Cholesterol regulates membrane binding and aggregation by annexin 2 at submicromolar Ca2+ concentration

Annexin 2 is a member of the annexin family which has been implicated in calcium-regulated exocytosis. This contention is largely based on Ca²⁺-dependent binding of the protein to anionic phospholipids. However, annexin 2 was shown to be associated with chromaffin granules in the presence of EGTA. A fraction of this bound annexin 2 was released by methyl-β-cyclodextrin, a reagent which depletes cholesterol from membranes. Restoration of the cholesterol content of chromaffin granule membranes with cholesterol/methyl-β-cyclodextrin complexes restored the Ca²⁺-independent binding of annexin 2. The binding of both, monomeric and tetrameric forms of annexin 2 was also tested on liposomes of different composition. In the absence of Ca²⁺, annexin 2, especially in its tetrameric form, bound to liposomes containing phosphatidylserine, and the addition of cholesterol to these liposomes increased the binding. Consistent with this observation, liposomes containing phosphatidylserine and cholesterol were aggregated by the tetrameric form of annexin 2 at submicromolar Ca²⁺ concentrations. These results indicate that the lipid composition of membranes, and especially their cholesterol content, is important in the control of the subcellular localization of annexin 2 in resting cells, at low Ca²⁺ concentration. Annexin 2 might be associated with membrane domains enriched in phosphatidylserine and cholesterol.     

The relationship between plasma membrane lipid composition and physical-chemical properties. III. Detailed physical and biochemical analysis of fatty acid-substituted EL4 plasma membranes

Murine leukemia EL4 cells were modified by supplementation of culture media with fatty acids for 24 h. A plasma membrane-enriched fraction was prepared from substituted and normal cells. Analyses were performed to determine fatty acyl composition, phospholipid headgroup composition and cholesterol content. The two major membrane phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were isolated by thin-layer chromatography and ESR measurements were done on liposomes prepared from these lipids as well as on the intact plasma membrane preparations. Slight perturbations in overall plasma membrane lipid composition were observed when EL4 cells were supplemented with a single exogenous fatty acid. This may be consistent with the idea that the incorporation of exogenous fatty acid induces compensatory changes in membrane lipid composition. On the other hand, we observed no significant difference in two ESR motional parameters between the unsubstituted control and various fatty acid-substituted plasma membranes. ESR measurements carried out on PE and PC liposomes derived from 17:0- and 18:2c-substituted membranes also failed to detect major differences between these liposomes and those made from normal EL4 phospholipids. In the case of liposomes prepared from 18:2t,-substituted membranes, the order parameter was significantly changed from the normal. However, the change was in opposite directions in PE and PC, perhaps accounting for the fact that no change parameter is seen in intact 18:2t-substituted plasma membrane. Measurements of order parameter (S) in mixed lipid vesicles showed that at up to 50 mol% mixture of a synthetic PC with plasma membrane PC, the value of S was only marginally different from that of the plasma membrane PC vesicles. We interpret these data as an indication that the two ESR parameters used are not sufficiently sensitive to detect changes due to modifications of the acyl chain composition of a complex biological membrane.     

Lipid peroxidation in hemoglobin-containing liposomes. Effects of membrane phospholipid composition and cholesterol content

The effects of phospholipid-oxidation state and vesicle composition on lipid peroxidation in hemolysate-containing liposomes (hemosomes) were studied by the thiobarbituric acid assay. Liposomes (hemosomes) were prepared from egg phosphatidylcholine (PC) with either low (PC0.08) or high (PC0.66) oxidation indices reflecting low and high conjugated diene/lipid hydroperoxy contents. Thiobarbituric acid reactivity was negligible over 6 h at 38 degrees C in buffer-containing (control) liposomes prepared from PC0.08, whereas it was slightly increased in those prepared from PC0.66. Encapsulated hemolysate had no effect in PC0.08 liposomes, but significantly increased thiobarbituric acid reactivity in those prepared from PC0.66. Inclusion of either phosphatidylethanolamine or phosphatidylinositol in the membrane further increased lipid peroxidation in hemosomes prepared from PC0.66, whereas phosphatidic acid and phosphatidylserine were inhibitory. Inclusion of cholesterol in the membrane had no effect in PC0.66 hemosomes, but significantly inhibited lipid peroxidation in the presence of phosphatidylethanolamine or phosphatidylinositol. The effects of phosphatidic acid and cholesterol were dose-dependent. Co-incorporation of cholesterol and phosphatidic acid or phosphatidylserine in the membrane resulted in almost complete elimination of hemoglobin (Hb)-induced lipid peroxidation. Lysophosphatidic acid had similar effect as phosphatidic acid, whereas lysophosphatidylserine exerted inhibition only in the presence of phosphatidylethanolamine. The rate of lipid peroxidation showed no correlation with the amount of encapsulated Hb, neither with the oxidation indices nor the polyunsaturated fatty acid contents of negatively charged phospholipids. The above findings suggest a possible role for the high cholesterol content and preferential localization of phosphatidylserine in the inner bilayer leaflet of erythrocyte membrane in protecting against Hb-induced lipid peroxidation in the membrane.     

