The interaction of liposomes containing intrinsic erythrocyte membrane proteins with lipid monolayers at air/water and oil/water interfaces

The main intrinsic membrane proteins of the human erythrocyte membrane, glycophorin and the anion transporter, were isolated by extraction with Triton X-100 and ion-exchange chromatography. After removal of detergent the extract consisted of proteolipid vesicles with a lipid:protein molar ratio in the range 50-60 and a diameter of the order of 200 nm. The interaction between these vesicles and dipalmitoylphosphatidylcholine (DPPC), cholesterol and cholesterol:DPPC (2:1 molar ratio) monolayers at air/water and n-decane/water interfaces has been studied. The vesicles interact with the monolayers, rapidly causing large increases in surface pressure. Limiting values of surface pressure, 39.4-43 mN . m-1 at air/water and 31.5-33.4 mN . m-1 at the n-decane/water interface, were reached at protein levels above 1 microgram . ml-1. At the air/water interface, and probably at the n-decane/water, surface pressure increases were limited by monolayer collapse. Compression isotherms and surface potential measurements indicated that material from the proteolipid vesicles entered the monolayer phase. In contrast to proteolipid vesicles, injection of protein-free liposomes beneath the monolayer resulted in smaller, slower increases in surface pressure. Thus, the presence of intrinsic membrane proteins in vesicles greatly facilitated the transfer of material into the lipid monolayer.     

Structural investigation on the adsorption of the MARCKS peptide on anionic lipid monolayers - effects beyond electrostatic

The presence of charged lipids in the cell membrane constitutes the background for the interaction with numerous membrane proteins. As a result, the valence of the lipids plays an important role concerning their lateral organization in the membrane and therefore the very manner of this interaction. This present study examines this aspect, particularly regarding to the interaction of the anionic lipid DPPS with the highly basic charged effector domain of the MARCKS protein, examined in monolayer model systems. Film balance, fluorescence microscopy and X-ray reflection/diffraction measurements were used to study the behavior of DPPS in a mixture with DPPC for its dependance on the presence of MARCKS (151-175). In the mixed monolayer, both lipids are completely miscible therefore DPPS is incorporated in the ordered crystalline DPPC domains as well. The interaction of MARCKS peptide with the mixed monolayer leads to the formation of lipid/peptide clusters causing an elongation of the serine group of the DPPS up to 7? in direction to surface normal into the subphase. The large cationic charge of the peptide pulls out the serine group of the interface which simultaneously causes an elongation of the phosphodiester group of the lipid fraction too. The obtained results were used to compare the interaction of MARCKS peptide with the polyvalent PIP(2) in mixed monolayers. On this way we surprisingly find out, that the relative small charge difference of the anionic lipids causes a significant different interaction with MARCKS (151-175). The lateral arrangement of the anionic lipids depends on their charge values and determines the diffusion of the electrostatic binding clusters within the membrane.     

Molecular organization of surfactin-phospholipid monolayers: Effect of phospholipid chain length and polar head

Mixed monolayers of the surface-active lipopeptide surfactin-C₁₅ and various lipids differing by their chain length (DMPC, DPPC, DSPC) and polar headgroup (DPPC, DPPE, DPPS) were investigated by atomic force microscopy (AFM) in combination with molecular modeling (Hypermatrix procedure) and surface pressure-area isotherms. In the presence of surfactin, AFM topographic images showed phase separation for each surfactin-phospholipid system except for surfactin-DMPC, which was in good agreement with compression isotherms. On the basis of domain shape and line tension theory, we conclude that the miscibility between surfactin and phospholipids is higher for shorter chain lengths (DMPC > DPPC > DSPC) and that the polar headgroup of phospholipids influences the miscibility of surfactin in the order DPPC > DPPE > DPPS. Molecular modeling data show that mixing surfactin and DPPC has a destabilizing effect on DPPC monolayer while it has a stabilizing effect towards DPPE and DPPS molecular interactions. Our results provide valuable information on the activity mechanism of surfactin and may be useful for the design of surfactin delivery systems.     

