Nobuto Arashiki

Membrane Peroxidation and Methemoglobin Formation are Both Necessary for Band 3 Clustering: Mechanistic Insights into Human Erythrocyte Senescence

Oxidative damage and clustering of band 3 in the membrane have been implicated in the removal of senescent human erythrocytes from the circulation at the end of their 120 day life span. However, the biochemical and mechanistic events leading to band 3 cluster formation have yet to be fully defined. Here we show that while neither membrane peroxidation nor methemoglobin (MetHb) formation on their own can induce band 3 clustering in the human erythrocytes, they can do so when acting in combination. We further show that binding of MetHb to the cytoplasmic domain of band 3 in peroxidized, but not in untreated, erythrocyte membranes induces cluster formation. Age-fractionated populations of erythrocytes from normal human blood, obtained by a density gradient procedure, have allowed us to examine a subpopulation, highly enriched in senescent cells. We have found that band 3 clustering is a feature of only this small fraction, amounting to ∼0.1% of total circulating erythrocytes. These senescent cells are characterized by an increased proportion of MetHb as a result of reduced nicotinamide adenine dinucleotide-dependent reductase activity and accumulated oxidative membrane damage. These findings have allowed us to establish that the combined effects of membrane peroxidation and MetHb formation are necessary for band 3 clustering, and this is a very late event in erythrocyte life. A plausible mechanism for the combined effects of membrane peroxidation and MetHb is proposed, involving high-affinity cooperative binding of MetHb to the cytoplasmic domain of oxidized band 3, probably because of its carbonylation, rather than other forms of oxidative damage. This modification leads to dissociation of ankyrin from band 3, allowing the tetrameric MetHb to cross-link the resulting freely diffusible band 3 dimers, with formation of clusters.

Ubiquitylation-independent ER-associated degradation of an AE1 mutant associated with dominant hereditary spherocytosis in cattle

Various mutations in the AE1 (anion exchanger 1, band 3) gene cause dominant hereditary spherocytosis, a common congenital hemolytic anemia associated with deficiencies of AE1 of different degrees and loss of mutant protein from red blood cell membranes. To determine the mechanisms underlying decreases in AE1 protein levels, we employed K562 and HEK293 cell lines and Xenopus oocytes together with bovine wild-type AE1 and an R664X nonsense mutant responsible for dominant hereditary spherocytosis to analyze protein expression, turnover, and intracellular localization. R664X-mutant protein underwent rapid degradation and caused specifically increased turnover and impaired trafficking to the plasma membrane of the wild-type protein through hetero-oligomer formation in K562 cells. Consistent with those observations, co-expression of mutant and wild-type AE1 reduced anion transport by the wild-type protein in oocytes. Transfection studies in K562 and HEK293 cells revealed that the major pathway mediating degradation of both R664X and wild-type AE1 employed endoplasmic reticulum (ER)-associated degradation through the proteasomal pathway. Proteasomal degradation of R664X protein appeared to be independent of both ubiquitylation and N-glycosylation, and aggresome formation was not observed following proteasome inhibition. These findings indicate that AE1 R664X protein, which is associated with dominant hereditary spherocytosis, has a dominant-negative effect on the expression of wild-type AE1.

The covalent modification of spectrin in red cell membranes by the lipid peroxidation product 4-hydroxy-2-nonenal

Spectrin strengthens the red cell membrane through its direct association with membrane lipids and through protein-protein interactions. Spectrin loss reduces the membrane stability and results in various types of hereditary spherocytosis. However, less is known about acquired spectrin damage. Here, we showed that alpha- and beta-spectrin in human red cells are the primary targets of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) by immunoblotting and mass spectrometry analyses. The level of HNE adducts in spectrin (particularly alpha-spectrin) and several other membrane proteins was increased following the HNE treatment of red cell membrane ghosts prepared in the absence of MgATP. In contrast, ghost preparation in the presence of MgATP reduced HNE adduct formation, with preferential beta-spectrin modification and increased cross-linking of the HNE-modified spectrins. Exposure of intact red cells to HNE resulted in selective HNE-spectrin adduct formation with a similar preponderance of HNE-beta-spectrin modifications. These findings indicate that HNE adduction occurs preferentially in spectrin at the interface between the skeletal proteins and lipid bilayer in red cells and suggest that HNE-spectrin adduct aggregation results in the extrusion of damaged spectrin and membrane lipids under physiological and disease conditions.