Hyperalphalipoproteinemic scavenger receptor BI knockout mice exhibit a disrupted epidermal lipid barrier

Scavenger receptor class B type I (SR-BI) mediates the selective uptake of cholesteryl esters (CE) from high-density lipoproteins (HDL). An impaired SR-BI function leads to hyperalphalipoproteinemia with elevated levels of cholesterol transported in the HDL fraction. Accumulation of cholesterol in apolipoprotein B (apoB)-containing lipoproteins has been shown to alter skin lipid composition and barrier function in mice. To investigate whether these hypercholesterolemic effects on the skin also occur in hyperalphalipoproteinemia, we compared skins of wild-type and SR-BI knockout (SR-BI^−/−) mice. SR-BI deficiency did not affect the epidermal cholesterol content and induced only minor changes in the ceramide subclasses. The epidermal free fatty acid (FFA) pool was, however, enriched in short and unsaturated chains. Plasma CE levels strongly correlated with epidermal FFA C18:1 content. The increase in epidermal FFA coincided with downregulation of cholesterol and FFA synthesis genes, suggesting a compensatory response to increased flux of plasma cholesterol and FFAs into the skin. Importantly, the SR-BI^−/− epidermal lipid barrier showed increased permeability to ethyl-paraminobenzoic acid, indicating an impairment of the barrier function. In conclusion, increased HDL-cholesterol levels in SR-BI^−/− mice can alter the epidermal lipid composition and lipid barrier function similarly as observed in hypercholesterolemia due to elevated levels of apoB-containing lipoproteins.     

Selective toxin-lipid membrane interactions of natural, haemolytic Scyphozoan toxins analyzed by surface plasmon resonance

A comparison of the molecular interaction of natural Scyphozoan lysins with their bioactivity in a haemolytic assay was performed by establishing an efficient, automatable and reproducible procedure for the measurement of protein-membrane interactions. The toxin-membrane interactions were analyzed utilising a chip-based technology with immobilized liposomes as artificial cell membranes. The technique was established with streptolysin O as a cholesterol-selective model toxin and its cholesterol-selectivity has been proven. The haemolytic potency of protein fractions derived from the venom of the jellyfish Aurelia aurita and Cyanea capillata was tested and EC50 values of 35.3 μg/mL and 43.1 μg/mL against sheep and 13.5 μg/mL and 8.8 μg/mL against rabbit erythrocytes were measured. Cell membrane binding as a first step in the haemolytic process was analyzed using the Biacore^® technology. Major cell membrane lipids (cholesterol, sphingomyelin and phosphatidylcholine) were immobilized as pure liposomes and in binary mixtures. A preference for cholesterol and sphingomyelin of both jellyfish species was demonstrated. The specificity of the method was proven with a non-haemolytic A. aurita protein fraction that did not express a lipid binding. Additionally, an inactivated C. capillata lysine with negligible haemolytic activity showed a remaining but reduced adsorption onto lipid layers. The binding level of the lytic venom fraction of these dominant boreal jellyfish species increased as a function of protein concentration. The binding strength was expressed in RU50 values ranging from 12.4 μg/mL to 35.4 μg/mL, which were in the same order of magnitude as the EC50 values in the haemolytic assay.     