Molecular organization of surfactin-phospholipid monolayers: effect of phospholipid chain length and polar head

Mixed monolayers of the surface-active lipopeptide surfactin-C(15) and various lipids differing by their chain length (DMPC, DPPC, DSPC) and polar headgroup (DPPC, DPPE, DPPS) were investigated by atomic force microscopy (AFM) in combination with molecular modeling (Hypermatrix procedure) and surface pressure-area isotherms. In the presence of surfactin, AFM topographic images showed phase separation for each surfactin-phospholipid system except for surfactin-DMPC, which was in good agreement with compression isotherms. On the basis of domain shape and line tension theory, we conclude that the miscibility between surfactin and phospholipids is higher for shorter chain lengths (DMPC>DPPC>DSPC) and that the polar headgroup of phospholipids influences the miscibility of surfactin in the order DPPC>DPPE>DPPS. Molecular modeling data show that mixing surfactin and DPPC has a destabilizing effect on DPPC monolayer while it has a stabilizing effect towards DPPE and DPPS molecular interactions. Our results provide valuable information on the activity mechanism of surfactin and may be useful for the design of surfactin delivery systems.     

Calcium-activated membrane interaction of the islet amyloid polypeptide: implications in the pathogenesis of type II diabetes mellitus

The role played by Ca²⁺ ions in the interaction of the human islet amyloid polypeptide (hIAPP) with model membranes has been investigated by differential scanning calorimetry (DSC) and circular dichroism (CD) experiments. In particular, the interaction of hIAPP and its rat isoform (rIAPP) with zwitterionic dipalmitoyl-phosphatidylcholine (DPPC), negatively charged dipalmitoyl-phosphatidylserine (DPPS) vesicles and with a 3:1 mixtures of them, has been studied in the presence of Ca²⁺ ions. The experiments have evidenced that amorphous, soluble hIAPP assemblies interact with the hydrophobic core of DPPC bilayers. Conversely, the presence of Ca²⁺ ions is necessary to activate a preferential interaction of hIAPP with the hydrophobic core of DPPS membranes. These findings support the hypothesis that an impaired cellular homeostasis of Ca²⁺ ions may promote the insertion of hIAPP into the hydrophobic core of carrier vesicles which is thought to contribute to an eventual intracellular accumulation of β-sheet rich hIAPP aggregates.     

Calcium ion interactions with insoluble phospholipid monolayer films at the A/W interface. External reflection-absorption IR studies.

External reflection Fourier transform infrared (FT-IR) experiments are reported for insoluble monomolecular films of an equimolar mixture of 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1,2-dipalmitoylphosphatidylserine (DPPS) at the A/W interface as a function of surface pressure and Ca2+ ion presence. The separate components showed a surface pressure-induced conformational ordering of the acyl chains. The conformational ordering occurred more cooperatively for the DPPS. Acyl chain perdeuteration of the DPPC permitted the observation of the response of the individual components in the binary mixture to changes in surface tension and to the presence of Ca2+. Plots of surface pressure versus CH2 or CD2 stretching frequencies were analyzed with a two-state model. At each surface pressure within the two-state region, the fraction of disordered form was the same for each lipid component, suggesting that they are well mixed on the surface. Calcium ion (5 mM in the subphase) produces almost no effect on the pressure-induced acyl chain ordering of the DPPC in a single component film, whereas the same levels of Ca2+ induce acyl chain ordering at all surface pressures in both components of the binary mixture. Thus, unlike the bulk phase mixture of DPPC/DPPS, the binary lipids in this mixed monolayer film appear to retain their miscibility in the presence of Ca2+. Finally, Ca(2+)-induced dehydration of the phosphate group was observed through characteristic frequency shifts in the asymmetric PO2- stretching mode.     

