Background \ud \ud Stomatocytoses are a group of inherited autosomal dominant hemolytic anemias and include overhydrated hereditary stomatocytosis, dehydrated hereditary stomatocytosis, hereditary cryohydrocytosis and familial pseudohyperkalemia. \ud \ud Design and Methods \ud \ud We report a novel variant of hereditary stomatocytosis clue to a de novo band 3 mutation (p. G796R-band3 CEINGE) associated with a dyserythropoietic phenotype. Band 3 genomic analysis, measurement at of hematologic parameters and red cell indices and morphological analysis of bone marrow were carried out. We then evaluated the red cell membrane permeability and ion transport systems by functional studies of the patients erythrocytes and Xenopus oocytes transfected with mutated band 3. We analyzed the red cell membrane tyrosine phosphorylation profile and the membrane association of the tyrosine kinases Syk and Lyn from the Src-family-kinase group, since the activity of the membrane cation transport pathways is related to cyclic phosphorylation-dephosphorylation events. \ud \ud Results \ud \ud The patient showed mild hemolytic anemia with circulating stomatocytes together with signs of dyserythropoiesis. Her red cells displayed increased Na(+) content with decreased K(+) content and abnormal membrane cation transport activities. Functional characterization of band 3 CEINGE in Xenopus oocytes showed that the mutated band 3 is converted from being an anion exchanger (Cl(-), HCO(3)(-)) to being a cation pathway for Na(+) and K(+). Increased tyrosine phosphorylation of some red cell membrane proteins was observed in diseased erythrocytes. Syk and Lyn membrane association was increased in the patient's red cells compared to in normal controls, indicating perturbation of phospho-signaling pathways involved in cell volume regulation events. \ud \ud Conclusions \ud \ud Band 3 CEINGE alters function from that of anion exchange to cation transport, affects the membrane tyrosine phosphorylation profile, in particular of band 3 and stomatin, and its presence during red cell development likely contributes to dyserythropiesis
Protein tyrosine phosphatases (PTPs) are crucial components of cellular signal transduction pathways. We report here that red blood cells (RBCs) from mice lacking PTPε (Ptpre −/− ) exhibit abnormal morphology and increased Ca 2+ -activated-K + channel activity, which was partially blocked by the Src-Family-Kinases (SFKs) inhibitor PP1. In Ptpre −/− mouse RBCs, the activity of Fyn and Yes, two SFKs, were increased, suggesting a functional relationship between SFKs, PTPε and Ca 2+ -activated-K + -channel. The absence of PTPε markedly affected the RBC membrane tyrosine (Tyr-) phosphoproteome, indicating a perturbation of RBCs signal transduction pathways. Using signaling network computational analysis of the Tyr-phosphoproteomic data, we identified 7 topological clusters. We studied cluster 1, containing Syk-Tyr-kinase: Syk-kinase activity was higher in wild-type than in Ptpre −/− RBCs, validating the network computational analysis and indicating a novel signaling pathway, which involves Fyn and Syk in regulation of red cell morphology.
Sickle cell disease (SCD) is an autosomal recessive genetic red cell disorder characterized by the production of a defective form of hemoglobin, hemoglobin-S, that is worldwide-distributed. The acute clinical manifestations of SCD are related to hemoglobin cyclic-polymerization and to the generation of rigid, dense red blood cells (RBCs). We studied RBCs membrane proteome from human sickle RBCs, fractioned according to density compared to normal RBCs. 2-DE followed by MS analysis was carried out. We identified 65 proteins differently expressed, divided into five major clusters according to their functions: (i) membrane-cytoskeleton proteins; (ii) metabolic enzymes; (iii) ubiquitin-proteasome-system; (iv) flotillins; (v) chaperones. HSP27, HSP70 and peroxiredoxin-II (Prx-II) showed the most relevant changes. They were differently recruited to sickle RBCs membrane in response to in vitro hypoxia. Potential markers were then validated in a transgenic-mouse model for SCD, the SAD mice, exposed to hypoxia mimicking acute SCD vaso-occlusive-crisis (VOCs); we found that HSP70 and HSP27 bound to RBCs membrane respectively after 12 h and 48 h of hypoxia, while Prx-II membrane binding was modulated during hypoxia. Our data indicate that HSP27 and HSP70 play a novel role as RBCs membrane protein protectors and as possibly new markers of severity of RBCs membrane damage during acute VOCs.
The K-Cl cotransport (KCC) is an electroneutral-gradient-driven-membrane transport system, which is involved in regulation of red cell volume. Although the regulatory cascade of KCC is largely unknown, a signaling pathway involving phosphatases and kinases has been proposed. Here, we investigated the expression and the activity of protein phosphatase 1(PP-1) isoforms in mouse red cells, focusing on two models of abnormally activated KCC: mice genetically lacking the two Src-family tyrosine kinases, Hck and Fgr, (hck-/-fgr-/-) and the SAD transgenic sickle-cell-mice. The PP-1alpha, PP-1gamma, PP-1delta isoforms were expressed at similar levels in wild-type, hck-/-fgr-/- and SAD mouse erythrocytes and in each case were predominantly localized to cytoplasm. The PP-1alpha activity was significantly higher in both membrane and cytosol fractions of hck-/-fgr-/- and of SAD erythrocytes than in those of wild-type red cells, suggesting PP-1alpha as a target of the Hck and Fgr kinases. The PP2, a specific inhibitor of Src-family kinase, significantly increased KCC activity in wild-type mouse red cells, but failed to modify the already increased KCC activity in SAD erythrocytes. The lag-time for activation of KCC was considerably reduced in both hck-/-fgr-/- and SAD erythrocytes, suggesting that the rate limiting activation steps in both strains are freed from their tonic inhibition. Sulfhydryl reduction by dithiothreitol (DTT) lowered KCC activity only in SAD red cells, but did not affect the PP2-treated erythrocytes. These data suggest up-regulation of KCC in SAD red cells is mainly secondary to oxidative damage, which most likely reduces or removes the tonic KCC inhibition resulting from PP-1alpha activity controlled in turn by Src-family kinases.
