Interaction of Clostridium perfringens epsilon-toxin with biological and model membranes: A putative protein receptor in cells

Manni, Marco M.; Sot, Jesús; Goñi, Félix M.

doi:10.1016/j.bbamem.2014.11.028

Cited by 22 publications

(23 citation statements)

References 35 publications

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“…These findings are of particular relevance to studies using GPMVs for drug encapsulation (27,28), therapeutic targeting (26,27), and studies of membrane permeation (26,(29)(30)(31)(32). Our suggestion for possible approaches to avoid leakage in GPMVs may be of value therein.…”

Section: Discussionmentioning

confidence: 68%

“…These vesicles have also been used in combination with mass spectrometric lipidomics to characterize the detailed and comprehensive lipid profiles of PMs and their remodeling during various cellular processes (22)(23)(24). Recently, GPMVs have also been employed to study mechanisms of viral binding and fusion to the PM (25), as well as toxin binding (26).…”

Section: Introductionmentioning

confidence: 99%

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Cell-derived plasma membrane vesicles are permeable to hydrophilic macromolecules

Skinkle

Levental

2019

Preprint

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Giant Plasma Membrane Vesicles (GPMVs) are a widely used model system for biochemical and biophysical analysis of the isolated mammalian plasma membrane (PM). A core advantage of these vesicles is that they maintain the native lipid and protein diversity of the plasma membrane while affording the experimental flexibility of synthetic giant vesicles. In addition to fundamental investigations of PM structure and composition, GPMVs have been used to evaluate the binding of proteins and small molecules to cell-derived membranes, and the permeation of drug-like molecules through them. An important assumption of such experiments is that GPMVs are sealed; i.e. that permeation occurs by diffusion through the hydrophobic core rather than through hydrophilic pores. Here we demonstrate that this assumption is often incorrect. We find that most GPMVs isolated using standard preparations are passively permeable to various hydrophilic solutes as large as 40 kDa, in contrast to synthetic giant unilamellar vesicles (GUVs). We attribute this leakiness to relatively large and heterogeneous pores formed by rupture of vesicles from cells. These pores are stable and persist throughout experimentally relevant time scales. Finally, we identify preparation conditions that minimize poration and allow evaluation of sealed GPMVs. These unexpected observations of GPMV poration are of critical importance for interpreting experiments utilizing GPMVs as plasma membrane models, particularly for drug permeation and membrane asymmetry. STATEMENT OF SIGNIFICANCE:A critical assumption in using Giant Plasma Membrane Vesicles to study membrane penetration and interactions is that these vesicles maintain the permeability barrier of the native membrane from which they form. Using large fluorescently-labeled hydrophilic probes, we demonstrate that this assumption is often incorrect and conclude that macromolecular solutes permeate GPMVs through stable pores formed during shear-induced rupture of vesicles from cells. Using these insights into the mechanisms of poration, we demonstrate an approach to isolate sealed GPMVs.

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Section: Discussionmentioning

confidence: 68%

Section: Introductionmentioning

confidence: 99%

Cell-derived plasma membrane vesicles are permeable to hydrophilic macromolecules

Skinkle

Levental

2019

Preprint

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“…This finding is consistent with those of previous studies, which have shown that there is a putative protein receptor located at detergent resistant membrane (DRMs) in the apical cell membrane. 7,24,29,39 However, previous studies also showed that epsilon toxin was capable of forming channels in lipid bilayers without the need of a receptor, although with less efficiency. 21,25 We also found that rETX could occasionally form slight heptamer in PBS without cells.…”

Section: Discussionmentioning

confidence: 99%

F199E substitution reduced toxicity ofClostridium perfringensepsilon toxin by depriving the receptor binding capability

Kang

Gao

Yao

et al. 2017

Human Vaccines & Immunotherapeutics

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Epsilon toxin (ETX), a potent toxin, is produced by types B and D strains of Clostridium perfringens, which could cause severe diseases in humans and domestic animals. Mutant rETX F199E was previously demonstrated to be a good vaccine candidate. However, the mechanism concerned remains unknown. To clarify how F199E substitution reduced ETX toxicity, we performed a series of experiments. The results showed that the cell-binding and pore-forming ability of rETX F199E was almost abolished. We speculated that F199E substitution reduced toxicity by depriving the receptor binding capability of ETX, which contributed to the hypothesis that domain I of ETX is responsible for cell binding. In addition, our data suggested that ETX could cause Ca 2C release from intracellular Ca 2C stores, which may underlie an alternate pathway leading to cell death. Furthermore, ETX induced crenation of the MDCK cells was observed, with sags and crests first appearing on the surface of condensed MDCK cells, according to scanning electron microscopy. The data also demonstrated the safety and potentiality of rETX F199E as a vaccine candidate for humans. In summary, findings of this work potentially contribute to a better understanding of the pathogenic mechanism of ETX and the development of vaccine against diseases caused by ETX, using mutant proteins.

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“…Several washing and centrifugation steps were conducted to eliminate dithiothreitol and paraformaldehyde traces. Finally, GPMV were resuspended in 500 µL GPMV buffer [28].…”

Section: Gpmv (Bleb) Formationmentioning

confidence: 99%

Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells

Monasterio

Jiménez‐Rojo

García-Arribas

et al. 2020

IJMS

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This study was aimed at preparing and characterizing plasma membranes (PM) from Chinese Hamster Ovary (CHO) cells. Two methods of PM preparation were applied, one based on adhering cells to a poly-lysine-coated surface, followed by hypotonic lysis and removal of intracellular components, so that PM patches remain adhered to each other, and a second one consisting of bleb induction in cells, followed by separation of giant plasma membrane vesicles (GPMV). Both methods gave rise to PM in sufficient amounts to allow biophysical and biochemical characterization. Laurdan generalized polarization was used to measure molecular order in membranes, PM preparations were clearly more ordered than the average cell membranes (GP ≈0.450 vs. ≈0.20 respectively). Atomic force microscopy was used in the force spectroscopy mode to measure breakthrough forces of PM, both PM preparations provided values in the 4–6 nN range, while the corresponding value for whole cell lipid extracts was ≈2 nN. Lipidomic analysis of the PM preparations revealed that, as compared to the average cell membranes, PM were enriched in phospholipids containing 30–32 C atoms in their acyl chains but were relatively poor in those containing 34–40 C atoms. PM contained more saturated and less polyunsaturated fatty acids than the average cell membranes. Blebs (GPMV) and patches were very similar in their lipid composition, except that blebs contained four-fold the amount of cholesterol of patches (≈23 vs. ≈6 mol% total membrane lipids) while the average cell lipids contained 3 mol%. The differences in lipid composition are in agreement with the observed variations in physical properties between PM and whole cell membranes.

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Interaction of Clostridium perfringens epsilon-toxin with biological and model membranes: A putative protein receptor in cells

Cited by 22 publications

References 35 publications

Cell-derived plasma membrane vesicles are permeable to hydrophilic macromolecules

Cell-derived plasma membrane vesicles are permeable to hydrophilic macromolecules

F199E substitution reduced toxicity ofClostridium perfringensepsilon toxin by depriving the receptor binding capability

Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells

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