Egg defenses of Pomacea canaliculata: Identification of Kunitz-type inhibitor sequence of PcOvo

ITUARTE S,; BROLA,T.; DREON MS,; HERAS, H

Rosario

Congreso; L Reunión SAIByBM; 2014

SAIByBM

Pomacea canaliculata eggs have antipredator defenses including antinutritive, antidigestive and neurotoxic proteins. PcOvo is the most abundant factor, providing eggs with warning coloration and antidigestive properties; several authors have also ascribed a proteinase inhibitor activity to it. Recently, we sequenced three of its subunits. Here we identified the major proteinase inhibitor activity of the egg as a separate protein, and report 3 new PcOvo subunits. Using 2DE and MS identification followed by a search in P. canaliculata transcriptome, 3 more subunits of PcOvo were identified. Sequence analysis showed that the 6 subunits share at least 30% identity indicating they ptobably arised by duplications. Remarkably no proteinase inhibitor domain was detected in PcOvo. Analysis of egg fluid fractions identified the proteinase inhibitor activity in a fraction named PcPV3. N-terminal sequencing and transcriptome analysis confirmed this fraction contains a 22 kDa Kunitz-type inhibitor. Finally, cross linking experiments were assayed to evaluate PcOvo interaction with trypsin. Our results show that the multifunctional PcOvo is a defensive reserve protein with warning signal and antinutritive properties, while the PcPV3 fraction provides antidigestive properties to the eggs, via Kunitz-type protease inhibition. PcOVO subunits would be the product of gene duplications.