Induction of fusion in aggregated and nonaggregated liposomes bearing cationic detergents

The addition of polyanionic polymers such as poly(aspartic acid) (PASP), DNA or dextran sulfate to liposomes composed of phosphatidylcholine (PC) and cholesterol (CHOL) and bearing the quaternary ammonium detergent [[[(1,1,3,3-tetramethylbutyl)cresoxy]ethoxy]ethyl]dimethy lbe nzylammonium hydroxide (DEBDA[OH]) resulted in liposome aggregation and fusion. Liposome-liposome fusion was studied by using fluorescently labeled liposomes and fluorescence-dequenching (DQ) methods. Addition of monoanions, such as aspartate or acetate, to liposomes bearing DEBDA[OH] caused neither their aggregation nor liposome-liposome fusion. Aggregation of liposomes bearing DEBDA[OH] by the binding pair avidin-biotin did not result in their fusion. Fusion in such aggregated liposomes was observed by the addition of chaotropic anions, such as nitrate or thiocyanate, or by PASP. A variety of other quaternary ammonium detergents behaved similarly to DEBDA[OH] in their ability to confer fusogenic properties upon PC/chol liposomes. The relevance of these findings to the mechanism of liposome-liposome fusion is discussed.     

Membrane fusion activity of influenza virus. Effects of gangliosides and negatively charged phospholipids in target liposomes

Fusion of influenza virus with liposomes composed of negatively charged phospholipids differs from fusion with biological membranes or zwitterionic liposomes with ganglioside receptors [Stegmann, T., Hoekstra, D., Scherphof, G., & Wilschut, J. (1986) J. Biol. Chem. 261, 10966-10969]. In this study, we investigated how the kinetics and extent of fusion of influenza virus, monitored with a fluorescence resonance energy-transfer assay, are influenced by the surface charge and the presence of receptors on liposomal membranes. The results were analyzed in terms of mass action kinetic model, providing separate rate constants for the initial virus-liposome adhesion, or aggregation, and for the actual fusion reaction. Incorporation of increasing amounts of cardiolipin (CL) or phosphatidylserine (PS) into otherwise zwitterionic phosphatidylcholine (PC)/phosphatidylethanolamine (PE) vesicles results in a gradual shift of the pH threshold of fusion to neutral, relative to the pH threshold obtained with PC/PE vesicles containing the ganglioside GD1a, while also the rate of fusion increases. This indicates the emergence of a fusion mechanism not involving the well-documented conformational change in the viral hemagglutinin (HA). However, only with pure CL liposomes this nonphysiological fusion reaction dominates the overall fusion process; with pure PS or with zwitterionic vesicles containing CL or PS, the contribution of the nonphysiological fusion reaction is small. Accordingly, preincubation of the virus alone at low pH results in a rapid inactivation of the viral fusion capacity toward all liposome compositions studied, except pure CL liposomes. The results of the kinetic analyses show that with pure CL liposomes the rates of both virus-liposome adhesion and fusion are considerably higher than with all other liposome compositions studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of lipid composition on HVJ-mediated fusion of glycophorin liposomes to erythrocytes

We previously reported that liposomes containing glycophorin or gangliosides, both of which were isolated from human erythrocytes, are efficiently fused to erythrocyte membranes in the presence of HVJ (Umeda, M. et al., J. Biochem. 94, 1955-1966 (1983), and Virology 133, 172-182 (1984]. In the present work, the effect of lipid composition in glycophorin liposomes on their sensitivity to fusion with erythrocytes was studied. Very little fusion occurred when glycophorin liposomes composed of dipalmitoylphosphatidylcholine-dicetylphosphate (9:1), dimyristoylphosphatidylcholine-dicetylphosphate (9:1), or egg yolk phosphatidylcholine-dicetylphosphate (9:1) were incubated with human erythrocytes in the presence of HVJ at 37 degrees C. Addition of cholesterol into these liposomal membranes greatly enhanced the sensitivity of the liposomes to fusion. The presence of phosphatidic acid and phosphatidylethanolamine in liposomes also enhanced the sensitivity, whereas the presence of lysophosphatidylcholine had no significant effect on the ability of the liposomes to fuse. The fusion efficiency of liposomes was also enhanced by the presence of glucosylceramide. Change of lipid composition in liposomes had, however, no appreciable influence on the HVJ-mediated binding of liposomes to erythrocytes, suggesting that the interaction between HANA protein of HVJ and glycophorin in liposomes was not affected by the lipid composition of the liposomes.     

