Structural analysis of the protein/lipid complexes associated with pore formation by the bacterial toxin pneumolysin

Pneumolysin, a major virulence factor of the human pathogen Streptococcus pneumoniae, is a soluble protein that disrupts cholesterol-containing membranes of cells by forming ring-shaped oligomers. Magic angle spinning and wideline static (31)P NMR have been used in combination with freeze-fracture electron microscopy to investigate the effect of pneumolysin on fully hydrated model membranes containing cholesterol and phosphatidylcholine and dicetyl phosphate (10:10:1 molar ratio). NMR spectra show that the interaction of pneumolysin with cholesterol-containing liposomes results in the formation of a nonbilayer phospholipid phase and vesicle aggregation. The amount of the nonbilayer phase increases with increasing protein concentration. Freeze-fracture electron microscopy indicates the coexistence of aggregated vesicles and free ring-shaped structures in the presence of pneumolysin. On the basis of their size and analysis of the NMR spectra it is concluded that the rings are pneumolysin oligomers (containing 30-50 monomers) complexed with lipid (each with 840-1400 lipids). The lifetime of the phospholipid in either bilayer-associated complexes or free pneumolysin-lipid complexes is > 15 ms. It is further concluded that the effect of pneumolysin on lipid membranes is a complex combination of pore formation within the bilayer, extraction of lipid into free oligomeric complexes, aggregation and fusion of liposomes, and the destabilization of membranes leading to formation of small vesicles.     

Characterization of recombinant murine binder of sperm protein homolog 1 and its role in capacitation

Sperm capacitation is a maturation step that is deemed to be essential for sperm to fertilize an oocyte. A family of proteins, the binder of sperm (BSP), are known to bind choline phospholipids on sperm membranes and promote capacitation in bulls and boars. Recently, BSP-homologous genes have been identified in the epididymal tissues of human (BSPH1) and mouse (Bsph1, Bsph2). The aim of this study was to determine the binding characteristics of the murine binder of sperm protein homolog 1 (BSPH1) and evaluate its effects on sperm capacitation. Since it is not possible to purify the native BSP proteins from human and mouse in sufficient quantity, a murine recombinant BSPH1 (rec-BSPH1) was produced and used for the functional studies. Similarly to BSP proteins from other species, rec-BSPH1 bound to gelatin, heparin, phosphatidylcholine liposomes, and sperm. Both native BSPH1 and rec-BSPH1 were detected on the head and the midpiece region of sperm, although a stronger signal was detected on the midpiece region when sperm were incubated in a capacitating media containing bovine serum albumin. More importantly, murine rec-BSPH1 was able to capacitate sperm, but was unable to induce the acrosome reaction. These results show that murine epididymal BSPH1 shares many biochemical and functional characteristics with BSP proteins secreted by seminal vesicles of ungulates, and suggest that it might play a similar role in sperm functions.     

Electron Cryo‐Microscopy Reveals Mechanism of Action of Propranolol on Artificial Membranes

The pharmacological activity of several amphiphilic drugs is often related to their ability to interact with biological membranes. Propranolol is an efficient multidrug resistance (MDR) modulator; it is a nonselective β‐blocker and is thought to reduce hypertension by decreasing the cardiac frequency and thus blood pressure. It is used in drug delivery studies in order to treat systemic hypertension. We are interested in the interaction of propranolol with artificial membranes, as liposomes of controllable size are used as biocompatible and protective structures to encapsulate labile molecules, such as proteins, nucleic acids or drugs, for pharmaceutical, cosmetic or chemical applications. We present here a study of the interaction of propranolol, a cationic surfactant, with pure egg phosphatidylcholine (EPC) vesicles. The gradual transition from liposome to micelle of EPC vesicles in the presence of propranolol was monitored by time‐resolved electron cryo‐microscopy (cryo‐EM) under different experimental conditions. The liposome–drug interaction was studied with varying drug/lipid (D/L) ratios and different stages were captured by direct thin‐film vitrification. The time‐series cryo‐EM data clearly illustrate the mechanism of action of propranolol on the liposome structure: the drug disrupts the lipid bilayer by perturbing the local organization of the phospholipids. This is followed by the formation of thread‐like micelles, also called worm‐like micelles (WLM), and ends with the formation of spherical (globular) micelles. The overall reaction is slow, with the process taking almost two hours to be completed. The effect of a monovalent salt was also investigated by repeating the lipid–surfactant interaction experiments in the presence of KCl as an additive to the lipid/drug suspension. When KCl was added in the presence of propranolol the overall reaction was the same but with slower kinetics, suggesting that this monovalent salt affects the general lipid‐to‐micelle transition by stabilizing the membrane, presumably by binding to the carbonyl chains of the phosphatidylcholine.     

