Adenosine signaling inhibits erythropoiesis and promotes myeloid differentiation.

Intracellular uptake of adenosine is essential for optimal erythroid commitment and differentiation of hematopoietic progenitor cells. The role of adenosine signaling is well documented in the regulation of blood flow, cell proliferation, apoptosis, and stem cell regeneration. However, the role of adenosine signaling in hematopoiesis remains unclear. In this study, we show that adenosine signaling inhibits the proliferation of erythroid precursors by activating the p53 pathway and hampers the terminal erythroid maturation. Furthermore, we demonstrate that the activation of specific adenosine receptors promotes myelopoiesis. Overall, our findings indicate that extracellular adenosine could be a new player in the regulation of hematopoiesis.

Erythrocyte type 1 equilibrative nucleoside transporter expression in sickle cell disease and sickle cell trait.

Hypoxia-mediated red blood cell (RBC) sickling is central to the pathophysiology of sickle cell disease (SCD). The signalling nucleoside adenosine is thought to play a significant role in this process. This study investigated expression of the erythrocyte type 1 equilibrative nucleoside transporter (ENT1), a key regulator of plasma adenosine, in adult patients with SCD and carriers of sickle cell trait (SCT). Relative quantitative expression analysis of erythrocyte ENT1 was carried out by Western blot and flow cytometry. Patients with SCD with steady state conditions, either with SS or SC genotype, untreated or under hydroxycarbamide (HC) treatment, exhibited a relatively high variability of erythrocyte ENT1, but with levels not significantly different from normal controls. Most strikingly, expression of erythrocyte ENT1 was found to be significantly decreased in patients with SCD undergoing painful vaso-occlusive episode and, unexpectedly, also in healthy SCT carriers. Promoting hypoxia-induced adenosine signalling, the reduced expression of erythrocyte ENT1 might contribute to the pathophysiology of SCD and to the susceptibility of SCT individuals to altitude hypoxia or exercise to exhaustion.

Inherited glycosylphosphatidylinositol defects cause the rare Emm-negative blood phenotype and developmental disorders.

Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors >150 proteins to the cell surface. Pathogenic variants in several genes that participate in GPI biosynthesis cause inherited GPI deficiency disorders. Here, we reported that homozygous null alleles of PIGG, a gene involved in GPI modification, are responsible for the rare Emm-negative blood phenotype. Using a panel of K562 cells defective in both the GPI-transamidase and GPI remodeling pathways, we show that the Emm antigen, whose molecular basis has remained unknown for decades, is carried only by free GPI and that its epitope is composed of the second and third ethanolamine of the GPI backbone. Importantly, we show that the decrease in Emm expression in several inherited GPI deficiency patients is indicative of GPI defects. Overall, our findings establish Emm as a novel blood group system, and they have important implications for understanding the biological function of human free GPI.

The equilibrative nucleoside transporter ENT1 is critical for nucleotide homeostasis and optimal erythropoiesis.

The tight regulation of intracellular nucleotides is critical for the self-renewal and lineage specification of hematopoietic stem cells (HSCs). Nucleosides are major metabolite precursors for nucleotide biosynthesis and their availability in HSCs is dependent on their transport through specific membrane transporters. However, the role of nucleoside transporters in the differentiation of HSCs to the erythroid lineage and in red cell biology remains to be fully defined. Here, we show that the absence of the equilibrative nucleoside transporter (ENT1) in human red blood cells with a rare Augustine-null blood type is associated with macrocytosis, anisopoikilocytosis, an abnormal nucleotide metabolome, and deregulated protein phosphorylation. A specific role for ENT1 in human erythropoiesis was demonstrated by a defective erythropoiesis of human CD34+ progenitors following short hairpin RNA-mediated knockdown of ENT1. Furthermore, genetic deletion of ENT1 in mice was associated with reduced erythroid progenitors in the bone marrow, anemia, and macrocytosis. Mechanistically, we found that ENT1-mediated adenosine transport is critical for cyclic adenosine monophosphate homeostasis and the regulation of erythroid transcription factors. Notably, genetic investigation of 2 ENT1null individuals demonstrated a compensation by a loss-of-function variant in the ABCC4 cyclic nucleotide exporter. Indeed, pharmacological inhibition of ABCC4 in Ent1-/- mice rescued erythropoiesis. Overall, our results highlight the importance of ENT1-mediated nucleotide metabolism in erythropoiesis.

