Membrane interactions of hemoglobin variants, HbA, HbE, HbF and globin subunits of HbA: Effects of aminophospholipids and cholesterol

The interaction of hemoglobin with phospholipid bilayer vesicles (liposomes) has been analyzed in several studies to better understand membrane-protein interactions. However, not much is known on hemoglobin interactions with the aminophospholipids, predominantly localized in the inner leaflet of erythrocytes, e.g., phosphatidylserine (PS), phosphatidylethanolamine (PE) in membranes containing phosphatidylcholine (PC). Effects of cholesterol, largely abundant in erythrocytes, have also not been studied in great details in earlier studies. This work therefore describes the study of the interactions of different hemoglobin variants HbA, HbE and HbF and the globin subunits of HbA with the two aminophospholipids in the presence and absence of cholesterol. Absorption measurements indicate preferential oxidative interaction of HbE and alpha-globin subunit with unilamellar vesicles containing PE and PS compared to normal HbA. Cholesterol was found to stabilize such oxidative interactions in membranes containing both the aminophospholipids. HbE and alpha-globin subunits were also found to induce greater leakage of membrane entrapped carboxyfluorescein (CF) using fluorescence measurements. HbE was found to induce fusion of membrane vesicles containing cholesterol and PE when observed under electron microscope. Taken together, these findings might be helpful in understanding the oxidative stress-related mechanism(s) involved in the premature destruction of erythrocytes in peripheral blood, implicated in the hemoglobin disorder, HbE/beta-thalassemia.     

Faster heme loss from hemoglobin E than HbS,in acidic pH: Effect of aminophospholipids

We report studies on loss of heme at or below pH 3.0 from two clinically important hemoglobin variants, HbE and HbS, in the presence and absence of phopholipid membranes. The kinetics of heme loss has been studied at pH 3.0 to simulate the same at a faster rate than at physiological pH, for spectroscopic investigation. Results obtained from the study clearly establish the probable fate of the lost heme to partition into the phospholipid bilayer independent of the pH range. This is also of particular importance to membranes containing the aminophospholipid and cholesterol which are predominantly localized in the inner leaflet of erythrocytes. Absorption measurements indicated such loss of heme when the Soret peak at 415 nm blue-shifted to 380 nm at pH 3.0. The extent of this blue shift decreased from 35 nm to ~15 nm in the presence of small unilammelar vesicles of both dimyristoyl- and dioleoyl-based phosphatidylcholine and phosphatidylethanolamine, indicating partitioning of the released heme in the membrane bilayer. The kinetics of heme loss was faster from HbE than HbA and HbS, obeying first-order reaction kinetics. Released heme could be involved in the premature destruction of erythrocytes in hemoglobin disorders.     

Differential Thermal Stability and Oxidative Vulnerability of the Hemoglobin Variants,HbA2 and HbE

Apart from few early biophysical studies, the relative thermal instability of HbE has been only shown by clinical investigations. We have compared in vitro thermal stability of HbE with HbA₂ and HbA using optical spectroscopy. From absorption measurements in the soret region, synchronous fluorescence spectroscopy and dynamic light scattering experiments, we have found thermal stability of the three hemoglobin variants following the order HbE<HbA<HbA₂ in terms of structural unfolding and aggregation pattern. We have found formation of intermolecular dityrosine fluorophores with characteristic fluorescence signature, at pH >11.0 in all the three variants. Under oxidative stress conditions in presence of hydrogen peroxide, HbE has been found to be more vulnerable to aggregation compared to HbA and HbA₂. Taken together, these studies have shown thermal and oxidative instability of HbE and points towards the role of HbE in the upregulation of redox regulators and chaperone proteins in erythrocyte proteome of patients suffering from HbEbeta thalassemia.     

