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How different sterols contribute to saponin tolerant plasma membranes in sea cucumbers.
Claereboudt EJS
,
Eeckhaut I
,
Lins L
,
Deleu M
.
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Sea cucumbers produce saponins as a chemical defense mechanism, however their cells can tolerate the cytotoxic nature of these chemicals. To elucidate the molecular mechanisms behind this tolerance a suite of complementary biophysical tools was used, firstly using liposomes for in vitro techniques then using in silico approaches for a molecular-level insight. The holothuroid saponin Frondoside A, caused significantly less permeabilization in liposomes containing a Δ7 holothuroid sterol than those containing cholesterol and resulted in endothermic interactions versus exothermic interactions with cholesterol containing liposomes. Lipid phases simulations revealed that Frondoside A has an agglomerating effect on cholesterol domains, however, induced small irregular Δ7 sterol clusters. Our results suggest that the structural peculiarities of holothuroid sterols provide sea cucumbers with a mechanism to mitigate the sterol-agglomerating effect of saponins, and therefore to protect their cells from the cytotoxicity of the saponins they produce.
PhD position Belgian National Fund for Scientific Research | Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture (Training Fund for Research in Industry and Agriculture), WISD Holothuriculture project (Ref: 29101409) Fonds De La Recherche Scientifique - FNRS (Belgian National Fund for Scientific Research), PDR T.1003.14 Fonds De La Recherche Scientifique - FNRS (Belgian National Fund for Scientific Research), Senior Research Associate position Fonds De La Recherche Scientifique - FNRS (Belgian National Fund for Scientific Research), Senior Research Associate Position Fonds De La Recherche Scientifique - FNRS (Belgian National Fund for Scientific Research)
Figure 1. Raw thermograms and calculated thermodynamic characteristics of ITC experiments conducted with the holothuroid saponin Frondoside A and various liposome compositions. (A) Holothuroid-like liposomes composed of DMPC:D(C16:1)PC: Δ7 sterol (5:2:3) (B) Sterol free liposomes composed of DMPC:D(C16:1)PC (3:1) (C) and fish-like liposomes D(C16:1)PC:DOPC:Chol (5:2:3). Graphic representation of the thermodynamic values (D) Enthalpy (E) Entropy (F) Free Gibbs Energy, associated with the different ITC experiments. Error bars are standard deviation of replicates (n = 3). Lowercase letters depict significant differences calculated through a pairwise comparison using t-test.
Figure 2. Mean relative leakage of calcein from liposomes of different composition in the presence of the holothuroid saponin Frondoside A. Saponin concentration was 0.4 mM. Fish-like liposomes are composed of D(C16:1)PC:DOPC:Chol (5:2:3); holothuroid-like liposomes are composed of DMPC:D(C16:1)PC: Δ7 sterol (5:2:3) and sterol free liposomes are composed of DMPC:D(C16:1)PC (3:1). Error bars are standard deviation of replicates. Lowercase letters depict significant differences calculated through a pairwise comparison using t-test.
Figure 3. Structural formulae and 3D models of the sterols used in the present study. (A) Cholesterol (B) 5α-Cholest-7-en-3β-ol (Δ7-sterol). Green: carbon atoms; grey: hydrogen; red: oxygen. The 3D conformations of the two sterols were very different. The Δ7 double bond caused a bend in the molecule generating a “L” shaped sterol. The Δ7 sterol is therefore shorter and wider compared to the Δ5 cholesterol that is relatively elongated. When the interfacial area is calculated, this structural difference is clearly highlighted (51 Å for cholesterol and 78 Å for Δ7).
Figure 4. Structure of frondoside A. (A) Structural formula; (B) 3D model of the holothuroid saponin Frondoside A. Green: carbon; grey: hydrogen; red: oxygen, blue: sodium; yellow: sulfur. Frondoside A had an overall conical shape, with the saccharadic moiety as the base, and the aliphatic chain on the triterpene moiety as the point. The ester bonds of the xylose monomer accommodating the sulphate group were the points of inflection between the two moieties.
Figure 5. Results from IMPALA simulation of Frondoside A traversing an implicit membrane 36Å thick. (A) The “energy-like” profile of the saponin traversing the implicit bilayer. The X-axis corresponds to the position of the center of mass along the Z-axis. The vertical lines represent the same planar surfaces depicted in B. (B) The most stable position of the saponin into the implicit bilayer. The different planar surfaces represent the water/membrane interface (pink), the lipid polar head/alkyl chain interface (purple), and the center of the bilayer (yellow). Carbons atoms are dark grey, Hydrogen light grey, Oxygen atoms are red, Sulfur yellow and Sodium light blue.
Figure 6. Energy of interaction of Frondoside A and various membrane lipids calculated as described in methods section. Dark grey represents the hydrophobic component and the light grey the polar component of the energies. DPPC: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DMPC: 1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine, DOPC: 1,2-Dioleoyl-sn-glycero-3-phosphocholine, Chol: Cholesterol, Δ7-sterol: 5α-Cholest-7-en-3β-ol.
Figure 7. Monolayer simulations of lipid-saponin systems. Each panel is a 200 × 200 grid where each pixel is a molecule. These are obtained by a Monte Carlo minimization for different molecular systems of DMPC, Cholesterol, Δ7-sterol, Frondoside A, α-Hederin and Hederagenin. The molar ratios have been selected to be the same as systems published in28. Cholesterol is represented in yellow, DMPC in teal, Frondoside A in red, Δ7-sterol in green, α-Hederin in purple, and Hederagenin in orange. Panel C and D are adapted adapted with permission from Supporting information of Lorent, J.; Lins, L; Domenech, O.; Quetin-Leclercq, J., Brasseur R. and and Mingeot-Leclercq, M.P. Domain Formation and Permeabilization Induced by the Saponin α-Hederin and Its Aglycone Hederagenin in a Cholesterol-Containing Bilayer. Langmuir
30, 4556–4569 (2014). Copyright (2018) American Chemical Society.
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