Effects of sphingomyelin on melittin pore formation

The effect of sphingomyelin (SM), one of the main lipids in the external monolayer of erythrocyte plasma membrane, on the ability of the hemolytic peptide melittin to permeabilize liposomes was investigated. The peptide induced contents efflux in large unilamellar vesicles (LUV) composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/SM (1:1 mole ratio), at lower (>1:10,000) peptide-to-lipid mole ratios than in pure POPC (>1:1000) or POPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) (1:1 mole ratio) (>1:300) vesicles. Analysis of the leakage data according to a kinetic model of pore formation showed a good fit for hexameric-octameric pores in SM-containing vesicles, whereas mediocre fits and lower surface aggregation constants were obtained in POPC and POPC/POPG vesicles. Disturbance of lateral separation into solid (s_o) and liquid-disordered (l_d) phases in POPC/SM mixtures increased the peptide-dose requirements for leakage. Inclusion of cholesterol (Chol) in POPC/SM mixtures under conditions inducing lateral separation of lipids into liquid-ordered (l_o) and l_d phases did not alter the number of melittin peptides required to permeabilize a single vesicle, but increased surface aggregation reversibility. Partitioning into liposomes or insertion into lipid monolayers was not affected by the presence of SM, suggesting that: (i) melittin accumulated at comparable doses in membranes with different SM content, and (ii) differences in leakage were due to promotion of melittin transmembrane pores under coexistence of s_o-l_d and l_o-l_d phases. Our results support the notion that SM may regulate the stability of size-defined melittin pores in natural membranes.     

On the mechanism of pore formation by melittin

The mechanism of pore formation of lytic peptides, such as melittin from bee venom, is thought to involve binding to the membrane surface, followed by insertion at threshold levels of bound peptide. We show that in membranes composed of zwitterionic lipids, i.e. phosphatidylcholine, melittin not only forms pores but also inhibits pore formation. We propose that these two modes of action are the result of two competing reactions: direct insertion into the membrane and binding parallel to the membrane surface. The direct insertion of melittin leads to pore formation, whereas the parallel conformation is inactive and prevents other melittin molecules from inserting, hence preventing pore formation.     

Kinetics of melittin induced pore formation in the membrane of lipid vesicles.

We have investigated the permeabilization of POPC unilamellar vesicle bilayers upon the addition of melittin. This process was measured in an early time range of a few minutes by means of monitoring the release of an entrapped marker, the self-quenching fluorescent dye carboxyfluorescein. Pore formation is indicated by an apparent 'all-or-none' efflux out of individual vesicles and a higher than linear dependence on melittin concentration. Applying a recently developed evaluation procedure, the data are readily converted into the gross number of pores per vesicle formed within the elapsed measuring time t. The results can be generally described in terms of a fast initial rate of pore formation that slows down to a much lower value after a period of about 1 to 2 minutes, following a single exponential time course. The three rate parameters involved are shown to be power functions of the concentration of melittin that is actually associated with the vesicle membrane. These findings are in excellent quantitative agreement with a proposed scheme of reaction steps where the formation of lipid associated peptide dimers becomes rate determining once an initial fast deposit is exhausted.     

Free energy analysis of membrane pore formation process in the presence of multiple melittin peptides

Understanding the molecular mechanism underlying pore formation in lipid membranes by antimicrobial peptides is of great importance in biological sciences as well as in drug design applications. Melittin has been widely studied as a pore forming peptide, though the molecular mechanism for pore formation is still illusive. We examined the free energy barrier for the creation of a pore in lipid membranes with and without multiple melittin peptides. It was found that six melittin peptides significantly stabilized a pore, though a small barrier (a few k_BT) for the formation still existed. With five melittin peptides or fewer, the pore formation barrier was much higher, though the established pore was in a local energy minimum. Although seven melittins effectively reduced the free energy barrier, a single melittin peptide left the pore after a long time MD simulation probably because of the overcrowded environment around the bilayer pore. Thus, it is highly selective for the number of melittin peptides to stabilize the membrane pore, as was also suggested by the line tension evaluations. The free energy cost required to insert a single melittin into the membrane is too high to explain the one-by-one insertion mechanism for pore formation, which also supports the collective melittin mechanism for pore formation.     