Mass action analysis of kinetics and extent of fusion between Sendai virus and phospholipid vesicles

The kinetics and extent of fusion between Sendai virus particles and liposomes were investigated with an assay for lipid mixing based on the relief of self-quenching of fluorescence. The measurements, which were carried out at pH 7.4 and 5.0, included liposomes of three compositions, cardiolipin (CL), CL/dioleoylphosphatidylcholine (CL/DOPC 1:1), and phosphatidylserine (PS). Liposomal lipid concentrations varied from 2.5 to 50 microM. In addition, the effect of low concentrations of the dehydrating agent poly(ethylene glycol) (PEG) on fusion between the virus and the liposomes at pH 7.4 was studied. The results were analyzed in terms of a mass action kinetic model which views the overall fusion reaction as a sequence of a second-order process of virus-liposome adhesion or aggregation, followed by the first-order fusion reaction itself. The fusion products were shown to consist of a single virus particle and several liposomes. Analytical solutions were found for the final extent of fusion and increase in fluorescence intensity following the fusion of fluorescently labeled virus particles with liposomes. The final extents of fluorescence intensity were explained by assuming an essentially irreversible binding of liposomes to inactive virus particles. The percents of active virus particles and the rate constants of fusion and aggregation were larger at pH 5 than at pH 7.4, increased when PEG was included in the medium, and varied with liposomal lipid composition according to the sequence CL greater than CL/DOPC greater than PS.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Polyamines as modulators of membrane fusion: aggregation and fusion of liposomes

We have studied the effect of the polyamines (spermine, spermidine, and putrescine) on the aggregation and fusion of large (approximately 100 nm in diameter) unilamellar liposomes in the presence of 100 mM NaCl, pH 7.4. Liposome fusion was monitored by the Tb/dipicolinic acid fluorescence assay for the intermixing of internal aqueous contents, and the release of contents was followed by carboxyfluorescein fluorescence. Spermine and spermidine at physiological concentrations aggregated liposomes composed of pure phosphatidylserine (PS) or phosphatidate (PA) and mixtures of PA with phosphatidylcholine (PC) but did not induce any fusion. However, liposomes composed of mixtures of acidic phospholipids, cholesterol, and a high mole fraction of phosphatidylethanolamine could be induced to fuse by spermine and spermidine in the absence of divalent cations. Putrescine alone in the physiological concentration range was ineffective for both aggregation and fusion of these liposomes. Liposomes made of pure PC did not aggregate in the presence of polyamines. Addition of aggregating concentrations of spermine caused a drastic increase in the rate of Ca(2+)-induced fusion of PA liposomes and a large decrease in the threshold Ca(2+) concentration required for fusion. This effect was less pronounced in the case of PS or PA/PC vesicles. Preincubation of PA vesicles with spermine before the addition of Ca(2+) resulted in a 30-fold increase in the initial rate of fusion. We propose that polyamines may be involved in the regulation of membrane fusion phenomena accompanying cell growth, cell division, exocytosis, and fertilization.     

山莨菪碱促进心磷脂脂质体膜融合

前文曾报道,山莨菪碱能诱发心磷脂脂质体形成六角形Ⅱ结构,本文采用荧光共振能量转移技术研究了山莨菪碱对心磷脂脂体融合的影响,实验结果表明,山莨菪碱能诱发心磷脂脂质体融合并能促进由C_2~(2+)诱发的融合速率.     

Conformationally changed cytochrome c-mediated fusion of enzyme- and substrate-containing liposomes.

