ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes.

Spin-labeled analogs of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine have been used to study phospholipid transverse diffusion and asymmetry in the human erythrocyte membrane. Ascorbate reduction was used to assess the transbilayer distribution of the labels. All three spin-labeled phospholipids initially incorporated into the outer leaflet of the membrane. On fresh erythrocytes at 5 degrees C, the phosphatidylcholine label remained mainly in the outer leaflet. In contrast, the phosphatidylserine and phosphatidylethanolamine labels underwent rapid transverse diffusion that led to their asymmetric distribution in favor of the inner leaflet. The latter effect was reversibly inhibited after ATP depletion of the erythrocytes and could be reproduced on resealed erythrocyte ghosts only if hydrolyzable Mg-ATP was included in the internal medium. It is suggested that an ATP-driven transport of amino phospholipids toward the inner leaflet could be the major cause of the phospholipid asymmetry in the erythrocyte membrane. It is also proposed that the same mechanism could explain the ATP requirement of the maintenance of the erythrocyte membrane discoid shape.     

Transmembrane mobility of phospholipids in sickle erythrocytes: effect of deoxygenation on diffusion and asymmetry

We studied the effect of sickling on the transmembrane reorientation and distribution of phospholipids in the red blood cells of patients homozygous for sickle cell anemia (SS). To this purpose, we followed the redistribution kinetics of trace amounts of spin-labeled analogues of natural phospholipids first introduced in the membrane outer leaflet of normal or sickle erythrocytes exposed to air or nitrogen. Deoxygenation had no effect on the lipid redistribution kinetics in normal (AA) cell membranes. At atmospheric pO2, unfractionated SS cells were not different from normal cells. However, on deoxygenation inducing sickling, phosphatidylcholine passive diffusion was accelerated and the rate of the adenosine triphosphate-dependent transport of aminophospholipids was reduced, especially for phosphatidylserine. The stationary distribution of the aminophospholipids between the two leaflets was slightly less asymmetric, a phenomenon more pronounced with phosphatidylethanolamine. These changes were rapidly reversible on reoxygenation. When SS cells were separated by density, both dense and light cells exhibited the properties cited above. However, dense cells exposed to air possessed a lower aminophospholipid transport rate. These data favor the relationship between aminophospholipid translocase activity and phospholipid transmembrane asymmetry. Sickle cell disease is the first case of aminophospholipid translocase pathology.     

Reconstitution of ATP-dependent aminophospholipid translocation in proteoliposomes.

In addition to ion-pumping ATPases, most plasma membranes of animal cells contain a Mg2+ ATPase activity, the function of which is unknown. This enzyme, of apparent molecular mass 110 kDa, was purified from human erythrocyte membranes by a series of column chromatographic procedures after solubilization in Triton X-100. When reincorporated into artificial bilayers formed from phosphatidylcholine, it was able to transport a spin-labeled phosphatidylserine analogue from the inner to the outer membrane leaflet provided Mg2+ ATP was present in the incubation mixture. The ATP-dependent transport of the phosphatidylethanolamine analogue required the presence of an anionic phospholipid (e.g., phosphatidylinositol) in the outer membrane leaflet. In contrast the transmembrane distribution of spin-labeled phosphatidylcholine was unaffected in the same experimental conditions. This transmembrane movement of aminophospholipid analogues was inhibited by treatment of the proteoliposomes with a sulfhydryl reagent. We conclude that the Mg2+ ATPase is sufficient for the biochemical expression of the aminophospholipid translocase activity, which is responsible for the inward transport of phosphatidylserine and phosphatidylethanolamine within the erythrocyte membrane. The presence of this transport activity in many animal cell plasma membranes provides a function for the Mg2+ ATPase borne by these membranes.     

Asymmetric lateral mobility of phospholipids in the human erythrocyte membrane.