A comparative study of the action of melittin on sphingomyelin and phosphatidylcholine bilayers

To investigate whether lipid solubilization is of relevance in describing the interaction between melittin and biological membranes, we studied melittin-induced polymorphism using model membranes composed of the biological lipid sphingomyelin (bovine brain). The behavior of the system was monitored by solid state ³¹P-NMR and turbidity measurements and compared to the peptides well-characterized action on the synthetic lipid dipalmitoylphosphatidylcholine. It was found that melittin-induced macroscopic changes of sphingomyelin membranes are qualitatively the same as in the case of dipalmitoylphosphatidylcholine bilayers. The sphingomyelin/melittin system is thus proposed to show a reversible vesicle-to-disc transition (fluid-to-gel phase) through an intermediate fusion or aggregation event centered at the main transition temperature, T_m, as reported in the case of saturated phosphatidylcholine. In the case of spontaneous disc formation at 37 °C, the lipid-to-peptide molar ratio in the discoidal objects was determined to be approximately 20 for dipalmitoylphosphatidylcholine and about 12 in the case of natural sphingomyelin. Melittin partition coefficients between membranes and the aqueous medium at 37 °C were found to be 6.1±0.8 mm ^–1 and 3.7±0.4 mm ^–1 for sphingomyelin and dipalmitoylphosphatidylcholine, respectively. For very high peptide quantities (lipid-to-peptide molar ratio, R_i≤5) mixed micelles are formed over the entire temperature range (20° to 60 °C) for both kinds of lipids.     

The single-giant unilamellar vesicle method reveals lysenin-induced pore formation in lipid membranes containing sphingomyelin

Lysenin is a sphingomyelin (SM)-binding pore-forming toxin. To reveal the interaction of lysenin with lipid membranes, we investigated lysenin-induced membrane permeation of a fluorescent probe, calcein, through dioleoylphosphatidylcholine(DOPC)/SM, DOPC/SM/cholesterol(chol), and SM/chol membranes, using the single-giant unilamellar vesicle (GUV) method. The results clearly show that lysenin formed pores in all the membranes, through which membrane permeation of calcein occurred without disruption of GUVs. The membrane permeation began stochastically, and the membrane permeability coefficient increased over time to reach a maximum, steady value, Ps, which persisted for a long time(100--500 s), indicating that the pore concentration increases over time and finally reaches its steady value, NP s . The Ps values increased as the SM/lysenin ratio decreased, and at low concentrations of lysenin, the Ps values of SM/DOPC/chol (42/30/28)GUVs were much larger than those of SM/DOPC (58/42) GUVs. The dependence of Ps on the SM/lysenin ratio for these membranes was almost the same as that of the fraction of sodium dodecyl sulfate (SDS)-resistant lysenin oligomers, indicating that NP s increases as the SDS-resistant oligomer fraction increases. On the other hand, lysenin formed pores in GUVs of SM/chol(60/40) membrane, which is in a homogeneous liquid-ordered phase, indicating that the phase boundary is not necessary for pore formation. The Ps values of SM/chol (60/40) GUVs were smaller than those of SM/DOPC/chol (42/30/28) GUVs even though the SDS-resistant oligomer fractions were similar for both membranes, suggesting that not all of the oligomers can convert into a pore. On the basis of these results, we discuss the elementary processes of lysenin-induced pore formation.     

Effects of chemical modification of sphingomyelin ammonium group on formation of liquid-ordered phase

Sphingomyelin (SM) and cholesterol form microdomains called lipid rafts in cellular membranes. To develop a versatile fluorescent lipid probe, chemical modifications to both the hydrophobic and hydrophilic portions of SM are essential. Few reports describing SM probes with a fluorophore at the polar head group have been published. This study examined the effect of substitution on an ammonium moiety of SM on the membrane properties of SM. Two SM analogs with small propargyl and allyl groups on the quaternary nitrogen atom were synthesized and subjected to analysis using differential scanning calorimetry, fluorescent anisotropy, detergent solubilization, surface pressure, and density measurements. Results demonstrated that the two SM analogs retained the membrane properties of SM, including formation of an ordered phase and the ability to interact with cholesterol. A dansyl-substituted SM was prepared for fluorescent measurements. Dansyl-SM showed less of a propensity to form microdomains. These findings imply the potential application of N-substituted SMs as a raft-specific molecular probe.     