15N chemically induced dynamic nuclear polarization during reaction of N-acetyl-L-tyrosine with the nitrating systems nitrite/hydrogen peroxide/horseradish peroxidase and nitrite/hypochloric acid

While the Maillard reaction of amino acids and proteins as well as its consequences in vivo has been thoroughly investigated, little attention has so far been paid to the glycation of aminophospholipids such as phosphatidylethanolamine (PE) or phosphatidylserine (PS), which are essential for structure and functionality of biological membranes. PE-derived glucosylamines (Schiff-PEs) and aminoketoses (Amadori-PEs) have now for the first time been simultaneously identified and quantified in erythrocytes from diabetics and healthy individuals by liquid chromatography-electrospray mass spectrometry (LC-(ESI)MS). The amounts of glycated PE (gPE) were significantly higher in diabetics (0.18-34.2 mol% Schiff-PE and 0.047-0.375 mol% Amadori-PE) than in controls (0.12-3.99 mol% Schiff-PE and 0.018-0.055 mol% Amadori-PE). A positive correlation between fructosylated hemoglobin (HbA(1c)) and the gPE levels was established. No advanced glycation endproducts (AGEs) like 5-hydroxymethylpyrrole-2-carbaldehyde (pyrrole-PE), carboxymethyl (CM-PE), or carboxyethyl (CE-PE) derivatives were detected. To investigate the influence of gPE on lipid peroxidation of biological membranes, liposomes consisting of soy-PE and synthetically prepared Amadori-PE (16:0-16:0) were incubated for several days and the formation of oxidation products was monitored. It could be shown that Amadori-PE extensively promotes lipid peroxidation even in the absence of transition metal ions like Cu(2+) and Fe(3+). Oxidative damage to membrane lipids therefore is supposed to be at least partially caused by the glycation of aminophospholipids.     

Lipid composition of subcellular particles from sheep platelets. Location of phosphatidylethanolamine and phosphatidylserine in plasma membranes and platelet liposomes

The lipid composition of whole sheep platelets and their subcellular fractions was determined. The basic lipids show similar distributions in granules, microsomes, plasma membranes and whole platelets. Phospholipid (about 70% of total lipids) and cholesterol (25% of total lipids) are the principal lipid components. Free cholesterol represents about 98% of the total, whereas cholesteryl ester is a minor component. The phospholipid composition found in intact platelets and their subcellular particles is about: 35% phosphatidylethanolamine (PE), 30% phosphatidylcholine (PC), 20% sphingomyelin and 15% phosphatidylserine (PS). We also investigated aminophospholipid topology in intact platelet plasma membranes and platelet liposomes by using the nonpenetrating chemical probe trinitrobenzenesulfonic acid (TNBS), because they are the major components of total lipids. In intact platelets, PS is not accessible to TNBS during the initial 15 min of incubation, whereas 18% PE is labelled after 15 min. In contrast, in phospholipid extracted from platelets 80% PE and 67% PS react with TNBS within 5 min, while 27 and 25% PE and 15 and 19% PS from liposomes and isolated plasma membranes, respectively, were modified after 15 min of incubation. In view of this chemical modification, it is concluded that 22% of PE and less than 1% of PS are located on the external surface of intact platelet plasma membranes. The asymmetric orientation of aminophospholipids is similar between liposomes and isolated plasma membrane. PS (23 and 28%) and PE (34 and 31%) are scarcely represented outside the bilayer. The data found are consistent with the nonrandom phospholipid distribution of blood cell surface membranes.     

