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

et al. (2013) Novel Animal Defenses against Predation: A Snail Egg Neurotoxin Combining Lectin and Pore-Forming Chains That Resembles Plant Defense and Bacteria Attack Toxins. PLoS ONE 8(5): e63782. doi:10.1371/journal.pone.0063782 Novel Animal Defenses against Predation: A Snail Egg Neurotoxin Combining Lectin and Pore-Forming Chains That Resembles Plant Defense and Bacteria Attack Toxins Marcos Sebastia n Dreon 0 Mara Victoria Frassa 0 Marcelo Ceoln 0 Santiago Ituarte 0 Jian-Wen Qiu 0 Jin Sun 0 Patricia E. Ferna ndez 0 Horacio Heras 0 F. Gisou van der Goot, Ecole Polytechnique Federale de Lausanne, Switzerland 0 1 Instituto de Investigaciones Bioqu micas de La Plata (INIBIOLP), Universidad Nacional de La Plata (UNLP) - Consejo Nacional de Investigaciones Cient ficas y Te cnicas (CONICET CCT-La Plata), La Plata, Argentina, 2 Instituto de Investigaciones F sico-Qu micas, Teo ricas y Aplicadas (INIFTA), UNLP - CONICET CCT-La Plata, La Plata, Argentina, 3 Ca tedra de Bioqu mica y Biolog a Molecular, Facultad de Ciencias. Me dicas, UNLP, La Plata, Argentina, 4 Department of Biology, Hong Kong Baptist University , Hong Kong , P. R. China , 5 Instituto de Patolog a B. Epstein, Ca tedra de Patolog a General Veterinaria , Facultad Cs. Veterinarias, UNLP, La Plata, Argentina, 6 Facultad de Ciencias Naturales y Museo, UNLP, La Plata , Argentina 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 ribosomeinactivating proteins and bacterial botulinum toxins. The protomer has therefore a novel AB toxin combination of a MACPFlike 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. - . These authors contributed equally to this work. Escaping predation is essential to survival. To reduce predation, organisms have developed an array of chemical and physical defensive strategies, but predators in turn have evolved adaptive mechanisms to overcome these defenses in a battle of coevolving prey defenses and predator counter-defenses [1]. Eggs are perhaps the most endangered stage in the life cycle of an animal. Motionless and often conspicuous, eggs are highly vulnerable to both predators and parasites [2] and their high nutritional value makes them subject to intense predation. Consequently, many invertebrates defend their eggs by endowing them with deterrent chemicals as has been well documented in insects that sequester toxic compounds from plants [35] and in some terrestrial and marine gastropods [6,7]. There are, however, a few eggs that are winning in the arms-race and escape intense predation such as the aerial egg clutches from the aquatic apple snail Pomacea canaliculata (Lamarck, 1822) (Caenogastropoda, Ampullariidae) which, while filled with large amounts of carbohydrates and storage proteins (perivitellins) [8], have only one reported predator worldwide, the fire ant Solenopsis geminata (Fabricius, 1804) [9]. Females from this freshwater apple snail deposit clutches of hundreds of pink-reddish eggs that are unusual in two respects: they are cemented outside the water and they are brightly colored [10,11]. The conspicuous egg coloration presumably advertises to visual-hunting predators the presence of egg defenses (aposematic or warning coloration), a strategy used by noxious organisms to visually communicate their toxicity or distastefulness to potential predators [12]. Biochemical evidence indicates that apple snail perivitellins are involved in egg defense against predation [13]. These proteins, synthesized in the female albumen gland, are deposited around the fertilized ovocyte as part of the perivitelline fluid [14]. Two of these perivitellins, PcPV2 (previously called PV2) and ovorubin (PcOvo), are neurotoxic and diminish rat growth rates, respectively [8,13,1517]. PcPV2 is a neurotoxin with a strong lethal effect on the central nervous system of mice that induces changes in calcium homeostasis and results in the apoptosis of selected neuron populations. Structural studies have shown that PcPV2 is a globular, compact and well-folded glycolipoprotein, with 2.5% w/w carbohydrates [18]. This 400 kDa oligomer is an octamer of four 98 kDa heterodimers, each composed of a 67 kDa heavy chain (PcPV2-67) and a 31 kDa light chain (PcPV2-31). The heavy and light chains are held together by disulfide bonds and the heterodimers are assembled into native PcPV2 by non-covalent forces [18]. PcPV2 is the only reported genetically encoded toxin located inside an egg [17]. PcPV2 is synthesized and secreted together with the other identified component of the apple snail egg defenses, the carotenoprotein PcOvo. This protein is a multifunctional perivitellin that is massively accumulated in the perivitelline fluid and is involved in protection against abiotic stressors [19], as well as against predators. Besides providing conspicuous coloration to eggs, PcOvo defends the embryos against predators by reducing the digestibility and nutritional quality of the eggs [13,20]. The recently described proteome of P. canaliculata perivitellin fluid revealed more potential defensive proteins [21]. Unlike this comprehensive body of information on PcOvo, information is lacking about the capacity of th (...truncated)