Molecular and structural characterization of a novel high-prevalence antigen of the Augustine blood group system.

BACKGROUND: An antibody directed against a high-prevalence red blood cell (RBC) antigen was detected in a 67-year-old female patient of North African ancestry with a history of a single pregnancy and blood transfusion. So far, the specificity of the proband's alloantibody remained unknown in our immunohematology reference laboratory. STUDY DESIGN AND METHODS: Whole-exome sequencing (WES) was performed on the proband's DNA. The reactivity to the SLC29A1-encoded ENT1 adenosine transporter was investigated by flow cytometry analyses of ENT1-expressing HEK293 cells, and RBCs from Augustine-typed individuals. Erythrocyte protein expression level, nucleoside-binding capacity, and molecular structure of the proband's ENT1 variant were further explored by western blot, flow cytometry, and molecular dynamics calculations, respectively. RESULTS: A missense variant was identified in the SLC29A1 gene, which encodes the Augustine blood group system. It arises from homozygosity for a rare c.242A > G missense mutation that results in a nonsynonymous p.Asn81Ser substitution within the large extracellular loop of ENT1. Flow cytometry analyses demonstrated that the proband's antibody was reactive against HEK-293 cells transfected with control but not proband's SLC29A1 cDNA. Consistent with this finding, proband's antibody was found to be reactive with At(a-) (AUG:-2), but not AUG:-1 (null phenotype) RBCs. Data from structural analysis further supported that the proband's p.Asn81Ser variation does not alter ENT1 binding of its specific inhibitor NBMPR. CONCLUSION: Our study provides evidence for a novel high-prevalence antigen, AUG4 (also called ATAM after the proband's name) in the Augustine blood group system, encoded by the rare SLC29A1 variant allele AUG*04 (c.242A > G, p.Asn81Ser).

Lack of the multidrug transporter MRP4/ABCC4 defines the PEL-negative blood group and impairs platelet aggregation.

The rare PEL-negative phenotype is one of the last blood groups with an unknown genetic basis. By combining whole-exome sequencing and comparative global proteomic investigations, we found a large deletion in the ABCC4/MRP4 gene encoding an ATP-binding cassette (ABC) transporter in PEL-negative individuals. The loss of PEL expression on ABCC4-CRISPR-Cas9 K562 cells and its overexpression in ABCC4-transfected cells provided evidence that ABCC4 is the gene underlying the PEL blood group antigen. Although ABCC4 is an important cyclic nucleotide exporter, red blood cells from ABCC4null/PEL-negative individuals exhibited a normal guanosine 3',5'-cyclic monophosphate level, suggesting a compensatory mechanism by other erythroid ABC transporters. Interestingly, PEL-negative individuals showed an impaired platelet aggregation, confirming a role for ABCC4 in platelet function. Finally, we showed that loss-of-function mutations in the ABCC4 gene, associated with leukemia outcome, altered the expression of the PEL antigen. In addition to ABCC4 genotyping, PEL phenotyping could open a new way toward drug dose adjustment for leukemia treatment.

Regulation of globin-heme balance in Diamond-Blackfan anemia by HSP70/GATA1.

Diamond-Blackfan anemia (DBA) is a congenital erythroblastopenia that is characterized by a blockade in erythroid differentiation related to impaired ribosome biogenesis. DBA phenotype and genotype are highly heterogeneous. We have previously identified 2 in vitro erythroid cell growth phenotypes for primary CD34+ cells from DBA patients and following short hairpin RNA knockdown of RPS19, RPL5, and RPL11 expression in normal human CD34+ cells. The haploinsufficient RPS19 in vitro phenotype is less severe than that of 2 other ribosomal protein (RP) mutant genes. We further documented that proteasomal degradation of HSP70, the chaperone of GATA1, is a major contributor to the defect in erythroid proliferation, delayed erythroid differentiation, increased apoptosis, and decreased globin expression, which are all features of the RPL5 or RPL11 DBA phenotype. In the present study, we explored the hypothesis that an imbalance between globin and heme synthesis may be involved in pure red cell aplasia of DBA. We identified disequilibrium between the globin chain and the heme synthesis in erythroid cells of DBA patients. This imbalance led to accumulation of excess free heme and increased reactive oxygen species production that was more pronounced in cells of the RPL5 or RPL11 phenotype. Strikingly, rescue experiments with wild-type HSP70 restored GATA1 expression levels, increased globin synthesis thereby reducing free heme excess and resulting in decreased apoptosis of DBA erythroid cells. These results demonstrate the involvement of heme in DBA pathophysiology and a major role of HSP70 in the control of balanced heme/globin synthesis.

