Novel Animal Defenses against Predation: A Snail Egg Neurotoxin Combining Lectin and Pore-Forming Chains That Resembles Plant Defense and Bacteria Attack Toxins

Abstract

Although most eggs are intensely predated, the aerial egg clutches from the aquatic snail Pomacea canaliculata have only one reported predator due to unparalleled biochemical defenses. These include two storage-proteins: ovorubin that provides a conspicuous (presumably warning) coloration and has antinutritive and antidigestive properties, and PcPV2 a neurotoxin with lethal effect on rodents. We sequenced PcPV2 and studied whether it was able to withstand the gastrointestinal environment and reach circulation of a potential predator. Capacity to resist digestion was assayed using small-angle X-ray scattering (SAXS), fluorescence spectroscopy and simulated gastrointestinal proteolysis. PcPV2 oligomer is antinutritive, withstanding proteinase digestion and displaying structural stability between pH 4.0–10.0. cDNA sequencing and protein domain search showed that its two subunits share homology with membrane attack complex/perforin (MACPF)-like toxins and tachylectin-like lectins, a previously unknown structure that resembles plant Type-2 ribosome-inactivating proteins and bacterial botulinum toxins. The protomer has therefore a novel AB toxin combination of a MACPF-like chain linked by disulfide bonds to a lectin-like chain, indicating a delivery system for the former. This was further supported by observing PcPV2 binding to glycocalix of enterocytes in vivo and in culture, and by its hemaggutinating, but not hemolytic activity, which suggested an interaction with surface oligosaccharides. PcPV2 is able to get into predator’s body as evidenced in rats and mice by the presence of circulating antibodies in response to sublethal oral doses. To our knowledge, a lectin-pore-forming toxin has not been reported before, providing the first evidence of a neurotoxic lectin in animals, and a novel function for ancient and widely distributed proteins. The acquisition of this unique neurotoxic/antinutritive/storage protein may confer the eggs a survival advantage, opening new perspectives in the study of the evolution of animal defensive strategies.

Insights into Embryo Defenses of the Invasive Apple Snail Pomacea canaliculata: Egg Mass Ingestion Affects Rat Intestine Morphology and Growth

“…A functional study performed after its ingestion by rats showed that PcPV2 has the ability to withstand protease digestion, displaying structural stability within the pH range of the gastrointestinal tract of rats. Moreover, this toxic lectin binds to the glycocalyx of rat enterocytes in vivo and to Caco-2 cells in culture [8]. This interaction is also in agreement with the high cytotoxic effect of the snail PVF on Caco-2 cells observed in this study.…”

Egg perivitelline fluid proteome of a freshwater snail: Insight into the transition from aquatic to terrestrial egg deposition

“…We focused the analysis on Group B homologs, which comprised the subclade B of seven genes with high expression in the albumen gland and their coding proteins detected in the PVF. Remarkably, the two PV2 subunits of P. canaliculata which exert neurotoxicity to mice 14,15 were not detected in P. diffusa PVF ( Figure S3, supporting information). 42 Studies of venom proteins showed that positive selection sites might be adaptive to predation and defense because most of the adaptive sites are located on the functional domains.…”

“…Remarkably, the two PV2 subunits of P. canaliculata which exert neurotoxicity to mice were not detected in P. diffusa PVF (Table ). The acquisition of neurotoxic PV2 in the terrestrial eggs of Pomacea may be an important biochemical defense mechanism against terrestrial predators .…”

“…Among two major perivitelline groups, PV1 is an oligomer composed of several subunits and plays several critical roles: photoprotection, antioxidant, and nutrient provision in P. canaliculata and P. maculate , and immune response in P. scalaris . PV2, which constitutes around 7.5% of total PVF protein ? is a protein complex composed of two subunits: a membrane attack complex/perforin (MACPF) protein as the toxic subunit, and a tachylectin‐like protein as the delivery subunit, which are linked with a disulfide bridge . Laboratory experiments have shown that PV2 is neurotoxic to mice in P. canaliculata and probably is also a neurotoxin in P. maculata .…”

“…14 Laboratory experiments have shown that PV2 is neurotoxic to mice in P. canaliculata and probably is also a neurotoxin in P. maculata. 7,14,15 However, the neurotoxicity was not observed in the PVF of P. scalaris, 7,10 suggesting that PV2-active toxins may not be present in all species of Pomacea. Since there are multiple copies of MACPF and tachylectin genes in Pomacea, 16 to avoid confusion we named the PV2 toxin associated subunits as PV2-MACPF and PV2tachylectin, and other paralogs as MACPF-like and tachylectinlike proteins, respectively.…”