An Unrecognized Function of Cholesterol: Regulating the Mechanism Controlling Membrane Phospholipid Asymmetry

An asymmetric distribution of phospholipids in the membrane bilayer is inseparable from physiological functions, including shape preservation and survival of erythrocytes, and by implication other cells. Aminophospholipids, notably phosphatidylserine (PS), are confined to the inner leaflet of the erythrocyte membrane lipid bilayer by the ATP-dependent flippase enzyme, ATP11C, counteracting the activity of an ATP-independent scramblase. Phospholipid scramblase 1 (PLSCR1), a single-transmembrane protein, was previously reported to possess scrambling activity in erythrocytes. However, its function was cast in doubt by the retention of scramblase activity in erythrocytes of knockout mice lacking this protein. We show that in the human erythrocyte PLSCR1 is the predominant scramblase and by reconstitution into liposomes that its activity resides in the transmembrane domain. At or below physiological intracellular calcium concentrations, total suppression of flippase activity nevertheless leaves the membrane asymmetry undisturbed. When liposomes or erythrocytes are depleted of cholesterol (a reversible process in the case of erythrocytes), PS quickly appears at the outer surface, implying that cholesterol acts in the cell as a powerful scramblase inhibitor. Thus, our results bring to light a previously unsuspected function of cholesterol in regulating phospholipid scrambling.

Band 3 phosphorylation induces irreversible alterations of stored red blood cells

