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Maintenance and regulation of asymmetric phospholipid distribution in human erythrocyte membranes

implications for erythrocyte functions

Arashiki, Nobuto; Takakuwa, Yuichi

Current Opinion in Hematology: May 2017 - Volume 24 - Issue 3 - p 167–172
doi: 10.1097/MOH.0000000000000326

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 μmol/l) and is exposed to the outer leaflet under elevated Ca2+ concentrations (>1 μmol/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.

Department of Biochemistry, School of Medicine, Tokyo Women's Medical University, Tokyo, Japan

Correspondence to Yuichi Takakuwa, Department of Biochemistry, School of Medicine, Tokyo Women's Medical University, 8–1 Kawada-cho, Shinjuku-ku, Tokyo 162–8666, Japan. Tel: +81 3 5269 7415;. e-mail:

This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

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In all biological membranes, phospholipids are asymmetrically distributed in the lipid bilayers and they contribute to cell functions. Specifically, phosphatidylserine has several physiological functions; for example, it is involved in blood coagulation and acts as a phagocytic signaling molecule when it is transported from the inner to outer leaflet. Phosphatidylserine needs to be precisely maintained in the inner leaflet in static conditions under low Ca2+ concentrations and is exposed to the outer leaflet in active conditions with elevated Ca2+. In this review, we focus on the correlation between phosphatidylserine distribution and functions in human erythrocyte membranes. We examine how and why phosphatidylserine is maintained in the inner leaflet under physiological conditions and exposed to the outer leaflet when cells become senescent.

Box 1

Box 1

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In human erythrocytes, phosphatidylserine is exclusively present in the inner leaflet of the membrane lipid bilayer (Fig. 1). This asymmetric distribution of phosphatidylserine is essential for normal functions of erythrocytes. First, phosphatidylserine interacts with spectrin, a cytoskeletal protein component of erythrocyte membranes, to maintain cell shape and membrane mechanical functions, such as the deformability and stability of erythrocytes [1] necessary for traversing narrow capillaries and the splenic sinuses [2], similar to other vertical protein–protein interactions [3]. Second, phosphatidylserine binds to target residues to protect spectrin from glycation [1], which dramatically decreases membrane deformability. Third, preventing phosphatidylserine surface exposure is critical for erythrocyte survival until the end of their 120-day lifespan, at which point it paradoxically functions as a phagocytic signal (an ‘eat-me signal’) for erythrocyte removal by splenic macrophages. Indeed, in these cells, the Ca2+ concentration is elevated to activate lipid scrambling and consequent phosphatidylserine surface exposure, which is recognized as an ‘eat-me signal’ by macrophages [4]. Incidentally, clustered band 3, another eat-me signal, also appears [5] (Fig. 1). In addition to its role in normal senescent cells, premature phosphatidylserine exposure by sickle erythrocytes and thalassemic erythrocytes results in a shortened life span and is associated with hemolytic anemia in these disorders [6]. Therefore, the maintenance and regulation of the asymmetric phosphatidylserine distribution are important for both erythrocyte survival and death. Although it is believed that the phosphatidylserine distribution is determined by flippase and scramblase activity, the molecular identities of these activities in human erythrocytes have only recently been determined. Furthermore, the relative contributions of flippase and scramblase for maintaining the asymmetric distribution of phosphatidylserine under physiological and pathological states as well as phosphatidylserine exposure for cell death are not well understood.



