The interaction of an antimicrobial decapeptide with phospholipid vesicles

Previously, by using combinatorial peptide libraries, we have identified activity-optimized decapeptide (KSL, KKVVFKVKFK-NH(2)), which exhibited a broad spectrum of the activity against bacteria and fungi without hemolytic activity. In order to examine lipid requirements and to understand the mode of KSL action, we investigated interactions of the peptide with vesicles consisting of various lipid compositions. KSL increased the permeability of negatively charged but not zwitterionic phospholipid membranes, and the leakage was independent on the size of encapsulated molecules (calcein, 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS)/N,N'-p-xylene bis(pyridinium) bromide (DPX), and fluorescein isothiocyanate (FITC)-dextran with different molecular weight), indicating that the peptide did not form pores or channels in this leakage process. KSL ability to permeabilize vesicles with negatively charged surface was dramatically reduced upon the addition of zwitterionic phospholipid rather than cholesterol, which revealed that the surface charge of lipid membranes played a major role in the activity and selectivity of KSL. Moreover, KSL diastereomer did not increase the permeability of negatively charged vesicles, indicating that the secondary structure of KSL was also required for membrane perturbation activity. Interestingly, KSL had an ability to cause aggregation and subsequent fusion of the acidic vesicles, which seemed to be related to the biological action. Structural studies performed by circular dichroism (CD) spectroscopy indicated that in the presence of acidic vesicles, the beta sheet structure of KSL must be required for the ability to (1) induce a leakage of dye from the acidic vesicles (2) to fuse the acidic vesicles.     

Immune lysis of reconstituted myelin basic protein--lipid vesicles and myelin vesicles

Complement-mediated lysis of reconstituted lipid-myelin basic protein (BP) vesicles and myelin vesicles due to antibody raised against BP and isolated myelin is measured by determination of the amount of a water-soluble spin label, tempocholine chloride, released from the vesicles. The response is shown to be antigen-specific, antibody-dependent, and complement mediated. The relative response to different anti-BP antibody samples is similar to that determined by radioimmunoassay procedures. In contrast to immunoassays with BP in aqueous solution, this method measures immune recognition of the protein in either a synthetic or a natural membranous environment. This is important because this protein has been shown to have a different conformation when bound to lipid bilayers than in aqueous solution and its conformation depends on lipid composition. It is also a more rapid method because no separation of spin label still trapped in the vesicles and that released due to immune lysis is required. In synthetic membranes consisting of sphingomyelin, cholesterol, and an acidic lipid, either phosphatidylglycerol, phosphatidic acid, or phosphatidylserine, the response was greatest when the acidic lipid was phosphatidic acid. The response did not depend significantly on the antigen concentration expressed as molar ratio of BP to sphingomyelin, over the range 0.15:600 to 2:600, although it decreased at molar ratios less than 0.15:600. The antigen density required for immune lysis of vesicles containing this protein antigen is similar to that reported elsewhere for lipid antigens, although the time required for maximal lysis was greater. Both anti-BP and anti-myelin antibodies caused a greater specific complement-mediated response with synthetic vesicles than with myelin vesicles, which may be due to the different lipid and/or protein composition of myelin. Response was also obtained with the myelin vesicles, however, indicating that some determinants of BP can be recognized on the surface of the bilayer in isolated myelin by anti-BP.     

Membrane-bound basic peptides sequester multivalent (PIP2), but not monovalent (PS), acidic lipids

Several biologically important peripheral (e.g., myristoylated alanine-rich C kinase substrate) and integral (e.g., the epidermal growth factor receptor) membrane proteins contain clusters of basic residues that interact with acidic lipids in the plasma membrane. Previous measurements demonstrate that the polyvalent acidic lipid phosphatidylinositol 4,5-bisphosphate is bound electrostatically (i.e., sequestered) by membrane-adsorbed basic peptides corresponding to these clusters. We report here three experimental observations that suggest monovalent acidic lipids are not sequestered by membrane-bound basic peptides. 1), Binding of basic peptides to vesicles does not decrease when the temperature is lowered below the fluid-to-gel phase transition. 2), The binding energy of Lys-13 to lipid vesicles increases linearly with the fraction of monovalent acidic lipids. 3), Binding of basic peptides to vesicles produces no self-quenching of fluorescent monovalent acidic lipids. One potential explanation for these results is that membrane-bound basic peptides diffuse too rapidly for the monovalent lipids to be sequestered. Indeed, our fluorescence correlation spectroscopy measurements show basic peptides bound to phosphatidylcholine/phosphatidylserine membranes have a diffusion coefficient approximately twofold higher than that of lipids, and those bound to phosphatidylcholine/phosphatidylinositol 4,5-bisphosphate membranes have a diffusion coefficient comparable to that of lipids.     