Docking and fast fusion of synaptobrevin vesicles depends on the lipid compositions of the vesicle and the acceptor SNARE complex-containing target membrane

The influence of the lipid environment on docking and fusion of synaptobrevin 2 (Syb2) vesicles with target SNARE complex membranes was examined in a planar supported membrane fusion assay with high time-resolution. Previously, we showed that approximately eight SNARE complexes are required to fuse phosphatidylcholine (PC) and cholesterol model membranes in ∼20 ms. Here we present experiments, in which phosphatidylserine (PS) and phosphatidylethanolamine (PE) were added to mixtures of PC/cholesterol in different proportions in the Syb2 vesicle membranes only or in both the supported bilayers and the Syb2 vesicles. We found that PS and PE both reduce the probability of fusion and that this reduction is fully accounted for by the lipid composition in the vesicle membrane. However, the docking efficiency increases when the PE content in the vesicle (and target membrane) is increased from 0 to 30%. The fraction of fast-activating SNARE complexes decreases with increasing PE content. As few as three SNARE complexes are sufficient to support membrane fusion when at least 5% PS and 10% PE are present in both membranes or 5% and 30% PE are present in the vesicle membrane only. Despite the smaller number of required SNAREs, the SNARE activation and fusion rates are almost as fast as previously reported in reconstituted PC/cholesterol bilayers, i.e., of 10 and ∼20 ms, respectively.     

Spectrin interactions with globin chains in the presence of phosphate metabolites and hydrogen peroxide: implications for thalassaemia

We have shown the differential interactions of the erythroid skeletal protein spectrin with the globin subunits of adult haemoglobin (HbA); these indicate a preference for alpha-globin over that for beta-globin and intact HbA in an adenosine 5'-triphosphate (ATP)-dependent manner. The presence of Mg/ATP led to an appreciable decrease in the binding affinity of the alpha-globin chain to spectrin and the overall yield of globin-spectrin cross-linked complexes formed in the presence of hydrogen peroxide. Similar effects were also seen in the presence of 2-,3-diphosphoglycerate (2,3 DPG), the other important phosphate metabolite of erythrocytes. The binding affinity and yield of cross-linked high molecular weight complexes (HMWCs) formed under oxidative conditions were significantly higher in alpha-globin compared with intact haemoglobin, HbA and the beta-globin chain. The results of this study indicate a possible correlation of the preferential spectrin binding of the alpha-globin chain over that of the beta-globin in the haemoglobin disorder beta-thalassaemia.     

Aminophospholipid translocase in the plasma membrane of Friend erythroleukemic cells can induce an asymmetric topology for phosphatidylserine but not for phosphatidylethanolamine

The ATP-dependent translocation of phospholipids in the plasma membrane of intact Friend erythroleukemic cells (FELCs) was studied in comparison with that in the membrane of mature murine erythrocytes. This was done by following the fate of radiolabeled phospholipid molecules, previously inserted into the outer monolayer of the plasma membranes by using a non-specific lipid transfer protein. The transbilayer equilibration of these probe molecules was monitored by treating the cells--under essentially non-lytic conditions--with phospholipases A2 of different origin. Rapid reorientations of the newly introduced aminophospholipids in favour of the inner membrane leaflet were observed in fresh mouse erythrocytes; the inward translocation of phosphatidylcholine (PC) in this membrane proceeded relatively slow. In FELCs, on the other hand, all three glycerophospholipids equilibrated over both halves of the plasma membrane very rapidly, i.e. within 1 h; nevertheless, an asymmetric distribution in favour of the inner monolayer was only observed for phosphatidylserine (PS). Lowering the ATP-level in the FELCs caused a reduction in the rate of inward translocation of both aminophospholipids, but not of that of PC, indicating that this translocation of PS and phosphatidylethanolamine (PE) is clearly ATP-dependent. Hence, the situation in the plasma membrane of the FELC is rather unique in a sense that, though an ATP-dependent translocase is present and active both for PS and PE, its activity results in an asymmetric distribution of PS, but not of PE. This remarkable situation might be the consequence of the fact that, in contrast to the mature red cell, this precursor cell still lacks a complete membrane skeletal network.     