Lateral mobility and organization of phospholipids and proteins in rat myocyte membranes. Effects of aging and manipulation of lipid composition

The effects of aging and of liposome treatment on the lateral mobility of phospholipids and proteins in the plasma membrane of cultured rat heart myocytes were studied by fluorescence photobleaching recovery. Both the mobile fraction (R) and the lateral diffusion coefficient (D) of the fluorescent phospholipid N-4-nitrobenzo-2-oxa-1,3-diazolyl phosphatidylethanolamine were found to depend on the culture's age. Aged myocyte cultures (15 days old) demonstrated higher R and lower D as compared with young ones (5 days old). Treatment of aged cultures with phosphatidylcholine (PC) liposomes, which increases the PC/sphingomyelin (SM) ratio and decreases the cholesterol level, reversed the D value to the level observed in young cultures and decreased R below the value encountered in young cells. Treatments with SM liposomes (which induce cholesterol depletion without altering the PC/SM ratio) and with PC/cholesterol (1:0.9) liposomes (which increase the PC/SM ratio without cholesterol depletion) have indicated that the PC-liposome effect is due to changes in both the PC/SM ratio and in the cholesterol level. Analogous experiments on the mobility of succinyl-concanavalin A receptors yielded similar effects on R, without altering the D value. The changes in the D and R values of the markers studied are most likely initiated by the observed alterations in the myocyte lipid composition under the conditions employed. The possible involvement of changes in the organization of membrane lipids in domains in the observed phenomena is discussed.     

Modulation of vitronectin receptor binding by membrane lipid composition.

The vitronectin (Vn) receptor belongs to the integrin family of proteins and although its biochemical structure is fully characterized little is known about its binding affinity and specificity. We report here that Vn receptor binding to different matrix proteins is influenced by the surrounding lipid composition of the membrane. Human placenta affinity purified Vn receptor was inserted into liposomes of different composition: (i) phosphatidylcholine (PC); (ii) PC+phosphatidylethanolamine (PE); (iii) PC+PE+phosphatidylserine (PS) + phosphatidylinositol (PI) + cholesterol (chol). The amount of purified material that could be incorporated into the three lipid vesicle preparations was proportional to the efficiency of the vesicle formation that increased from PC (38%) to PC+PE and PC+PE+PS+PI+chol (about 50%) vesicles. Electron microscopy analysis showed that the homogeneity and size of the three liposome preparations were comparable (20-nm diameter) but their binding capacity to a series of substrates differed widely. Vn receptor inserted in PC liposomes bound only Vn, but when it was inserted in PC+PE and PC+PE+PS+PI+chol liposomes it also attached to von Willebrand factor (vWF) and fibronectin (Fn). Vn receptor had higher binding capacity for substrates when it was inserted in PC+PE+PS+PI+chol than PC+PE liposomes. Antibodies to Vn receptor blocked Vn receptor liposome binding to Vn, vWF, and Fn. The intrinsic emission fluorescence spectrum of the Vn receptor reconstituted in PC+PE+PS+PI+chol liposomes was blue-shifted in relation to PC liposomes, suggesting a conformational change of the receptor in the membranes. These data provide direct evidence that the Vn receptor is "promiscuous" and can associate with Vn, vWF and Fn. The nature of the membrane lipid composition surrounding the receptor could thus influence its binding affinity, possibly by changing its conformation or exposure or both.     

The thermodynamic parameters of the interaction of concanavalin A with glycosyl-free liposomes: a microcalorimetric study