Study of the interaction between the antitumour protein alpha-sarcin and phospholipid vesicles.

alpha-Sarcin is a single polypeptide chain protein which exhibits antitumour activity by degrading the larger ribosomal RNA of tumour cells. We describe the interaction of a alpha-sarcin with lipid model systems. The protein specifically interacts with negatively-charged phospholipid vesicles, resulting in protein-lipid complexes which can be isolated by ultracentrifugation in a sucrose gradient. alpha-Sarcin causes aggregation of such vesicles. The extent of this interaction progressively decreases when the molar ratio of phosphatidylcholine increases in acidic vesicles. The kinetics of the vesicle aggregation induced by the protein have been measured. This process is dependent on the ratio of alpha-sarcin present in the protein-lipid system. A saturation plot is observed from phospholipid vesicles-protein titrations. The saturating protein/lipid molar ratio is 1:50. The effect produced by the antitumour protein on the lipid vesicles is dependent on neither the length nor the degree of unsaturation of the phospholipid acyl chain. However, the aggregation is dependent on temperature, being many times higher above the phase transition temperature of the corresponding phospholipid than below it. The effects of pH and ionic strength have also been considered. An increase in the ionic strength does not abolish the protein-lipid interaction. The effect of pH may be related to conformational changes of the protein. Binding experiments reveal a strong interaction between alpha-sarcin and acidic vesicles, with Kd = 0.06 microM. The peptide bonds of the protein are protected against trypsin hydrolysis upon binding to acidic vesicles. The interaction of the protein with phosphatidylglycerol vesicles does not modify the phase transition temperature of the lipid, although it decreases the amplitude of the change of fluorescence anisotropy associated to the co-operative melting of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labelled vesicles. The results are interpreted in terms of the existence of both electrostatic and hydrophobic components for the interaction between phospholipid vesicles and the antitumour protein.     

Acidic vesicles in living hyphae of an arbuscular mycorrhizal fungus, Gigaspora margarita

Localization and movement of organelles in living hyphae of an arbuscular mycorrhizal fungus, Gigaspora margarita, were observed using a combination of fluorescent probes and laser-scanning confocal microscopy. Dense, evenly distributed acidic vesicles were visible in germ tubes and extraradical hyphae using DIC with the fluorescent acidotropic probe LysoTracker. These vesicles were distinct from both tubular vacuoles stained with DFFDA and lipid bodies stained with BODIPY 493/503 or Nile Red. Tubular vacuole bundles appeared to be influenced by the bidirectional cytoplasmic streaming of acidic vesicles and lipid bodies. Movement of the acidic vesicles occurred bidrectionally at different rates. The size and distribution of lipid bodies were variable. Based on our observations, the function of these organelles is discussed in relation to nutrient translocation in arbuscular mycorrhizas. Abbreviations: AM – arbuscular mycorrhiza; DAPI – 4′,6-diamidino-2-phenylindole; DIC – differential interference contrast; BODIPY 493/503 – 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene; DMSO – dimethyl sulfoxide; FITC – fluorescein isothiocynate; caboxy-DFFDA – Oregon Green 488 carboxylic acid diacetate.     