Mimetic Membrane System to Carry Multiple Antigenic Proteins from Leishmania amazonensis

Liposomes have long been used as models for lipid membranes and for the reconstitution of a single or multiple proteins. Also, liposomes have adjuvant activity in vaccines against several protozoan or bacterial organisms. Thus, the main objective of the present study was to obtain a crude extract of detergent-solubilized proteins of Leishmania amazonensis amastigotes and reconstitute them into liposomes. Neutral and zwiterionic detergents were less efficient than an ionic detergent. In order to obtain efficient solubilization using only sodium dodecyl sulfate (SDS), the effects of detergent and protein concentration and incubation time were studied. The maximum of solubilized proteins was obtained instantaneously using a ratio of 0.5 mg/ml of protein to 0.1% (w/v) detergent at 4°C. Dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS) and cholesterol in a weight ratio of 5:1:4 were used for protein reconstitution into liposomes using the cosolubilization method, yielding 60% of incorporation. The incorporation of multiple parasite proteins results in a vesicular diameter of proteoliposomes of about 140 nm, presenting a final lipid weight ratio for DPPC, DPPS and cholesterol of 1:1:5, with high stability. The detergent-solubilized proteins of L. amazonensis amastigotes present in the proteoliposome, when analyzed by SDS-polyacrylamide gel electrophoresis, include a wide range of parasite-incorporated proteins. BALB/c mice inoculated with these proteoliposomes were able to produce antibodies against the proteins reconstituted in DPPC:DPPS:cholesterol liposomes and were partially resistant to infection with L. amazonensis promastigotes. These results indicate that this system can be used as a possible vaccine against L. amazonensis.     

Integration of ganglioside GT1b receptor into DPPE and DPPC phospholipid monolayers: an X-ray reflectivity and grazing-incidence diffraction study

Using synchrotron grazing-incidence x-ray diffraction (GIXD) and reflectivity, the in-plane and out-of-plane structures of mixed-ganglioside GT_1b-phospholipid monolayers were investigated at the air-liquid interface and compared with monolayers of the pure components. The receptor GT_1b is involved in the binding of lectins and toxins, including botulinum neurotoxin, to cell membranes. Monolayers composed of 20 mol % ganglioside GT_1b, the phospholipid dipalmitoyl phosphatidylethanolamine (DPPE), and the phospholipid dipalmitoyl phosphatidylcholine (DPPC) were studied in the gel phase at 23°C and at surface pressures of 20 and 40 mN/m, and at pH 7.4 and 5. Under these conditions, the two components did not phase-separate, and no evidence of domain formation was observed. The x-ray scattering measurements revealed that GT_1b was intercalated within the host DPPE/DPPC monolayers, and slightly expanded DPPE but condensed the DPPC matrix. The oligosaccharide headgroups extended normally from the monolayer surfaces into the subphase. This study demonstrated that these monolayers can serve as platforms for investigating toxin membrane binding and penetration.     

Role of pulmonary surfactant components in surface film formation and dynamics

Pulmonary surfactant is a mixture of lipids and proteins which is secreted by the epithelial type II cells into the alveolar space. Its main function is to reduce the surface tension at the air/liquid interface in the lung. This is achieved by forming a surface film that consists of a monolayer which is highly enriched in dipalmitoylphosphatidylcholine and bilayer lipid/protein structures closely attached to it. The molecular mechanisms of film formation and of film adaptation to surface changes during breathing in order to remain a low surface tension at the interface, are unknown. The results of several model systems give indications for the role of the surfactant proteins and lipids in these processes. In this review, we describe and compare the model systems that are used for this purpose and the progress that has been made. Despite some conflicting results using different techniques, we conclude that surfactant protein B (SP-B) plays the major role in adsorption of new material into the interface during inspiration. SP-C's main functions are to exclude non-DPPC lipids from the interface during expiration and to attach the bilayer structures to the lipid monolayer. Surfactant protein A (SP-A) appears to promote most of SP-B's functions. We describe a model proposing that SP-A and SP-B create DPPC enriched domains which can readily be adsorbed to create a DPPC-rich monolayer at the interface. Further enrichment in DPPC is achieved by selective desorption of non-DPPC lipids during repetitive breathing cycles.     