Effects of cholesterol on the divalent cation-mediated interactions of vesicles containing amino and choline phospholipids

We have used assays of lipid probe mixing, contents mixing and contents leakage to monitor the divalent cation-mediated interactions between lipid vesicles containing phosphatidylserine (PS) as a minority component together with mixtures of phosphatidylethanolamine (PE), phosphatidylcholine (PC) or sphingomyelin, and cholesterol in varying proportions. The initial rates of calcium- and magnesium-induced lipid probe quenching between vesicles, which reflect primarily the rates of vesicle aggregation, are strongly reduced as progressively higher proportions of PC or sphingomyelin are incorporated into PE/PS vesicles. The initial rates of divalent cation-induced contents mixing and contents leakage for PE/PS vesicles are also strongly reduced when choline phospholipids are incorporated into the vesicles in even low molar proportions. Sphingomyelin has a more potent inhibitory effect on these processes than does PC at an equal level in the vesicle membranes. The inclusion of cholesterol in these vesicles, at levels up to 1:2 moles sterol/mole phospholipid, has little effect on the rates of calcium- or magnesium-induced vesicle aggregation. However, cholesterol significantly enhances the initial rates of vesicle contents mixing and contents leakage in the presence of divalent cations when the vesicles contain choline as well as amino phospholipids. This effect is substantial only when the level of cholesterol exceeds the level of choline phospholipids in the vesicles. These results may have significance for the fusion of certain cellular membranes in mammalian cells, whose cytoplasmic faces have lipid compositions very similar to those of the vesicles examined in this study.     

Chaperone potential of erythroid spectrin: Effects of hemoglobin interaction,macromolecular crowders,phosphorylation and glycation

Spectrin, the major protein component of the erythrocyte membrane skeleton has chaperone like activity and is known to bind membrane phospholipids and hemoglobin. We have probed the chaperone activity of spectrin in presence of hemoglobin and phospholipid SUVs of different compositions to elucidate the effect of phospholipid/hemoglobin binding on chaperone function. It is seen that spectrin displays a preference for hemoglobin over other substrates leading to a decrease in chaperone activity in presence of hemoglobin. A competition is seen to exist between phospholipid binding and chaperone function of spectrin, in a dose dependent manner with the greatest extent of decrease being seen in case of phospholipid vesicles containing aminophospholipids e.g. PS and PE which may have implications in diseases like hereditary spherocytosis where mutation in spectrin is implicated in its detachment from cell membrane. To gain a clearer understanding of the chaperone like activity of spectrin under in-vivo like conditions we have investigated the effect of macromolecular crowders as well as phosphorylation and glycation states on chaperone activity. It is seen that the presence of non-specific, protein and non-protein macromolecular crowders do not appreciably affect chaperone function. Phosphorylation also does not affect the chaperone function unlike glycation which progressively diminishes chaperone activity. We propose a model where chaperone clients adsorb onto spectrin's surface and processes that bind to and occlude these surfaces decrease chaperone activity.     

Crystal structures of HbA2 and HbE and modeling of hemoglobin delta 4: interpretation of the thermal stability and the antisickling effect of HbA2 and identification of the ferrocyanide binding site in Hb

Hemoglobin A(2) (alpha(2)delta(2)) is an important hemoglobin variant which is a minor component (2-3%) in the circulating red blood cells, and its elevated concentration in beta-thalassemia is a useful clinical diagnostic. In beta-thalassemia major, where there is beta-chain production failure, HbA(2) acts as the predominant oxygen deliverer. HbA(2) has two more important features. (1) It is more resistant to thermal denaturation than HbA, and (2) it inhibits the polymerization of deoxy sickle hemoglobin (HbS). Hemoglobin E (E26K(beta)), formed as a result of the splice site mutation on exon 1 of the beta-globin gene, is another important hemoglobin variant which is known to be unstable at high temperatures. Both heterozygous HbE (HbAE) and homozygous HbE (HbEE) are benign disorders, but when HbE combines with beta-thalassemia, it causes E/beta-thalassemia which has severe clinical consequences. In this paper, we present the crystal structures of HbA(2) and HbE at 2.20 and 1.74 A resolution, respectively, in their R2 states, which have been used here to provide the probable explanations of the thermal stability and instability of HbA(2) and HbE. Using the coordinates of R2 state HbA(2), we modeled the structure of T state HbA(2) which allowed us to address the structural basis of the antisickling property of HbA(2). Using the coordinates of the delta-chain of HbA(2) (R2 state), we also modeled the structure of hemoglobin homotetramer delta(4) that occurs in the case of rare HbH disease. From the differences in intersubunit contacts among beta(4), gamma(4), and delta(4), we formed a hypothesis regarding the possible tetramerization pathway of delta(4). The crystal structure of a ferrocyanide-bound HbA(2) at 1.88 A resolution is also presented here, which throws light on the location and the mode of binding of ferrocyanide anion with hemoglobin, predominantly using the residues involved in DPG binding. The pH dependence of ferrocyanide binding with hemoglobin has also been investigated.     