The fluorescent phospholipid 1-acyl-2-[12-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)aminododecanoyl]phosphatidylcholine (NBD-phosphatidylcholine) and the corresponding aminophospholipid derivatives (NBD-phosphatidylethanolamine and NBD-phosphatidylserine) were introduced in the human erythrocyte membrane by a nonspecific phospholipid exchange protein purified from corn. The lateral mobility of the fluorescent phospholipids was measured by using an extension of the classical photobleaching recovery technique that takes advantage of a modulated fringe pattern and provides a high sensitivity. In intact erythrocytes and in ghosts resealed in the presence of ATP, the fluorescence-contrast curves after photobleaching decayed biexponentially corresponding to two lateral diffusion constants. With NBD-phosphatidylcholine, the majority of the signal corresponded to a "slow" component (1.08 X 10(-9) cm2/sec at 20 degrees C), whereas with the amino derivatives the majority of the signal corresponded to a "fast" component (5.14 X 10(-9) cm2/sec at 20 degrees C). If the ghosts were resealed without ATP, the fast component of the aminophospholipids disappeared. We interpret these results as follows: (i) Provided the cells or the ghosts contain ATP, the three fluorescent phospholipids distribute spontaneously between inner and outer leaflets as endogenous phospholipids, namely NBD-phosphatidylcholine is located in the outer leaflet, while both aminophospholipids are preferentially located in the inner leaflet. (ii) The viscosity of the inner leaflet of human erythrocyte membranes is lower than that of the outer leaflet.     

Measurement of outward translocation of phospholipids across human erythrocyte membrane.

Spin-labeled phospholipids have been used to study the outside----inside and inside----outside transport of phospholipids across the human erythrocyte membrane at 37 degrees C. As already shown, inward transport is much faster for aminophospholipids than for phosphatidylcholine. In addition, we show here that outward transport of the phosphatidylserine and phosphatidylethanolamine analogues is three to four times faster than that of phosphatidylcholine. Magnesium depletion of the erythrocytes considerably decreases the outward rate of both aminophospholipids to values close to that of phosphatidylcholine. These results suggest that the outward aminophospholipid translocation is, at least partly, protein mediated. The protein involved could be identical to the inward Mg-ATP-dependent aminophospholipid carrier.     

Phospholipid composition and organization in model β-thalassemic erythrocytes

The membrane phospholipid organization in human red blood cells (RBC) is rigidly maintained by a complex system of enzymes. However, several elements of this system are sensitive to oxidative damage. An important component in the destruction of β-thalassemic RBC is the generation of reactive oxygen species and the release of redox-active iron by the unpaired α-hemoglobin chains. Consequently, we hypothesized that the presence of this oxidative stress to the RBC membrane could lead to alterations in membrane lipid organization. Model β thalassemic RBC, prepared by the introduction of excess α-globin in the cell, have previously been shown to exhibit structural and functional changes almost identical to those observed in β-thalassemic cells. After 24 hr at 37°C, the model β thalassemic cells exhibited a significant loss of deformability, as measured by ektacytometric analysis, indicative of extensive membrane damage. However, a normal steady-state distribution of endogenous phospholipids was found, as evidenced by the accessibility of membrane phospholipids to hydrolysis by phospholipases. Similarly, the kinetics of transbilayer movement of spin-labeled phosphatidylserine (PS) and phosphatidylethanolamine (PE) in all samples was in the normal range and was not affected by the presence of excess α-globin chains. In contrast, a faster rate of spin-labeled phosphatidylcholine (PC) transbilayer movement was observed in these cells. While control RBC exhibited a complete loss of their initial (2 mol%) lysophosphatidylcholine (LPC) levels following 24 hr of incubation at 37°C, 1.5 mol% LPC was still present in model β-thalassemic cells, suggesting an altered phospholipid molecular species turnover, possibly as a result of an increased repair of oxidatively damaged phospholipids. © 1996 Wiley-Liss, Inc.     

