Genetic polymorphisms and expression of Rhesus blood group RHCE are associated with 2,3-bisphosphoglycerate in humans at high altitude.

Red blood cell (RBC) metabolic reprogramming upon exposure to high altitude contributes to physiological human adaptations to hypoxia, a multifaceted process critical to health and disease. To delve into the molecular underpinnings of this phenomenon, first, we performed a multi-omics analysis of RBCs from six lowlanders after exposure to high-altitude hypoxia, with longitudinal sampling at baseline, upon ascent to 5,100 m and descent to sea level. Results highlighted an association between erythrocyte levels of 2,3-bisphosphoglycerate (BPG), an allosteric regulator of hemoglobin that favors oxygen off-loading in the face of hypoxia, and expression levels of the Rhesus blood group RHCE protein. We then expanded on these findings by measuring BPG in RBCs from 13,091 blood donors from the Recipient Epidemiology and Donor Evaluation Study. These data informed a genome-wide association study using BPG levels as a quantitative trait, which identified genetic polymorphisms in the region coding for the Rhesus blood group RHCE as critical determinants of BPG levels in erythrocytes from healthy human volunteers. Mechanistically, we suggest that the Rh group complex, which participates in the exchange of ammonium with the extracellular compartment, may contribute to intracellular alkalinization, thus favoring BPG mutase activity.

A novel missense variant in ATP11C is associated with reduced red blood cell phosphatidylserine flippase activity and mild hereditary hemolytic anemia.

Adenosine Triphosphatase (ATPase) Phospholipid Transporting 11C gene (ATP11C) encodes the major phosphatidylserine (PS) flippase in human red blood cells (RBCs). Flippases actively transport phospholipids (e.g., PS) from the outer to the inner leaflet to establish and maintain phospholipid asymmetry of the lipid bilayer of cell membranes. This asymmetry is crucial for survival since externalized PS triggers phagocytosis by splenic macrophages. Here we report on pathophysiological consequences of decreased flippase activity, prompted by a patient with hemolytic anemia and hemizygosity for a novel c.2365C > T p.(Leu789Phe) missense variant in ATP11C. ATP11C protein expression was strongly reduced by 58% in patient-derived RBC ghosts. Furthermore, functional characterization showed only 26% PS flippase activity. These results were confirmed by recombinant mutant ATP11C protein expression in HEK293T cells, which was decreased to 27% compared to wild type, whereas PS-stimulated ATPase activity was decreased by 57%. Patient RBCs showed a mild increase in PS surface exposure when compared to control RBCs, which further increased in the most dense RBCs after RBC storage stress. The increase in PS was not due to higher global membrane content of PS or other phospholipids. In contrast, membrane lipid lateral distribution showed increased abundance of cholesterol-enriched domains in RBC low curvature areas. Finally, more dense RBCs and subtle changes in RBC morphology under flow hint toward alterations in flow behavior of ATP11C-deficient RBCs. Altogether, ATP11C deficiency is the likely cause of hemolytic anemia in our patient, thereby underlining the physiological role and relevance of this flippase in human RBCs.

Spectrin and Other Membrane-Skeletal Components in Human Red Blood Cells of Different Age.

BACKGROUND: Old human red blood cells (RBCs) have a reduced surface area with respect to young RBCs. If this decrease occurred through the release of vesicles similar to the spectrin-free vesicles that are shed in vitro under different experimental conditions or during storage, there would be no decrease of membrane-skeleton, but only of lipid bilayer surface area, during RBC ageing in vivo. However, we observed a decrease in spectrin and other membrane-skeletal proteins in old RBCs. Because RBCs contain components of the ubiquitin-proteasome system and other hydrolytic systems for protein degradation, we asked whether increased membrane-skeleton fragments could be detected in older RBCs. METHODS: Four different anti-spectrin antibodies and an antibody anti-ubiquitin conjugates were used to analyse, by Western blotting, fragments of spectrin and other proteins in RBCs of different age separated in density gradients and characterized for their protein 4.1a/4.1b ratio as a cell age parameter. RESULTS: spectrin fragments do exist in RBCs of all ages, they represent a minute fraction of all spectrin, are membrane-bound and not cytoplasmic and do not increase with cell age. Besides spectrin, other membrane-skeletal components decrease with cell age. CONCLUSION: Observed results challenge the commonly accepted view that decrease in cell membrane throughout RBC life in vivo occurs via the release of spectrin-free vesicles.