Lipid organization in erythrocyte membrane microvesicles.

The aminophospholipids of microvesicles released from human erythrocytes on storage or prepared from erythrocyte ghosts by shearing under pressure are susceptible to the action of 2,4,6-trinitrobenzenesulphonic acid. The aminophospholipids of the former vesicles are also susceptible to attack by phospholipase A2. Under the same conditions, the aminophospholipids of erythrocytes undergo little reaction. This suggests that the phospholipids in microvesicle membranes are more randomly distributed than those in erythrocyte membranes. Measurements have also been made of the ability of filipin to react with the cholesterol of sealed and unsealed erythrocyte ghosts and of microvesicles prepared from them. From the initial rates of reaction, it was concluded that there is no preferential transfer of cholesterol molecules from one side of the bilayer to the other during the formation of the microvesicles.     

Rearrangement of aminophospholipids in bilayers from sheep platelet plasma membranes and platelet liposomes by increasing their cholesterol levels

Phospholipid orientation in platelet plasma membranes and other blood cells, such as erythrocytes, appears to be rather similar. The negatively charged phospholipids are almost exclusively located on the inner leaflet of the bilayer. No phosphatidylserine is present on the outer membrane bilayer. The results of the present study, using a specific reagent for amino groups, trinitrobenzenesulfanilic acid, showed that in sheep platelet plasma membranes enriched with free exogenous cholesterol, an alteration in the aminophospholipid topology occurs, with a portion of phosphatidylserine moving from the inner to the outer side. A progressive appearance of aminophospholipids in the outer membrane bilayer was also observed in artificial vesicles prepared with total lipids from sheep platelets supplemented with increased amounts of free cholesterol.     

Kinetics of cholesterol and phospholipid exchange from membranes containing cross-linked proteins or cross-linked phosphatidylethanolamines

Mono- and dipalmitoylphosphatidylethanolamine derivatives have been synthesized and used to evaluate the role of cross-links between the amino groups of two phospholipid molecules in the rate of cholesterol movement between membranes. Incorporation of the cross-linked phospholipids into small unilamellar vesicles (the donor species) decreased the rate of spontaneous cholesterol exchange with acceptor membranes (small unilamellar vesicles or Mycoplasma gallisepticum cells). These results suggest that the cross-linking of aminophospholipids by reactive intermediates, which may be one of the degenerative transformations associated with peroxidation of unsaturated lipids and cellular aging, can inhibit cholesterol exchangeability in biological membranes. The rates of spontaneous [14C]cholesterol and protein-mediated 14C-labeled phospholipid exchange from diamide-treated mycoplasma and erythrocyte membranes have also been measured. The formation of extensive disulfide bonds in the membrane proteins of M. gallisepticum enhanced the 14C-labeled phospholipid exchange rate but did not affect the rate of [14C]cholesterol exchange. The rates of radiolabeled cholesterol and phospholipid exchange between erythrocyte ghosts and vesicles were both enhanced (but to different extents) when ghosts were treated with diamide. These observations suggest that diamide-induced oxidative cross-linking of sulfhydryl groups in membrane proteins does not lead to random defects in the lipid domain.     