The fusion between enzyme-containing liposomes and substrate-containing liposomes was studied, utilizing conformationally altered cytochrome c as fusion mediator under stress conditions. The liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and liposome aggregation and subsequent liposome fusion were induced by the addition of cytochrome c, which was partially denatured by 0.5 M guanidinium hydrochloride (GuHCl). In the presence of 0.5 M GuHCl, cytochrome c was found to have a significantly large local hydrophobicity which was determined with the aqueous two-phase partitioning method. Under these conditions, cytochrome c could efficiently bind to POPC bilayer membranes as quantitatively evaluated by immobilized liposome chromatography (ILC). The retardation of cytochrome c treated with 0, 0.5, and 1 M GuHCl on ILC could be correlated with the corresponding local hydrophobicity of cytochrome c. The enzymatic reaction triggered by liposome fusion involved the proteolytic enzyme alpha-chymotrypsin and its substrate succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-pNA), which were separately trapped in POPC liposomes. Addition of partially denatured cytochrome c (most likely in the molten globule state) to the mixture of enzyme- and substrate-containing liposomes resulted in the release of one of the hydrolysis products, p-nitroaniline, to the outer phase of the fused liposomes, indicating that the enzymatic reaction occurred during the liposome fusion process. Such a coupled fusion-reaction system may have specific advantages over the conventional fusion analysis and may find application as drug delivery system.     

Fusion of artificial lipid membranes induced by synthetic "fusion peptide" of arenaviruses

The fusing capacity of lipid membranes of a synthetic 23-member peptide was studied. This hydrophobic peptide represents an analog of a predicted functional site ("fusion peptide") of the GP2 envelope protein of the Lassa virus (family Arenaviridae). Fusion of small monolayer liposomes was detected by the method of resonance energy transfer between the fluorescent derivatives of the lipid, NBD-PE (donor) and Rd-PE (acceptor). Using this peptide, the pH-dependent fusing activity was found in liposomes having different phospholipid composition. The rate and efficiency of liposome fusion increased with a decrease in pH and the lipid/peptide ratio as well as with a temperature increase. The increase in the ionic strength and Ca2+ concentration in the reaction mixture led to the inhibition of the peptide-induced fusion of liposomes. Neither the phospholipid charge, nor the transmembrane proton gradient of liposomes had any appreciable effect on the kinetics of the peptide-induced fusion. Neutralization of the medium in the course of the fusion reaction sharply decelerated, whereas repeated acidification activated this process. This finding suggests that peptide protonation plays a role in fusion reactions. It was suggested that acidification causes conformational changes in the peptide structure, thus activating the peptide-induced fusion of liposomes. The fusing capacity of the predicted Lassa virus fusion peptide is similar to that of viruses characterized by a pH-dependent step at the initial stages of the viral infection.     

The Role of the Target Membrane Structure in Fusion with Sendai Virus

Fusion between membranes of Sendai virus and liposomes or human erythrocytes ghosts was studied using an assay for lipid mixing based on the relief of self-quenching of octadecylrhodamine (R18) fluorescence. We considered only viral fusion that reflects the biological activity of the viral spike glycoproteins. The liposomes were made of phosphatidylcholine, and the effects of including cholesterol, the sialoglycolipid GD1a, and/or the sialoglycoprotein glycophorin as receptors were tested. Binding of Sendai virus to those liposomes at 37 ?C was very weak. Fusion with the erythrocyte membranes occurred at a 30-fold faster rate than with the liposomes. Experiments with biological and liposomal targets of different size indicated that size did not account for differences in fusion efficiency.     

The role of the target membrane structure in fusion with Sendai virus

Fusion between membranes of Sendai virus and liposomes or human erythrocytes ghosts was studied using an assay for lipid mixing based on the relief of self-quenching of octadecylrhodamine (R18) fluorescence. We considered only viral fusion that reflects the biological activity of the viral spike glycoproteins. The liposomes were made of phosphatidylcholine, and the effects of including cholesterol, the sialoglycolipid GD1a, and/or the sialoglycoprotein glycophorin as receptors were tested. Binding of Sendai virus to those liposomes at 37 degrees C was very weak. Fusion with the erythrocyte membranes occurred at a 30-fold faster rate than with the liposomes. Experiments with biological and liposomal targets of different size indicated that size did not account for differences in fusion efficiency.     

Low-pH-Dependent Fusion of Sindbis Virus with Receptor-Free Cholesterol- and Sphingolipid-Containing Liposomes