Oxidative stress activates red cell adhesion to laminin in sickle cell disease.

Vaso-occlusive crises are the hallmark of sickle cell disease (SCD). They are believed to occur in two steps, starting with adhesion of deformable low-dense red blood cells (RBCs), or other blood cells such as neutrophils, to the wall of post-capillary venules, followed by trapping of the denser RBCs or leukocytes in the areas of adhesion because of reduced effective lumen-diameter. In SCD, RBCs are heterogeneous in terms of density, shape, deformability and surface proteins, which accounts for the differences observed in their adhesion and resistance to shear stress. Sickle RBCs exhibit abnormal adhesion to laminin mediated by Lu/BCAM protein at their surface. This adhesion is triggered by Lu/BCAM phosphorylation in reticulocytes but such phosphorylation does not occur in mature dense RBCs despite firm adhesion to laminin. In this study, we investigated the adhesive properties of sickle RBC subpopulations and addressed the molecular mechanism responsible for the increased adhesion of dense RBCs to laminin in the absence of Lu/BCAM phosphorylation. We provide evidence for the implication of oxidative stress in post-translational modifications of Lu/BCAM that impact its distribution and cis-interaction with glycophorin C at the cell surface activating its adhesive function in sickle dense RBCs.

ABCG2 Is Overexpressed on Red Blood Cells in Ph-Negative Myeloproliferative Neoplasms and Potentiates Ruxolitinib-Induced Apoptosis.

Myeloproliferative neoplasms (MPNs) are a group of disorders characterized by clonal expansion of abnormal hematopoietic stem cells leading to hyperproliferation of one or more myeloid lineages. The main complications in MPNs are high risk of thrombosis and progression to myelofibrosis and leukemia. MPN patients with high risk scores are treated by hydroxyurea (HU), interferon-α, or ruxolitinib, a tyrosine kinase inhibitor. Polycythemia vera (PV) is an MPN characterized by overproduction of red blood cells (RBCs). ABCG2 is a member of the ATP-binding cassette superfamily transporters known to play a crucial role in multidrug resistance development. Proteome analysis showed higher ABCG2 levels in PV RBCs compared to RBCs from healthy controls and an additional increase of these levels in PV patients treated with HU, suggesting that ABCG2 might play a role in multidrug resistance in MPNs. In this work, we explored the role of ABCG2 in the transport of ruxolitinib and HU using human cell lines, RBCs, and in vitro differentiated erythroid progenitors. Using stopped-flow analysis, we showed that HU is not a substrate for ABCG2. Using transfected K562 cells expressing three different levels of recombinant ABCG2, MPN RBCs, and cultured erythroblasts, we showed that ABCG2 potentiates ruxolitinib-induced cytotoxicity that was blocked by the ABCG2-specific inhibitor KO143 suggesting ruxolitinib intracellular import by ABCG2. In silico modeling analysis identified possible ruxolitinib-binding site locations within the cavities of ABCG2. Our study opens new perspectives in ruxolitinib efficacy research targeting cell types depending on ABCG2 expression and polymorphisms among patients.

CORS (CROM20): A new high-prevalence antigen in the Cromer blood group system.

The Cromer blood group system consists of 19 antigens (16 of high prevalence and 3 of low prevalence). This study describes the identification and characterization of a new Cromer high-prevalence antigen, named CORS. The CORS-negative proband carries a c.713G>A substitution in the CD55 gene, resulting in the substitution of glycine 238 into a glutamic acid (p.Gly238Glu).

Antioxidant and Membrane Binding Properties of Serotonin Protect Lipids from Oxidation.