Influence of the bovine seminal plasma protein PDC-109 on the physical state of membranes.

PDC-109 is the main component of bovine seminal plasma and has been suggested to play an important role in the genesis of bovine sperm cells. Here, the effect of binding of PDC-109 to membranes on the structure and physical properties of the lipid phase was investigated. For that, ESR measurements were undertaken on model membranes (lipid vesicles) and on biological membranes (epididymal spermatozoa) by employing various spin-labeled phospholipids. We found that PDC-109 alters the membrane structure of lipid vesicles as well as of bovine epididymal spermatozoa in that the mobility of spin-labeled phospholipids was reduced in the presence of the protein. This immobilizing effect of the protein was not restricted to analogues of phosphatidylcholine but was also detected with spin-labeled phosphatidylethanolamine. However, the extent of immobilization was lower for phosphatidylethanolamine compared with phosphatidylcholine, supporting the lipid headgroup specificity of the protein. Besides phospholipid headgroups, the physical state of membrane lipids is also important for the interaction of PDC-109 with membranes, in that, e.g., the immobilizing effect of the protein on labeled lipids was larger in membranes above the phase transition temperature compared with the effect below this temperature. The results are of relevance for understanding the physiological role of PDC-109 in the genesis of sperm cells.     

Bovine seminal plasma phospholipid-binding proteins stimulate phospholipid efflux from epididymal sperm.

Several studies have shown that sperm capacitation was accompanied by a change in the lipid composition of the sperm membrane. In cattle, the major proteins of (bovine)seminal plasma (BSP proteins: BSP-A1/A2, BSP-A3, and BSP-30-kDa) potentiate sperm capacitation induced by high-density lipoprotein (HDL). Our recent studies indicate that these proteins and HDL stimulate sperm cholesterol efflux during capacitation. In order to gain more insight into the mechanisms of BSP-mediated sperm capacitation, we studied whether or not BSP proteins induce phospholipid efflux from epididymal sperm membrane. By direct determination of choline phospholipids on unlabeled epididymal sperm, the results show that sperm incubated in the presence of BSP-A1/A2 protein lost 34.4% of their choline phospholipids compared with the control (11.5%). Similar results were obtained using labeled epididymal sperm. Labeling was carried out by incubating washed epididymal sperm for 1 h with medium containing [(3)H]palmitic acid. The majority of the label was incorporated into sperm phosphatidylcholine. Studies of sperm phospholipid efflux were done by incubating the labeled sperm with purified BSP proteins, delipidated BSA, or bovine seminal ribonuclease (RNase, control protein). When labeled ([(3)H]phospholipid) epididymal sperm were incubated with BSP proteins (20-120 microg/ml) for 8 h, the sperm lost [(3)H]phospholipid in a dose-dependent manner (maximum efflux of approximately 30%). After the incubation with BSP proteins, the efflux particles were fractionated by size-exclusion chromatography. Analysis of the fractions obtained showed that the [(3)H]phospholipid was associated with BSP proteins. BSA (6 mg/ml) stimulated a specific phospholipid efflux of approximately 22%. In contrast, bovine RNase (120 microg/ml) did not stimulate phospholipid efflux. These results indicate that BSP proteins participate in the sperm cholesterol and phospholipid efflux that occurs during capacitation.     