Behavior of a GPI-anchored protein in phospholipid monolayers at the air-water interface

The interaction between alkaline phosphatase (AP), a glycosylphosphatidylinositol (GPI)-anchored protein (AP-GPI), and phospholipids was monitored using Langmuir isotherms and PM-IRRAS spectroscopy. AP-GPI was injected under C16 phospholipid monolayers with either a neutral polar head (1,2-dipalmitoyl-sn-glycero-3-phosphocholine monohydrate (DPPC)) or an anionic polar head (1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS)). The increase in molecular area due to the injection of protein depended on the surface pressure and the type of phospholipid. At all surface pressures, it was highest in the case of DPPS monolayers. The surface elasticity coefficient E, determined from the pi-A diagrams, allowed to deduct that the AP-GPI-phospholipid mixtures presented a molecular arrangement less condensed than the corresponding pure phospholipid films. PM-IRRAS spectra suggested different protein-lipid interactions as a function of the nature of the lipids. AP-GPI modified the organization of the DPPS deuterated chains whereas AP-GPI affected only the polar group of DPPC at low surface pressure (8 mN/m). Different protein hydration layers between the DPPC and DPPS monolayers were suggested to explain these results. PM-IRRAS spectra of AP-GPI in the presence of lipids showed a shape similar to those collected for pure AP-GPI, indicating a similar orientation of AP-GPI in the presence or absence of phospholipids, where the active sites of the enzyme are turned outside of the membrane.     

Lipid membranes exposed to dispersions of phytantriol and monoolein cubosomes: Langmuir monolayer and HeLa cell membrane studies

The interactions of liquid-crystalline nanoparticles based on lipid-like surfactants, glyceryl monooleate, monoolein (GMO) and 1,2,3-trihydroxy-3,7,11,15-tetramethylhexadecane, phytantriol (PT) with selected model lipid membranes prepared by Langmuir technique were compared. Monolayers of DPPC, DMPS and their mixture DPPC:DMPS 87:13 mol% were used as simple models of one leaflet of a cell membrane. The incorporation of cubosomes into the lipid layers spread at the air-water interface was followed by surface-pressure measurements and Brewster angle microscopy. The cubosome - membrane interactions lead to the fluidization of the model membranes but this effect depended on the composition of the model membrane and on the type of cubosomes. The interactions of PT cubosomes with lipid layers, especially DMPS-based monolayer were stronger compared with those of GMO-based nanoparticles. The kinetics of incorporation of cubosomal material into the lipid layer was influenced by the extent of hydration of the polar headgroups of the lipid: faster in the case of smaller, less hydrated polar groups of DMPS than for strongly hydrated uncharged choline of DPPC. The membrane disrupting effect of cubosomes increased at longer times of the lipid membrane exposure to the cubosome solution and at larger carrier concentrations. Langmuir monolayer observations correspond well to results of studies of HeLa cells exposed to cubosomes. The larger toxicity of PT cubosomes was confirmed by MTS. Their ability to disrupt lipid membranes was imaged by confocal microscopy. On the other hand, PT cubosomes easily penetrated cellular membranes and released cargo into various cellular compartments more effectively than GMO-based nanocarriers. Therefore, at low concentrations, they may be further investigated as a promising drug delivery tool.     

Proton transport at the monolayer-water interface.

It is shown that when monolayers of stearic acid, palmitic acid, DPPC, or DPPS are compressed above some critical area Ac a lateral conduction mechanism is initiated at the monolayer/water interface. The interfacial conductance increases on further increasing the molecular packing density in the monolayer. All compounds also show major changes in surface potential at Ac the potential becoming more positive in all cases. It is argued that this is a consequence of structural reorganisation at the headgroup/water interface causing a significant reduction in the local permittivity. The critical area, Ac, is approximately double the molecular areas estimated from the pressure-area isotherm, and experiments with stearic acid monolayers show that Ac decreases significantly when the chaotropic ion SCN-, which is known to disrupt the molecular structure of water, is added to the subphase. It is likely, therefore, that the structural changes occurring at Ac involve the formation of a hydrogen bonded network between monolayer headgroups and adjacent water molecules at the monolayer/water interface. It is suggested that the conduction mechanism initiated at Ac arises from proton hopping along this hydrogen-bond network.     