Sizing membrane pores in lipid vesicles by leakage of co-encapsulated markers: pore formation by melittin.

Many toxins and antimicrobial peptides permeabilize membrane vesicles by forming multimeric pores. Determination of the size of such pores is an important first step for understanding their structure and the mechanism of their self-assembly. We report a simple method for sizing pores in vesicles based on the differential release of co-encapsulated fluorescently labeled dextran markers of two different sizes. The method was tested using the bee venom peptide melittin, which was found to form pores of 25-30 A diameter in palmitoyloleoylphosphatidylcholine (POPC) vesicles at a lipid-to-peptide ratio of 50. This result is consistent with observations on melittin pore formation in erythrocytes (Katsu, T., C. Ninomiya, M. Kuroko, H. Kobayashi, T. Hirota, and Y. Fujita 1988. Action mechanism of amphipathic peptides gramicidin S and melittin on erythrocyte membrane Biochim. Biophys. Acta. 939:57-63).     

Differential localization of sphingomyelin synthase isoforms in neurons regulates sphingomyelin cluster formation

Sphingomyelin (SM) plays important roles in regulating structure and function of plasma membrane, but how intracellular localization of SM is regulated in neuronal cells is not understood. Here we show that two isoforms of SM synthase (SMS) are differentially expressed in neuronal subtypes and that only SMS2 proteins localize in neurites of hippocampal neurons. Moreover, SMS proteins induce Lysenin-binding SM clusters exclusively in their vicinity although neurons hardly contain such cluster under control condition. These findings indicate three important notions about SM metabolism in neurons. First, the activity of SMS is the rate-limiting step of SM cluster formation. Second, the SM content or clustering can be modulated by SMS activity. Third, SMS1 and SMS2 play distinct roles in regulating local SM clustering. Particularly, SMS2, rather than SMS1, is likely to be the major enzyme that is important for SM synthesis in the long neurites and its tip, the growth cone.     

Pore formation and translocation of melittin.

Melittin, a bee venom, is a basic amphiphilic peptide, which mainly acts on the lipid matrix of membranes, lysing various cells. To elucidate the molecular mechanism, we investigated its interactions with phospholipid vesicles. The peptide formed a pore with a short lifetime in the membrane, as revealed by the release of an anionic fluorescent dye, calcein, from the liposomes. Our new double-labeling method clarified that the pore size increased with the peptide-to-lipid ratio. Upon the disintegration of the pore, a fraction of the peptides translocated across the bilayer. The pore formation was coupled with the translocation, which was proved by three fluorescence experiments recently developed by our laboratory. A novel model for the melittin pore formation was discussed in comparison with other pore-forming peptides.     

Investigation of toroidal pore and oligomerization by melittin using transmission electron microscopy

We studied the effects of melittin on various cell wall components and vesicles of various lipid compositions. To interact with the cytoplasmic membrane, melittin must traverse the cell wall, which is composed of oligosaccharides. Here, we found that melittin had a strong affinity for chitin, peptidoglycan, and lipopolysaccharide. We further examined the influence of lipid compositions on the lysis of the membranes by melittin. The result showed that melittin bound better to negatively charged than to zwitterionic lipid vesicles but was more potent at inducing leakage from zwitterionic lipid vesicles. Our studies further indicated that the oligomeric state of melittin varied between tetramers and octamers during the formation of toroidal pores. Dextran leakage experiments confirmed the formation and dimension of these toroidal pores. Finally, transmission electron microscopy revealed that melittin formed pores via peptide oligomerization by the toroidal pore-forming mechanism. The toroidal pores composed of 7-8 nm diameter rings that encircled 3.5-4.5 nm diameter cavities on zwitterionic lipid vesicles.     