Interaction of lipid-bound myelin basic protein with actin filaments and calmodulin

Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocytes (OLs) and is believed to be responsible for adhesion of these surfaces in the multilayered myelin sheath. MBP in solution has been shown by others to bind to both G- and F-actin, to bundle F-actin filaments, and to induce polymerization of G-actin. Here we show that MBP bound to acidic lipids can also bind to both G- and F-actin and cause their sedimentation together with MBP-lipid vesicles. Thus it can simultaneously utilize some of its basic residues to bind to the lipid bilayer and some to bind to actin. The amount of actin bound to the MBP-lipid vesicles decreased with increasing net negative surface charge of the lipid vesicles. It was also less for vesicles containing the lipid composition predicted for the cytosolic surface of myelin than for PC vesicles containing a similar amount of an acidic lipid. Calmodulin caused dissociation of actin from MBP and of the MBP-actin complex from the vesicles. However, it did not cause dissociation of bundles of actin filaments once these had formed as long as some MBP was still present. These results suggest that MBP could be a membrane actin-binding protein in OLs/myelin and its actin binding can be regulated by calmodulin and by the lipid composition of the membrane. Actin binding to MBP decreased the labeling of MBP by the hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine (TID), indicating that it decreased the hydrophobic interactions of MBP with the bilayer. This change in interaction of MBP with the bilayer could then create a cytosol to membrane signal caused by changes in interaction of the cytoskeleton with the membrane.     

Photolabeling of myelin basic protein in lipid vesicles with the hydrophobic reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine

The hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine([125I]TID) was used to label myelin basic protein or polylysine in aqueous solution and bound to lipid vesicles of different composition. Although myelin basic protein is a water soluble protein which binds electrostatically only to acidic lipids, unlike polylysine it has several short hydrophobic regions. Myelin basic protein was labeled to a significant extent by TID when in aqueous solution indicating that it has a hydrophobic site which can bind the reagent. However, myelin basic protein was labeled 2-4-times more when bound to the acidic lipids phosphatidylglycerol, phosphatidylserine, phosphatidic acid, and cerebroside sulfate than when bound to phosphatidylethanolamine, or when in solution in the presence of phosphatidylcholine vesicles. It was labeled 5-7-times more than polylysine bound to acidic lipids. These results suggest that when myelin basic protein is bound to acidic lipids, it is labeled from the lipid bilayer rather than from the aqueous phase. However, this conclusion is not unequivocal because of the possibility of changes in the protein conformation or degree of aggregation upon binding to lipid. Within this limitation the results are consistent with, but do not prove, the concept that some of its hydrophobic residues penetrate partway into the lipid bilayer. However, it is likely that most of the protein is on the surface of the bilayer with its basic residues bound electrostatically to the lipid head groups.     

Phosphorylation reverses the membrane association of peptides that correspond to the basic domains of MARCKS and neuromodulin.

Several groups have observed that phosphorylation causes the MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) protein to move off cell membranes and phospholipid vesicles. Our working hypothesis is that significant membrane binding of MARCKS requires both hydrophobic insertion of the N-terminal myristate into the bilayer and electrostatic association of the single cluster of basic residues in the protein with acidic lipids and that phosphorylation reverses this electrostatic association. Membrane binding measurements with myristoylated peptides and phospholipid vesicles show this hydrophobic moiety could, at best, barely attach proteins to plasma membranes. We report here membrane binding measurements with basic peptides that correspond to the phosphorylation domains of MARCKS and neuromodulin. Binding of these peptides increases sigmoidally with the percent acidic lipid in the phospholipid vesicle and can be described by a Gouy-Chapman/mass action theory that explains how electrostatics and reduction of dimensionality produce apparent cooperativity. The electrostatic affinity of the MARCKS peptide for membranes containing 10% acidic phospholipids (10(4) M-1 = chi/[P], where chi is the mole ratio of peptide bound to the outer monolayer of the vesicles and [P] is the concentration of peptide in the aqueous phase) is the same as the hydrophobic affinity of the myristate moiety for bilayer membranes. Phosphorylation decreases the affinity of the MARCKS peptide for membranes containing 15% acidic lipid about 1000-fold and produces a rapid (t1/2 < 30 s) dissociation of the peptide from phospholipid vesicles.     

Interaction of pertussis toxin with cells and model membranes.

