Lipid-polyethylene glycol interactions: II. Formation of defects in bilayers

Summary Polyethylene glycol, a known cell fusogen, is found to induce the formation of structural defects in egg phosphatidylcholine multilamellar vesicles, as shown by freeze-fracture microscopy.³¹P NMR spectra of these vesicles reveal the existence of a nonbilayer (isotropic) phase. The observed disruption in the bilayers is believed to be associated with an intermediate stage of membrane fusion.Abbreviations PEG Polyethylene glycol - IMP Intramembranous particle - PC Phosphatidylcholine - PS Phosphatidylserine - SUV Small unilamellar vesicles - MLV Multilamellar vesicles - DPPC Dipalmitoyl phosphatidylcholine - DSC Differential scanning calorimetry - DMPC Dimyristoylphosphatidylcholine - T _c Phase transition temperature     

Action of polyethylene glycol on the fusion of human erythrocyte membranes

Summary Factors affecting the polyethylene glycol (PEG)-induced membrane fusion were examined. Human erythrocyte membrane ghosts, cytoskeleton-free vesicles budded from erythrocytes, mechanically disrupted erythrocyte vesicles, and recombinant vesicles from glycophorin and egg phosphatidylcholine were used as models. Fusion was monitored by darkfield light microscopy and by freeze-fracture electron microscopy. Osmotic swelling was found necessary for fusion between membrane ghosts following PEG treatment. The sample with the highest fusion percentage was sealed ghosts incubated in hypotonic media after at least 5 min of treatment in <25% PEG. At similar osmolarity, glycerol, dextran and PEG produced progressively more pronounced intramembranous particle (IMP) patching, correlating with their increasing fusion percentages. The patching of IMP preceded cell-cell contact, and occurred without direct PEG-protein interaction. The presence of cytoskeletal elements in small vesicles had no significant effect on fusion, nor on the aggregation of intramembranous particle (IMP) upon PEG treatment. Disrupting the membrane by lysolecithin, dimethylsulfoxide, retinol or mild sonication resulted in the fragmentation of ghosts without an increase in fusion percentage. The purity of the commercial PEG used had no apparent effect on fusion. We concluded that the key steps in PEG-induced fusion of cell membrane are the creation of IMP-free zones, and the osmotic swelling of cells after the formation of bilayer contacts during the PEG treatment. Cell cytoskeleton affects PEG-induced fusion only to the extent of affecting IMP patching.     

Modulation of protein aggregation by polyethylene glycol conjugation: GCSF as a case study

Polyethylene glycol (PEG) conjugation to proteins has emerged as an important technology to produce drug molecules with sustained duration in the body. However, the implications of PEG conjugation to protein aggregation have not been well understood. In this study, conducted under physiological pH and temperature, N-terminal attachment of a 20 kDa PEG moiety to GCSF had the ability to (1) prevent protein precipitation by rendering the aggregates soluble, and (2) slow the rate of aggregation relative to GCSF. Our data suggest that PEG-GCSF solubility was mediated by favorable solvation of water molecules around the PEG group. PEG-GCSF appeared to aggregate on the same pathway as that of GCSF, as evidenced by (a) almost identical secondary structural transitions accompanying aggregation, (b) almost identical covalent character in the aggregates, and (c) the ability of PEG-GCSF to rescue GCSF precipitation. To understand the role of PEG length, the aggregation properties of free GCSF were compared to 5kPEG-GCSF and 20kPEG-GCSF. It was observed that even 5kPEG-GCSF avoided precipitation by forming soluble aggregates, and the stability toward aggregation was vastly improved compared to GCSF, but only marginally less stable than the 20kPEG-GCSF. Biological activity measurements demonstrated that both 5kPEG-GCSF and 20kPEG-GCSF retained greater activity after incubation at physiological conditions than free GCSF, consistent with the stability measurements. The data is most compatible with a model where PEG conjugation preserves the mechanism underlying protein aggregation in GCSF, steric hindrance by PEG influences aggregation rate, while aqueous solubility is mediated by polar PEG groups on the aggregate surface.     

