Oxidized and poorly glycosylated band 3 is selectively phosphorylated by Syk kinase to form large membrane clusters in normal and G6PD-deficient red blood cells

Pantaleo, Antonella; Ferru, Emanuela; Giribaldi, Giuliana; Mannu, Franca; Carta, Francesco; Mattè, Alessandro; Franceschi, Lucia De; Turrini, Franco

doi:10.1042/bj20081557

Cited by 95 publications

(154 citation statements)

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“…19 Moreover phosphorylation of band 3 has already been demonstrated to be increased in thalassemia, although its functional effects have not been investigated. In accordance with a previous report, 20 Figure 2A,B shows that oxidized and clustered band 3 is strongly hyper-phosphorylated in splenectomized TI patients in comparison with the situation in non-splenectomized patients. Figure 2C shows that the amount of MP released from TI-RBC strictly correlates with the level of band 3 tyrosine phosphorylation (correlations are significant in both splenectomised and nonsplenectomized patients).…”

Section: E Ferru Et Alsupporting

confidence: 79%

“…Standard hypotonic membranes were prepared, as previously described, 20 and stored frozen at -80°C until use. Membrane protein content was quantified using the DC Protein assay (Biorad, Hercules, CA, USA).…”

Section: Red Blood Cell Membrane Preparationmentioning

confidence: 99%

“…RBC appear to possess a mechanism able to recruit Syk kinase to a fraction of less glycosylated band 3 molecules capable of forming disulfide dimers. 20 In turn, the affinity of the phosphorylated band 3 molecules for ankyrin is drastically decreased, their lateral mobility is increased and they have a greater propensity to form large clusters inducing vesiculation. 19 In thalassemias, it has been previously demonstrated that HMC bind to band 3 causing free iron accumulation and free radical production, 7,21 but their role in inducing band 3 phosphorylation and membrane destabilization has never been investigated.…”

Section: Introductionmentioning

confidence: 99%

“…11 The composition and pathogenic roles of MP have been extensively studied in various diseases, such as ischemia, diabetes and atherosclerosis, revealing complex pathogenic roles in modulating nitric oxide and prostacyclin production, stimulating cytokine release, inducing tissue factor expression, as well as monocyte chemotaxis and adherence to the endothelium. [12][13][14][15][16][17][18] Recent studies on the mechanisms of redox regulation of RBC membrane stability 19,20 indicate that oxidative stress induces a phosphorylative response that specifically involves two tyrosine residues located in the cytoplasmic domain of band 3. 20 Band 3 is the most abundant RBC membrane protein, and represents one of the major components of the junctional complexes that connect the lipid bilayer to the cytoskeleton.…”

Section: Introductionmentioning

confidence: 99%

“…[12][13][14][15][16][17][18] Recent studies on the mechanisms of redox regulation of RBC membrane stability 19,20 indicate that oxidative stress induces a phosphorylative response that specifically involves two tyrosine residues located in the cytoplasmic domain of band 3. 20 Band 3 is the most abundant RBC membrane protein, and represents one of the major components of the junctional complexes that connect the lipid bilayer to the cytoskeleton. We previously found that the oxidation of two cysteine residues in the band 3 cytoplasmic domain leads to the docking of Syk kinase.…”

Section: Introductionmentioning

confidence: 99%

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Thalassemic erythrocytes release microparticles loaded with hemichromes by redox activation of p72Syk kinase

Ferru

Pantaleo

Carta³

et al. 2013

Haematologica

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© F e r r a t a S t o r t i F o u n d a t i o ndifferent genetic and clinical status provided new insights into the pathogenic role of circulating MP and possible interventions to control their amount in thalassemia. MethodsUnless otherwise stated, all materials were obtained from Sigma-Aldrich, St. Louis, MO, USA. Additional information about the methods are provided in the Online Supplementary Data. Treatment of red blood cellsVenous blood was drawn from 12 healthy individuals, eight non-splenectomized TI patients and six splenectomized TI patients. All subjects provided written, informed consent before entering the study. The study was approved by the local Ethics Committee and conducted in accordance with Good Clinical Practice guidelines and the Declaration of Helsinki.Clinical analyses were performed by routine laboratory tests at the Policlinico Hospital (Milan, Italy). Blood anti-coagulated with heparin was stored in citrate-phosphate-dextrose with adenine (CPDA-1) prior to its use. RBC were washed three times with phosphate-buffered saline (PBS: 137mM NaCl, 2.7mM KCl, 8.1mM K 2 HPO 4 , 1.5mM KH 2 PO 4 , pH 7.4) containing glucose 5 mM to obtain packed RBC.To stimulate HMC formation, RBC from healthy donors were suspended at a hematocrit of 30% and incubated with different concentrations (0, 0.25, 0.5, 1, 1.5, 2 mM) of phenylhydrazine at 37 °C for 4 h. To test the antioxidant effect in MP release we incubated 200 μM of 2-mercaptoethanol in the presence of 1 mM phenylhydrazine. When necessary, RBC were pretreated with Syk kinase inhibitors (10 μM Syk inhibitors II and 10 μM Syk inhibitors IV, Calbiochem, Darmstadt, Germany), for 1 h at 37 °C in the dark. Each reaction was terminated by three washes with PBS-glucose. For all protocols described, untreated controls were processed identically except that the stimulant/inducer was omitted from the incubation. Red blood cell membrane preparationStandard hypotonic membranes were prepared, as previously described, 20 and stored frozen at -80°C until use. Membrane protein content was quantified using the DC Protein assay (Biorad, Hercules, CA, USA). Analysis of microparticlesThe MP in plasma were analyzed by flow cytometry using a modification of a previously described method:22 25 μL of plasma diluted 1:1 with PBS-glucose 5mM were analyzed using anti-CD41 (BD, Franklin Lakes, NJ, USA) and anti-glycophorin-A (Dako, Denmark), both diluted 1:10. Assay of hemichromesHMC were quantified by measuring heme absorbance (Abs) using the following equation:HMC= -133xAbs577 -114xAbs630 + 233xAbs560, and expressed as nMoles/mL of solubilized membranes. 23 Microparticle isolationTo induce MP release in vitro, RBC from each volunteer and phenylhydrazine-treated RBC in PBS (30% hematocrit) were incubated as previously described. 24 Electrophoresis and immunoblottingMembrane and MP proteins were solubilized in Laemmli buffer 25 under reducing [2% (w/v) dithiothreitol] or non-reducing conditions at a volume ratio of 1:1. Sodium dodecylsulfate polyacrylamide gel electropho...

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Section: E Ferru Et Alsupporting

confidence: 79%

Section: Red Blood Cell Membrane Preparationmentioning

confidence: 99%