Ethanol specifically alters the synthesis, acylation and transbilayer movement of aminophospholipids in rat-liver microsomes

By experimenting with the aminoalcohols [3-3H]serine and [2-14C]ethanolamine we have been able to relate the effects of ethanol upon the biosynthesis of radioactive aminophospholipids (APL) in rat-liver microsomes and their distribution within the bilayer. The translocation of newly synthesized molecules of aminophospholipids labeled with different fatty acids was also investigated. The synthesis of phosphatidylserine (PS) and phosphatidylethanolamine (PE) by base-exchange reaction (BES) was inhibited in membranes exposed to ethanol in direct response to its concentration. In addition, 100 mM ethanol specifically inhibited the transport of newly synthesized PS to the inner leaflet, resulting in similar levels of PS in both leaflets of the bilayer. The inhibition of PE synthesis by ethanol caused a decrease in its distribution in both inner and outer leaflets. An in vitro study of the incorporation of radioactive palmitate and oleate into the PS and PE of microsomes incubated with ethanol showed a decrease in the radioactivity levels of PE, suggesting that ethanol was specifically inhibiting the corresponding acyltransferase. It specifically altered the transbilayer movement of newly acylated phospholipids, modifying the distribution of palmitoyl- and oleoyl-acylated PS and PE in both leaflets. These results demonstrate for the first time that ethanol interferes with both the synthesis and intramembrane transport of aminophospholipids in endoplasmic reticulum (ER) membranes. Bearing in mind that if a membrane is to function properly its structure must be in optimum condition; it is evident that the observed processes may be responsible to some degree for the pathophysiological effects of alcohol upon cells.     

Capacitation induces cyclic adenosine 3',5'-monophosphate-dependent,but apoptosis-unrelated,exposure of aminophospholipids at the apical head plasma membrane of boar sperm cells

The capacitating agent bicarbonate/CO(2) has been shown to induce profound changes in the architecture and dynamics within the sperm's plasma membrane lipid bilayer via a cAMP-dependent protein phosphorylation signaling pathway. Here we have investigated the effect of bicarbonate on surface exposure of endogenous aminophospholipids in boar spermatozoa, detecting phosphatidylserine (PS) with fluorescein-conjugated annexin V and phosphatidylethanolamine (PE) with fluorescein-conjugated streptavidin/biotinylated Ro-09-0198. Flow cytometric analyses revealed that incubation with 15 mM bicarbonate induced 30%-70% of live acrosome-intact cells to expose PE very rapidly; this exposure was closely related to a decrease in lipid packing order as detected by enhanced binding of merocyanine 540. PS exposure was detectable in the same proportion of cells, though its expression was slower. Confocal microscopy revealed that exposure of aminophospholipids in intact cells was restricted to the anterior acrosomal region of the head plasma membrane. Aminophospholipid exposure, merocyanine stainability, and a subsequent migration of cholesterol to the apical region of the head plasma membrane, were all under the control of the cAMP-dependent protein phosphorylation pathway. The close coupling of decreased lipid packing order with exposure of PE led us to conclude that bicarbonate was inducing phospholipid scrambling (i.e., collapse of asymmetric transverse distribution), and that the scrambling was a prerequisite for cholesterol relocation. There was no evidence whatever that the bicarbonate-induced scrambling was an apoptotic process. It was not accompanied by major loss of viability or by DNA degeneration or by loss of mitochondrial function, and it could not be blocked by the broad-specificity caspase inhibitors zVAD-fmk and BocD-fmk. In the absence of bicarbonate, scrambling could not be induced by the apoptotic agents UV, staurosporine, or cycloheximide. Bicarbonate-induced phospholipid scrambling thus appears to be an important and early physiological event in the capacitation process.     

