[Show abstract][Hide abstract] 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.
PLoS ONE 01/2013; 8(5):e63782. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is little information on the egg proteins of gastropod mollusks. Here we focus on PV2, a novel neurotoxin from snail eggs, studying its size, shape, structure, and stability, using small angle X-ray scattering (SAXS), absorption and fluorescence spectroscopy, circular dichroism, electron microscopy and partial proteolysis. Results indicate that PV2 is a compact and well folded oligomer of 130x44 A. It is an octamer of four 98 kDa heterodimers composed of 67 and 31 kDa subunits. Subunits are held together by disulfide bonds. Dimers are assembled into native PV2 by non-covalent forces. The larger subunit is more susceptible to proteolysis, indicating it is less compactly folded and/or more exposed. Quenching of tryptophan fluorescence showed a single class of tryptophyl side chains occluded in hydrophobic regions. Native structure shows loss of secondary structure (alpha+beta) at 6 M urea or 60-70 degrees C; the effects on the quaternary structure suggest an unfolding without disassembling of the protein. The 3D model of PV2 presented here is the first for an egg proteinaceous neurotoxin in animals.
Biochimica et Biophysica Acta 03/2010; 1804(7):1492-9. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: While many invertebrates sequester toxic compounds to endow eggs with chemical defences, here we show, for the first time to our knowledge, the identification of a neurotoxin of proteinaceous nature localized inside an egg. Egg extracts from the freshwater apple snail Pomacea canaliculata displayed a neurotoxic effect in mice upon intraperitoneal injection (i.p.) (LD50, 96h 2.3mg/kg). Egg protein and total lipids were analysed separately and the only fraction displaying a highly toxic effect (LD50, 96h 0.25mg/kg, i.p.) was further purified to homogeneity as an oligomeric glyco-lipoprotein of 400kDa and two subunits biochemically and immunologically indistinguishable from the previously described perivitellin PV2. The neurotoxin was heat sensitive and there was evidence of circulating antibody response to sublethal i.p. doses on mice. Clinical signs, histopathological and immunocytochemical studies revealed damage mostly in mice spinal cord. Experiments showed chromatolysis and a decreased response to calbindin D-28K associated with a significant increase of TUNEL-positive cells in the dorsal horn neurons. These results suggest that calcium buffering and apoptosis may play a role in the neurological disorders induced by the toxin in mammalian central nervous system. This is the first report of a mollusc neurotoxin genetically encoded outside the cone-snail species.
[Show abstract][Hide abstract] ABSTRACT: Dysdercus Guerin Méneville, 1831 comprises insect species that are often serious pests of cotton both in the Old and New World, representing the only taxon from Pyrrhocoridae in the Neotropical Region. The genus is cytologically characterized by possession of holokinetic chromosomes and a prereductional type of meiosis. So far, only seven species from the Old World and five species from the Neotropical Region have been cytogenetically described. In the present report we compare the male meiosis from both natural and inbred populations of Dysdercus chaquensis Freiberg, 1948. Our results demonstrated that even though both populations share the same diploid chromosome number, the presence of a diffuse stage was found to be committed to the wild population of the species. Furthermore, the diffuse stage was found in a high frequency in all analysed wild specimens. indicating the long duration of this period among the regular meiosis of D. chaquensis. Taking into account that the diffuse stage is connected with an intense and long period of cellular growth, and with an important transcriptional activity, the absence of this stage in all the inbred specimens of D. chaquensis could be related with the lack of unfavourable physiological conditions due to the environmental uniformity along seven years of inbreeding.