Lipid model membranes for drug interaction study

The present work shows a structural study on the process of incorporation of a hydrophobic drug, Ellipticine (ELPT), into lipid model membranes for drug targeting purpose. The ELPT is an alkaloid that showed an anti-proliferation activity against several types of tumor cells and against the HIV1 virus. We used the zwitterionic lipid dipalmitoyl phosphatidylcholine (DPPC) and four different anionic lipids: cardiolipin (CL), dipalmitoyl phosphatidic acid (DPPA), dipalmitoyl phosphatidylglycerol (DPPG) and dipalmitoyl phosphatidylserine (DPPS), both spread on a Langmuir monolayer and deposited on a solid substrate to mimic a model membrane and study the interaction with the drug ELPT. X-ray reflectivity results pointed toward an increase in drug loading efficiency up to 13.5% mol/mol of ELPT into mixed systems DPPC/CL. This increase in loading efficiency was also accompanied by a slight distortion in the stacking of the bilayers less evidenced after optimization of the molar ratio between the co-lipids. Grazing incidence X-ray diffraction measurements revealed an in-plane lattice distortion due to the presence of hydrocarbon chain backbone ordering in pure systems of DPPC doped with ELPT. The same was not observed in mixed membranes with DPPC/CL and DPPC/DPPA.     

Membrane binding of a lipidated N-Ras protein studied in lipid monolayers

The adsorption of doubly lipidated full-length N-Ras protein on 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) monolayers was studied by lateral pressure analysis, grazing incidence X-ray diffraction (GIXD), and specular reflectivity (XR). N-Ras protein adsorbs to the DPPC monolayer (lateral pressure of 20 mN/m) from the subphase thereby increasing the lateral pressure in the monolayer by 4 mN/m. The protein insertion does not alter the tilt angle and structure of the lipid molecules at the air/water interface but influences the electron density profile of the monolayer. Further, electron density differences into the subphase were observed. The Fresnel normalized reflectivity could be reconstructed in the analysis using box models yielding electron density profiles of the DPPC monolayer in the absence and in the presence of N-Ras protein. The electron density profiles of the DPPC monolayer in the presence of Ras showed clear intensity variations in the headgroup/glycerol/upper chain region, the so-called interface region where previous bilayer studies had confirmed Ras binding. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Incorporation of dehydrodieugenol,a neolignan isolated from Nectandra leucantha (Lauraceae), in lipid Langmuir monolayers as biomembrane models

Dehydrodieugenol, a neolignan isolated from the Brazilian plant Nectandra leucantha (Lauraceae) with reported antiprotozoal and anticancer activity, was incorporated in Langmuir monolayers of selected lipids as cell membrane models, aiming to comprehend its action mechanism at the molecular level. The interaction of this compound with the lipids dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylserine (DPPS), and dipalmitoylphosphatidylglycerol (DPPG) was inferred through tensiometry, infrared spectroscopy, and Brewster angle microscopy. The interactions had different effects depending on the chemical nature of the lipid polar head, with expansion for DPPC monolayers, condensation for DPPE, and expansion (at low surface pressures) followed by the overlap of the isotherms (at high surface pressure values) for DPPS and DPPG. Effects caused by dehydrodieugenol in the negatively charged lipids were distinctive, which was also reflected in the hysteresis assays, surface potential-area isotherms, and rheological measurements. Infrared spectroscopy indicated that the drug interaction with the monolayer affects not only the polar groups, but also the acyl lipid chains for all lipids. These results pointed to the fact that the interaction of the drug with lipid monolayers at the air-water interface is modulated by the lipid composition, mainly considering the polar head of the lipids, as well as the hydrophobicity of the lipids and the drug. As negatively charged lipids pointed to distinctive interaction, we believe this can be related to the antiprotozoal and anticancer properties of the compound.     