Identification of a functional role for lipid asymmetry in biological membranes: Phosphatidylserine-skeletal protein interactions modulate membrane stability

Asymmetric distribution of phospholipids is ubiquitous in the plasma membranes of many eukaryotic cells. The majority of the aminophospholipids are located in the inner leaflet whereas the cholinephospholipids are localized predominantly in the outer leaflet. Several functional roles for asymmetric phospholipid distribution in plasma membranes have been suggested. Disruption of lipid asymmetry creates a procoagulant surface on platelets and serves as a trigger for macrophage recognition of apoptotic cells. Furthermore, the dynamic process of phospholipid translocation regulates important cellular events such as membrane budding and endocytosis. In the present study, we used the red cell membrane as the model system to explore the contribution of phospholipid asymmetry to the maintenance of membrane mechanical properties. We prepared two different types of membranes in terms of their phospholipid distribution, one in which phospholipids were scrambled and the other in which the asymmetric distribution of phospholipids was maintained and quantitated their mechanical properties. We documented that maintenance of asymmetric distribution of phospholipids resulted in improved membrane mechanical stability. The greater difficulty in extracting the spectrin-actin complex at low-ionic strength from the membranes with asymmetric phospholipid distribution further suggested the involvement of interactions between aminophospholipids in the inner leaflet and skeletal proteins in modulating mechanical stability of the red cell membrane. These findings have enabled us to document a functional role of lipid asymmetry in regulating membrane material properties.     

Altered plasma membrane phospholipid organization in Plasmodium falciparum-infected human erythrocytes

The intraerythrocytic development of the malaria parasite is accompanied by distinct morphological and biochemical changes in the host cell membrane, yet little is known about development-related alterations in the transbilayer organization of membrane phospholipids in parasitized cells. This question was examined in human red cells infected with Plasmodium falciparum. Normal red cells were infected with strain FCR3 or with clonal derivatives that either produce (K+) or do not produce (K-) knobby protuberances on the infected red cells. Parasitized cells were harvested at various stages of parasite development, and the bilayer orientation of red cell membrane phospholipids was determined chemically using 2,4,6-trinitrobenzene sulphonic acid (TNBS) or enzymatically using bee venom phospholipase A2 (PLA2) and sphingomyelinase C (SMC). We found that parasite development was accompanied by distinct alterations in the red cell membrane transbilayer distribution of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Increases in the exoplasmic membrane leaflet exposure of PE and PS were larger in the late-stage parasitized cells than in the early-stage parasitized cells. Similar results were obtained for PE membrane distribution using either chemical (TNBS) or enzymatic (PLA2 plus SMC) methods, although changes in PS distribution were observed only with TNBS. Uninfected cohort cells derived from mixed populations of infected and uninfected cells exhibited normal patterns of membrane phospholipid organization. The observed alterations in P falciparum-infected red cell membrane phospholipid distribution, which is independent of the presence or absence of knobby protuberances, might be associated with the drastic changes in cell membrane permeability and susceptibility to early hemolysis observed in the late stages of parasite development.     

A unique phospholipid organization in bovine erythrocyte membranes

Ruminant erythrocytes are remarkable for their choline-phospholipid anomalies; namely, low or absent phosphatidylcholine (PC) along with high sphingomyelin levels. Here, we report another anomaly in bovine erythrocytes that affects aminophospholipids: phosphatidylethanolamine (PE) shows an extreme asymmetry, with only 2% of the total present in the outer leaflet. Furthermore, we found that phospholipase A(2), an enzyme located on the external surface of the erythrocytes, shows higher activity against PC than against PE. In addition, we observed that acylation of PE is by far the most important biosynthetic event in this system. We propose that deacylation of PE and PC by phospholipase A(2) to generate lysocompounds, followed by selective reacylation of lyso-PE in the inner leaflet, can account for the compositional and architectural peculiarities of bovine erythrocyte membranes.     

Enzymatic synthesis and rapid translocation of phosphatidylcholine by two methyltransferases in erythrocyte membranes.