The interaction of the Bax C-terminal domain with negatively charged lipids modifies the secondary structure and changes its way of insertion into membranes

Fourier transform infrared spectroscopy (FTIR) was used to study the secondary structure of peptides which imitate the amino acid sequences of the C-terminal domain of the pro-apoptotic protein Bax (Bax-C) when incorporated into different lipid vesicles with or without negatively charged phospholipids. The infrared spectroscopy results showed that while the beta-sheet components are predominant in the membrane-free Bax-C secondary structure as well as in the presence of phosphatidylcholine vesicles, the peptide changes its secondary structure in the presence of negatively charged membranes, including phospholipids such as phosphatidylglycerol or phosphatidylinositol, depending on both the lipid composition and their molar ratio. The negative charges in the model membrane surface caused a marked change from beta-sheet to alpha-helix structure. Moreover, using attenuated total reflection infrared spectroscopy (ATR-FTIR), we investigated the orientation of Bax-C alpha-helical structures with respect to the normal to the internal reflection element. The orientation of Bax-C in membranes was also affected by negatively charged lipids, the presence of phosphatidylglycerol reduced the angle it forms with the normal to the germanium plate from 45 degrees in phosphatidylcholine to 27 degrees in phosphatidylglycerol vesicles. These results highlight the importance of lipid-protein interaction for the correct folding of membrane proteins and they suggest that the C-terminal domain of Bax will only span membranes with a net negative charge in their surface.     

Low-density lipoprotein fraction from hen's egg yolk decreases the binding of the major proteins of bovine seminal plasma to sperm and prevents lipid efflux from the sperm membrane

For sperm preservation, semen is generally diluted with extender containing egg yolk (EY), but the mechanisms of sperm protection by EY are unclear. The major proteins of bull seminal plasma (BSP proteins: BSP-A1/A2, BSP-A3, and BSP-30-kDa) bind to sperm surface at ejaculation and stimulate cholesterol and phospholipid efflux from the sperm membrane. Since EY low-density lipoprotein fraction (LDF) interacts specifically with BSP proteins, it is proposed that the sequestration of BSP proteins in seminal plasma by EY-LDF represents the major mechanism of sperm protection by EY. In order to gain further insight into this mechanism, we investigated the effect of seminal plasma, EY, and EY-LDF on the binding of BSP proteins to sperm and the lipid efflux from the sperm membrane. As shown by immunodetection, radioimmunoassays, and lipid analysis, when semen was incubated undiluted or diluted with control extender (without EY or EY-LDF), BSP proteins bound to sperm in a time-dependent manner, and there is a continuous cholesterol and phospholipid efflux from the sperm membrane. In contrast, when semen was diluted with extender containing EY or EY-LDF, there was 50%-80% fewer BSP proteins associated with sperm and a significant amount of lipid added to sperm membrane during incubation. In addition, sperm function analysis showed that the presence of EY or EY-LDF in the extender preserved sperm motility. These results show that LDF is the constituent of EY that prevents binding of the BSP proteins to sperm and lipid efflux from the sperm membrane and is beneficial to sperm functions during sperm preservation.     

Differential interaction of equinatoxin II with model membranes in response to lipid composition

Equinatoxin II is a 179-amino-acid pore-forming protein isolated from the venom of the sea anemone Actinia equina. Large unilamellar vesicles and lipid monolayers of different lipid compositions have been used to study its interaction with membranes. The critical pressure for insertion is the same in monolayers made of phosphatidylcholine or sphingomyelin (approximately 26 mN m(-1)) and explains why the permeabilization of large unilamellar vesicles by equinatoxin II with these lipid compositions is null or moderate. In phosphatidylcholine-sphingomyelin (1:1) monolayers, the critical pressure is higher (approximately 33 mN m(-1)), thus permitting the insertion of equinatoxin II in large unilamellar vesicles, a process that is accompanied by major conformational changes. In the presence of vesicles made of phosphatidylcholine, a fraction of the protein molecules remains associated with the membranes. This interaction is fully reversible, does not involve major conformational changes, and is governed by the high affinity for membrane interfaces of the protein region comprising amino acids 101-120. We conclude that although the presence of sphingomyelin within the membrane creates conditions for irreversible insertion and pore formation, this lipid is not essential for the initial partitioning event, and its role as a specific receptor for the toxin is not so clear-cut.     

