Chemical defenses of sea cucumbers : interactions between saponins and model plasma membranes

Saponins are a very diverse class of secondary metabolites found in plants and some marine invertebrates. They are amphiphilic molecules composed of a hydrophilic sugar moiety, and a hydrophobic steroid/triterpenic-like part known as the aglycone. Saponins are studied for their pharmacological properties such as their anti-fungal, anti-microbial and anti-tumoral activity. Holothuroids, or sea cucumbers, produce saponins as a chemical defense against predators and parasites, but interestingly, are immune to the cytotoxic nature of these chemicals. This immunity is extremely poorly understood. The standing hypothesis, based purely on observation, is that the rare Δ7 and Δ9(11) sterols that replace cholesterol in the cellular plasma membranes of sea cucumbers are responsible for this immunity, however, the molecular mechanism remains obscure. The aim of this study was to elucidate the mechanisms behind the immunity of holothuroid to the cytotoxic saponins (e.g. Frondoside A) they produce but also to describe, at a molecular level, the interactions that occur between model plasma membranes and saponins. This investigation was conducted using complementary biophysical tools, using both in silico approaches such as the Hypermatrix, IMPALA and Big Monolayer simulation models and with an in vitro technique called Isothermal Titration Calorimetry (ITC). The Hypermatrix method calculates energies of interaction between a central molecule and a surrounding lipid monolayer, allowing to determine if certain interactions are more favorable than others. The Big Monolayer uses these energies to simulate a monolayer composed of different proportions of lipids and saponin. ITC is a technique used to describe interactions in a thermodynamic framework, and allows for the enthalpy, entropy, free Gibbs energy and the binding constant of a particular interaction to be determined from a recorded thermogram. The structural differences of the holothuroid sterols were first described and compared to the mammalian membrane sterol: cholesterol. Structural differences of saponins and sterols were associated with different interaction affinities and mechanisms. Saponin-lipid (both phospholipids and sterols) interactions were mainly apolar in nature. Interactions with phospholipids were more favorable than with sterols, and among the sterols, saponins interacted more favorably with cholesterol than the holothuroid Δ7 and Δ9(11) sterols. Liposomes containing cholesterol resulted in exothermic interactions with Frondoside A whereas liposomes containing the Δ7 sterol were endothermic with the same saponin. Big Monolayer simulations using experimental settings previously developed for plant saponins revealed that the holothuroid saponin Frondoside A has an agglomerating effect on cholesterol domains, similarly to the plant saponin. However when interacting with the Δ7 sterols, the sterol domains were fragmented into small clusters. The coevolution of a saponin-Δ7 sterol pair may be an adaptation required for holothuroid membranes to inhibit the formation of large membrane disruptive sterol domains in the presence of saponin. Key-words: Saponin, holothuroid, Holothuria scabra, model plasma membrane, membrane interactions, sterol, biophysics, ITC