The use of fluorescence energy transfer to distinguish between poly(ethylene glycol)-induced aggregation and fusion of phospholipid vesicles

Two fluorescence energy transfer assays for phospholipid vesicle-vesicle fusion have been developed, one of which is also sensitive to vesicle aggregation. Using a combination of these assays it was possible to distinguish between vesicle aggregation and fusion as induced by poly(ethylene glycol) PEG 8000. The chromophores used were 1-(4′-carboxyethyl)-6-diphenyl-trans-1,3,5-hexatriene as fluorescent ‘donor’ and 1-(4′-carboxyethyl)-6-(4″-nitro)diphenyl-trans-1,3,5-hexatriene as ‘acceptor’. These acids were appropriately esterified giving fluorescent phospholipid and triacylglycerol analogues. At 20°C poly(ethylene glycol) 8000 (PEG 8000) caused aggregation of l-α-dipalmitoylphosphatidylcholine (DPPC) vesicles without extensive fusion up to a concentration of about 35% (w/w). Fusion occurred above this poly(ethylene glycol) concentration. The triacylglycerol probes showed different behaviour from the phospholipids: while not exchangeable through solution in the absence of fusogen, they appeared to redistribute between bilayers under aggregating conditions. DPPC vesicles aggregated with < 35% poly(ethylene glycol) could not be disaggregated by dilution, as monitored by the phospholipid probes. However, DPPC vesicles containing approx. 5% phosphatidylserine which had been aggregated by poly(ethylene glycol) could be disaggregated by either dilution or sonication. Phospholipid vesicles aggregated by low concentrations of poly(ethylene glycol) appear to fuse to multilamellar structures on heating above the lipid phase transition temperature.     

Effects of poly(ethylene glycol) on liposomes and erythrocytes: Permeability changes and membrane fusion

Poly(ethylene glycol) 6000 induced a concentration-dependent, time-dependent decrease in the latency of the reaction between Arsenazo III sequestered in liposomes and extraliposomal Ca²⁺. This was mediated by a gross change in liposomal permeability, i.e. by a release of Arsenazo III from liposomes rather than simply by an entry of Ca²⁺. The loss of latency was strongly temperature-dependent, and it was markedly diminished on increasing the cholesterol content of the liposomes. It was apparently not due to an osmotic stress of the polymer. The high activation energy found (63 kJ · mol^?1) is thought to indicate that the loss of latency resulted from local discontinuities in the lipid bilayers, caused by dehydration, rather than from partial or total lysis. Related microscopy experiments indicated that the polymer also caused the liposomes to fuse, and it is suggested that membrane fusion may have occurred at the sites of dehydration-induced discontinuities in adjacent bilayers, in addition the polymer was found to enhance the permeability of hen erythrocytes to Ca²⁺ in a manner that was comparable to its effect on liposomal latency, and it is proposed that cell fusion induced by poly(ethylene glycol) may occur at the sites of similarly induced discontinuities in the phospholipid bilayers of two closely adjacent cells.     

Alterations in phospholipid polymorphism by polyethylene glycol

Summary The fusogen polyethylene glycol is shown to alter the polymorphism of dimyristoyl phosphatidylcholine, soybean phosphatidylethanolamine, bovine phosphatidylserine, egg phosphatidylcholine/cholesterol mixture, dilinoleoylphosphatidylethanolamine/palmitoyl-oleoylphosphatidylcholine mixture, and egg lysolecithin. Suspension of these lipids in 50% polyethylene glycol (mol wt=6000) reduces both the lamellar and the hexagonal II repeat spacings as measured by X-ray diffraction. An increase in the gel to liquid crystalline and bilayer to hexagonal transition temperatures are observed by freeze-fracture, X-ray diffraction, differential scanning calorimetry and³¹P NMR. Freeze-fracture electron micrographs revealed different bilayer defects depending on the physical states of the lipid. Lipidic particles in mixtures containing unsaturated phosphatidylethanolamine is eliminated. Some of the influences of polyethylene glycol on lipids may be explained by its dehydrating effect. However, other nonfusogenic dehydrating agents failed to produce similar results. These findings are consistent with the proposal that close bilayer contact and the formation of bilayer defects are associated with the fusogenic properties of polyethylene glycol.     