Membrane Remodelling and Vesicle Formation During Ageing of Human Red Blood Cells.

BACKGROUND/AIMS: A high surface-to-volume ratio and a spectrin membrane-skeleton (MS) confer to the mammalian red blood cells (RBCs) their characteristic deformability, mechanical strength and structural stability. During their 120 days of circulatory life in humans, RBCs decrease in size, while remaining biconcave disks, owing to a coordinated decrease in membrane surface area and cell water. It is generally believed that part of the membrane is lost with the shedding of spectrin-free vesicles of the same type that can be obtained in vitro by different treatments. If this were true, an excess of MS would arise in old RBCs, with respect to the lipid bilayer. Aim of this paper was to investigate this aspect. METHODS: Quantification of spectrin by electrophoretic methods was carried out in RBCs of different age. RESULTS: Spectrin decreases, on a per cell basis, with RBC ageing. On the other hand, the membrane raft protein marker flotillin-2, while decreasing in the membrane of old cells, was found to be strongly depleted in the membrane of in vitro-induced vesicles. CONCLUSION: Part of the membrane-skeleton is probably lost together with part of the lipid bilayer in a balanced way. These findings point to a mechanism for the in vivo release of membrane that is different from that which is known to occur in vitro.

Membrane rafts of the human red blood cell.

The cell type of election for the study of cell membranes, the mammalian non-nucleated erythrocyte, has been scarcely considered in the research of membrane rafts of the plasma membrane. However, detergent-resistant-membranes (DRM) were actually first described in human erythrocytes, as a fraction resisting solubilization by the nonionic detergent Triton X-100. These DRMs were insoluble entities of high density, easily pelleted by centrifugation, as opposed to the now accepted concept of lipid raft-like membrane fractions as material floating in low-density regions of sucrose gradients. The present article reviews the available literature on membrane rafts/DRMs in human erythrocytes from an historical point of view, describing the experiments that provided the solution to the above described discrepancy and suggesting possible avenue of research in the field of membrane rafts that, moving from the most studied model of living cell membrane, the erythrocyte's, could be relevant also for other cell types.

Freely turning over palmitate in erythrocyte membrane proteins is not responsible for the anchoring of lipid rafts to the spectrin skeleton: a study with bio-orthogonal chemical probes.

Erythrocyte lipid rafts are anchored to the underlying spectrin membrane skeleton [A. Ciana, C. Achilli, C. Balduini, G. Minetti, On the association of lipid rafts to the spectrin skeleton in human erythrocytes, Biochim. Biophys. Acta 1808 (2011) 183-190]. The nature of this linkage and the molecules involved are poorly understood. The interaction is sensitive to the increase in pH and ionic strength induced by carbonate. Given the role of palmitoylation in modulating the partitioning of certain proteins between various sub-cellular compartments and the plasma membrane, we asked whether palmitoylation of p55, a peripheral protein located at the junctional complex between spectrin-actin-protein 4.1 that anchors the membrane skeleton to the lipid bilayer via the transmembrane protein glycophorin C, could contribute to the anchoring of lipid rafts to the membrane skeleton. We adopted a new, non-radioactive method for studying protein palmitoylation, based on bio-orthogonal chemical analogues of fatty acids, containing an omega-alkynyl group, to metabolically label cell proteins, which are then revealed by a "click chemistry" reaction of the alkynyl moiety with an azide-containing reporter tag. We show that the membrane localization and palmitoylation levels of p55 did not change after carbonate treatment. 2-bromopalmitate and cerulenin, two known palmitoylation inhibitors, completely inhibited p55 palmitoylation, and protein palmitoyl thioesterase-1 (PPT1) reduced it, without affecting the association between lipid rafts and membrane-skeleton, indicating, on the one hand, that p55 palmitoylation is enzymatic, and, on the other, that it is not involved in the modulation of the linkage of lipid rafts to the membrane-skeleton.

Survival and senescence of human young red cells in vitro.