The Influence of Natural Lipid Asymmetry upon the Conformation of a Membrane-inserted Protein (Perfringolysin O)

Eukaryotic membrane proteins generally reside in membrane bilayers that have lipid asymmetry. However, in vitro studies of the impact of lipids upon membrane proteins are generally carried out in model membrane vesicles that lack lipid asymmetry. Our recently developed method to prepare lipid vesicles with asymmetry similar to that in plasma membranes and with controlled amounts of cholesterol was used to investigate the influence of lipid composition and lipid asymmetry upon the conformational behavior of the pore-forming, cholesterol-dependent cytolysin perfringolysin O (PFO). PFO conformational behavior in asymmetric vesicles was found to be distinct both from that in symmetric vesicles with the same lipid composition as the asymmetric vesicles and from that in vesicles containing either only the inner leaflet lipids from the asymmetric vesicles or only the outer leaflet lipids from the asymmetric vesicles. The presence of phosphatidylcholine in the outer leaflet increased the cholesterol concentration required to induce PFO binding, whereas phosphatidylethanolamine and phosphatidylserine in the inner leaflet of asymmetric vesicles stabilized the formation of a novel deeply inserted conformation that does not form pores, even though it contains transmembrane segments. This conformation may represent an important intermediate stage in PFO pore formation. These studies show that lipid asymmetry can strongly influence the behavior of membrane-inserted proteins.     

Cytochemical study of liposome and lipid vesicle phagocytosis

Electron microscopy cytochemistry has been used to study the cytoplasmic location of liposomes and lipid vesicles following specific antibody-dependent phagocytosis. The vesicle compositions were 94–99 mol% ‘fluid’ lipid (egg phosphatidylcholine or dimyristoylphosphatidylcholine at 37°C or ‘solid’ lipid (dipalmitoylphosphatidylcholine at 37°C). In some cases, 4 mol% phosphatidylserine was included in the vesicle membrane so as to vary the surface charge density. These vesicles undergo specific antibody-dependent phagocytosis by RAW264 macrophages when the lipid membranes contain 1–2 mol% dinitrophenyl lipid hapten in the presence of rabbit anti-dinitrophenyl IgG antibody. Internalized lipid vesicles can be visualized with the electron microscope when ferritin is trapped in the internal aqueous compartments prior to internalization. The lipid vesicles were demonstrated to be internal to the macrophage plasma membranes by selectively staining the plasma membranes with Ruthenium red. The cytoplasmic location of vesicles and liposomes was studied by electron microscopic staining for activities of the following enzymes: (1) acid phosphatase; (2) inorganic trimetaphosphatase; (3) adenosine triphosphatase; and (4) glucose-6-phosphatase. The first two enzymatic activities were found in association with ferritin-containing vesicles after antibody-dependent phagocytosis, showing the formation of vesicle-containing phagolysosomes. Adenosine triphosphatase and glucose-6-phosphatase were primary not associated with the vesicles, suggesting a minimal association of vesicles with plasma membrane, Golgi, endoplasmic reticulum and perinuclear cisternae. Phagosome-lysosome fusion did not appear to depend on the type of target lipid vesicle or liposome, on the ‘fluidity’ of the target membrane, or the presence of phosphatidylserine in the target membrane.     

Biophysical study of the perturbation of model membrane structure caused by seminal plasma protein PDC-109

PDC-109, the major heparin-binding protein of bull seminal plasma, binds specifically to sperm choline lipids at ejaculation and mediates capacitation by stimulating cholesterol and phospholipid efflux. We carried out a biophysical study to investigate the membrane perturbation effect caused by PDC-109. Binding of PDC-109 to phosphatidylcholine model membranes was maximal at a 12:1 phosphatidylcholine to protein molar ratio. The process was independent of the membrane structure and involved a slight conformational change of the protein, compatible with an increased exposure to the solvent. PDC-109 binding to dimyristoylphosphatidylcholine prevented lipid molecules from participating in the gel-to-liquid phase transition, due to enhancement of both acyl chain disorder and interfacial hydration. Visualization of the lipid-protein complexes by electron microscopy showed surface irregularities and the presence of 10-nm particles. Permeability assays confirmed the PDC-109-induced disruption of the vesicles. This effect was not modified by heparin. However, presence of cholesterol inhibited the process in a concentration-dependent manner.     