Cholesterol affects divalent cation-induced fusion and isothermal phase transitions of phospholipid membranes

The influence of cholesterol on divalent cation-induced fusion and isothermal phase transitions of large unilamellar vesicles composed of phosphatidylserine (PS) was investigated. Vesicle fusion was monitored by the terbium/dipicolinic acid assay for the intermixing of internal aqueous contents, in the temperature range 10-40 degrees C. The fusogenic activity of the cations decreases in the sequence Ca2+ greater than Ba2+ greater than Sr2+ much greater than Mg2+ for cholesterol concentrations in the range 20-40 mol%, and at all temperatures. Increasing the cholesterol concentration decreases the initial rate of fusion in the presence of Ca2+ and Ba2+ at 25 degrees C, reaching about 50% of the rate for pure PS at a mole fraction of 0.4. From 10 to 25 degrees C, Mg2+ is ineffective in causing fusion at all cholesterol concentrations. However, at 30 degrees C, Mg2+-induced fusion is observed with vesicles containing cholesterol. At 40 degrees C, Mg2+ induces slow fusion of pure PS vesicles, which is enhanced by the presence of cholesterol. Increasing the temperature also causes a monotonic increase in the rate of fusion induced by Ca2+, Ba2+ and Sr2+. The enhancement of the effect of cholesterol at high temperatures suggests that changes in hydrogen bonding and interbilayer hydration forces may be involved in the modulation of fusion by cholesterol. The phase behavior of PS/cholesterol membranes in the presence of Na+ and divalent cations was studied by differential scanning calorimetry. The temperature of the gel-liquid crystalline transition (Tm) in Na+ is lowered as the cholesterol content is increased, and the endotherm is broadened. Addition of divalent cations shifts the Tm upward, with a sequence of effectiveness Ba2+ greater than Sr2+ greater than Mg2+. The Tm of these complexes decreases as the cholesterol content is increased. Although the transition is not detectable for cholesterol concentrations of 40 and 50 mol% in the presence of Na+, Sr2+ or Mg2+, the addition of Ba2+ reveals endotherms with Tm progressively lower than that observed at 30 mol%. Although the presence of cholesterol appears to induce an isothermal gel-liquid crystalline transition by decreasing the Tm, this change in membrane fluidity does not enhance the rate of fusion, but rather decreases it. The effect of cholesterol on the fusion of PS/phosphatidylethanolamine (PE) vesicles was investigated by utilizing a resonance energy transfer assay for lipid mixing. The initial rate of fusion of PS/PE and PS/PE/cholesterol vesicles is saturated at high Mg2+ concentrations. With Ca2+, saturation is not observed for cholesterol-containing vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fusion of liposomes containing a novel cationic lipid, N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium: induction by multivalent anions and asymmetric fusion with acidic phospholipid vesicles

The fusion behavior of large unilamellar liposomes composed of N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA) and either phosphatidylcholine (PC) or phosphatidylethanolamine (PE) has been investigated by a fluorescence resonance energy transfer assay for lipid mixing, dynamic light scattering, and electron microscopy. Polyvalent anions induced the fusion of DOTMA/PE (1:1) liposomes with the following sequence of effectiveness: citrate greater than EDTA greater than phosphate, in the presence 100 mM NaCl, pH 7.4. Sulfate, dipicolinate, and acetate were ineffective. DOTMA/PC (1:1) vesicles were completely refractory to fusion in the presence of multivalent anions in the concentration range studied, consistent with the inhibitory effect of PC in divalent cation induced fusion of negatively charged vesicles. DOTMA/PE vesicles could fuse with DOTMA/PC vesicles in the presence of high concentrations of citrate, but not of phosphate. Mixing of DOTMA/PE liposomes with negatively charged phosphatidylserine (PS)/PE or PS/PC (1:1) vesicles resulted in membrane fusion in the absence of multivalent anions. DOTMA/PC liposomes also fused with PS/PE liposomes and, to a limited extent, with PS/PC liposomes. These observations suggest that the interaction of the negatively charged PS polar group with the positively charged trimethylammonium of DOTMA is sufficient to mediate fusion between the two membranes containing these lipids and that the nature of the zwitterionic phospholipid component of these vesicles is an additional determinant of membrane fusion.     