There is controversy as to whether the cell entry mechanism of Sindbis virus (SIN) involves direct fusion of the viral envelope with the plasma membrane at neutral pH or uptake by receptor-mediated endocytosis and subsequent low-pH-induced fusion from within acidic endosomes. Here, we studied the membrane fusion activity of SIN in a liposomal model system. Fusion was followed fluorometrically by monitoring the dilution of pyrene-labeled lipids from biosynthetically labeled virus into unlabeled liposomes or from labeled liposomes into unlabeled virus. Fusion was also assessed on the basis of degradation of the viral core protein by trypsin encapsulated in the liposomes. SIN fused efficiently with receptor-free liposomes, consisting of phospholipids and cholesterol, indicating that receptor interaction is not a mechanistic requirement for fusion of the virus. Fusion was optimal at pH 5.0, with a threshold at pH 6.0, and undetectable at neutral pH, supporting a cell entry mechanism of SIN involving fusion from within acidic endosomes. Under optimal conditions, 60 to 85% of the virus fused, depending on the assay used, corresponding to all of the virus bound to the liposomes as assessed in a direct binding assay. Preincubation of the virus alone at pH 5.0 resulted in a rapid loss of fusion capacity. Fusion of SIN required the presence of both cholesterol and sphingolipid in the target liposomes, cholesterol being primarily involved in low-pH-induced virus-liposome binding and the sphingolipid catalyzing the fusion process itself. Under low-pH conditions, the E2/E1 heterodimeric envelope glycoprotein of the virus dissociated, with formation of a trypsin-resistant E1 homotrimer, which kinetically preceded the fusion reaction, thus suggesting that the E1 trimer represents the fusion-active conformation of the viral spike.     

Fusogenic activity of PEGylated pH-sensitive liposomes

The aim of this study was to investigate the fusogenic properties of poly(ethylene glycol) (PEG)ylated dioleoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) liposomes. These pH-sensitive liposomes were prepared by incorporating two different PEG lipids: distearoylphosphatidylethanolamine (DSPE)-PEG???? was mixed with the liposomal lipids using the conventional method, whereas sterol-PEG???? was inserted into the outer monolayer of preformed vesicles. Both types of PEGylated liposomes were characterized and compared for their entrapment efficiency, zeta potential and size, and were tested in vitro for pH sensitivity by means of proton-induced leakage and membrane fusion activity. To mimic the routes of intracellular delivery, fusion between pH-sensitive liposomes and liposomes designed to simulate the endosomal membrane was studied. Our investigations confirmed that DOPE/CHEMS liposomes were capable of rapidly releasing calcein and of fusing upon acidification. However, after incorporation of DSPE-PEG???? or sterol-PEG???? into the membrane, pH sensitivity was significantly reduced; as the mol ratio of PEG-lipid was increased, the ability to fuse was decreased. Comparison between two different PEGylated pH-sensitive liposomes showed that only vesicles containing 0.6 mol% sterol-PEG???? in the outer monolayer were still capable of fusing with the endosome-like liposomes and showing leakage of calcein at pH 5.5.     

Low pH fusion of mouse liver nuclei with liposomes bearing covalently bound lysozyme

Lysozyme covalently bound to liposomes induces the fusion of liposomes with isolated mouse liver nuclei. The fusion behavior is very similar to the case of erythrocyte ghosts (Arvinte, T., Hildenbrand, K., Wahl, P. and Nicolau, C. (1986) Proc. Natl. Acad. Sci. USA 83, 962-966). Kinetic studies showed that membrane lipid mixing was completed within 15 min, as indicated from the resonance energy transfer (RET) measurements. For the resonance energy transfer kinetic measurements the liposomes contained L-alpha-dipalmitoylphosphatidylethanolamine (DPPE), labeled at the free amino group with the energy donor 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) or with the energy acceptor tetramethylrhodamine. The lipid mixing at equilibrium was studied by the fluorescence recovery after photobleaching technique (FRAP). Liposomes (with/without lysozyme) containing Rh-labeled DPPE in their membranes were incubated with nuclei at 37 degrees C, pH 5.2, for 30 min. After washing of nuclei by three centrifugations, 60-70% of the initial amount of labeled DPPE was associated with the nuclei in the case of liposomes bearing lysozyme and only 7-10% in the case of liposomes without lysozyme. For the nuclei incubated with liposomes having lysozyme, about 70% of the total Rh-labeled lipids present in the nuclei diffused in the nuclear membrane(s) (lateral diffusion constant of D = (1.4 +/- 0.5) X 10(-9) cm2/s). By encapsulating fluorescein isothiocyanate-labeled dextran of 150 kDa molecular mass into the liposomes and using a microfluorimetric method, it was shown that after the fusion a part of the liposome contents is found in the nuclei interior. In this lysozyme-induced fusion process between liposomes and nuclei or erythrocyte ghosts, the binding of lysozyme to the glycoconjugates contained in the biomembranes at acidic pH seems to be the determining step which explains the high fusogenic property of the liposomes bearing lysozyme.     