Syntaxin Is Efficiently Excluded from Sphingomyelin-enriched Domains in Supported Lipid Bilayers Containing Cholesterol

Formation of a trans-complex between the three SNARE proteins syntaxin, synaptobrevin and SNAP-25 drives membrane fusion. The structure of the core SNARE complex has been studied extensively. Here we have used atomic force microscopy to study the behavior of recombinant syntaxin 1A both in detergent extracts and in a lipid environment. Full-length syntaxin in detergent extracts had a marked tendency to aggregate, which was countered by addition of munc-18. In contrast, syntaxin lacking its transmembrane region was predominantly monomeric. Syntaxin could be integrated into liposomes, which formed lipid bilayers when deposited on a mica support. Supported bilayers were decorated with lipid vesicles in the presence, but not the absence, of full-length syntaxin, indicating that formation of syntaxin complexes in trans could mediate vesicle docking. Syntaxin complexes remained at the sites of docking following detergent solubilization of the lipids. Raised lipid domains could be seen in bilayers containing sphingomyelin, and these domains were devoid of syntaxin and docked vesicles in the presence, but not the absence, of cholesterol. Our results demonstrate that syntaxin is excluded from sphingomyelin-enriched domains in a cholesterol-dependent manner.     

Cholesterol attenuates and prevents bilayer damage and breakdown in lipoperoxidized model membranes. A spin labeling EPR study

The stabilizing effect of cholesterol on oxidized membranes has been studied in planar phospholipid bilayers and multilamellar 1-palmitoyl-2-linoleoyl-phosphatidylcholine vesicles also containing either 1-palmitoyl-2-glutaroyl-phosphatidylcholine or 1-palmitoyl-2-(13-hydroxy-9,11-octadecanedienoyl)-phosphatidylcholine oxidized phosphatidylcholine in variable ratio. Lipid peroxidation-dependent membrane alterations in the absence and in the presence of cholesterol were analyzed using Electron Paramagnetic Resonance spectroscopy of the model membranes spin labelled with either cholestane spin label (3-DC) or phosphatidylcholine spin label (5-DSPC). Cholesterol, added to lipid mixtures up to 40% final molar ratio, decreased the inner bilayer disorder as compared to cholesterol-free membranes and strongly reduced bilayer alterations brought about by the two oxidized phosphatidylcholine species. Furthermore, Sepharose 4B gel-chromatography and cryo electron microscopy of aqueous suspensions of the lipid mixtures clearly showed that cholesterol is able to counteract the micelle forming tendency of pure 1-palmitoyl-2-glutaroyl-phosphatidylcholine and to sustain multilamellar vesicles formation. It is concluded that membrane cholesterol may exert a beneficial and protective role against bilayer damage caused by oxidized phospholipids formation following reactive oxygen species attack to biomembranes.     

Reincorporation of adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier into phospholipid membranes. Phospholipid-protein interaction as studied by phosphorus-31 nuclear magnetic resonance and electron microscopy