The synthesis of phosphatidylcholine from phosphatidylethanolamine is carried out by two methyltransferases in erythrocyte membranes. The first enzyme uses phosphatidylethanolamine as a substrate, requires Mg2+, and has a high affinity for methyl donor, S-adenosyl-L-methionine. The second enzyme methylates phosphatidyl-N-monomethylethanolamine to phosphatidylcholine and has a low affinity for S-adenosyl-L-methionine. The first enzyme is localized on the cytoplasmic side of the membrane and the second enzyme faces the external surface. This asymmetric arrangement of the two enzymes across the membrane makes possible the stepwide methylation of phosphatidylethanolamine localized on the cytoplasmic side and facilitates the rapid transmembrane transfer of the final product, phosphatidylcholine, to the external surface of the membrane. A mechanism for an enzyme-mediated flip-flop of phospholipids from the cytoplasmic to the outer surface of erythrocyte membranes is described.     

Rapid transmembrane movement of newly synthesized phospholipids during membrane assembly.

The transbilayer distribution of phospholipids in Bacillus megaterium is asymmetrical, with twice as much phosphatidylethanolamine internally as externally (Rothaman, J. E. & Kennedy, E. P. (1977) J. Mol. Biol. 110,603-618). We now report that the biosynthesis of phosphatidylethanolamine is also asymmetrical. Newly synthesized phosphatidylethanolamine was found first on the cytoplasmic side of the membrane of pulse-labeled cells and later was redistributed until the specific radioactivity of the outer face became equal to that of the inner face of the bilayer. The rate of transmembrane movement is at least 30,000 times faster than the rate of spontaneous diffusion (flip-flop) of phosphatidylethanolamine across artificial phospholipid bilayers, indicating that transmembrane movement must be a facilitated process in living cells, perhaps involving membrane proteins.     

Impaired redistribution of aminophospholipids with distinctive cell shape change during Ca2+-induced activation of platelets from a patient with Scott syndrome

We have investigated phospholipid redistribution, membrane vesicle shedding, shape change, and granule release following A23187 activation of platelets from a patient with Scott syndrome, characterized by impaired transmembrane migration of phosphatidylserine (PS) accompanied by haemorrhagic complications, and two of her children. Electron spin resonance spectroscopy measurement of phospholipids redistribution showed that the internalization of PS was unaffected by the disorder but, after activation, PS exposure was significantly reduced in platelets from the homozygous-type patient. Vesicle shedding was also reduced in these platelets. However, the slow redistribution of phosphatidylcholine was similar to that observed in normal platelets. When treated with calpeptin, platelets from the homozygous-type patient, unlike normal or heterozygous Scott syndrome platelets, showed a smoothly rounded shape without filopods after activation. Following A23187 activation of normal platelets, filopod formation was consecutive to the re-exposition of aminophospholipids on the outer leaflet of the plasma membrane, and the existence of a floppase (outward aminoPLs translocase) has been suggested. In homozygous Scott syndrome platelets the deficiency in PS re-exposition, the absence of filopod formation, and low vesicle shedding are correlated with each other, and argue in favour of a disruption of the proposed floppase activity.     

Evidence against phospholipid asymmetry in intracellular membranes from liver.

We have studied the distribution of phospholipids across the membrane of microsomal vesicles and Golgi-derived secretory vesicles from rat liver by the use of phospholipases. Model studies on single-bilayer phospholipid vesicles showed that phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) cleaved at least 80% of the lipids on the outer surface of such vesicles without significant attack on the inner surface. In microsomal vesicles approximately 40% of the outer surface phospholipids were cleaved before the enzyme gained access to the interior of the vesicles. The same conclusion was reached for Golgi vesicles. By following the degradation of the three major phospholipids in intact microsomes and in extracted lipids we found that the same fraction of each of these phospholipids was exposed on the outer surface of the microsomal vesicles. Corresponding experiments with Golgi vesicles showed that distinctly different fractions of phosphatidylcholine and phosphatidylethanolamine were present on the surface of these vesicles. However, the difference was accounted for by enrichment of phosphatidylcholine in intravesicular particles rather than by asymmetry across the vesicle membrane. The results from specific hydrolysis of phosphatidylinositol confirmed an essentially symmetric distribution of this phospholipid across the microsomal and the Golgi vesicle membranes.     