Serotonin (5-hydroxytryptamine, 5-HT) is a well-known neurotransmitter that is involved in a growing number of functions in peripheral tissues. Recent studies have shown nonpharmacological functions of 5-HT linked to its chemical properties. Indeed, it was reported that 5-HT may, on the one hand, bind lipid membranes and, on the other hand, protect red blood cells through a mechanism independent of its specific receptors. To better understand these underevaluated properties of 5-HT, we combined biochemical, biophysical, and molecular dynamics simulations approaches to characterize, at the molecular level, the antioxidant capacity of 5-HT and its interaction with lipid membranes. To do so, 5-HT was added to red blood cells and lipid membranes bearing different degrees of unsaturation. Our results demonstrate that 5-HT acts as a potent antioxidant and binds with a superior affinity to lipids with unsaturation on both alkyl chains. We show that 5-HT locates at the hydrophobic-hydrophilic interface, below the glycerol group. This interfacial location is stabilized by hydrogen bonds between the 5-HT hydroxyl group and lipid headgroups and allows 5-HT to intercept reactive oxygen species, preventing membrane oxidation. Experimental and molecular dynamics simulations using membrane enriched with oxidized lipids converge to further reveal that 5-HT contributes to the termination of lipid peroxidation by direct interaction with active groups of these lipids and could also contribute to limit the production of new radicals. Taken together, our results identify 5-HT as a potent inhibitor of lipid peroxidation and offer a different perspective on the role of this pleiotropic molecule.

Hematin loses its membranotropic activity upon oligomerization into malaria pigment.

Malaria is an infectious disease caused by Plasmodium type parasites transmitted by the bites of infected female anopheles mosquitoes. The malaria parasite multiplies in red blood cells where it degrades hemoglobin. This degradation of hemoglobin proteins releases hematin, an iron-containing porphyrin, which provokes membrane disruption and lysis. The malaria parasite blocks hematin-induced lysis by biocrystallization, a process that converts hematin into insoluble and chemically inert crystals. Hematin molecules are especially prone to self-assembly as dimers, oligomers and aggregates depending on environmental conditions (pH, solvent, temperature, concentration, ionic strength). Considering the different forms of hematin-based assemblies, it is still unclear which are the ones able to interact with membranes. We have prepared hematin under different conditions to form hematin-based assemblies and to measure their ability to interact and to disorganize membranes. Our results show that different forms of hematin molecules are able to penetrate lipid membranes. Interestingly, this membrane activity is spontaneously inhibited at acidic pH and it can be restored under neutral pH. By contrast, the oligomers of ß-hematin were found to be completely harmless toward lipid membranes. Finally, the AFM visualization of hematin interaction with supported lipid bilayers showed for the first time its preferential interaction with defaults in membranes, at the boundaries between two distinct lipid phases. The superficial adsorption of aggregates on membranes and the absence of effect due to oligomers were also confirmed with AFM.

The human Kell blood group binds the erythroid 4.1R protein: new insights into the 4.1R-dependent red cell membrane complex.

Protein 4.1R plays an important role in maintaining the mechanical properties of the erythrocyte membrane. We analysed the expression of Kell blood group protein in erythrocytes from a patient with hereditary elliptocytosis associated with complete 4.1R deficiency (4.1(-) HE). Flow cytometry and Western blot analyses revealed a severe reduction of Kell. In vitro pull down and co-immunoprecipitation experiments from erythrocyte membranes showed a direct interaction between Kell and 4.1R. Using different recombinant domains of 4.1R and the cytoplasmic domain of Kell, we demonstrated that the R(46) R motif in the juxta-membrane region of Kell binds to lobe B of the 4.1R FERM domain. We also observed that 4.1R deficiency is associated with a reduction of XK and DARC (also termed ACKR1) proteins, the absence of the glycosylated form of the urea transporter B and a slight decrease of band 3. The functional alteration of the 4.1(-) HE erythrocyte membranes was also determined by measuring various transport activities. We documented a slower rate of HCO3 (-) /Cl(-) exchange, but normal water and ammonia transport across erythrocyte membrane in the absence of 4.1. These findings provide novel insights into the structural organization of blood group antigen proteins into the 4.1R complex of the human red cell membrane.

Atomic force microscopy of model lipid membranes.

Supported lipid bilayers (SLBs) are biomimetic model systems that are now widely used to address the biophysical and biochemical properties of biological membranes. Two main methods are usually employed to form SLBs: the transfer of two successive monolayers by Langmuir-Blodgett or Langmuir-Schaefer techniques, and the fusion of preformed lipid vesicles. The transfer of lipid films on flat solid substrates offers the possibility to apply a wide range of surface analytical techniques that are very sensitive. Among them, atomic force microscopy (AFM) has opened new opportunities for determining the nanoscale organization of SLBs under physiological conditions. In this review, we first focus on the different protocols generally employed to prepare SLBs. Then, we describe AFM studies on the nanoscale lateral organization and mechanical properties of SLBs. Lastly, we survey recent developments in the AFM monitoring of bilayer alteration, remodeling, or digestion, by incubation with exogenous agents such as drugs, proteins, peptides, and nanoparticles.