Secondary structure, phospholipid membrane interactions, and fusion activity of two glutamate-rich analogs of influenza hemagglutinin fusion peptide

Two synthetic mutants of influenza HA2 fusion peptide (residues 1-25), containing Glu on the polar (residues 4,8-E5(4,8)) or the hydrophobic (residues 3,7-E5(3,7)) face of the amphipathic helix, were synthesized and labeled with NBD at the N-terminus. Introduction of Glu residues into the fusion peptide leads to increased sensitivity of various biochemical properties to pH compared to the wild type. The E5 peptides showed a decrease of alpha-helix content and increase of beta-sheet structure. Lipid binding was diminished, but not abolished even at high pH. The E5 analogs penetrate the lipid bilayer less deeply than the wild type, especially at high pH. The N-terminal half of the peptide showed significant variation of the depth of the penetration into the lipid bilayer. Both E5 peptides were fusion active. The properties of E5(3,7) were more affected by the Glu substitution and showed greater variation with pH than E5(4,8).     

Ca2+-independent,protein-mediated fusion of chromaffin granule ghosts with liposomes

We have investigated the interaction between isolated membrane vesicles from chromaffin granules and large unilamellar phospholipid vesicles (liposomes). Mixing of membrane lipids has been monitored continuously, utilizing the fluorescence resonance energy transfer assay described by Struck et al. ((1982) Biochemistry 20, 4093–4099). To demonstrate coalescence of the internal vesicle volumes the transfer of colloidal gold from the liposomes to the interior of the granule membrane vesicles has been examined. Efficient fusion of the liposomes with the granule membranes was observed. Significant fusion occurred in the absence of Ca²⁺, although the extent of interaction was enhanced in its presence. The sensitivity of the interaction to pretreatment of the granule membranes with trypsin showed the fusion reaction to be a protein-mediated process.     

Interaction of acidic liposomes with red blood cells: Induction of endocytosis and shedding of particles

Calcium ions induced tight binding of massive amounts of liposomes containing cardiolipin, phosphatidylethanolamine and phosphatidylcholine to erythrocytes. Initially, liposome-liposome fusion occurred and only subsequently the resulting large structures adhered to cells. Large clumps composed of liposomes and cells were formed. Upon prolonged incubation, the clumps were dissipated spontaneously and excess liposomes were released. A constant amount of phospholipid (15–25 nmol/10⁸ cells) were incorporated into the cell membranes. Upon disaggregation, the cells shed erythrocyte particles. The latter were isolated and shown to contain lipids from both cellular and liposomal origin. The particles lacked spectrins and contained variable amounts of band 3 content. Liposomes induced endocytosis in reticulocytes but not in mature erythrocytes. In most cases, the liposomes themselves were not engulfed by the cells and remained attached to their surface. The relevance of this phenomenon to delivery of liposome contents into cells is discussed.     

Aggregation of ligand-modified liposomes by specific interactions with proteins. I: Biotinylated liposomes and avidin

The aggregation of biotin-modified phospholipid vesicles (liposomes) induced by binding the protein avidin in solution is analyzed experimentally and theoretically. Avidin has four binding sites that can recognize biotin specifically, and is able to cross-link the liposomes to form large aggregates. The aggregation kinetics were followed using quasi-elastic light scattering (QLS) to measure the mean particle size, and by measuring the solution turbidity. The rate and extent of aggregation were determined as a function of vesicle concentration, protein concentration, and the biotin density on the surface of the liposomes. A model based on Smoluchowski kinetics, fractal concepts, and Rayleigh and Mie light scattering theory was developed to analyze the experimental observations. Small aggregates (<7800 A diameter) may be treated as globular; however, the fractal nature of larger particles must be taken into account. Parameters in the model are taken from molecular simulations, or fit to the experimental observations. The aggregation kinetics are primarily determined by the biotin density on the liposome surface, the stoichiometric ratio of avidin molecules to liposomes, and the liposome concentration. Good agreement is found between the model and the experimental results. (c) 1996 John Wiley & Sons, Inc.     

Association of bacteriorhodopsin with lipid-impregnated filters. Evidence for fusion of bacteriorhodopsin-containing vesicles with the lipid phase of the filter

Bacteriorhodopsin vesicles were associated with cellulose-nitrate filters impregnated with a solution of phospholipids in hexadecane. The generation of (photo)potentials upon illumination of the filter was studied in the absence and presence of ionophores, phospholipase A₂, EDTA or polyene antibiotics.From these experiments the following conclusions are drawn.