BACKGROUND: A number of experimental investigations in vivo suggest that in humans a decrease of circulating erythrocyte number ensues whenever erythropoietin (EPO) plasma level decreases. Since the process seems to selectively eliminate young red cells (neocytes), it has been named neocytolysis. The experimental models in vivo have revealed and documented multiple forms of neocytolysis but have not fully elucidated the specificity of the target red cells and the relation with EPO level changes. In an attempt to better characterize the neocytolytic process, we have undertaken an in vitro investigation on age-ranked human red cells. METHODS: By centrifugation on Percoll density gradient we separated the red cells population into three subsets, neocytes, middle-aged and old. Then we comparatively investigated the kinetics of survival of the subsets cultured under different conditions: with medium alone, with 10% autologous plasma, with EPO, alone or in combination with autologous monocytes. RESULTS: Neocytes showed a viability and a survival rate lower than the other red cells when cultured in medium or with 10% plasma. EPO at physiological doses increased their survival rate, but not that of the other subsets. This effect was enhanced by co-culture with monocytes. CONCLUSION: Likely neocytes are more sensitive than the other RBCs subsets to presence or absence of survival signals, such as EPO or plasma or monocytes derived factors. These observations could provide an insight into the link between the decrease in EPO plasma level and the reduction of circulating red cells mass and account for the specificity of neocytes clearance.

Amyloid-beta (25-35) peptide induces the release of pro-matrix metalloprotease 9 (pro-MMP-9) from human neutrophils.

Alzheimer's disease (AD) is a degenerative process of the brain, leading to increasing impairment of cognitive functions, and is associated with accumulation in the brain of several amyloid-beta (Aß) peptides (as amyloid plaques), including Aß25-35. Neutrophils, the most abundant immune cell type infiltrated in the brain of AD patients, accumulate behind amyloid plaques. Aß peptides can trigger activation of chemotaxis and oxidative burst in neutrophils, suggesting a role in modulating the neuroinflammation process. We have shown that Aß25-35 can induce the release from human neutrophils of pro-MMP-9, a metalloprotease involved in the onset of inflammation, corroborating the hypothesis of the involvement of infiltrated neutrophils in the inflammatory processes, which occur in the AD brain.

Biocompatibility of functionalized boron phosphate (BPO4) nanoparticles for boron neutron capture therapy (BNCT) application.

Boron neutron capture therapy (BNCT) is a radiotherapy treatment based on the accumulation in the tumor of a (10)B-containing drug and subsequent irradiation with low energy neutrons, which bring about the decay of (10)B to (7)Li and an α particle, causing the death of the neoplastic cell. The effectiveness of BNCT is limited by the low delivery and accumulation of the used boron-containing compounds. Here we report the development and the characterization of BPO4 nanoparticles (NPs) as a novel possible alternative drug for BNCT. An extensive analysis of BPO4 NP biocompatibility was performed using both mature blood cells (erythrocytes, neutrophils and platelets) and a model of hematopoietic progenitor cells. A time- and concentration-dependent cytotoxicity study was performed on neoplastic coloncarcinoma and osteosarcoma cell lines. BPO4 functionalization with folic acid, introduced to improve the uptake by tumor cells, appeared to effectively limit the unwanted effects of NPs on the analyzed blood components. FROM THE CLINICAL EDITOR: Boron neutron capture therapy (BNCT) is a radiotherapy treatment modality based on the accumulation of a (10)B-containing drug and subsequent irradiation with low energy neutrons, inducing the decay of (10)B to (7)Li and an α particle, causing neoplastic cell death. This team of authors reports on a folic acid functionalized BPO4 nanoparticle with improved characteristics compared with conventional BNCT approaches, as demonstrated in tumor cell lines, and hopefully to be followed by translational human studies.

On the association of lipid rafts to the spectrin skeleton in human erythrocytes.

Lipid rafts are local inhomogeneities in the composition of the plasma membrane of living cells, that are enriched in sphingolipids and cholesterol in a liquid-ordered state, and proteins involved in receptor-mediated signalling. Interactions between lipid rafts and the cytoskeleton have been observed in various cell types. They are isolated as a fraction of the plasma membrane that resists solubilization by nonionic detergents at 4°C (detergent-resistant membranes, DRMs). We have previously described that DRMs are anchored to the spectrin-based membrane skeleton in human erythrocytes and can be released by increasing the pH and ionic strength of the solubilization medium with sodium carbonate. It was unexplained why this carbonate treatment was necessary and why this requirement was not reported by other workers in this area. We show here that when contaminating leukocytes are present in erythrocyte preparations that are subjected to detergent treatment, the isolation of DRMs can occur without the requirement for carbonate treatment. This is due to the uncontrolled breakdown of erythrocyte membrane components by hydrolases that are released from contaminating neutrophils that lead to proteolytic disruption of the supramolecular assembly of the membrane skeleton. Results presented here corroborate the concept that DRMs are anchored to the membrane skeleton through electrostatic interactions that most likely involve the spectrin molecule.