Combined phosphorus-31 nuclear magnetic resonance (31P NMR) and electron microscopic studies were performed on the ADP/ATP carrier protein from beef heart mitochondria. The protein was incorporated into phospholipids by addition of Triton-protein micelles to a lipid suspension or to the dry lipid. All of the phospholipid (egg phosphatidylcholine or mixtures of egg phosphatidylcholine and egg phosphatidylethanolamine) that contributed to the observed 31P NMR signal under these conditions appeared to be in a bilayer configuration. Freeze-fracturing and negative-staining electron microscopy showed unilamellar vesicles and multilayers. An isotropic signal could be attributed to vesicle rotation, judging from its sensitivity to increasing viscosity. The presence of small vesicles was also noticeable in the 31P NMR spectra of planar oriented membranes. In the presence of phosphatidylethanolamine, aggregation of protein particles was observed. Gel chromatography of the protein-Triton-phospholipid mixture revealed that, before Triton removal, large amounts of protein are associated with multibilayers. Separation of loaded and unloaded membranes by centrifugation in D2O showed that, upon stepwise addition, protein incorporates preferentially into unloaded liposomes. From these findings a mechanism of protein reincorporation was deduced.     

Identification of a Plasmodium falciparum Phospholipid Transfer Protein

Infection of erythrocytes by the human malaria parasite Plasmodium falciparum results in dramatic modifications to the host cell, including changes to its antigenic and transport properties and the de novo formation of membranous compartments within the erythrocyte cytosol. These parasite-induced structures are implicated in the transport of nutrients, metabolic products, and parasite proteins, as well as in parasite virulence. However, very few of the parasite effector proteins that underlie remodeling of the host erythrocyte are functionally characterized. Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes. In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles. Furthermore, assays using HL60 cells containing radiolabeled phospholipids indicated that orthologs of PFA0210c can also transfer phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Biochemical and immunochemical analysis showed that PFA0210c associates with membranes in infected erythrocytes at mature stages of intracellular parasite growth. Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite. Together these data suggest that PFA0210c plays a role in the formation of the membranous structures and nutrient phospholipid transfer in the malaria-parasitized erythrocyte.     

Bovine brain phosphatidylinositol transfer protein. Effects of pH, ionic strength and lipid composition on transfer activity

Phosphatidylinositol and phosphatidylcholine are transferred between bilayer membranes in the presence of a specific phosphatidylinositol transfer protein isolated from bovine brain. The effects of pH, ionic strength and lipid composition on the rate of transfer of these phospholipids between small unilamellar vesicles have been investigated. At low ionic strength, phosphatidylinositol transfer between vesicles prepared from phosphatidylcholine and 5 mol% phosphatidylinositol was maximal at about pH 5 and moderately dependent on hydrogen ion concentration in more alkaline regions. A similar dependence on pH was noted for phosphatidylcholine transfer between membranes containing phosphatidylcholine or mixtures of phosphatidylcholine and 5 mol% phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine or stearylamine. The rate of transfer between anionic vesicles was somewhat higher than that between neutral or cationic vesicles. At higher ionic strength the transfer reactions in neutral and alkaline regions were less sensitive to pH. Phospholipid transfers between vesicles containing 5 mol% of anionic lipid increased sharply as ionic strength decreased below 0.1. In contrast, phosphatidylcholine transfer between membranes which contained only zwitterionic phospholipids or 5 mol% stearylamine was unaffected by variations of ionic strength. Irrespective of the lipid composition of membranes, pH affected both the apparent Km and Vmax, while ionic strength generally affected the apparent Vmax. These results indicate a significant role of electrostatic interactions in the phospholipid transfer catalyzed by phosphatidylinositol transfer protein.     

Binding of bovine seminal plasma protein BSP-A1/-A2 to model membranes: Lipid specificity and effect of the temperature

Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins. The affinity of the protein BSP-A1/-A2 for lipid membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and POPC containing 30% (mol/mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or cholesterol, has been investigated by the isothermal titration calorimetry (ITC). This study confirms the association of these proteins to lipid bilayers, and provides a direct characterization of this exothermic process, at 37 °C. The measurements indicate that the protein affinity for lipid bilayers is modulated by the lipid composition, the lipid/protein ratio, and the temperature. The saturation lipid/protein ratio was increased in the presence of cholesterol and, to a lesser extent, of phosphatidylethanolamine, suggesting that it is modulated by the lipid acyl chain order. For all the investigated systems, the binding of BSP-A1/-A2 could not be modeled using a simple partitioning of the proteins between the aqueous and lipid phases. The existence of "binding sites", and lipid phase separations is discussed. The decrease of temperature, from 37 to 10 °C, converts the exothermic association of the proteins to the POPC bilayers to an endothermic process. A complementary 1-D and 2-D infrared spectroscopy study excludes the thermal denaturation of BSP-A1/-A2 as a contributor in the temperature dependence of the protein affinity for lipid bilayers. The reported findings suggest that changes in the affinity of BSP-A1/-A2 for lipid bilayers could be involved in modulating the association of these proteins to sperm membranes as a function of space and time; this would consequently modulate the extent of lipid extraction, including cholesterol, at a given place and given time.     