1. 1. Upon illumination of the filter, bacteriorhodopsin pumps protons into aqueous compartments located in the filter.

2. 2. These aqueous compartments possibly do not originate from the compartments enclosed by the bacteriorhodopsin vesicles. Evidence is obtained that aqueous compartments are present in the surface layers of the lipid-impregnated filters.

3. 3. The results are explained most easily by a mechanism, whereby fusion occurs between the vesicles and the lipids of the filter.

Depletion interactions and modulation of DNA‐intercalators binding: Opposite behavior of the “neutral” polymer poly(ethylene‐glycol)

In this work we have investigated the role of high molecular weight poly(ethylene‐glycol) 8000 (PEG 8000) in modulating the interactions of the DNA molecule with two hydrophobic compounds: Ethidium Bromide (EtBr) and GelRed (GR). Both compounds are DNA intercalators and are used here to mimic the behavior of more complex DNA ligands such as chemotherapeutic drugs and proteins whose domains intercalate DNA. By means of single‐molecule stretching experiments, we have been able to show that PEG 8000 strongly shifts the binding equilibrium between the intercalators and the DNA even at very low concentrations (1% in mass). Additionally, microcalorimetry experiments were performed to estimate the strength of the interaction between PEG and the DNA ligands. Our results suggest that PEG, depending on the system under study, may act as an “inert polymer” with no enthalpic contribution in some processes but, on the other hand, it may as well be an active (non‐neutral) osmolyte in the context of modulating the activity of the reactants and products involved in DNA‐ligand interactions. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 227–233, 2016.     

Utilization of Polyphenyl and Polyphenyl-related Compounds by Microorganisms. Part I

Two hundreds and fifty eight strains of microorganisms have been isolated from 526 samples (soil, leaf and river water gathered from 17 prefectures) by repeating liquid enrichment culture techniques in the medium containing biphenyl, diphenylmethane, diphenylethane or terphenyl, as the sole source of carbon.

In the course of investigation, several strains were found to produce a large amount of γ-benzoylbutyric acid from biphenyl. Furthermore these strains utilized p-Cl-biphenyl and produced p-Cl-benzoic acid in good yield.

Microorganisms obtained were almost short rod, motile bacteria, and fungi were also found from the screening medium of diphenylethane.     

Interactions between PEG and type I soluble tumor necrosis factor receptor: modulation by pH and by PEGylation at the N terminus

The effects of polyethylene glycol (PEG) on protein structure and the molecular details that regulate its association to polypeptides are largely unknown. These issues were addressed using type I soluble tumor necrosis factor receptor (sTNF-RI) as a model system. Changes in solution viscosity established that a truncated form of sTNF-RI bound free PEG in a pH-dependent manner. Above pH 5.3, the viscosity escalated as the pH increased, while no effect occurred below pH 5.0. Conjugation of 2 kD, 5 kD, or 20 kD PEG to the N terminus attenuated the viscosity at the higher pH values. Tryptophan phosphorescence spectroscopy correlated changes in the protein structure about Trp-107, at the C terminus, with the pH-dependent and PEGylation-dependent attenuation of the viscosity. The results indicate that specific interactions between PEG and the truncated form of sTNF-RI are elicited by an increased flexibility of the truncated protein combined perhaps with removal of steric or charge barriers. Covalently bound PEG at the N terminus reduced the protein affinity for the free polymer and induced a more rigid and polar configuration around Trp-107. Deprotonation of His-105, which is perpendicular to Trp-107, was integral to the binding mechanism producing a pH-dependent switching mechanism. These findings stress the importance of surface charge and structural plasticity in determining macromolecular binding affinities and demonstrate the ability of conjugated PEG to modify the localized surface structure in proteins away from the site of conjugation.     

Interactions between human defensins and lipid bilayers: evidence for formation of multimeric pores.