Continuous Percoll Gradient Centrifugation of Erythrocytes-Explanation of Cellular Bands and Compromised Age Separation.

(1) Background: When red blood cells are centrifuged in a continuous Percoll-based density gradient, they form discrete bands. While this is a popular approach for red blood cell age separation, the mechanisms involved in banding were unknown. (2) Methods: Percoll centrifugations of red blood cells were performed under various experimental conditions and the resulting distributions analyzed. The age of the red blood cells was measured by determining the protein band 4.1a to 4.1b ratio based on western blots. Red blood cell aggregates, so-called rouleaux, were monitored microscopically. A mathematical model for the centrifugation process was developed. (3) Results: The red blood cell band pattern is reproducible but re-centrifugation of sub-bands reveals a new set of bands. This is caused by red blood cell aggregation. Based on the aggregation, our mathematical model predicts the band formation. Suppression of red blood cell aggregation reduces the band formation. (4) Conclusions: The red blood cell band formation in continuous Percoll density gradients could be explained physically by red blood cell aggregate formation. This aggregate formation distorts the density-based red blood cell age separation. Suppressing aggregation by osmotic swelling has a more severe effect on compromising the RBC age separation to a higher degree.

In Vitro Erythropoiesis at Different pO2 Induces Adaptations That Are Independent of Prior Systemic Exposure to Hypoxia.

Hypoxia is associated with increased erythropoietin (EPO) release to drive erythropoiesis. At high altitude, EPO levels first increase and then decrease, although erythropoiesis remains elevated at a stable level. The roles of hypoxia and related EPO adjustments are not fully understood, which has contributed to the formulation of the theory of neocytolysis. We aimed to evaluate the role of oxygen exclusively on erythropoiesis, comparing in vitro erythroid differentiation performed at atmospheric oxygen, a lower oxygen concentration (three percent oxygen) and with cultures of erythroid precursors isolated from peripheral blood after a 19-day sojourn at high altitude (3450 m). Results highlight an accelerated erythroid maturation at low oxygen and more concave morphology of reticulocytes. No differences in deformability were observed in the formed reticulocytes in the tested conditions. Moreover, hematopoietic stem and progenitor cells isolated from blood affected by hypoxia at high altitude did not result in different erythroid development, suggesting no retention of a high-altitude signature but rather an immediate adaptation to oxygen concentration. This adaptation was observed during in vitro erythropoiesis at three percent oxygen by a significantly increased glycolytic metabolic profile. These hypoxia-induced effects on in vitro erythropoiesis fail to provide an intrinsic explanation of the concept of neocytolysis.

Hemoglobin is an oxygen-dependent glutathione buffer adapting the intracellular reduced glutathione levels to oxygen availability.

Fast changes in environmental oxygen availability translate into shifts in mitochondrial free radical production. An increase in intraerythrocytic reduced glutathione (GSH) during deoxygenation would support the detoxification of exogenous oxidants released into the circulation from hypoxic peripheral tissues. Although reported, the mechanism behind this acute oxygen-dependent regulation of GSH in red blood cells remains unknown. This study explores the role of hemoglobin (Hb) in the oxygen-dependent modulation of GSH levels in red blood cells. We have demonstrated that a decrease in Hb O2 saturation to 50% or less observed in healthy humans while at high altitude, or in red blood cell suspensions results in rising of the intraerythrocytic GSH level that is proportional to the reduction in Hb O2 saturation. This effect was not caused by the stimulation of GSH de novo synthesis or its release during deglutathionylation of Hb's cysteines. Using isothermal titration calorimetry and in silico modeling, we observed the non-covalent binding of four molecules of GSH to oxy-Hb and the release of two of them upon deoxygenation. Localization of the GSH binding sites within the Hb molecule was identified. Oxygen-dependent binding of GSH to oxy-Hb and its release upon deoxygenation occurred reciprocally to the binding and release of 2,3-bisphosphoglycerate. Furthermore, noncovalent binding of GSH to Hb moderately increased Hb oxygen affinity. Taken together, our findings have identified an adaptive mechanism by which red blood cells may provide an advanced antioxidant defense to respond to oxidative challenges immediately upon deoxygenation.

Application of gelatin zymography for evaluating low levels of contaminating neutrophils in red blood cell samples.

