Lysozyme induced fusion of negatively charged phospholipid vesicles

Posse, E; Vinals, Alberto Lopez; Arcuri, Beatriz F. de; Farı́as, Ricardo N.; Morero, Roberto D.

doi:10.1016/0005-2736(90)90370-4

Cited by 25 publications

(9 citation statements)

References 20 publications

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“…In contrast, covalent conjugation to negatively charged liposomes proceeded readily. All in all, these observations are in excellent agreement with published experimental work on the interaction of lysozymes and other proteins with liposomes [ 40 , 41 , 42 ]. Also in agreement with our observations, Pejawar-Gaddy et al have reported that the inclusion of DOTAP to double-functionalised, DOPC-based interbilayer-cross-linked multilamellar vesicles (ICMVs) improved the conjugation efficiency of Env trimers from around 5% to slightly above 20% [ 17 ].…”

Section: Resultssupporting

confidence: 91%

“…Published experimental work on the conjugation of proteins on functionalised surfaces and the interaction of proteins with lipid membranes suggest that appropriate surface concentrations of protein are a prerequisite for conjugation reactions to proceed efficiently [ 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ]. This is particularly relevant for conjugation reactions performed at low(er) molar bulk concentrations of protein.…”

Section: Introductionmentioning

confidence: 99%

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Conjugation of Native-Like HIV-1 Envelope Trimers onto Liposomes Using EDC/Sulfo-NHS Chemistry: Requirements and Limitations

et al. 2020

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The display of native-like human immunodeficiency virus type 1 envelope (HIV-1 Env) trimers on liposomes has gained wide attention over the last few years. Currently, available methods have enabled the preparation of Env-liposome conjugates of unprecedented quality. However, these protocols require the Env trimer to be tagged and/or to carry a specific functional group. For this reason, we have investigated N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide/N-Hydroxysulfosuccinimide (EDC/Sulfo-NHS) chemistry for its potential to covalently conjugate tag-free, non-functionalized native-like Env trimers onto the surface of carboxyl-functionalized liposomes. The preservation of the liposome’s physical integrity and the immunogen’s conformation required a fine-tuned two-step approach based on the controlled use of β-mercaptoethanol. The display of Env trimers was strictly limited to activated liposomes of positive charge, i.e., liposomes with a positive zeta potential that carry amine-reactive Sulfo-NHS esters on their surface. In agreement with that, conjugation was found to be highly ionic strength- and pH-dependent. Overall, we have identified electrostatic pre-concentration (i.e., close proximity between negatively charged Env trimers and positively charged liposomes established through electrostatic attraction) to be crucial for conjugation reactions to proceed. The present study highlights the requirements and limitations of potentially scalable EDC/Sulfo-NHS-based approaches and represents a solid basis for further research into the controlled conjugation of tag-free, non-functionalized native-like Env trimers on the surface of liposomes, and other nanoparticles.

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Section: Resultssupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Conjugation of Native-Like HIV-1 Envelope Trimers onto Liposomes Using EDC/Sulfo-NHS Chemistry: Requirements and Limitations

et al. 2020

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“…33,34 Recently, lysozyme has been reported to induce pH-dependent fusion of phosphatidylserine vesicles below pH 5.0 with protein penetration inside the lipid bilayer; 35 however, other authors have reported that lysozyme shows fusion efficiency over a broad pH range with extensive mixing of phospholipids, but not vesicle content, suggesting vesicle aggregation without effective fusion. 36 The present investigation was aimed at improving the knowledge of the effects of different HEWL concentrations adsorbed onto lipid vesicles containing various phospholipids, mixed in different ratios to vary the density of the surface negative charge. In particular our purpose was to gain a deeper understanding on the structural changes that occur simultaneously in the protein and the vesicle membrane upon interaction.…”

Section: Introductionmentioning

confidence: 99%

Lysozyme interaction with negatively charged lipid bilayers: protein aggregation and membrane fusion