Vesicles with charged domains

This work summarizes results obtained on membranes composed of the ternary mixture dioleoylphosphatidylglycerol (DOPG), egg sphingomyelin (eSM) and cholesterol (Chol). The membrane phase state as a function of composition is characterized from data collected with fluorescence microscopy on giant unilamellar vesicles. The results suggest that the presence of the charged DOPG significantly decreases the composition region of coexistence of liquid ordered and liquid disordered phases as compared to that in the ternary mixture of dioleoylphosphatidycholine, sphingomyelin and cholesterol. The addition of calcium chloride to DOPG:eSM:Chol vesicles, and to a lesser extent the addition of sodium chloride, leads to the stabilization of the two-phase coexistence region, which is expressed in an increase in the miscibility temperature. On the other hand, addition of the chelating agent EDTA has the opposite effect, suggesting that impurities of divalent cations in preparations of giant vesicles contribute to the stabilization of charged domains. We also explore the behavior of these membranes in the presence of extruded unilamellar vesicles made of the positively charged lipid dioleoyltrimethylammoniumpropane (DOTAP). The latter can induce domain formation in DOPG:eSM:Chol vesicles with initial composition in the one-phase region.     

Lipid phase coexistence favors membrane insertion of equinatoxin-II, a pore-forming toxin from Actinia equina

Equinatoxin-II is a eukaryotic pore-forming toxin belonging to the family of actinoporins. Its interaction with model membranes is largely modulated by the presence of sphingomyelin. We have used large unilamellar vesicles and lipid monolayers to gain further information about this interaction. The coexistence of gel and liquid-crystal lipid phases in sphingomyelin/phosphatidylcholine mixtures and the coexistence of liquid-ordered and liquid-disordered lipid phases in phosphatidylcholine/cholesterol or sphingomyelin/phosphatidylcholine/cholesterol mixtures favor membrane insertion of equinatoxin-II. Phosphatidylcholine vesicles are not permeabilized by equinatoxin-II. However, the localized accumulation of phospholipase C-generated diacylglycerol creates conditions for toxin activity. By using epifluorescence microscopy of transferred monolayers, it seems that lipid packing defects arising at the interfaces between coexisting lipid phases may function as preferential binding sites for the toxin. The possible implications of such a mechanism in the assembly of a toroidal pore are discussed.     

Influence of the bovine seminal plasma protein PDC-109 on cholesterol in the presence of phospholipids

The interaction of the major bovine seminal plasma protein PDC-109 with cholesterol was studied by employing spin-labelled analogues. It could be shown that PDC-109 does not interact directly with cholesterol molecules. However, in the presence of phospholipids we found a strong reduction of cholesterol motion by PDC-109. The fraction of immobilized cholesterol was largest for phosphorylcholine-containing lipids. This is consistent with the preferential interaction between PDC-109 and phosphatidylcholine. It is concluded that a stronger association and interaction of PDC-109 with phosphatidylcholine leads to an enhanced fraction of immobilized cholesterol analogues, but not to a phospholipid-dependent specific interaction between the protein and cholesterol. Moreover, the interaction of PDC-109 with various spin-labelled analogues of phosphatidylcholine (lysoPC, diacylPC) was investigated. In membranes of lipid vesicles the protein caused an immobilization of the phosphatidylcholine analogues mainly in the outer membrane leaflet, with no differences between diacylPC and lysoPC. The results are of relevance for understanding the physiological role of PDC-109 in the genesis of sperm cells.