

Regulation of Membrane-Cytoskeletal Interactions by Tyrosine Phosphorylation of Erythrocyte Band 3

Il dominio citoplasmatico della banda 3 funge da centro di organizzazione della membrana eritrocitaria e costituisce il principale substrato della tirosina chinasi del globulo rosso. La fosforilazione in tirosina della banda 3 \ue8 indotta da vari stimoli fisiologici, compresi l'invasione del parassita della malaria, la perdita di volume cellulare, il normale invecchiamento cellulare e stress ossidativo (talassemie, anemia falciforme, G6PDH carenza, ecc.). Nel tentativo di caratterizzare le conseguenze biologiche della fosforilazione in tirosina della banda 3, abbiamo indagato sui cambiamenti funzionali del polipeptide che accompagnano la sua fosforilazione. Dimostriamo che la fosforilazione in tirosina promuove la dissociazione della banda 3 dallo scheletro spettrina-actina, come evidenziato da: i) una diminuzione di affinit\ue0 con l\u2019anchirina, in studi sul legame, ii) un aumento di estraibilit\ue0, mediante detergente, della banda 3 dalle membrane, iii) un aumento della cross-linkabilit\ue0 della banda 3 mediante BS3, iv) cambiamenti significativi nella morfologia degli eritrociti, e v) aumento del tasso di diffusione della banda 3 in cellule intatte. La dissociazione della banda 3 dal citoscheletro, a causa della rottura del legame con l\u2019anchirina e l\u2019adducina, pu\uf2 facilitare cambiamenti nelle propriet\ue0 di membrana. Ci\uf2 sostiene l\u2019ipotesi che la fosforilazione in tirosina della banda 3 possa avere un ruolo nell\u2019attivazione dei cambiamenti adattativi dell\u2019eritrocita, che permettono alla cellula di rispondere alle sollecitazioni precedentemente menzionate.The cytoplasmic domain of band 3 serves as a center of erythrocyte membrane organization and constitutes the major substrate of erythrocyte tyrosine kinases. Tyrosine phosphorylation of band 3 is induced by several physiological stimuli including malaria parasite invasion, cell shrinkage, normal cell aging, and oxidant stress (thalassemias, sickle cell disease, G6PDH deficiency, etc). In an effort to characterize the biologic sequelae of band 3 tyrosine phosphorylation, we have looked for changes in the polypeptide\u2019s function that accompany its phosphorylation. We report that tyrosine phosphorylation promotes dissociation of band 3 from the spectrin-actin skeleton as evidenced by: i) a decrease in ankyrin affinity in direct binding studies, ii) an increase in detergent extractability of band 3 from ghosts, iii) a rise in band 3 cross-linkability by BS3, iv) significant changes in erythrocyte morphology, and v) elevation of the rate of band 3 diffusion in intact cells. Because release of band 3 from its ankyrin and adducin linkages to the cytoskeleton can facilitate changes in multiple membrane properties, tyrosine phosphorylation of band 3 is argued to enable adaptive changes in erythrocyte biology that permit the cell to respond to the above stresses

May band 3 hyper-phosphorylation have a functional role in microcyte formation in heterozygous thalassemias?

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