Lack of the human choline transporter-like protein SLC44A2 causes hearing impairment and a rare red blood phenotype.

Blood phenotypes are defined by the presence or absence of specific blood group antigens at the red blood cell (RBC) surface, due to genetic polymorphisms among individuals. The recent development of genomic and proteomic approaches enabled the characterization of several enigmatic antigens. The choline transporter-like protein CTL2 encoded by the SLC44A2 gene plays an important role in platelet aggregation and neutrophil activation. By investigating alloantibodies to a high-prevalence antigen of unknown specificity, found in patients with a rare blood type, we showed that SLC44A2 is also expressed in RBCs and carries a new blood group system. Furthermore, we identified three siblings homozygous for a large deletion in SLC44A2, resulting in complete SLC44A2 deficiency. Interestingly, the first-ever reported SLC44A2-deficient individuals suffer from progressive hearing impairment, recurrent arterial aneurysms, and epilepsy. Furthermore, SLC44A2null individuals showed no significant platelet aggregation changes and do not suffer from any apparent hematological disorders. Overall, our findings confirm the function of SLC44A2 in hearing preservation and provide new insights into the possible role of this protein in maintaining cerebrovascular homeostasis.

The potent antimalarial peptide cyclosporin A induces the aggregation and permeabilization of sphingomyelin-rich membranes.

Cyclosporin A (CsA) is a hydrophobic peptide drug produced by the fungus Tolypocladium inflatum. CsA is commonly used as an immunosuppressive drug, but it also has antimalarial activity. The immunosuppressive activity of CsA is clearly due to its association with specific proteins of immune cells such as cyclophilins. By contrast, the antimalarial properties of this peptide are completely independent of the association with a parasite's cyclophilins. Because of its hydrophobicity, CsA may interact with biological membranes, which may participate in its therapeutic effect. Recently, we have shown a marked preference of CsA for insertion into sphingomyelin (SM) monolayers. In this article, we measure for the first time the ability of CsA to induce permeabilization and aggregation and to change the lipid order, especially in the presence of SM. Calcein-release experiments permitted us to show that CsA causes the leakage of the fluorescent probe from SM-rich liposomes by 40% and PC liposomes by 11%, suggesting a lipid-selective effect. Electron microscopy and dynamic light scattering experiments confirmed the different interaction of CsA with SM and PC vesicles: it formed much larger aggregates with SM than with PC. Our results taken together suggest that CsA could specifically weaken and aggregate SM-rich membranes, which could in turn explain why CsA is efficient in the treatment of malaria. Indeed, CsA could inhibit the development of Plasmodium by permeabilizing and aggregating the SM-rich membrane network formed by the parasite during its intraerythrocytic growth cycle.

Sickle Cell Trait Modulates the Proteome and Phosphoproteome of Plasmodium falciparum-Infected Erythrocytes.

The high prevalence of sickle cell disease in some human populations likely results from the protection afforded against severe Plasmodium falciparum malaria and death by heterozygous carriage of HbS. P. falciparum remodels the erythrocyte membrane and skeleton, displaying parasite proteins at the erythrocyte surface that interact with key human proteins in the Ankyrin R and 4.1R complexes. Oxidative stress generated by HbS, as well as by parasite invasion, disrupts the kinase/phosphatase balance, potentially interfering with the molecular interactions between human and parasite proteins. HbS is known to be associated with abnormal membrane display of parasite antigens. Studying the proteome and the phosphoproteome of red cell membrane extracts from P. falciparum infected and non-infected erythrocytes, we show here that HbS heterozygous carriage, combined with infection, modulates the phosphorylation of erythrocyte membrane transporters and skeletal proteins as well as of parasite proteins. Our results highlight modifications of Ser-/Thr- and/or Tyr- phosphorylation in key human proteins, such as ankyrin, ß-adducin, ß-spectrin and Band 3, and key parasite proteins, such as RESA or MESA. Altered phosphorylation patterns could disturb the interactions within membrane protein complexes, affect nutrient uptake and the infected erythrocyte cytoadherence phenomenon, thus lessening the severity of malaria symptoms.