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Flippases include 14 members of the P-IV ATPase family of proteins and are composed of 10 transmembrane domains. They contribute to the localization of phosphatidylserine in the inner leaflet of cellular membranes, including red blood cell membranes via the ATP-dependent active transport of aminophospholipids, such as phosphatidylserine, from the outer to inner leaflet (flip) [7], enabling the localization of these phospholipids in the inner leaflet. Recently, we identified ATP11C as a major flippase of human erythrocytes in an analysis of a male patient presenting congenital mild hemolytic anemia with an ATP11C mutation on the X chromosome [8▪] (Fig. 2a). The patient's red blood cells exhibited a normal biconcave shape, but flippase activity was 10% of that observed in control cells. Even in the near absence of flippase activity, phosphatidylserine still localized to the inner leaflet of the vast majority of erythrocyte membranes, suggesting that flippase activity per se is not essential for the maintenance of phosphatidylserine in the inner leaflet under physiological conditions. The low levels of remaining activity are probably enabled by ATP11A, another member of the P-IV ATPase family that is expressed during the terminal differentiation stages of erythroblasts. The maintenance of phosphatidylserine in the inner leaflet at erythroblast stages is mediated by ATP11A, leading to normal erythropoiesis in this patient. His mother, a heterozygote for this mutation with no symptoms related to hemolytic anemia, has both flippase-active and inactive red blood cells at an approximately 1 : 1 ratio as a result of X chromosome inactivation at an early erythroblast stage, suggesting that the removal or hemolytic rates of the latter are very similar to those of the former. However, in the patient's senescent erythrocytes (0.1% of cells), phosphatidylserine was subtly exposed to the outer leaflet, corresponding to ‘mild’ hemolytic anemia. The patient did not exhibit other clinical symptoms, suggesting that in other cells, different members of the P-IV ATPase family play compensatory roles in the maintenance of the internal distribution of phosphatidylserine. Atp11c mutant mice exhibit anemia with stomatocytosis, B-cell deficiency, cholestasis, and liver tumors, different from the phenotypes associated with human ATP11C deficiency [9–11]. In a mutant mouse with stomatocytic anemia, flippase activity in erythroblasts decreased by approximately 50% relative to that of wild-type mouse cells, but a decrease was not detected in mature erythrocytes [9]. According to our study, at the early differentiation stages, mouse erythroblasts express Atp8a1 and Atp11c at similar levels, whereas at the terminal stage, Atp8a1 is preferentially expressed and Atp11c expression is decreased [8▪], implying that mature erythrocytes in mice possess Atp8a1 as a major flippase and Atp11c contributes only to erythroblast flippase activity. Indeed, Atp8a1 was separated from mouse erythrocytes [12]. Thus, anemia in Atp11c mutant mice occurs via impaired erythropoiesis. As there are many differences between human and mouse erythrocytes, such as hematopoietic organs, erythrocyte lifespan, and expressed proteins [13], careful interpretation of results obtained using mouse erythrocytes is necessary for applications to human erythrocytes. However, this patient should be monitored for the emergence of a B-cell deficiency, cholestasis, and/or liver tumor in the future.



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Scramblase bidirectionally and unselectively transports phospholipids between the lipid bilayer in Ca2+-dependent and ATP-independent manners, resulting in the transfer of phosphatidylserine from the inner to outer leaflet. As candidate scramblase molecules, phospholipid scramblase 1 (PLSCR1) was identified 20 years ago and transmembrane protein 16F (TMEM16F) and XK-related protein 8 (XKR8) were recently identified [14]. In red blood cells, numerous studies of PLSCR1 have contributed to our understanding scramblase functions and molecules [15]. However, the scramblase function of PLSCR1 has been questioned based on the observation that PLSCR1 knockout mice exhibit normal phosphatidylserine exposure under high calcium concentrations [16]. We recently identified PLSCR1 as a human red blood cell scramblase in an analysis focused on the inhibitory effect of cholesterol, which accounts for half of the total lipids in the red blood cell membrane [17▪]. The scramblase in red blood cells from which about half of cholesterol was removed is dramatically activated, even at low physiological Ca2+ concentrations (0.5 or 1 μmol/l). Both recombinant full-length PLSCR1 and the partial peptide composed of its sole transmembrane region scrambled various phospholipids in the liposome without cholesterol, but not with cholesterol (Fig. 2b). The single transmembrane protein PLSCR1 was detected in the self-assembled state (5–8 mer) on the red blood cell membrane both with and without cholesterol, indicating that it exists constantly in an active form. A hydrophilic region in the middle portion of the PLSCR1 transmembrane domain is critical for glycerophospholipid transfer (scrambling), as evidenced by the disruption of phosphatidylcholine transport when a substitution with a hydrophobic amino acid is introduced. Self-assembled multimers and this hydrophilic region enable unselective and ATP-independent phospholipid transport (scrambling) by forming a pore for the transport of the phospholipid polar head. If red blood cells had less cholesterol, similar to the levels observed in other typical cells (20%), phosphatidylserine would be easily exposed at the cell surface by slight elevations in Ca2+ when they pass through narrow capillary vessels, resulting in their early removal or hemolysis. PLSCR1 belongs to the PLSCR family composed of five molecules (PLSCR1-5) [14]. Based on RNAseq analyses of PLSCR family genes, human erythroblasts predominantly express PLSCR1, whereas mouse erythroblasts predominantly express Plscr3, with low levels of Plscr1[17▪]. As expected, erythrocytes of Plscr1 knockout mice exhibit normal scrambling activity as described above. Past research on ATP11C and PLSCR1 emphasize the obvious difference between humans and mice.