The nonspecific interaction of the mitogenic lectin Concanavalin A (Con A) with glycosyl-free liposomes of various composition has been investigated by microcalorimetric titration measurements. The results obtained show the following features of main interest: (1) the affinity constants (K_a) of the interaction of Con A with liposomal bilayers are in the order of magnitude 10⁵–10⁶M^?1. The reaction enthalpies (ΔH) are positive, and small (approximately 0.1 KJ mol^?1 lipid), compared to the free energy terms (?ΔG = 30–40 KJ mol^?1 lipid). All lectin–lipid interactions are strongly entropy-controlled (ΔH/TΔS < 1.0). These thermodynamic features are characteristic for hydrophobic interaction processes. (2) The liposomal head-group charge does not significantly affect the lipid-affinity of Con A. Electrostatic forces thus appear to play a minor role in lectin–lipid interactions. (3) The lipid affinity of Con A is sensitive to the fluidity of the liposomal bilayers, increasing with increasing fluidity. Below the gel to liquid-crystal phase transition temperature, the lectin binding to liposomal bilayers is inhibited. (4) The binding isotherms, corresponding to the interaction of Con A with liposomes, composed of tightly packed, saturated phospholipids, exhibit pronounced positive cooperativity. This phenomenon is absent in the binding curves, corresponding to the interaction of Con A with more fluid liposomal bilayers. (5) The Con A specific inhibitor α-D -methylmannopyranoside (50 mM) drastically increases the molar reaction enthalpy. The K_a term is significantly reduced in presence of the inhibitor sugar. Urea induces analogous changes in the thermodynamic parameters of the lectin–lipid interaction. The effects of α-D -methylmannopyranoside are thus not Con A specific, but are attributable to solvent effects. (6) It was shown that the binding of one Con A molecule affects a large number (approximately 1000) of phospholipid molecules in the liposomal bilayer. (7) The affinity constants (K_a) of the interaction of Con A with glycosyl-free lipids are smaller by a factor of approximately 10, compared to the K_a terms, reported for Con A binding to biological membranes. The presence of glycosidic receptor groups thus controls the specificity of lectin–membrane interactions, whereas the nonspecific lectin–lipid interactions appear to represent the main driving force for the strong attachment of the lectin to membrane surfaces.     

Lipid Binding to Amyloid β-Peptide Aggregates: Preferential Binding of Cholesterol as Compared with Phosphatidylcholine and Fatty Acids

Abstract: Amyloid β-peptide (Aβ) aggregates are one of the key neuropathological characteristics of Alzheimer's disease. Aβ belongs to a group of proteins that aggregate and form β-sheets, and some of these proteins bind cholesterol and other lipids. The purpose of the experiments reported here was to determine if cholesterol, fatty acids, and phosphatidylcholine (PC) would bind to Aβ_1–40 and if such binding would be dependent on aggregation of Aβ_1–40. Lipid binding was determined using fluorescent-labeled lipids. Incubation of Aβ_1–40 for 0, 1, 3, 6, 21, and 24 h resulted in aggregation of the peptide with formation of dimers, trimers (1–24 h), and polymers (6–24 h) as determined by sodium dodecyl sulfate-gel electrophoresis. No change in the fluorescence of the lipids was observed when lipids were added to Aβ_1–40 that had been incubated for 0, 1, or 3 h. However, the fluorescence intensities of cholesterol, saturated fatty acids, and PC were significantly increased (p < 0.0001) when added to Aβ_1–40 that had been incubated for 6, 21, and 24 h in which Aβ_1–40 polymers were detected. The binding affinity of cholesterol to Aβ_1–40 polymers (K_D of 3.24 ± 0.315 × 10^?9M) was markedly higher as compared with the other lipids (stearic acid, 9.42 ± 0.41 × 10^?8M; PC, 7.07 ± 0.12 × 10^?7M). The results of this study indicate that Aβ_1–40 polymers bind lipids and have a higher affinity for cholesterol than PC or saturated fatty acids. Aggregated Aβ_1–40 may affect lipid transport between cells or remove specific lipids from membranes, and such effects could contribute to neuronal dysfunction.     

Effects of changed lipid composition on responses of liposomes to various odorants: possible mechanism of odor discrimination

In a previous paper [Nomura, T., & Kurihara, K. (1987) Biochemistry (preceding paper in this issue)], we showed that azolectin liposomes are depolarized by various odorants and there is a good correlation between the responses in the liposomes and the frog or porcine olfactory responses. In this study, we examined effects of changed lipid composition on responses of liposomes to various odorants. The membrane potential changes in response to odorants were monitored with the fluorescent dye 3,3'-dipropylthiocarbocyanine iodide [diS-C3(5)]. Egg phosphatidylcholine (PC) liposomes showed depolarizing responses to nine odorants among ten odorants tested. The magnitudes of depolarization by alcohols were similar to those in azolectin liposomes, but those by other odorants were much less than those in azolectin liposomes. Addition of sphingomyelin (SM) to PC led to an increase in the magnitude of depolarization by most odorants. Addition of phosphatidylethanolamine (PE) to PC (PE/PC = 0.25) led to depolarizing responses to four odorants among six odorants tested, and a further increase in PE content (PE/PC = 0.54) led to depolarizing responses only to two odorants. Addition of SM to the lipids of this composition of PC and PE [SM/(PC + PE) = 0.22] led to depolarizing responses to four odorants again. Liposomes made of a mixture of SM, PE, and PC exhibited depolarizing responses to four odorants tested, and addition of cholesterol to the lipids [cholesterol/(PC + PE + SM) = 0.05 and 0.11] led to depolarizing responses only to two and one odorant, respectively. Thus, changes in lipid composition of liposomes led to great changes in specificity of the responses to odorants.(ABSTRACT TRUNCATED AT 250 WORDS)     