et al. 2012

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We report on the interaction of hen egg-white lysozyme (HEWL) with lipid vesicles in terms of surfaceinduced protein conformational variation and subsequent aggregation. In particular, we investigated the variations of the secondary structure of native lysozyme in the presence of liposomes with different surface charge density, resulting from different molar ratios of the zwitterionic POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and the negatively charged POPG (1-palmitoyl-2-oleoyl-snglycero-3-phospho-(1 0 -rac-glycerol)). It is well known that the main driving force involved in the interaction between globally anionic liposomes and lysozyme is electrostatic compensation, which, in some cases, produces extended aggregation. Moreover the presence of membranes can induce unfolding in the protein. In order to understand the main determinants of such phenomena, we probed simultaneously lysozyme-induced vesicle fusion events, variations in the secondary structure of the protein and its effect on liposomal membrane fluidity. We found that above a charge-density threshold, the association with vesicles results in modifications of the native structure associated with a decrease of liposomal membrane fluidity. Electron microscopy images revealed that the above described interactions result in mesoscopic structural changes, i.e. liposome clustering and fusion, together with the appearance of elongated structures, reminiscent of fibrillar aggregates. Additionally, a confocal microscopy analysis revealed that upon interaction with giant unilamellar vesicles (GUVs) of the same lipid composition where the above interactions were observed, a prompt insertion of lysozyme in the membrane occurs, leading to vesicle clustering, with the appearance of elongated structures where both the lipid and the protein are present.

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“…The structure and physicochemical properties of lysozyme have been thoroughly characterized and this protein continues to be widely employed as a model in fundamental studies assessing protein adsorption at interfaces (1), lipid-protein interactions (2)(3)(4)(5)(6), membrane fusion (7)(8)(9)(10), and structural requirements for bactericidal action (11)(12)(13), as well as protein folding, aggregation, and amyloid fibrillogenesis (14)(15)(16). There are a number of reports on the lipid-binding properties of lysozyme (5)(6)(7)(8)(9)17,18). In brief, membrane association of lysozyme is thought to involve both electrostatic and hydrophobic interactions (4,9,17).…”

Section: Introductionmentioning

confidence: 99%

“…Lysozyme (pI ;11.0) bears a net positive charge over a broad pH range and, accordingly, has a high affinity for anionic phospholipids. The primary role of electrostatic interactions in the membrane association of lysozyme has been demonstrated by monitoring changes in the electrophoretic mobility of lipid vesicles after protein adsorption (6,17), examining the effects of pH, ionic strength, and charge state of chemically modified lysozyme on its lipid binding (6,7), and comparing the effects of lysozyme on the aggregation and fusion of negatively charged and neutral liposomes (9). There is also evidence demonstrating the involvement of hydrophobic interaction in the membrane association of lysozyme.…”

Section: Introductionmentioning

confidence: 99%

Binding of Lysozyme to Phospholipid Bilayers: Evidence for Protein Aggregation upon Membrane Association

Gorbenko

Ioffe

Kinnunen

2007

Biophysical Journal

133

102

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Biological functions of lysozyme, including its antimicrobial, antitumor, and immune-modulatory activities have been suggested to be largely determined by the lipid binding properties of this protein. To gain further insight into these interactions on a molecular level the association of lysozyme to liposomes composed of either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-phosphatidylserine, or bovine heart cardiolipin was studied by a combination of fluorescence techniques. The characteristics of the adsorption of lysozyme to lipid bilayers were investigated using fluorescein 5'-isothiocyanate labeled protein, responding to membrane association by a decrease in fluorescence. Upon increasing the content of anionic phospholipids in lipid vesicles, the binding isotherms changed from Langmuir-like to sigmoidal. Using adsorption models based on scaled particle and double-layer theories, this finding was rationalized in terms of self-association of the membrane-bound protein. The extent of quenching of lysozyme tryptophan fluorescence by acrylamide decreased upon membrane binding, revealing a conformational transition for the protein upon its surface association, resulting in a diminished access of the fluorophore to the aqueous phase. Steady-state fluorescence anisotropy of bilayer-incorporated probe 1,6-diphenyl-1,3,5-hexatriene was measured at varying lipid-to-protein molar ratios. Lysozyme was found to increase acyl-chain order for liposomes with the content of acidic phospholipid exceeding 10 mol %. Both electrostatic and hydrophobic protein-lipid interactions can be concluded to modulate the aggregation behavior of lysozyme when bound to lipid bilayers. Modulation of lysozyme aggregation propensity by membrane binding may have important implications for protein fibrillogenesis in vivo. Disruption of membrane integrity by the aggregated protein species is likely to be the mechanism responsible for the cytotoxicity of lysozyme.

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Lysozyme induced fusion of negatively charged phospholipid vesicles

Cited by 25 publications

References 20 publications

Conjugation of Native-Like HIV-1 Envelope Trimers onto Liposomes Using EDC/Sulfo-NHS Chemistry: Requirements and Limitations

Conjugation of Native-Like HIV-1 Envelope Trimers onto Liposomes Using EDC/Sulfo-NHS Chemistry: Requirements and Limitations

Lysozyme interaction with negatively charged lipid bilayers: protein aggregation and membrane fusion

Binding of Lysozyme to Phospholipid Bilayers: Evidence for Protein Aggregation upon Membrane Association

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