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Normally, phospholipid scrambling occurs only in senescent cells; accordingly, phosphatidylserine exposure has been described as an ‘eat-me signal’ for senescent cell recognition and subsequent phagocytosis by splenic macrophages. Phosphatidylserine exposure is facilitated by the activation of scramblase activity under high Ca2+ concentrations. However, such activity is either suppressed or minimized in most erythrocytes, in which cholesterol is more abundant compared with other cells and Ca2+ concentrations are maintained at low, submicromolar levels by Ca2+ ATPase [18]. Cholesterol does not affect the self-assembly of PLSCR1 molecules (Fig. 2b). Additional studies are needed to determine the precise molecular mechanisms for cholesterol inhibition of scrambling, including whether cholesterol binds directly to PLSCR1 or indirectly affects scrambling activity. The ATP concentration in senescent red blood cells is maintained at the submillimolar level [18], which is sufficient to activate the flippase ATP11C, whose Michaelis constant Km is in the submillimolar range [19]. However, it should be emphasized that the contribution of the flippase ATP11C is less than that of the inhibition of scrambling activity (Fig. 3). These observations indicate that the Ca2+ concentration is a key factor for phosphatidylserine exposure in senescent red blood cells. The precise molecular mechanism for Ca2+ activation of cholesterol-inhibited scrambling via PLSCR1 is under investigation.



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Recent evidence has indicated that human red blood cells employ a survival strategy in which phosphatidylserine exposure to the outer leaflet is absolutely prevented by cholesterol at low physiological Ca2+ concentrations, irrespective of the ATP-driven flippase ATP11C (Fig. 3). The strategy for human erythrocyte survival is reasonable from the perspective of ATP metabolism. Similar to other cells, ATP is indispensable for red blood cell functions, including active transport by several ATPases and the regulation of protein functions via phosphorylation. Different from other cells, ATP is very precious in erythrocytes, since it is only synthesized by glycolysis owing to a lack of mitochondria. It is, therefore, critical that cholesterol inhibits PLSCR1 and simultaneously minimizes ATP consumption by ATP11C. This may explain why human erythrocyte membranes contain large amounts of cholesterol, representing up to about half of the total lipids. Owing to cholesterol inhibition, PLSCR1 is utilized as an erythrocyte scramblase, instead of TMEM16F and XKR8 used in other cells. When erythrocytes become senescent, the Ca2+ concentration is gradually elevated to activate PLSCR1, allowing macrophage phagocytosis. It is not clear how Ca2+ binds to PLSCR1 molecules for activation, even in the presence of cholesterol.

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We acknowledge Hitoshi Kanno at Tokyo Women's Medical University for the analysis of patient erythrocytes lacking ATP11C and Narla Mohandas at New York Blood Center for useful discussions concerning the implications of PS distributions. We would like to thank Editage ( for English language editing.

Funding received for this work: This work was supported by JSPS KAKENHI grant numbers 22591112 and 25460375.

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Financial support and sponsorship

This work was supported by JSPS KAKENHI grant numbers 22591112 and 25460375.

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Conflicts of interest

There are no conflicts of interest.

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Papers of particular interest, published within the annual period of review, have been highlighted as:

  • ▪ of special interest
  • ▪▪ of outstanding interest
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ATP11C; cholesterol; human erythrocyte; phosphatidylserine; phospholipid scramblase 1

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