A spectroscopic study of the membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39)

The membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39), which selectively activates the parathyroid hormone 2 (PTH2) receptor (PTH2-R), has been studied by fluorescence and NMR spectroscopic techniques. Membrane binding would be the first step of a potential membrane-bound activation pathway which has been discussed for a number of neuropeptides and G-protein coupled receptors (GPCRs). Here, the orientation of TIP39 on the surface of membrane mimicking dodecyl-phosphocholine (DPC) micelles was monitored by Photo-CIDNP (chemically-induced dynamic nuclear polarization) NMR which indicates that both Trp25 and Tyr29 face the membrane surface. However, the PTH2 receptor is located in the hypothalamus membrane, for which a more realistic model is required. Therefore, liposomes containing different mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) and cholesterol were used for fluorescence and solid-state NMR spectroscopy. Fluorescence spectroscopy showed that a large proportion of TIP39 added to these liposomes binds to the membrane surface. Proton-decoupled ³¹P-MAS NMR is used to investigate the potential role of individual lipid headgroups in peptide binding. Significant line-broadening in POPC/cholesterol and POPC/POPS liposomes upon TIP39 association supports a surface binding model and indicates an interaction which is slightly mediated by the presence of POPS and cholesterol. Furthermore, smoothed order parameter profiles obtained from ²H powder spectra of liposomes containing POPC-d31 as bulk lipid in addition to POPS and cholesterol show that TIP39 does not penetrate beyond the headgroup region. Spectra of similar bilayers with POPS-d31 show a small increase in segmental chain order parameters which is interpreted as a small but specific interaction between the peptide and POPS. Our data demonstrate that TIP39 belongs to a class of signaling peptides that associate weakly with the membrane surface but do not proceed to insert into the membrane hydrophobic compartment.     

Mechanism of the interaction of hydrophobically-modified poly-(N-isopropylacrylamides) with liposomes

Interactions of hydrophobically-modified poly-(N-isopropylacrylamides) (HM PNIPAM) with phospholipid liposomes were studied as a function of the lipid type, the lipid bilayer fluidity, and the polymer conformation. Fluorescence experiments monitoring non-radiative energy transfer (NRET), between naphthalene attached to the HM PNIPAM and 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into the lipid bilayer, confirmed the direct penetration of hydrophobic anchor groups linked to the polymer into the liposome hydrophobic core. Contraction of the polymer backbone above the lower critical solution temperature (LCST) resulted in a partial withdrawal of the anchor groups from the lipid bilayer. Analysis of polymer/lipid mixtures by centrifugation and quasi-elastic light scattering (QELS) revealed the polymer-induced fission of liposomes in the liquid-crystalline state, resulting in the formation of vesicles 150–230 nm in diameter. The process is reversible and upon transition of the bilayer into the gel state these vesicles are converted into larger aggregates. According to the results of gel-filtration experiments the HM PNIPAM is in dynamic exchange between the liquid-crystalline lipid bilayer and the water solution, while the binding to the bilayer in the gel state is more static in nature. The binding constant for mixture of HM PNIPAM with DMPC liposomes, evaluated from the centrifugation experiments, was found to be 120 M⁻¹.     