The interaction of pertussis toxin (PT) with cells and model membranes was investigated by examining PT-induced intoxication of Chinese hamster ovary cells and by studying the binding of PT and its subunits to phospholipid vesicles. Since certain bacterial toxins require an acidic environment for efficient interaction with membranes and subsequent entry into the cell, the requirement for an acidic environment for PT action was examined. PT, unlike bacterial toxins such as diphtheria toxin, did not require an acidic environment for efficient intoxication of Chinese hamster ovary cells. Potential modes by which PT might interact with biological membranes were studied by examining the binding of PT to a model membrane system. PT was found to be capable of interacting with phospholipid vesicles, however, efficient binding of the toxin to the vesicles occurred only in the presence of both ATP and reducing agent. The A subunit portion of the toxin bound preferentially to the vesicles while little binding of the B oligomer portion of PT to the model membranes was observed. Isolated A subunit, in the absence of the B oligomer, also bound to the vesicles with optimal binding occurring in the presence of reducing agent. After cleavage of the A subunit by trypsin, probably at Arg-181, Arg-182, and/or Arg-193, large fragments which lacked the C-terminal portion of the A subunit of PT no longer associated with the lipid vesicles. These results suggest that the A subunit of PT can interact directly with a lipid matrix and, if freed from the constraints imposed by the B oligomer, may be capable of interacting with cellular membranes.     

The effect of polypeptide-lipid interactions on the ultraviolet spectrum of the olefinic bonds of the lipid

The effect of associating acidic and basic polypeptides with dilute suspensions of vesicles composed of various unsaturated phospholipids was assessed with regard to optical density and ultraviolet absorption. Associating basic polypeptides with phosphatidylserine, phosphatidylethanolamine, and phosphatidylglycerol vesicles, or acidic polypeptide with phosphatidylcholine vesicles, caused an increase in the optical density of the preparations, with no measurable effect on the intensity of the ultraviolet spectrum of the olefinic bonds of the lipid. Associating basic polypeptides with phosphatidylcholine vesicles, in addition to causing similar increases in optical density, resulted in a large decrease in the intensity of ultraviolet absorption by the olefinic bonds. This implies that the interaction between the basic polypeptides and phosphatidylcholine vesicles results in major alterations in the microenvironment of the olefinic bonds, which would require intimate association of the polypeptide with the ninth carbon region of the acyl chains. These observations support the conclusion, drawn from our earlier studies, that the association of basic polypeptides and liquid crystalline phase phosphatidylcholine vesicles is peculiar in that it involves a major hydrophobic component.     

pH-dependent membrane fusion and vesiculation of phospholipid large unilamellar vesicles induced by amphiphilic anionic and cationic peptides.

We studied fusion induced by a 20-amino acid peptide derived from the amino-terminal segment of hemagglutinin of influenza virus A/PR/8/34 [Murata, M., Sugahara, Y., Takahashi, S., & Ohnishi, S. (1987) J. Biochem. (Tokyo) 102, 957-962]. To extend the study, we have prepared several water-soluble amphiphilic peptides derived from the HA peptide; the anionic peptides D4, E5, and E5L contain four and five acidic residues and the cationic peptide K5 has five Lys residues in place of the five Glu residues in E5. Fusion of egg phosphatidylcholine large unilamellar vesicles induced by these peptides is assayed by two different fluorescence methods, lipid mixing and internal content mixing. Fusion is rapid in the initial stage (12-15% within 20 s) and remains nearly the same or slightly increasing afterward. The anionic peptides cause fusion at acidic pH lower than 6.0-6.5, and the cationic peptide causes fusion at alkaline pH higher than 9.0. Leakage and vesiculation of vesicles are also measured. These peptides are bound and associated with vesicles as shown by Ficoll discontinuous gradients and by the blue shift of tryptophan fluorescence. They take an alpha-helical structure in the presence of vesicles. They become more hydrophobic in the pH regions for fusion. When the suspension is made acidic or alkaline, the vesicles aggregate, as shown by the increase in light scattering. The fusion mechanism suggests that the amphiphilic peptides become more hydrophobic by neutralization due to protonation of the carboxyl groups or deprotonation of the lysyl amino groups, aggregate the vesicles together, and interact strongly with lipid bilayers to cause fusion. At higher peptide concentrations, E5 and E5L cause fusion transiently at acidic pH followed by vesiculation.     