Phosphatidylserine as a determinant of reticuloendothelial recognition of liposome models of the erythrocyte surface.

Liposomes formulated to resemble the outer leaflet of the erythrocyte membrane were found to substantially avoid recognition and clearance by the reticuloendothelial system. When these models of the erythrocyte surface were modified by the incorporation of greater than 2 mol % of phosphatidylserine (PtdSer), their ability to remain in the circulation of mice was greatly reduced. To examine whether this altered behavior was the consequence of an alteration in bilayer organization induced by PtdSer, a method utilizing the fluorescent dye merocyanine 540 was used to assess the packing of external phospholipids. No significant difference in overall membrane lipid organization was detected between liposomes containing 2 or 3 mol % of PtdSer, at which dramatic differences in recognition and clearance occurred. These results exclude alterations in phospholipid packing as an indirect cause of increased clearance of PtdSer-containing liposomes and implicate PtdSer directly in recognition by the reticuloendothelial system.     

糖尿病患者红细胞膜磷脂成分的改变

应用高效液相色谱法分析７２例ＮＩＤＤＭ患者红细胞膜磷脂成分。结果显示，ＮＩＤＤＭ患者红细胞膜磷脂酰乙醇胺、磷脂酰肌醇、磷脂酰丝氨酸、磷脂酰胆碱、神经鞘磷脂均显著降低，而溶血磷脂酰胆碱显著增高，并且红细胞膜磷脂成分的改变与空腹血糖、糖化血红蛋白、血脂及过氧化脂质有关。提示糖尿病代谢紊乱与脂质过氧化可能是导致红细胞膜磷脂成分改变的重要因素。     

Cytoplasmic remodeling of erythrocyte raft lipids during infection by the human malaria parasite Plasmodium falciparum

Studies of detergent-resistant membrane (DRM) rafts in mature erythrocytes have facilitated identification of proteins that regulate formation of endovacuolar structures such as the parasitophorous vacuolar membrane (PVM) induced by the malaria parasite Plasmodium falciparum. However, analyses of raft lipids have remained elusive because detergents interfere with lipid detection. Here, we use primaquine to perturb the erythrocyte membrane and induce detergent-free buoyant vesicles, which are enriched in cholesterol and major raft proteins flotillin and stomatin and contain low levels of cytoskeleton, all characteristics of raft microdomains. Lipid mass spectrometry revealed that phosphatidylethanolamine and phosphatidylglycerol are depleted in endovesicles while phosphoinositides are highly enriched, suggesting raft-based endovesiculation can be achieved by simple (non-receptor-mediated) mechanical perturbation of the erythrocyte plasma membrane and results in sorting of inner leaflet phospholipids. Live-cell imaging of lipid-specific protein probes showed that phosphatidylinositol (4,5) bisphosphate (PIP(2)) is highly concentrated in primaquine-induced vesicles, confirming that it is an erythrocyte raft lipid. However, the malarial PVM lacks PIP(2), although another raft lipid, phosphatidylserine, is readily detected. Thus, different remodeling/sorting of cytoplasmic raft phospholipids may occur in distinct endovacuoles. Importantly, erythrocyte raft lipids recruited to the invasion junction by mechanical stimulation may be remodeled by the malaria parasite to establish blood-stage infection.     

Isolation of a Chinese hamster ovary cell mutant defective in intramitochondrial transport of phosphatidylserine.