Liposomes composed of unsaturated lipids for membrane modification of human erythrocytes

Abstract

Previous studies have shown that certain saturated lipids protect red blood cells (RBCs) during hypothermic storage but provide little protection during freezing or freeze-drying, whereas various unsaturated lipids destabilize RBCs during hypothermic storage but protect during freezing and freeze-drying. The protective effect of liposomes has been attributed to membrane modifications. We have previously shown that cholesterol exchange and lipid transfer between liposomes composed of saturated lipids and RBCs critically depends on the length of the lipid acyl chains. In this study the effect of unsaturated lipids with differences in their number of unsaturated bonds (18:0/18:1, 18:1/18:1, 18:2/18:2) on RBC membrane properties has been studied. RBCs were incubated in the presence of liposomes and both the liposomal and RBC fraction were analyzed by Fourier transform infrared spectroscopy (FTIR) after incubation. The liposomes caused an increase in RBC membrane conformational disorder at suprazero temperatures. The fluidizing effect of the liposomes on the RBC membranes, however, was found to be similar for the different lipids irrespective of their unsaturation level. The gel to liquid crystalline phase transition temperature of the liposomes increased after incubation with RBCs. RBC membrane fluidity increased linearly during the first 8 hours of incubation in the presence of liposomes. The increase in RBC membrane fluidity was found to be temperature dependent and displayed Arrhenius behaviour between 20 and 40°C, with an activation energy of 88 kJ mol^-1. Taken together, liposomes composed of unsaturated lipids increase RBC membrane conformational disorder, which could explain their cryoprotective action.     

Evaluation of Staphylococcus aureus DNA aptamer by enzyme‐linked aptamer assay and isothermal titration calorimetry

To monitor the specificity of Staphylococcus aureus aptamer (SA‐31) against its target cell, we used enzyme‐linked aptamer assay. In the presence of target cell, horseradish peroxidase–conjugated streptavidin bound to biotin‐labeled SA‐31 showed specific binding to S   aureus among 3 different bacteria with limit of detection of 10³ colony‐forming unit per milliliter. The apparent K _a was 1.39 μM⁻¹ ± 0.3 μM⁻¹. The binding of SA‐31 to membrane proteins extracted from cell surface was characterized using isothermal titration calorimetry, and the effect of changes in binding temperature and salt concentrations of binding buffer was evaluated based on thermodynamic parameters (K _a, ΔH , and ΔG ). Since binding of aptamer to its targets solely depends on its 3‐dimensional structure under experimental conditions used in selection process, the change in temperature and ion concentration changed the affinity of SA‐31 to its target on surface of bacteria. At 4°C, SA‐31 did not show an affinity to its target with poor heat change upon injection of membrane fraction to aptamer solution. However, the apparent association constants of SA‐31 slightly varied from K _a = 1.56 μM⁻¹ ± 0.69 μM⁻¹ at 25°C to K _a = 1.03 μM⁻¹ ± 0.9 μM⁻¹ at 37°C. At spontaneously occurring exothermic binding reactions, affinities of S  aureus aptamer to its target were also 9.44 μM⁻¹ ± 0.38 μM⁻¹ at 50mM, 1.60 μM⁻¹ ± 0.11 μM⁻¹ at 137mM, and 3.28 μM⁻¹ ± 0.46 μM⁻¹ at 200 mM of salt concentration. In this study, it was demonstrated that enzyme‐linked aptamer assay and isothermal titration calorimetry were useful tools for studying the fundamental binding mechanism between a DNA aptamer and its target on the outer surface of S  aureus .     

Effect of cholesterol content on affinity and stability of factor VIII and annexin V binding to a liposomal bilayer membrane

To investigate the effect of cholesterol composition on the binding of factor VIII (FVIII) and annexin V (AV) to membranes, liposomal membranes with phospholipid bilayers of various compositions of phosphatidylcholine (PC), phosphatidylserine (PS), and cholesterol were constructed. A surface plasmon resonance (SPR) biosensor system was employed to measure the equilibrium and rate constants of the bindings. As expected, PS was found to play a dominant role in the binding of AV; its binding level was directly proportional to the PS composition in a liposome. The binding levels of FVIII and AV to liposome increased with an increase in cholesterol composition in liposome. It seemed to suggest that cholesterol in liposome acts as a ‘phospholipid arrangement’ factor by inducing the formation of PS-rich microdomains. However, in the absence of PS (20% on a mole basis), cholesterol could not exert the binding enhancement effect, which again confirmed the critical role of PS in the bindings. Stability of the AV binding was significantly improved by the increase in cholesterol content; for AV, the dissociation rate constant was decreased approximately fivefold, from 1.7 × 10^?3 s^?1 in the absence of cholesterol to 3.3 × 10^?4 s^?1 in the presence of only 10% cholesterol. But, for FVIII the binding stability was not so much influenced by the cholesterol addition (up to 50% on a mole basis). In summary, by using liposomes on an SPR system, we were able to demonstrate quantitatively the apparent effects of cholesterol on the binding affinity and stability of the membrane-binding proteins.     