Intervesicular exchange of lipids with weak acid and weak base characteristics: influence of transmembrane pH gradients

Transmembrane pH gradients have previously been shown to induce an asymmetric transmembrane distribution of simple lipids that exhibit weak acid or basic characteristics (Hope, M.J. and Cullis, P.R. (1987) J. Biol. Chem. 262, 4360-4366). In the present study we have examined the influence of proton gradients on the inter-vesicular exchange of stearylamine and oleic acid. We show that vesicles containing stearylamine immediately aggregate with vesicles containing phosphatidylserine and that disaggregation occurs subsequently as stearylamine equilibrates between the two vesicle populations. Despite visible flocculation during the aggregation phase, vesicle integrity is maintained. Stearylamine is the only lipid to exchange, fusion does not occur and vesicles are able to maintain a proton gradient. When stearylamine is sequestered to the inner monolayer in response to a transmembrane pH gradient (inside acidic) aggregation is not observed and diffusion of stearylamine to acceptor vesicles is greatly reduced. The ability of delta pH-dependent lipid asymmetry to modulate lipid exchange is also demonstrated for fatty acids. Oleic acid can be induced to transfer from one population of vesicles to another by maintaining a basic interior pH in the acceptor vesicles. Moreover, it is shown that the same acceptor vesicles are capable of depleting serum albumin of bound fatty acid. These results are discussed with respect to the mechanism and modulation of lipid flow between membranes both in vitro and in vivo.     

Regulation of plasma membrane Ca(2+)-ATPase activity by acetylated tubulin: influence of the lipid environment

We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca(2+)-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300μg/ml) in combination with acidic lipids at concentrations >10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50μg/ml tubulin, increased PMCA activity >12-fold, whereas tubulin alone at high concentration (≥300μg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca(2+) transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (<50μg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.     

SecA protein needs both acidic phospholipids and SecY/E protein for functional high-affinity binding to the Escherichia coli plasma membrane.

Translocation of preproteins across the Escherichia coli inner membrane requires acidic phospholipids. We have studied the translocation of the precursor protein proOmpA across inverted inner membrane vesicles prepared from cells depleted of phosphatidylglycerol and cardiolipin. These membranes support neither translocation nor the translocation ATPase activity of the SecA subunit of preprotein translocase. We now report that inner membrane vesicles which are depleted of acidic phospholipids are unable to bind SecA protein with high affinity. These membranes can be restored to translocation competence by fusion with liposomes containing phosphatidylglycerol, suggesting that the defect in SecA binding is a direct effect of phospholipid depletion rather than a general derangement of inner membrane structure. Reconstitution of SecY/E, the membrane-embedded domain of translocase, into proteoliposomes containing predominantly a single synthetic acidic lipid, dioleoylphosphatidylglycerol, allows efficient catalysis of preprotein translocation.     

Lipid interaction of Pseudomonas aeruginosa exotoxin A. Acid-triggered permeabilization and aggregation of lipid vesicles.

We have investigated the interaction of Pseudomonas exotoxin A with small unilamellar vesicles comprised of different phospholipids as a function of pH, toxin, and lipid concentration. We have found that this toxin induces vesicle permeabilization, as measured by the release of a fluorescent dye. Permeabilization is due to the formation of ion-conductive channels which we have directly observed in planar lipid bilayers. The toxin also produces vesicle aggregation, as indicated by an increase of the turbidity. Aggregation and permeabilization have completely different time course and extent upon toxin dose and lipid composition, thus suggesting that they are two independent events. Both time constants decrease by lowering the pH of the bulk phase or by introducing a negative lipid into the vesicles. Our results indicate that at least three steps are involved in the interaction of Pseudomonas exotoxin A with lipid vesicles. After protonation of one charged group the toxin becomes competent to bind to the surface of the vesicles. Binding is probably initiated by an electrostatic interaction because it is absolutely dependent on the presence of acidic phospholipids. Binding is a prerequisite for the subsequent insertion of the toxin into the lipid bilayer, with a special preference for phosphatidylglycerol-containing membranes, to form ionic channels. At high toxin and vesicle concentrations, bound toxin may also induce aggregation of the vesicles, particularly when phosphatidic acid is present in the lipid mixture. A quenching of the intrinsic tryptophan fluorescence of the protein, which is induced by lowering the pH of the solution, becomes more drastic in the presence of lipid vesicles. However, this further quenching takes so long that it cannot be a prerequisite to either vesicle permeabilization or aggregation. Pseudomonas exotoxin A shares many of these properties with other bacterial toxins like diphtheria and tetanus toxin.     