Effects of peptide dimerization on pore formation: Antiparallel disulfide-dimerized magainin 2 analogue

To elucidate the effects of peptide dimerization on pore formation by magainin 2 (MG2), a covalently linked antiparallel dimer of the MG2 analogue [(F5Y, L6C, F16W, I20C-MG2)(2): II] was synthesized based on the dimer structure revealed by our NMR study. The interactions of the dimer with lipid bilayers were investigated by CD and fluorescence in comparison with a monomer analogue (F5Y, F16W-MG2: I). Similar to I, II was found to form a peptide-lipid supramolecular complex pore accompanied with lipid flip-flop and peptide translocation. The pore formed by II was characterized by a slightly larger pore diameter and a threefold longer lifetime than that of I, although the pore formation rate of the dimer was lower than that of the monomer. The coexistence of the dimer and the monomer exhibited slight but significant synergism in membrane permeabilization, which was maximal at a monomer/dimer ratio of 3. Therefore, we concluded that a pentameric pore composed of one pore-stabilizing dimer and three monomers maximized the overall leakage activity in keeping with our kinetic prediction.     

Effects of terbium on the hemocyanin pore formation rate in phosphatidylcholine planar bilayers

Megathura crenulata hemocyanin forms ionic channels in planar lipid bilayer membranes. It was found that hemocyanin is more potent as a channel former if TbCl₃ is added to the bathing solution. Furthermore membranes separating symmetrical TbCl₃ solutions show a pore formation rate which depends exponentially on the applied voltage, positive potentials favouring the insertion of new channels. The slope of this voltage dependence, which gives a measure of the effective charge displaced during the incorporation of one channel, increases and saturates with TbCl₃ concentration. The dose response curve indicates that binding of Tb³⁺ to the phosphatidylcholine bilayer is involved in creating the effective charge.     

Lipid‐dependent pore formation by antimicrobial peptides arenicin‐2 and melittin demonstrated by their proton transfer activity

This work presents a comparative study of proton transfer activity (PTA) of two cationic (+6) antimicrobial peptides, β‐structural arenicin‐2 and α‐helical melittin. A new approach was proposed for the detection of passive proton transfer by using proteoliposomes containing bacteriorhodopsin, which creates a small light‐induced electrochemical proton gradient ?ΔpH. Addition of several nanomoles of the peptides lowers ?ΔpH that is proximately indicative of the pore formation. The quantitative analysis of sigmoidal dependences of ?pH on the peptides concentration was carried out using liposomes prepared from PC, PC/PE, PC/PE/PI and PC/PG. Substitution of PC‐containing liposomes with PE‐containing ones, having negative spontaneous curvature, reduced the PTA of α‐helical melittin and increased that of β‐structural arenicin‐2. This result indicates an essential difference in the pore formation by these peptides. Further increase of PTA in response to arenicin‐2 (in contrast to melittin) was observed in the liposomes prepared from PC/PE/PI. The data analysis leads to the conclusion that PTA is influenced by (i) efficiency of the pore assemblage, which depends on the structure of pore‐forming peptides, and the spontaneous curvature of lipids and (ii) the presence of mobile protons in the polar head groups of phospholipids. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Stability of a melittin pore in a lipid bilayer: a molecular dynamics study

We have investigated the configuration and the stability of a single membrane pore bound by four melittin molecules and embedded in a fully hydrated bilayer lipid membrane. We used molecular dynamics simulations up to 5.8 ns. It is found that the initial tetrameric configuration decays with increasing time into a stable trimer and one monomer. This continuous transformation is accompanied by a lateral expansion of the aqueous pore exhibiting a final size comparable to experimental findings. The expansion-induced formation of an interface between the pore-lining acyl chains of the lipids and the pore water ("hydrophobic pore") is transformed into an energetically more favorable toroidal pore structure where some lipid heads are translocated from the rim to the central part of the interface ("hydrophilic pore"). The expansion of the pore is supported by the electrostatic repulsion among the alpha-helices. It is hypothesized that pore growth, and hence cell lysis, is induced by a melittin-mediated line tension of the pore.     