Bimodal solubilization of phospholipid-cholesterol vesicles in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) solutions. Formation of filamentous and helical microstructures depends on negatively charged lipids

Phospholipid/cholesterol vesicles were solu-bilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Above 30 mol% cholesterol (Ch) in the lipid vesicles several remarkable changes of the solubilization process were observed. (i) Two modes of solubilization: The effective detergent to lipid ratio R^c(M) for the formation of mixed micelles decreased from R^c(M) = 43 ± 3 at low lipid concentrations, [L]≤ 0.15 mm, to R^c(M) = 2.4 ± 0.3 above [L] = 0.5 mm (40 mol% Ch, T = 20 °C). (ii) At subsolubilizing CHAPS concentrations, filamentous and helical microstructures were formed, similar to those which were observed in native and model bile. (iii) The number of observed fibers was about two orders of magnitude higher in the presence of the negatively charged lipids phosphatidylglycerol (PG) and phosphatidic acid (PA) compared to the zwitterionic phosphatidylcholine (PC). Fiber formation began after 16–18 h using PG and PA compared to 3–4 days in the presence of PC. Screening of the charged lipids by NaCl effectively reduced the formation of fibers. Assuming binding of Na⁺ to the charged lipid aggregates, an intrinsic binding constant K_int = 0.6 M^–1 was determined by applying the Gouy-Chapman theory. After the addition of CHAPS to PG/Ch vesicles, a fast initial solubilization of the vesicles (<1 min) to mixed micelles (r_h = 2.3 ± 0.2 nm) and small vesicles (r_h = 23 ± 1 nm) was observed, followed by an intermediate period of 2 h, after which the formation of fibers occurred (>15 h). The microstructures are visualized by darkfield and electron microscopy. The method of vesicle solubilization is compared to the dilution of concentrated micellar solutions, which is usually applied to model bile systems. Received: 28 May 1996 / Accepted: 26 July 1996     

Cooperative binding promotes demand-driven recruitment of AnxA8 to cholesterol-containing membranes

The functionality of cellular membranes is critically determined by their lipid composition. Within the endolysosomal system, cholesterol is mainly found in more peripheral compartments. In contrast, cholesterol levels are low in late endosomes/lysosomes (LEL), and the occurrence of enlarged pools of this lipid is commonly linked to endolysosomal dysfunction. Here, we show that Annexin A8 (AnxA8), a member of the annexin family of Ca^2 +-dependent membrane-binding proteins, participates in the endosomal regulation of cholesterol homeostasis. Depletion of AnxA8 caused accumulation of cholesterol in LEL, and pharmacological inhibition of the LEL cholesterol export recruited AnxA8 to the cholesterol-laden LEL. Biophysical analysis revealed that cholesterol enhanced the Ca^2 +-dependent affinity of AnxA8 to lipid bilayers, and induced positive cooperativity of membrane binding. Our findings identify AnxA8 as a regulator of LEL cholesterol balance and point to altered membrane binding cooperativity induced by aberrant lipid composition in the target membrane as a means to control the demand-driven recruitment of this cytosolic regulatory protein.     

Partial area of cholesterol in monounsaturated diacylphosphatidylcholine bilayers

The influence of cholesterol on the structural parameters of phosphatidylcholine bilayers is studied by small-angle neutron scattering on unilamellar liposomes. Monounsaturated diacylphosphatidylcholines diCn:1PC with the length of acyl chains n = 14, 18 and 22 carbons are used. We confirm that the bilayer thickness increases with increasing concentration of cholesterol for all studied diCn:1PCs. However, partial areas per diCn:1PC and cholesterol molecule on lipid–water interface are found not to depend of cholesterol concentration. The partial area per cholesterol molecule is 0.24 nm². In addition, the partial area per diC18:1PC is larger than that for diC14:1PC and diC22:1PC.     