Quantification of alpha-synuclein binding to lipid vesicles using fluorescence correlation spectroscopy

Alpha-synuclein (alphaS) is a soluble synaptic protein that is the major proteinaceous component of insoluble fibrillar Lewy body deposits that are the hallmark of Parkinson's disease. The interaction of alphaS with synaptic vesicles is thought to be critical both to its normal function as well as to its pathological role in Parkinson's disease. We demonstrate the use of fluorescence correlation spectroscopy as a tool for rapid and quantitative analysis of the binding of alphaS to large unilamellar vesicles of various lipid compositions. We find that alphaS binds preferentially to vesicles containing acidic lipids, and that this interaction can be blocked by increasing the concentration of NaCl in solution. Negative charge is not the only factor determining binding, as we clearly observe binding to vesicles composed entirely of zwitterionic lipids. Additionally, we find enhanced binding to lipids with less bulky headgroups. Quantification of the protein-to-lipid ratio required for binding to different lipid compositions, combined with other data in the literature, yields an upper bound estimate for the number of lipid molecules required to bind each individual molecule of alphaS. Our results demonstrate that fluorescence correlation spectroscopy provides a powerful tool for the quantitative characterization of alphaS-lipid interactions.     

A study of the regional effects of alpha-synuclein on the organization and stability of phospholipid bilayers

Associations between the protein alpha-synuclein (alpha-syn) and presynaptic vesicles have been implicated in synaptic plasticity and neurotransmitter release and may also affect how the protein aggregates into fibrils found in Lewy bodies, the cellular inclusions associated with neurodegenerative diseases. This work investigated how alpha-syn interacts with model phospholipid membranes and examined what effect protein binding has upon the physical properties of lipid bilayers. Wide line 2H and 31P NMR spectra of phospholipid vesicles revealed that alpha-syn associates with membranes containing lipids with anionic headgroups and can disrupt the integrity of the lipid bilayer, but the protein has little effect on membranes of zwitterionic phosphatidylcholine. A peptide, alpha-syn(10-48), which corresponds to the lysine-rich N-terminal region of alpha-syn, was found to associate with lipid headgroups with a preference for a negative membrane surface charge. Another peptide, alpha-syn(120-140), which corresponds to the glutamate-rich C-terminal region, also associates weakly with lipid headgroups but with a slightly higher affinity for membranes with no net surface charge than for negatively charged membrane surfaces. Binding of alpha-syn(10-48) and alpha-syn(120-140) to the lipid vesicles did not disrupt the lamellar structure of the membranes, but both peptides appeared to induce the lateral segregation of the lipids into clusters of acidic lipid-enriched and acidic lipid-deficient domains. From these findings, it is speculated that the N-terminal and C-terminal domains of full-length alpha-syn might act in concert to organize the membrane components during normal protein function and perhaps play a role in presynaptic vesicle synthesis, maintenance, and fusion.     

Aqueous two-phase polymer partitioning of lipid vesicles of defined size and composition

The kinetics of the partitioning of lipid vesicles containing acidic phospholipids in aqueous two-phase polymer systems are dependent upon the vesicle size; the larger the vesicles, the more readily they absorb to the interfaces between the two polymer phases and hence are cleared from the top phase as phase separation proceeds. The partitioning of neutral lipid vesicles is principally to the bulk interface and is the same in phase systems of both low and high electrostatic potential difference between the two phases (delta psi). The incorporation of negatively charged lipids has two effects upon partition. First, vesicles with negatively charged lipids exhibit increased bottom phase partitioning in phases of low delta psi due to an enhanced wetting of the charged lipids by the lower phase. Second, the presence of a negatively charged group on the vesicle surface results in increased partition to the interface and top phase in phase systems of high delta psi. Differences observed in the partition of vesicles containing various species of negatively charged lipid thus reflect a competition between these two opposing factors.     

