CONICET | Buscador de Institutos y Recursos Humanos

Cellular membranes are crucial for life and animals have evolved many effective strategies for damaging them. We found that the reduced number of predators of the eggs from the invasive snails Pomacea canaliculata and P. maculata is largely explained by the occurrence of noxious defensive proteins. In particular, a 400-KDa tetrameric protein, named PV2, that triggers neurological symptoms in mice. The aim of this study was to look further into the structural aspects of this toxin in P. maculata (PmPV2), and its interaction with membranes.Here we show that PmPV2 is a pore-forming toxin (PFT) combining two immune proteins: a tachylectin module linked to a membrane attack complex/perforin (MACPF) module. Homology modeling and bioinformatics indicated that the tachylectin domain belongs to the HydWA family, with a 6 blade β-propeller structure. The MACPF fold has the characteristic twisted and bent β-sheet core with two flanking transmembrane helixes and it is fused with a novel C-terminal accessory domain restricted to invertebrates MACPFs. Through Atomic Force Microscopy (AFM), we observed that PmPV2 inserts into POPC/Cho lipid bilayers and oligomerizes into ring-like structures compatible with pores, causing membrane thinning. We tested the effect of PmPV2 on intestinal cell cultures, since enterocytes would be the first cells the toxin would encounter when ingested by a predator. Viability of Caco-2 cell cultures decreased in a dose-dependent manner, while patch-clamp experiments indicated alterations in the plasma membrane conductance, compatible with pores of ~ 14 nm. The toxin has a remarkable structural stability in a wide range of pH values (2.0-10.0) as determined by fluorescence spectroscopy, small angle X ray scattering and circular dichroism suggesting it can withstand the passage through gut.This study provides the first evidence into the molecular mechanism of membrane interaction and pore formation for an animal toxin with a MACPF domain