Effects of enrichment of fibroblasts with unesterified cholesterol on the efflux of cellular lipids to apolipoprotein A-I.

This study elucidates the factors underlying the enhancement in efflux of human fibroblast unesterified cholesterol and phospholipid (PL) by lipid-free apolipoprotein (apo) A-I that is induced by cholesterol enrichment of the cells. Doubling the unesterified cholesterol content of the plasma membrane by incubation for 24 h with low density lipoprotein and lipid/cholesterol dispersions increases the pools of PL and cholesterol available for removal by apoA-I from about 0.8-5%; the initial rates of mass release of cholesterol and PL are both increased about 6-fold. Expression of the ATP binding cassette transporter A1 (ABCA1) is critical for this increased efflux of lipids, and cholesterol loading of the fibroblasts over 24 h increases ABCA1 mRNA about 12-fold. The presence of more ABCA1 and cholesterol in the plasma membrane results in a 2-fold increase in the level of specific binding of apoA-I to the cells with no change in binding affinity. Characterization of the species released from either control or cholesterol-enriched cells indicates that the plasma membrane domains from which lipids are removed are cholesterol-enriched with respect to the average plasma membrane composition. Cholesterol enrichment of fibroblasts also affects PL synthesis, and this leads to enhanced release of phosphatidylcholine (PC) relative to sphingomyelin (SM); the ratios of PC to SM solubilized from control and cholesterol-enriched fibroblasts are approximately 2/1 and 5/1, respectively. Biosynthesis of PC is critical for this preferential release of PC and the enhanced cholesterol efflux because inhibition of PC synthesis by choline depletion reduces cholesterol efflux from cholesterol-enriched cells. Overall, it is clear that enrichment of fibroblasts with unesterified cholesterol enhances efflux of cholesterol and PL to apoA-I because of three effects, 1) increased PC biosynthesis, 2) increased PC transport via ABCA1, and 3) increased cholesterol in the plasma membrane.     

Cholesterol affects spectrin-phospholipid interactions in a manner different from changes resulting from alterations in membrane fluidity due to fatty acyl chain composition

We previously showed that erythrocyte and brain spectrins bind phospholipid vesicles and monolayers prepared from phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (Review: A.F. Sikorski, B. Hanus-Lorenz, A. Jezierski, A. R. Dluzewski, Interaction of membrane skeletal proteins with membrane lipid domain, Acta Biochim. Polon. 47 (2000) 565). Here, we show how changes in the fluidity of the phospholipid monolayer affect spectrin-phospholipid interaction. The presence of up to 10%-20% cholesterol in the PE/PC monolayer facilitates the penetration of the monolayer by both types of spectrin. For monolayers constructed from mixtures of PI/PC and cholesterol, the effect of spectrins was characterised by the presence of two maxima (at 5 and 30% cholesterol) of surface pressure for erythroid spectrin, and a single maximum (at 20% cholesterol) for brain spectrin. The binding assay results indicated a small but easily detectable decrease in the affinity of erythrocyte spectrin for FAT-liposomes prepared from a PE/PC mixture containing cholesterol, and a 2- to 5-fold increase in maximal binding capacity (B_max) depending on the cholesterol content. On the other hand, the results from experiments with a monolayer constructed from homogenous synthetic phospholipids indicated an increase in Δπ change with the increase in the fatty acyl chain length of the phospholipids used to prepare the monolayer. This was confirmed by the results of a pelleting experiment. Adding spectrins into the subphase of raft-like monolayers constructed from DOPC, SM and cholesterol (1/1/1) induced an increase in surface pressure. The Δπ change values were, however, much smaller than those observed in the case of a natural PE/PC (6/4) monolayer. An increased binding capacity for spectrins of liposomes prepared from a “raft-like” mixture of lipids could also be concluded from the pelleting assay. In conclusion, we suggest that the effect of membrane lipid fluidity on spectrin-phospholipid interactions is not simple but depends on how it is regulated, i.e., by cholesterol content or by the chemical structure of the membrane lipids.