Optimizing and quantifying fusion of liposomes to mammalian sperm using resonance energy transfer and flow cytometric methods

BACKGROUND: Liposomes are used to carry pharmaceutical agents and to alter the lipid composition of cell membranes. This study compared resonance energy transfer (RET), fluorescence dequenching, and flow cytometry as monitors and quantifiers of fusion between liposomes and mammalian spermatozoa. METHODS: Preliminary experiments used RET to determine the optimum sperm concentration for fusion of DL-alpha-phosphatidylcholine dipalmitoyl (PC)/DL-alpha-phosphatidylethanolamine dipalmitoyl (PE) liposomes at 35 degrees C +/- 5 mM Ca2+. Microscopy confirmed the fusion of liposomes, not just adhesion (n = 3). Dequenching tested the time-dependent fusion of liposomes of two different lipid compositions to sperm, both, (n = 3) +/- 1 mM Ca2+ and (n = 3) without Ca2+ at two sperm concentrations. Finally, flow cytometry absolutely quantified the percentage of sperm fusing to liposomes at different liposome-to-sperm ratios (n = 4) and with sperm from different donors (n = 3). RESULTS: RET detected fusion of liposomes with sperm and microscopy confirmed the interaction to be true fusion. Dequenching detected more fusion of liposomes with sperm at 100 x 10(6) sperm per milliliter than at lower concentrations (P < 0.05). Fusion dynamics differed with lipid composition but Ca2+ had no effect. Flow cytometry reliably quantified the percentage of sperm fusing with liposomes, which varied from bull to bull (P < 0.05). CONCLUSION: Liposome fusion with mammalian sperm membranes can be quantified cytometrically and varies with lipid composition, sperm-to-liposome ratio, and individual animals.     

Interactions between pH-sensitive liposomes and model membranes

The structure and dynamics of two different pH-sensitive liposome systems were investigated by means of cryo-transmission electron microscopy and different photophysical techniques. Both systems consisted of dioleoylphosphatidylethanolamine (DOPE) and contained either oleic acid (OA) or a novel acid-labile polyethylene glycol-conjugated lipid (DHCho-MPEG5000) as stabiliser. Proton induced leakage, lipid mixing and structural changes were studied in the absence and presence of EPC liposomes, as well as in the presence of liposomes designed to model the endosome membrane. Neither DHCho-MPEG5000- nor OA-stabilised liposomes showed any tendency for fusion with pure EPC liposomes or endosome-like liposomes composed of EPC/DOPE/SM/Cho (40/20/6/34 mol.%). Our investigations showed, however, that incorporation of lipids from the pH-sensitive liposomes into the endosome membrane may lead to increased permeability and formation of non-lamellar structures. Taken together the results suggest that the observed ability of DOPE-containing liposomes to mediate cytoplasmic delivery of hydrophilic molecules cannot be explained by a mechanism based on a direct, and non-leaky, fusion between the liposome and endosome membranes. A mechanism involving destabilisation of the endosome membrane due to incorporation of DOPE, seems more plausible.     

Hepatitis C virus glycoproteins mediate low pH-dependent membrane fusion with liposomes

It has been suggested that the hepatitis C virus (HCV) infects host cells through a pH-dependent internalization mechanism, but the steps leading from virus attachment to the fusion of viral and cellular membranes remain uncharacterized. Here we studied the mechanism underlying the HCV fusion process in vitro using liposomes and our recently described HCV pseudoparticles (pp) bearing functional E1E2 envelope glycoproteins. The fusion of HCVpp with liposomes was monitored with fluorescent probes incorporated into either the HCVpp or the liposomes. To validate these assays, pseudoparticles bearing either the hemagglutinin of the influenza virus or the amphotropic glycoprotein of murine leukemia virus were used as models for pH-dependent and pH-independent entry, respectively. The use of assays based either on fusion-induced dequenching of fluorescent probes or on reporter systems, which produce fluorescence when the virus and liposome contents are mixed, allowed us to demonstrate that HCVpp mediated a complete fusion process, leading to the merging of both membrane leaflets and to the mixing of the internal contents of pseudoparticle and liposome. This HCVpp-mediated fusion was dependent on low pH, with a threshold of 6.3 and an optimum at about 5.5. Fusion was temperature-dependent and did not require any protein or receptor at the surface of the target liposomes. Most interestingly, fusion was facilitated by the presence of cholesterol in the target membrane. These findings clearly indicate that HCV infection is mediated by a pH-dependent membrane fusion process. This paves the way for future studies of the mechanisms underlying HCV membrane fusion.     