Defensins comprise a family of broad-spectrum antimicrobial peptides that are stored in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP-2, one of 4 human neutrophil defensins, permeabilizes large unilamellar vesicles formed from the anionic lipid palmitoyloleoylphosphatidylglycerol (POPG). As observed by others, we find that HNP-2 (net charge = +3) cannot bind to vesicles formed from neutral lipids. The binding of HNP-2 to vesicles containing varying amounts of POPG and neutral (zwitterionic) palmitoyloleoylphosphatidylcholine (POPC) demonstrates that binding is initiated through electrostatic interactions. Because vesicle aggregation and fusion can confound studies of the interaction of HNP-2 with vesicles, those processes were explored systematically by varying the concentrations of vesicles and HNP-2, and the POPG:POPC ratio. Vesicles (300 microM POPG) readily aggregated at HNP-2 concentrations above 1 microM, but no mixing of vesicle contents could be detected for concentrations as high as 2 microM despite the fact that intervesicular lipid mixing could be demonstrated. This indicates that if fusion of vesicles occurs, it is hemi-fusion, in which only the outer monolayers mix at bilayer contact sites. Under conditions of limited aggregation and intervesicular lipid mixing, the fractional leakage of small solutes is a sigmoidal function of peptide concentration. For 300 microM POPG vesicles, 50% of entrapped solute is released by 0.7 microM HNP-2. We introduce a simple method for determining whether leakage from vesicles is graded or all-or-none. We show by means of this fluorescence "requenching" method that native HNP-2 induces vesicle leakage in an all-or-none manner, whereas reduced HNP-2 induces partial, or graded, leakage of vesicle contents. At HNP-2 concentrations that release 100% of small (approximately 400 Da) markers, a fluorescent dextran of 4,400 Da is partially retained in the vesicles, and a 18,900-Da dextran is mostly retained. These results suggest that HNP-2 can form pores that have a maximum diameter of approximately 25 A. A speculative multimeric model of the pore is presented based on these results and on the crystal structure of a human defensin.     

Protection mechanisms of freely suspended animal cells (CRL 8018) from fluid-mechanical injury. Viscometric and bioreactor studies using serum, pluronic F68 and polyethylene glycol

We use bioreactor and viscometric studies to examine the mechanism by which three additives, fetal bovine serum (FBS), pluronic F68, and polyethylene glycol (PEG), protect the freely suspend CRL-8018 cells from damage due to interactions with bubbles in agitated bioreactors. In bioreactor studies, the protective effect of an addictive could be due to either changes in the ability of the cell resist shear (biological mechanism) or to changes in the medium properties that effect the level or frequency of forces experienced by the cells (physical mechanism). Bioreactor studies show that protection by all three addictives occurs whether the cells are grown in the presence of the addictives (long exposure) or the addictives are added to medium after the cells were exposed to detrimental agitation intensity (short exposure). In the viscometric studies, exposure of cells to laminar shear in the absence of gas-liquid interfaces assesses only the ability of the cells to resist a constant level of shear in a medium with or without an additive. Viscometric studies show that prolonged exposure to FBS makes the cells more shera tolerant, but that short (30-120 min) exposure to FBS does not affect their shear tolerance. We thus conclude that the protective effect of FBS in bioreactors id of both physical and biological nature. The biological contribution is metabolic in nature rather than fast acting. Viscometric studies show that either long or short exposure of the cells to either F68 or PEG does not make the cells more shear tolerant. WE therefore conclude that the protective effect of F68 and PEG does not make the cells more shear tolerant. We therefore conclude that the protective effect of F68 and PEG in bioreactors is physical in nature.     

Induction of microspore-derived embryos of Brassica napus L. with polyethylene glycol (PEG) as osmoticum in a low sucrose medium