Electrostatic and hydrophobic interactions of synapsin I and synapsin I fragments with phospholipid bilayers

Synapsin I, a major neuron-specific phosphoprotein, is localized on the cytoplasmic surface of small synaptic vesicles to which it binds with high affinity. It contains a collagenase-resistant head domain and a collagenase-sensitive elongated tail domain. In the present study, the interaction between synapsin I and phospholipid vesicles has been characterized, and the protein domains involved in these interactions have been identified. When lipid vesicles were prepared from cholesterol and phospholipids using a lipid composition similar to that found in native synaptic vesicle membranes (40% phosphatidylcholine, 32% phosphatidylethanolamine, 12% phosphatidylserine, 5% phosphatidylinositol, 10% cholesterol, wt/wt), synapsin I bound with a dissociation constant of 14 nM and a maximal binding capacity of about 160 fmol of synapsin I/microgram of phospholipid. Increasing the ionic strength decreased the affinity without greatly affecting the maximal amount of synapsin I bound. When vesicles containing cholesterol and either phosphatidylcholine or phosphatidylcholine/phosphatidylethanolamine were tested, no significant binding was detected under any conditions examined. On the other hand, phosphatidylcholine vesicles containing either phosphatidylserine or phosphatidylinositol strongly interacted with synapsin I. The amount of synapsin I maximally bound was directly proportional to the percentage of acidic phospholipids present in the lipid bilayer, whereas the Kd value was not affected by varying the phospholipid composition. A study of synapsin I fragments obtained by cysteine-specific cleavage showed that the collagenase-resistant head domain actively bound to phospholipid vesicles; in contrast, the collagenase-sensitive tail domain, though strongly basic, did not significantly interact. Photolabeling of synapsin I was performed with the phosphatidylcholine analogue 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl)diazirinyl]phenyl] [2-3H]undecanoyl]-sn-glycero-3-phosphocholine; this compound generates a highly reactive carbene that selectively interacts with membrane-embedded domains of membrane proteins. Synapsin I was significantly labeled upon photolysis when incubated with lipid vesicles containing acidic phospholipids and trace amounts of the photoactivatable phospholipid. Proteolytic cleavage of photolabeled synapsin I localized the label to the head domain of the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Scavenger receptor-mediated uptake and metabolism of lipid vesicles containing acidic phospholipids by mouse peritoneal macrophages

We studied the mechanism of uptake and metabolism of exogenous phospholipids in mouse peritoneal macrophages using vesicles composed of various phospholipids and cholesterol. Macrophages in culture were found to actively incorporate and metabolize phosphatidylcholine/cholesterol vesicles containing small amounts of acidic phospholipids such as phosphatidylserine, phosphatidylinositol, or phosphatidic acid and to store the fatty acyl chains and cholesterol in triacylglycerol and cholesteryl ester form in their cytosol. These cells exhibited massive amounts of oil red O-positive lipid droplets, a typical feature of foam cells. The metabolism of exogenous phospholipid vesicles was completely inhibited by chloroquine and cytochalasin B, suggesting that vesicle uptake occurs by endocytosis. A similar type of metabolism was observed in guinea pig peritoneal macrophages, macrophage cell line J774.1, but not in Swiss 3T3 fibroblasts. Competition studies using various ligands for the scavenger receptor showed that acetylated low density lipoprotein (acetyl-LDL), dextran sulfate, or fucoidan was able to compete for up to 60% of the binding of phosphatidylserine-containing vesicles, and that copper-oxidized LDL (oxidized LDL) competed for more than 90% of the vesicle binding. On the other hand, phosphatidylserine-containing vesicles was able to compete for more than 90% of the binding of acetyl-LDL. These results indicate that acidic phospholipids are recognized by the scavenger receptors on the surface of macrophages and that more than one scavenger receptor exists on mouse peritoneal macrophages, i.e. one capable of recognizing acetyl-LDL, oxidized LDL, and an array of acidic phospholipids on membranes, and the other recognizing both acidic phospholipids and oxidized LDL but not acetyl-LDL.