Effects of melittin on adipocyte metabolism unrelated to lysophospholipid accumulation

Melittin addition to rat or hamster adipocytes resulted in inhibition of lipolysis, cyclic AMP accumulation and glucose oxidation. Low concentrations of melittin were not insulin-like with respect to either stimulation of glucose metabolism or inhibition of lipolysis. Higher concentrations of melittin lysed adipocytes. In the presence of melittin, cellular phospholipids were released to the medium and hydrolyzed with little accumulation of lysophospholipids. Only in adipocytes incubated with melittin contaminated with phospholipase A2 was any appreciable accumulation of lysophospholipids seen and this was in the medium. These data suggest that the toxic effects of melittin on adipocytes are not due to the accumulation of lysophospholipids but rather to the loss of membrane phospholipids or alterations in membrane proteins.     

Onset of anthrax toxin pore formation

Protective antigen (PA) is the anthrax toxin protein recognized by capillary morphogenesis gene 2 (CMG2), a transmembrane cellular receptor. Upon activation, seven ligand-receptor units self-assemble into a heptameric ring-like complex that becomes endocytozed by the host cell. A critical step in the subsequent intoxication process is the formation and insertion of a pore into the endosome membrane by PA. The pore conversion requires a change in binding between PA and its receptor in the acidified endosome environment. Molecular dynamics simulations totaling approximately 136 ns on systems of over 92,000 atoms were performed. The simulations revealed how the PA-CMG2 complex, stable at neutral conditions, becomes transformed at low pH upon protonation of His-121 and Glu-122, two conserved amino acids of the receptor. The protonation disrupts a salt bridge important for the binding stability and leads to the detachment of PA domain II, which weakens the stability of the PA-CMG2 complex significantly, and subsequently releases a PA segment needed for pore formation. The simulations also explain the great strength of the PA-CMG2 complex achieves through extraordinary coordination of a divalent cation.     

Radiation-induced delay of nuclear pore formation

We have shown that radiation affects the nuclear envelope, a membrane structure-closely associated with DNA. The density of nuclear pores visible on freeze-etch surfaces decreased at a rate of 0.042 (pores/mum2)/100 rad with respect to unirradiated cells. This is interpreted as a radiation-induced delay in development of the nuclear envelope.     

Effects of melittin on lipid-protein interactions in sarcoplasmic reticulum membranes.

To investigate the physical mechanism by which melittin inhibits Ca-adenosine triphosphatase (ATPase) activity in sarcoplasmic reticulum (SR) membranes, we have used electron paramagnetic resonance spectroscopy to probe the effect of melittin on lipid-protein interactions in SR. Previous studies have shown that melittin substantially restricts the rotational mobility of the Ca-ATPase but only slightly decreases the average lipid hydrocarbon chain fluidity in SR. Therefore, in the present study, we ask whether melittin has a preferential effect on Ca-ATPase boundary lipids, i.e., the annular shell of motionally restricted lipid that surrounds the protein. Paramagnetic derivatives of stearic acid and phosphatidylcholine, spin-labeled at C-14, were incorporated into SR membranes. The electronic paramagnetic resonance spectra of these probes contained two components, corresponding to motionally restricted and motionally fluid lipids, that were analyzed by spectral subtraction. The addition of increasing amounts of melittin, to the level of 10 mol melittin/mol Ca-ATPase, progressively increased the fraction of restricted lipids and increased the hyperfine splitting of both components in the composite spectra, indicating that melittin decreases the hydrocarbon chain rotational mobility for both the fluid and restricted populations of lipids. No further effects were observed above a level of 10 mol melittin/mol Ca-ATPase. In the spectra from control and melittin-containing samples, the fraction of restricted lipids decreased significantly with increasing temperature. The effect of melittin was similar to that of decreased temperature, i.e., each spectrum obtained in the presence of melittin (10:1) was nearly identical to the spectrum obtained without melittin at a temperature approximately 5 degrees C lower. The results suggest that the principal effect of melittin on SR membranes is to induce protein aggregation and this in turn, augmented by direct binding of melittin to the lipid, is responsible for the observed decreases in lipid mobility. Protein aggregation is concluded to be the main cause of inactivation of the Ca-ATPase by melittin, with possible modulation also by the decrease in mobility of the boundary layer lipids.