Supposedly "homogeneous" red blood cell (RBC) samples are commonly obtained by "washing" whole blood free of plasma, platelets, and white cells with physiological solutions, a procedure that does not result, however, in sufficient removal of polymorphonuclear neutrophils (PMNs), leading to possible artifactual results. Pure RBC samples can be obtained only by leukodepletion procedures. Proposed here is a version of gelatin zymography adapted to detect matrix metalloproteinase 9 (MMP-9), selectively expressed by PMNs, in heterogeneous mixtures of RBCs and PMNs that can reveal contamination at levels as low as 1 PMN/106 RBCs.

Absence of neocytolysis in humans returning from a 3-week high-altitude sojourn.

AIMS: Total haemoglobin mass (tot-Hb) increases during high-altitude acclimatization. Normalization of tot-Hb upon descent is thought to occur via neocytolysis, the selective destruction of newly formed erythrocytes. Because convincing experimental proof of neocytolysis is lacking, we performed a prospective study on erythrocyte survival after a stay at the Jungfraujoch Research Station (JFJRS; 3450 m). METHODS: Newly formed erythrocytes of 12 male subjects (mean age 23.3 years) were age cohort labelled in normoxia (110 m) and during a 19-day high-altitude sojourn by ingestion of 13 C2- and 15 N-labelled glycine respectively. Elimination dynamics for erythrocytes produced in normoxia and at high altitude were measured by isotope ratio mass spectrometry of haem, by determining tot-Hb, reticulocyte counts, erythrocyte membrane protein 4.1a/4.1b ratio and by mathematical modelling. RESULTS: Tot-Hb increased by 4.7% ± 2.7% at high altitude and returned to pre-altitude values within 11 days after descent. Elimination of 13 C- (normoxia) and 15 N- (high altitude) labelled erythrocytes was not different. Erythropoietin levels and counts of CD71-positive reticulocytes decreased rapidly after descent. The band 4.1a/4.1b ratio decreased at altitude and remained low for 3-4 days after descent and normalized slowly. There was no indication of haemolysis. CONCLUSION: We confirm a rapid normalization of tot-Hb upon descent. Based on the lack of accelerated removal of age cohorts of erythrocytes labelled at high altitude, on patterns of changes in reticulocyte counts and of the band 4.1a/4.1b ratio and on modelling, this decrease did not occur via neocytolysis, but by a reduced rate of erythropoiesis along with normal clearance of senescent erythrocytes.

Effect of cholesterol depletion and temperature on the isolation of detergent-resistant membranes from human erythrocytes.

Transient lateral microdomains or lipid rafts play important roles in many physiological membrane-mediated cell processes. Detergent-resistant membranes (DRMs) are good models for the study of lipid rafts. Here we report that DRMs can be obtained by treating human erythrocytes with the nonionic detergents Triton X-100 or octaethylene glycol monododecyl ether (C(12)E(8)) at 37 degrees C, and by treatment at 4 degrees C of cholesterol-depleted erythrocytes. Electron paramagnetic resonance with spin labels inserted at different membrane depths (5- and 16-doxyl stearic acids, 5-SASL and 16-SASL) were used to measure the order parameter (S) of the cell membranes and DRMs. We previously reported significantly higher S values in DRMs with respect to intact erythrocyte membranes. Here we show that higher S values were still measurable in DRMs prepared from intact erythrocytes at 37 degrees C, or from cholesterol-depleted cells at 4 degrees C, for both detergents. For 5-SASL only, increased S values were measured in 4 degrees C DRMs obtained from cholesterol-depleted versus intact erythrocytes. Flotillin-2, a protein marker of lipid rafts, was found in DRMs from intact cells in trace amounts but it was sensitively increased in C(12)E(8) DRMs prepared at 4 degrees C from cholesterol-depleted erythrocytes, while the membrane-skeletal proteins spectrin and actin were excluded from both Triton X-100 and C(12)E(8) DRMs. However, contrary to the 4 degrees C treatment results, flotillin-2 and stomatin were not resistant to Triton X-100 and C(12)E(8) treatment at physiological temperature. The role of cholesterol in DRMs formation is discussed and the results presented provide further support for the use of C(12)E(8) to the study of DRMs.

A dynamic ratio of the alpha+ and alpha- isoforms of the tight junction protein ZO-1 is characteristic of Caco-2 cells and correlates with their degree of differentiation.