A CHO-K1 cell mutant with a specific decrease in cellular phosphatidylethanolamine (PE) level was isolated as a variant resistant to Ro09-0198, a PE-directed antibiotic peptide. The mutant was defective in the phosphatidylserine (PS) decarboxylation pathway for PE formation, in which PS produced in the endoplasmic reticulum is transported to mitochondria and then decarboxylated by an inner mitochondrial membrane enzyme, PS decarboxylase. Neither PS formation nor PS decarboxylase activity was reduced in the mutant, implying that the mutant is defective in some step of PS transport. The transport processes of phospholipids between the outer and inner mitochondrial membrane were analyzed by use of isolated mitochondria and two fluorescence-labeled phospholipid analogs, 1-palmitoyl-2-[N-[6(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-PS (C6-NBD-PS) and C6-NBD-phosphatidylcholine (C6-NBD-PC). On incubation with the CHO-K1 mitochondria, C6-NBD-PS was readily decarboxylated to C6-NBD-PE, suggesting that the PS analog was partitioned into the outer leaflet of mitochondria and then translocated to the inner mitochondrial membrane. The rate of decarboxylation of C6-NBD-PS in the mutant mitochondria was reduced to approximately 40% of that in the CHO-K1 mitochondria. The quantity of phospholipid analogs translocated from the outer leaflet of mitochondria into inner mitochondrial membranes was further examined by selective extraction of the analogs from the outer leaflet of mitochondria. In the mutant mitochondria, the translocation of C6-NBD-PS was significantly reduced, whereas the translocation of C6-NBD-PC was not affected. These results indicate that the mutant is defective in PS transport between the outer and inner mitochondrial membrane and provide genetic evidence for the existence of a specific mechanism for intramitochondrial transport of PS.     

Effect of phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine on the activated factor X-prothrombin system.

Membrane phospholipids are essential in blood coagulation reactions. The importance of negatively changed phosphatidylserine has been shown. The roles of other phospholipids in the blood coagulation system, however, are not clear. This study examined the effects of phosphatidylcholine on the blood coagulation system using liposomes containing varying concentrations of phosphatidylcholine in the presence of phosphatidylserine at a constant concentration. In addition, with phosphatidylserine and phosphatidylcholine at constant concentrations, the effects of phosphatidylethanolamine and lysophosphatidylcholine on the blood coagulation system were examined. Using an in vitro reconstructed system of the activated factor X-prothrombin system, blood coagulation was measured by the rate of thrombin formation after the addition of liposome preparations. The results showed suppression of the system by phosphatidylcholine and phosphatidylethanolamine and acceleration by lysophosphatidylcholine.The results of the present study suggest that the cell membrane, the 'location' of blood coagulation, is one of the regulatory factors, and that changes in phosphatidylcholine content and phospholipid composition of the cell membrane regulate the coagulation reaction.     

Ethanol-induced alterations in erythrocyte membrane phospholipid composition

Ethanol's effects on erythrocyte membrane lipid composition were examined in male squirrel monkeys divided into three groups receiving three different regimens: Controls were fed a chemically defined liquid diet, and low and high ethanol primates were given diets with vodka substituted isocalorically for 12% and 24% of calories, respectively. After membrane lipid extraction, phospholipid mass, class composition, and fatty acid profiles were measured in each group. Although there were no differences in the total phospholipid mass, the low ethanol primates had significantly elevated phosphatidylethanolamine in their membranes as compared with the other monkeys. Membrane phospholipid fatty acid profiles showed no differences among the three groups. There were also no differences in the animals' plasma liver enzymes. Results of this investigation suggest that, despite the absence of nutritional deficiencies and liver malfunction, low amounts (12%) of dietary ethanol cause elevations in phosphatidylethanolamine that may represent a specific change in the membrane's inner leaflet where this phospholipid is located. These results may have clinical significance because ethanol-induced modifications in membrane lipids may contribute to alterations in fluidity and lead to pathologic changes in function.