Multiphoton excitation fluorescence microscopy in planar membrane systems

The feasibility of applying multiphoton excitation fluorescence microscopy-related techniques in planar membrane systems, such as lipid monolayers at the air-water interface (named Langmuir films), is presented and discussed in this paper. The non-linear fluorescence microscopy approach, allows obtaining spatially and temporally resolved information by exploiting the fluorescent properties of particular fluorescence probes. For instance, the use of environmental sensitive probes, such as LAURDAN, allows performing measurements using the LAURDAN generalized polarization function that in turn is sensitive to the local lipid packing in the membrane. The fact that LAURDAN exhibit homogeneous distribution in monolayers, particularly in systems displaying domain coexistence, overcomes a general problem observed when “classical” fluorescence probes are used to label Langmuir films, i.e. the inability to obtain simultaneous information from the two coexisting membrane regions. Also, the well described photoselection effect caused by excitation light on LAURDAN allows: (i) to qualitative infer tilting information of the monolayer when liquid condensed phases are present and (ii) to provide high contrast to visualize 3D membranous structures at the film's collapse pressure. In the last case, computation of the LAURDAN GP function provides information about lipid packing in these 3D structures. Additionally, LAURDAN GP values upon compression in monolayers were compared with those obtained in compositionally similar planar bilayer systems. At similar GP values we found, for both DOPC and DPPC, a correspondence between the molecular areas reported in monolayers and bilayers. This correspondence occurs when the lateral pressure of the monolayer is 26 ± 2 mN/m and 28 ± 3 mN/m for DOPC and DPPC, respectively.     

Molecular dynamics simulation of dipalmitoylphosphatidylcholine membrane with cholesterol sulfate

Using the molecular dynamics simulation technique, we studied the changes occurring in a dipalmitoylphosphatidylcholine (DPPC):cholesterol (CH) membrane at 50 mol% sterol when cholesterol is replaced with cholesterol sulfate (CS). Our simulations were performed at constant pressure and temperature on a nanosecond time scale. We found that 1) the area per DPPC:CS heterodimer is greater than the area of the DPPC:CH heterodimer; 2) CS increases ordering of DPPC acyl chains, but to a lesser extent than CH; 3) the number of hydrogen bonds between DPPC and water is decreased in a CS-containing membrane, but CS forms more water hydrogen bonds than CH; and 4) the membrane dipole potential reverses its sign for a DPPC-CS membrane compared to a DPPC-CH bilayer. We also studied the changes occurring in lipid headgroup conformations and determined the location of CS molecules in the membrane. Our results are in good agreement with the data available from experiments.     

The psychotropic drug olanzapine (Zyprexa) increases the area of acid glycerophospholipid monolayers

The typical antipsychotics chlorpromazine (CPZ) and trifluoperazine (TFP) increase the mean molecular area (mma) of acidic, but not neutral, glycerophospholipids in monolayers at pH 7.36 measured by the Langmuir technique. The atypical antipsychotic olanzapine (OLP(1)) is structurally similar to TFP. We have therefore studied the effects of OLP on glycerophospholipid monolayers and in comparison with CPZ. Olanzapine (10 microM, in subphase, pH 7.36) influenced the isotherms (surface pressure versus mma) in monolayers of the neutral dipalmitoyl phosphatidylcholine (DPPC) and the acidic dipalmitoyl phosphatidylserine (DPPS) or 1-palmitoyl-2-oleoylphosphatidylserine (POPS) in the increasing order of mma: DPPS相似文献   

GPI-alkaline phosphatase insertion into phosphatidylcholine monolayers: phase behavior and morphology changes

GPI-anchored proteins are localized on the outer layer of plasma membranes and clustered in microdomains generally called lipid rafts. To study the interactions between the lipidic GPI-anchor of the protein and phospholipids, we used phosphatidylcholine monolayers at the air-water interface as a biomimetic membrane system and GPI-alkaline phosphatase prepared from bovine intestinal mucosa (GPI-BIAP) as an GPI-anchored protein model. The monolayer technique allowed us to define GPI-BIAP interaction with DPPC and POPC, lipids differing only by the presence of one unsaturation in their acyl chains. Meanwhile the exclusion pressures were similar for the two phospholipids, the comparison of the Langmuir isotherms (i.e., pressure/area diagrams) indicates that GPI-BIAP interacted differently with DPPC and POPC monolayers. BAM images, acquired in order to visualize the interface organization induced by GPI-BIAP incorporation, confirm these differences.