ZO-1 is a peripheral protein that plays a central role in the macromolecular assembly of tight junctions by interacting with integral proteins (occludin, claudins, JAMs) of the membrane of adjoining cells, with the actin cytoskeleton, and with nuclear factors. Human ZO-1 is expressed in all epithelia and some specialized endothelia as variable amounts of two related isoforms, which originate from the alternatively spliced mRNA transcripts alpha(+) and alpha(-) and whose specific differential role is still unknown. Moreover, little is known about the timing of expression of ZO-1 isoforms at the protein and mRNA level. This study shows that during growth of freshly plated Caco-2 cells, the alpha(+)/alpha(-) ratio increased as a result of simultaneous increase of alpha(+) and decrease of alpha(-). Differences in the isoform ratio also correlated with differences in epithelium differentiation. This was determined by aminopeptidase N measurements of cells grown on conventional substrates and on modified, micro/nano-patterned surfaces. A comparable shift of ZO-1 isoforms was not observed in other tumour cell lines of non-intestinal origin (A549, Calu-3). Pancreatic stem cells, propagated without exogenous differentiation stimuli, displayed a slight, stable prevalence of the alpha(-) isoform. Of the intestinal cell lines examined (Caco-2 and T84), only Caco-2 cells displayed a dramatic shift in isoform expression. This suggests that this tumour cell line retains to a higher degree a developmental programme related to the dynamic of enterocytic differentiation in vivo.

Mevalonate pathway, selenoproteins, redox balance, immune system, Covid-19: Reasoning about connections.

It has been proposed that a degraded immune system is (one of) the condition(s) that predispose certain subjects to fatal consequences from infection by SARS-CoV-2. It is unknown whether therapeutic regimens to which these patients may have been subjected to in the months/years preceding the infection could be immunocompromising. Statins are among the most widely prescribed cholesterol-lowering drugs. As competitive inhibitors of HMG-CoA-reductase, the key enzyme of the "mevalonate pathway" through which essential compounds, not only cholesterol, are synthesized, statins decrease the levels of cholesterol, and thus LDLs, as an innate defense mechanism, with controversial results in decreasing mortality from cardiovascular disease. Moreover, statins have pleiotropic, mostly deleterious effects on many cell types, including immune cells. In the attempt to decipher the enigma of SARS-CoV-2 infectivology, the hypothesis should be tested whether the population of subjects who succumbed to Covid-19 may have developed a compromised immunity at sub-clinical levels and have become more susceptible to fatal consequences from SARS-Cov-2 infection due to statin therapy.

Membrane Rearrangements in the Maturation of Circulating Human Reticulocytes.

Red blood cells (RBCs) begin their circulatory life as reticulocytes (Retics) after their egress from the bone marrow where, as R1 Retics, they undergo significant rearrangements in their membrane and intracellular components, via autophagic, proteolytic, and vesicle-based mechanisms. Circulating, R2 Retics must complete this maturational process, which involves additional loss of significant amounts of membrane and selected membrane proteins. Little is known about the mechanism(s) at the basis of this terminal differentiation in the circulation, which culminates with the production of a stable biconcave discocyte. The membrane of R1 Retics undergoes a selective remodeling through the release of exosomes that are enriched in transferrin receptor and membrane raft proteins and lipids, but are devoid of Band 3, glycophorin A, and membrane skeletal proteins. We wondered whether a similar selective remodeling occurred also in the maturation of R2 Retics. Peripheral blood R2 Retics, isolated by an immunomagnetic method, were compared with mature circulating RBCs from the same donor and their membrane protein and lipid content was analyzed. Results show that both Band 3 and spectrin decrease from R2 Retics to RBCs on a "per cell" basis. Looking at membrane proteins that are considered as markers of membrane rafts, flotillin-2 appears to decrease in a disproportionate manner with respect to Band 3. Stomatin also decreases but in a more proportionate manner with respect to Band 3, hinting at a heterogeneous nature of membrane rafts. High resolution lipidomics analysis, on the contrary, revealed that those lipids that are typically representative of the membrane raft phase, sphingomyelin and cholesterol, are enriched in mature RBCs with respct to Retics, relative to total cell lipids, strongly arguing in favor of the selective retention of at least certain subclasses of membrane rafts in RBCs as they mature from Retics. Our hypothesis that rafts serve as additional anchoring sites for the lipid bilayer to the underlying membrane-skeleton is corroborated by the present results. It is becoming ever more clear that a proper lipid composition of the reticulocyte is necessary for the production of a normal mature RBC.