Formation and function of phosphatidylserine and phosphatidylethanolamine in mammalian cells

Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are metabolically related membrane aminophospholipids. In mammalian cells, PS is required for targeting and function of several intracellular signaling proteins. Moreover, PS is asymmetrically distributed in the plasma membrane. Although PS is highly enriched in the cytoplasmic leaflet of plasma membranes, PS exposure on the cell surface initiates blood clotting and removal of apoptotic cells. PS is synthesized in mammalian cells by two distinct PS synthases that exchange serine for choline or ethanolamine in phosphatidylcholine (PC) or PE, respectively. Targeted disruption of each PS synthase individually in mice demonstrated that neither enzyme is required for viability whereas elimination of both synthases was embryonic lethal. Thus, mammalian cells require a threshold amount of PS. PE is synthesized in mammalian cells by four different pathways, the quantitatively most important of which are the CDP-ethanolamine pathway that produces PE in the ER, and PS decarboxylation that occurs in mitochondria. PS is made in ER membranes and is imported into mitochondria for decarboxylation to PE via a domain of the ER [mitochondria-associated membranes (MAM)] that transiently associates with mitochondria. Elimination of PS decarboxylase in mice caused mitochondrial defects and embryonic lethality. Global elimination of the CDP-ethanolamine pathway was also incompatible with mouse survival. Thus, PE made by each of these pathways has independent and necessary functions. In mammals PE is a substrate for methylation to PC in the liver, a substrate for anandamide synthesis, and supplies ethanolamine for glycosylphosphatidylinositol anchors of cell-surface signaling proteins. Thus, PS and PE participate in many previously unanticipated facets of mammalian cell biology. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

The curvature and cholesterol content of phospholipid bilayers alter the transbilayer distribution of specific molecular species of phosphatidylethanolamine

The curvature, cholesterol content, and transbilayer distribution of phospholipids significantly influence the functional properties of cellular membranes, yet little is known of how these parameters interact. In this study, the transbilayer distribution of phosphatidylethanolamine (PE) is determined in vesicles with large (98 nm) and small (19 nm) radii of curvature and with different proportions of PE, phosphatidylcholine, and cholesterol. It was found that the mean diameters of both types of vesicles were not influenced by their lipid composition, and that the amino-reactive compound 2,4,6-trinitrobenzenesulphonic acid (TNBS) was unable to cross the bilayer of either type of vesicle. When large vesicles were treated with TNBS, approximately 40% of the total membrane PE was derivatized; in the small vesicles 55% reacted. These values are interpreted as representing the percentage of total membrane PE residing in the outer leaflet of the vesicle bilayer. The large vesicles likely contained approximately 20% of the total membrane lipid as internal membranes. Therefore, in both types of vesicles, PE as a phospholipid class was randomly distributed between the inner and outer leaflets of the bilayer. The proportion of total PE residing in the outer leaflet was unaffected by changes in either the cholesterol or PE content of the vesicles. However, the transbilayer distributions of individual molecular species of PE were not random, and were significantly influenced by radius of curvature, membrane cholesterol content, or both. For example, palmitate- and docosahexaenoate-containing species of PE were preferentially located in the outer leaflet of the bilayer. Membrane cholesterol content affected the transbilayer distributions of stearate-, oleate-, and linoleate-containing species. The transbilayer distributions of palmitate-, docosahexaenoate-, and stearate-containing species were significantly influenced by membrane curvature, but only in the presence of high levels of cholesterol. Thus, differences in membrane curvature and cholesterol content alter the array of PE molecules present on the surfaces of phospholipid bilayers. In cells and organelles, these differences could have profound effects on a number of critical membrane functions and processes.