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.
IMPORTANCE OF THE PHOSPHATIDYLSERINE DISTRIBUTION IN HUMAN ERYTHROCYTE FUNCTIONS
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  necessary for traversing narrow capillaries and the splenic sinuses , similar to other vertical protein–protein interactions . Second, phosphatidylserine binds to target residues to protect spectrin from glycation , 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 . Incidentally, clustered band 3, another eat-me signal, also appears  (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 . 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.
ROLE OF ATP11C IN FLIPPASE ACTIVITY IN HUMAN ERYTHROCYTES
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) , 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 . 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 . 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 , 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.
ROLE OF PHOSPHOLIPID SCRAMBLASE 1 IN SCRAMBLING IN HUMAN ERYTHROCYTES
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 . In red blood cells, numerous studies of PLSCR1 have contributed to our understanding scramblase functions and molecules . 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 . 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) . 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.
MAINTENANCE AND REGULATION OF THE ASYMMETRIC PHOSPHATIDYLSERINE DISTRIBUTION OVER THE 120-DAY LIFESPAN
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 . 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 , which is sufficient to activate the flippase ATP11C, whose Michaelis constant Km is in the submillimolar range . 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.
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.
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 (http://www.editage.jp) for English language editing.
Funding received for this work: This work was supported by JSPS KAKENHI grant numbers 22591112 and 25460375.
Financial support and sponsorship
This work was supported by JSPS KAKENHI grant numbers 22591112 and 25460375.
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
- ▪▪ of outstanding interest
1. Manno S, Takakuwa Y, Mohandas N. Identification of a functional role for lipid asymmetry in biological membranes: phosphatidylserine
-skeletal protein interactions modulate membrane stability. Proc Natl Acad Sci U S A 2002; 99:1943–1948.
2. Manno S, Mohandas N, Takakuwa Y. ATP-dependent mechanism protects spectrin against glycation in human erythrocytes. J Biol Chem 2010; 285:33923–33929.
3. Mohandas N, Gallagher PG. Red cell membrane: past, present, and future. Blood 2008; 112:3939–3948.
4. Lauber K, Blumenthal SG, Waibel M, et al. Clearance of apoptotic cells: getting rid of the corpses. Mol Cell 2004; 14:277–287.
5. Arashiki N, Kimata N, Manno S, et al. Membrane peroxidation and methemoglobin formation are both necessary for band 3 clustering: mechanistic insights into human erythrocyte
senescence. Biochemistry 2013; 52:5760–5769.
6. Zwaal RF, Comfurius P, Bevers EM. Surface exposure of phosphatidylserine
in pathological cells. Cell Mol Life Sci 2005; 62:971–988.
7. Daleke DL. Regulation of phospholipid asymmetry in the erythrocyte membrane. Curr Opin Hematol 2008; 15:191–195.
8▪. Arashiki N, Takakuwa Y, Mohandas N, et al. ATP11C
is a major flippase in human erythrocytes and its defect causes congenital hemolytic anemia. Haematologica 2016; 101:559–565.
The study identified ATP11C as a major flippase in human erythrocyte membranes in analyses of patient erythrocytes with a 90% reduction in flippase activity. The limited symptoms, such as mild hemolysis, in this patient are discussed in terms of the correlation with scramblase activity.
9. Yabas M, Coupland LA, Cromer D, et al. Mice deficient in the putative phospholipid flippase ATP11C
exhibit altered erythrocyte shape, anemia, and reduced erythrocyte life span. J Biol Chem 2014; 289:19531–19537.
10. Siggs OM, Arnold CN, Huber C, et al. The P4-type ATPase ATP11C
is essential for B lymphopoiesis in adult bone marrow. Nat Immunol 2011; 12:434–440.
11. Siggs OM, Schnabl B, Webb B, Beutler B. X-linked cholestasis in mouse due to mutations of the P4-ATPase ATP11C
. Proc Natl Acad Sci U S A 2011; 108:7890–7895.
12. Soupene E, Kuypers FA. Identification of an erythroid ATP-dependent aminophospholipid transporter. Br J Haematol 2006; 133:436–438.
13. An X, Schulz VP, Li J, et al. Global transcriptome analyses of human and murine terminal erythroid differentiation. Blood 2014; 123:3466–3477.
14. Kodigepalli KM, Bowers K, Sharp A, Nanjundan M. Roles and regulation of phospholipid scramblases. FEBS Lett 2015; 589:3–14.
15. Sims PJ, Wiedmer T. Unraveling the mysteries of phospholipid scrambling. Thromb Haemost 2001; 86:266–275.
16. Zhou Q, Zhao J, Wiedmer T, et al. Normal hemostasis but defective hematopoietic response to growth factors in mice deficient in phospholipid scramblase 1
. Blood 2002; 99:4030–4038.
17▪. Arashiki N, Saito M, Koshino I, et al. An unrecognized function of cholesterol
: regulating the mechanism controlling membrane phospholipid asymmetry. Biochemistry 2016; 55:3504–3513.
The study defined PLSCR1 as a major scramblase in human erythrocyte membranes and demonstrated its suppression by membrane cholesterol under low Ca2+ conditions. The mechanisms for erythrocyte survival and death are discussed with respect to cholesterol and Ca2+ regulation.
18. Lutz HU, Bogdanova A. Mechanisms tagging senescent red blood cells for clearance in healthy humans. Front Physiol 2013; 4:387.
19. Coleman JA, Kwok MC, Molday RS. Localization, purification, and functional reconstitution of the P4-ATPase Atp8a2, a phosphatidylserine
flippase in photoreceptor disc membranes. J Biol Chem 2009; 284:32670–32679.