Liposome composition effects on lipid mixing between cells expressing influenza virus hemagglutinin and bound liposomes

The involvement of contacting and distal lipid monolayers in different stages of protein-mediated fusion was studied for fusion mediated by influenza virus hemagglutinin. Inclusion of non-bilayer lipids in the composition of the liposomes bound to hemagglutinin-expressing cells affects fusion triggered by low pH. Lysophosphatidylcholine added to the outer membrane monolayers inhibits fusion. The same lipid added to the inner monolayer of the liposomes promotes both lipid and content mixing. In contrast to the inverted cone-shaped lysophosphatidylcholine, lipids of the opposite effective shape, oleic acid or cardiolipin with calcium, present in the inner monolayers inhibit fusion. These results along with fusion inhibition by a bipolar lipid that does not support peeling of one monolayer of the liposomal membrane from the other substantiate the hypothesis that fusion proceeds through a local hemifusion intermediate. The transition from hemifusion to the opening of an expanding fusion pore allows content mixing and greatly facilitates lipid mixing between liposomes and cells.     

Effect of amphipathic peptides with different alpha-helical contents on liposome-fusion.

The peptide-induced fusion of neutral and acidic liposomes was studied in relation to the amphiphilicities evaluated by alpha-helical contents of peptides by means of a carboxyfluorescein leakage assay, light scattering, a membrane intermixing assay and electron microscopy. An amphipathic mother peptide, Ac-(Leu-Ala-Arg-Leu)3-NHCH3 (4(3], and its derivatives, [Pro6]4(3) (1), [Pro2,6]4(3) (2), and [Pro2,6,10]4(3) (3), which have very similar hydrophobic moments, caused a leakage of contents from small unilamellar vesicles composed of egg yolk phosphatidylcholine and egg yolk phosphatidic acid (3:1). The abilities of the peptides to induce the fusion of the acidic liposomes increased with increasing alpha-helical content: in acidic liposomes the helical contents were in the order of 4(3) greater than 1 greater than 2 greater than 3 (Lee et al. (1989) Chem. Lett., 599-602). Electron microscopic data showed that 1 caused a transformation of the small unilamellar vesicles (20-50 nm in diameter) to large ones (100-300 nm). Based on the fact that these peptides have very similar hydrophobic moments despite of decreasing in the mean residue hydrophobicities to some extent, it was concluded that the abilities of the peptides to induce the fusion of liposomes depend on the extent of amphiphilic conformation evaluated by alpha-helical contents of the peptides in the presence of liposomes. For neutral liposomes of egg yolk phosphatidylcholine, all the proline-containing peptides showed no fusogenic ability but weak leakage abilities, suggesting that the charge interaction between the basic peptides and acidic phospholipid is an important factor to induce the perturbation and fusion of the bilayer.     

Heterogeneity of early intermediates in cell-liposome fusion mediated by influenza hemagglutinin

To explore early intermediates in membrane fusion mediated by influenza virus hemagglutinin (HA) and their dependence on the composition of the target membrane, we studied lipid mixing between HA-expressing cells and liposomes containing phosphatidylcholine (PC) with different hydrocarbon chains. For all tested compositions, our results indicate the existence of at least two types of intermediates, which differ in their lifetimes. The composition of the target membrane affects the stability of fusion intermediates at a stage before lipid mixing. For less fusogenic distearoyl PC-containing liposomes at 4 degrees C, some of the intermediates inactivate, and no intermediates advance to lipid mixing. Fusion intermediates that formed for the more fusogenic dioleoyl PC-containing liposomes did not inactivate and even yielded partial lipid mixing at 4 degrees C. Thus, a more fusogenic target membrane effectively blocks nonproductive release of the conformational energy of HA. Even for the same liposome composition, HA forms two types of fusion intermediates, dissimilar in their stability and propensity to fuse. This diversity of fusion intermediates emphasizes the importance of local membrane composition and local protein concentration in fusion of heterogeneous biological membranes.