To the Editor: Red blood cells (RBCs) transfusion is a common practice in the global patient’s medical care. RBC concentrate is stored for up to 42 days after blood collection. Stored RBCs undergo abundant cellular and structural changes affecting their functional integrity and survival after transfusion and may be harmful for the blood recipient. Several randomized controlled trials compared transfusion of “fresher” versus “older” red cells but a correlation between the age of transfused RBCs and morbidity/mortality was not found to be conclusive. Storage lesions of RBCs leading to the progressive loss of biochemical, morphological, and mechanical properties are mostly due to irreversible changes in the membrane. These storage lesions are likely to affect RBC post-transfusion survival and increase the risks of adverse reactions in the recipients, especially in an inflammatory context. The underlying mechanisms leading to RBC membrane changes and vesiculation during storage are poorly known. We focused on the Band 3 anion exchanger, the most abundant protein of the RBC membrane, that plays a major role in membrane stability and deformability by linking the lipid bilayer to the skeleton via ankyrin R. Band 3 oligomerization plays a key role in the clearance of altered and old RBCs by forming senescence antigens recognized by naturally occurring autoantibodies (Nabs). Considering the absence of reliable marker of lesion storage, we aim to elucidate the molecular mechanisms leading to irreversible erythrocyte alterations during storage and to identify a relevant marker of “old” and damaged RBCs potentially harmful for patients. To investigate the oligomeric state of Band 3 in the RBC membrane, we performed the specific Eosin-5-maleimide (EMA) test used for the diagnosis of hereditary spherocytosis (Supporting Information Methods). Fluorescence intensities of EMA-labeled RBCs were measured for six blood bags at days 3, 14, 21, 28, 35, and 42 of storage (Figure 1A). We observed a progressive decrease of EMA labeling during storage with a maximum of 15% at day 42 (P< .001). This effect could be due to a decrease in Band 3 expression and/or to an increase in Band 3 oligomerization and mobility known to be due at least to Tyr phosphorylation. Then, we analyzed the phosphorylation state of Band 3 in RBC ghosts by Western blot analysis using an anti-phosphoTyr antibody. We observed an important increase in Band 3 phosphorylation between day 3 and day 42 of storage (Figure 1B, upper panel). Interestingly, we did not observe any change in the expression level of Band 3 after incubation of the same membrane with an antibody specific for the intracellular domain of Band 3 (Figure 1B, bottom panel). Similar results were obtained by flow cytometry analysis using an antibody against the extracellular domain of Band 3 (data not shown). Taken together, our findings strongly suggested that the decrease in EMA fluorescence was not due to the decrease of Band 3 expression but rather to an enhanced mobility following Band 3 phosphorylation. To further strengthen this hypothesis, we investigated the relationship between Band 3 phosphorylation and mobility by measuring the fluorescence of EMA-labeled RBCs treated with increasing concentrations of the phosphatase inhibitor O-vanadate. As expected, Band 3 tyrosine phosphorylation was induced and enhanced with increasing amounts of O-vanadate (Supporting Information Figure S1A) and this was associated with a decrease in EMA fluorescence (Supporting Information Figure S1B). Thus, it is likely that Band 3 phosphorylation during RBC storage induces its detachment from membrane skeleton, most probably by disruption from ankyrin R, increasing its mobility. To date, hyper-phosphorylated Band 3 was found to be associated with glucose-6-phosphate dehydrogenase deficiency (Ferru et al., 2011), hemoglobinopathies, and senescence of RBCs, all pathological conditions characterized by increased RBC oxidative stress and Band 3 clustering. To determine the impact of increased Band 3 oligomerization on its distribution and structure in the membrane, we took advantage of a unique antibody that specifically and selectively recognizes the clustered form of Band 3. We measured the binding capacity of this antibody to RBCs during storage by flow cytometry. A 14% increase in RBCs displaying Band 3 clusters was observed from day 3 to day 42 of storage (Figure 1C). This is of particular importance in the context of transfusion since Band 3 clustering was claimed to be the primary mechanism in the removal of RBCs from the circulation. This Band 3 alteration likely limits post-transfusion survival of RBCs. To explore the consequence of increased Band 3 phosphorylation and clustering during storage, we measured the release of microparticules (MPs), a well-known marker of irreversible lesions in the membrane in the same conditions of storage. Using flow cytometry and anti-Band 3 antibody, we found an important increase in the level of erythroid -positive MPs that all expressed Band 3 from day 28, with a 10-fold increase between day 28 and day 42 (Figure 1D). This strong elevation was associated with the increase of Band 3 phosphorylation. We hypothesized that it could represent the primary event leading to the formation of the “small cells” a sub-population of altered RBCs that accumulate during storage and are expected to be removed from the circulation in the hours following transfusion.

Maintenance and regulation of asymmetric phospholipid distribution in human erythrocyte membranes: implications for erythrocyte functions

Purpose of review The article summarizes new insights into the molecular mechanisms for the maintenance and regulation of the asymmetric distribution of phospholipids in human erythrocyte membranes. We focus on phosphatidylserine, which is primarily found in the inner leaflet of the membrane lipid bilayer under low Ca2+ conditions (<1 &mgr;mol/l) and is exposed to the outer leaflet under elevated Ca2+ concentrations (>1 &mgr;mol/l), when cells become senescent. Clarification of the molecular basis of phosphatidylserine flipping and scrambling is important for addressing long-standing questions regarding phosphatidylserine functions. Recent findings ATP11C, a P-IV ATPase, has been identified as a major flippase in analyses of patient erythrocytes with a 90% reduction in flippase activity. Phospholipid scramblase 1 (PLSCR1) has been defined as a Ca2+-activated scramblase that is completely suppressed by membrane cholesterol under low Ca2+ concentrations. Summary For survival, phosphatidylserine surface exposure is prevented by cholesterol-mediated suppression of PLSCR1 under low Ca2+ concentrations, irrespective of flipping by ATP11C. In senescent erythrocytes, PLSCR1 is activated by elevated Ca2+, resulting in phosphatidylserine exposure, allowing macrophage phagocytosis. These recent molecular findings establish the importance of the maintenance and regulation of phosphatidylserine distribution for both the survival and death of human erythrocytes.