Isolated microspores of Brassica napus were cultured on high concentrations of mannitol or polyethylene glycol (PEG 4000), with only a very limited amount of sucrose (0.08–0.1%) provided as carbohydrate source in the medium. While microspores cultured on high mannitol yielded no embryos and no embryogenic cell divisions were observed, microspores on high PEG developed into embryos within 2 weeks, and the embryo yield appeared comparable to that of the sucrose control. When placed under light, PEG embryos quickly changed color from yellow to dark green, while sucrose embryos first remained yellowish and then slowly changed color to pale green. Three-week-old PEG embryos were strikingly similar to immature zygotic embryos developed in ovulo, dissected at 14–15 days post-anthesis (DPA), while sucrose embryos differed from the latter in the size and shape, color and morphology of their cotyledons. These results demonstrate that in microspore embryogenesis of Brassica napus: (1) the level of metabolizable carbohydrate required for microspore embryo induction and formation appears to be substantially less than commonly used amounts, (2) sucrose as an osmoticum can be replaced with high-molecular-weight PEG. With further improvement the new method described here might be suitable for other Brassica species and would have a great potential application in breeding programs. Received: 29 May 1997 / Revision received: 12 August 1997 / Accepted: 2 September 1997     

Drought tolerance in faster- and slower-growing black spruce (Picea mariana) progenies: I. Stomatal and gas exchange responses to osmotic stress

Sixteen years growth in height and basal stem diameter of full-sib black spruce [ Picea mariana (Mill.) B. S. P.] progenies varied with soil moisture availability. The responses to water stress of two faster-growing progenies under drought were compared with two slower-growing progenies to determine the physiological basis of drought tolerance. Six-month-old seedlings were stressed using an osmoticum, polyethylene glycol-3350 (PEG). Seedlings were passed through a series of increasing concentration: 10, 18 and 25% PEG (w/v) each for 3 days to provide solution water potentials of -0.4, -1.0, and -2.0 MPa, respectively. The stress was then relieved by returning the seedlings to nutrient solution without PEG for 24 hours. Gas exchange and water relation parameters were similar in the 4 progenies prior to the imposition of the stress but varied during the stress and after stress relief. The two progenies which grew more vigorously on the driest site maintained significantly higher stomatal conductance, leaf transpiration rate, and net photosynthesis rate during mild (10% PEG) and moderate (18% PEG), but not severe (25% PEG) osmotic stress, and also recovered faster after release of the stress than the other two slower-growing progenies. Black spruce progenies did not differ in xylem water potential or water use efficiency. Progenies capable of faster growth under drought stress were thus characterized by a greater dehydration tolerance, rather than postponement, compared with slower-growing progenies.     

Liquid-phase peptide synthesis on polyethylene glycol (PEG) supports using strategies based on the 9-fluorenylmethoxycarbonyl amino protecting group: application of PEGylated peptides in biochemical assays.

Stepwise synthetic assembly of polypeptide chains reversibly linked to polyethylene glycol represents a hybrid between traditional solution and solid-phase chemistries and combines the inherent advantages of both approaches. The technical simplicity and scalability of the liquid-phase peptide synthesis method renders it particularly attractive for multiple parallel syntheses, combinatorial approaches and the large-scale preparation of peptides. The versatile protection strategy based on the N alpha-fluorenylmethoxycarbonyl group commonly used in solid-phase peptide synthesis was adapted to the liquid-phase approach. Fluoride ions were used rather than the conventional organic base piperidine for the repetitive amino-deprotection step. Using a range of acid- and base-labile linkers between the polymer and the peptide, it was shown that free and fully side-chain protected peptides can be obtained using our version of the liquid-phase peptide synthesis method. Protocols for simultaneous multiple syntheses requiring a minimum of equipment are presented and the use of polyethylene glycol-bound peptides in biochemical binding and functional assay systems is demonstrated.     

The influence of electrostatic interactions on partition in aqueous polyethylene glycol/dextran biphasic systems: Part II

In aqueous polyethylene glycol/dextran two-phase systems, the hydrophobicity, free volume, surface tension, and interfacial tension of the phases in equilibrium were measured as a function of pH and ionic strength. These parameters were found to change with pH, but the pattern and magnitude cannot explain the unusual partition of charged macromolecules, observed previously. The electrostatic potential difference was determined by a new experimental approach based on the measurement of the pH difference between the phases at equilibrium. In polyethylene glycol/dextran systems containing sodium chloride as ionized species, the electrostatic potential is not constant in the pH range 2 to 11. The partition behavior of charged macromolecules and its dependence on pH can be explained by the combined action of charge and phase potential. This conclusion was tested with poly-L-glutamate, which partitioned as predicted and in a pattern opposite to positively charged macro- molecules. (c) 1995 John Wiley & Sons, Inc.