BackgroundCystic fibrosis (CF) is one of the most common fatal autosomal recessive disorders in the Caucasian population caused by mutations of gene for the cystic fibrosis transmembrane conductance regulator (CFTR). New experimental therapeutic strategies for CF propose a diet supplementation to affect the plasma membrane fluidity and to modulate amplified inflammatory response. The objective of this study was to evaluate the efficacy of 5-methyltetrahydrofolate (5-MTHF) and vitamin B12 supplementation for ameliorating cell plasma membrane features in pediatric patients with cystic fibrosis.Methodology and Principal FindingsA single arm trial was conducted from April 2004 to March 2006 in an Italian CF care centre. 31 children with CF aged from 3 to 8 years old were enrolled. Exclusion criteria were diabetes, chronic infections of the airways and regular antibiotics intake. Children with CF were supplemented for 24 weeks with 5-methyltetrahydrofolate (5-MTHF, 7.5 mg /day) and vitamin B12 (0.5 mg/day). Red blood cells (RBCs) were used to investigate plasma membrane, since RBCs share lipid, protein composition and organization with other cell types. We evaluated RBCs membrane lipid composition, membrane protein oxidative damage, cation content, cation transport pathways, plasma and RBCs folate levels and plasma homocysteine levels at baseline and after 24 weeks of 5-MTHF and vitamin B12 supplementation. In CF children, 5-MTHF and vitamin B12 supplementation (i) increased plasma and RBC folate levels; (ii) decreased plasma homocysteine levels; (iii) modified RBC membrane phospholipid fatty acid composition; (iv) increased RBC K+ content; (v) reduced RBC membrane oxidative damage and HSP70 membrane association.Conclusion and Significance5-MTHF and vitamin B12 supplementation might ameliorate RBC membrane features of children with CF.Trial RegistrationClinicalTrials.gov NCT00730509
Stomatocytosis is an inherited autosomal dominant hemolytic anemia and includes overhydrated hereditary stomatocytosis (OHS), dehydrated hereditary stomatocytosis (DHS), hereditary cryohydrocytosis (CHC) and familial pseudohyperkalemia (FP). Here, we report a novel variant of hereditary stomatocytosis due to a de-novo band 3 mutation due to G>A transition at nucleotide 2500 in exon 17 (p. G796R, band3CEINGE) associated with dyserythropoietic phenotype. This 43-years-old Caucasian female (II-2) with unrelated parents was admitted to our hospital for mild anemia evaluation. The patient was in good health until 7 years when she frequently experienced asthenia. Anemia was first recognized at the age of eighth years with presence of jaundice and hyperchromic urine, but she had never received blood transfusions. We observed a mild hypochromic macrocytic anemia with a hemoglobin level of 11.5 g/dL, a mean cell volume (MCV) of 110 fL, and a mean hemoglobin concentration (MCH) of 36.1 pg, the reticulocyte count was 64 × 103/μL. There was a typical hemolytic features: high levels of indirect bilirubin (3.48 mg/dL) and lactate dehydrogenase ( 567 U/l, v.n. 240– 480 U/l ) with negativity at direct and indirect Coomb’s test. Spleen was enlarged and ultrasonography detected 15 cm of longitudinal size. She was cholecystectomized at the age of 14 years because of numerous symptomatic small stones. Serum iron, soluble transferrin receptor, serum ferritin and transferrin saturation levels were all increased, while the transferrin was in the normal range.Other blood tests including osmotic fragility with incubated and fresh erythrocytes, serum electrolytes, B12 and folate levels, erythrocyte enzyme levels, EMA test and Pink test were normal. Peripheral blood smear showed anisopoikilocytosis with rare stomatocytes and no spherocytes. Bone marrow aspirate showed remarkable dyserythropoiesis with increased number of erythroblasts and binucleate erythroblasts, basophilic erythroblasts with alterations, irregular nuclei maturation, intererythroblastic bridges and erythroblasts with basophilic stippling. She received since the age of 14 yrs a diagnosis for congenital dyserythropoietic anemia type I. Patients red cells showed increase Na+ content and decrease K+ content; reduced Na-K pump activity and increased Na-H exchange, NKCC cotransport and KCC cotransport activities. We then functionally characterized band 3 CEINGE in Xenopus oocytes, showing that the mutated band 3 is converted from anion exchanger (Cl−, HCO3 −) function to unregulated cation pathway for Na+ and K+. The mutated band 3 was also associated with increased tyrosine phosphorylation pattern of some red cell membrane proteins. During erythropoiesis band 3 protein is the last cytoskeletal protein to appear, thus the dyserythropoietic phenotype may be related to a possible role of the mutated band 3 in perturbation of cytoskeleton assembly in the late stage of erythropoiesis, allowing us to conclude for a new variant of stomatocytosis with dyserythropoietic phenotype.
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