Biochemical defences of Pomacea canaliculata eggs. Ovorubin as an antinutritive perivitelin

ITUARTE, S; DREÓN, MS; CEOLÍN, M; HERAS, H

Brest, Francia

Congreso; II Marine Mollusc Physiology Conference; 2008

IFREMER

The reproductive strategy of many species of the freshwater genus Pomacea involves cementing brightly coloured egg clutches on different substrata above the water line (Figure), thus exposing the embryos to sunlight, high temperatures and predators. The conspicuous coloration of Pomacea canaliculata eggs is thought to be a warning signal, but there is not evidence of the defensive compounds. It has been proposed that their perivitellins, besides the usual role as reserve proteins, are also involved in the adaptation of the embryo to the aerial conditions. The major egg protein is Ovorubin, a 300 KDa thermostable multifunctional protein, with protease inhibitor activity. This glyco-lipo-carotenoprotein also contains the carotenoid astaxanthin as a prosthetic group, a potent antioxidant responsible for the pink-reddish egg color. Here we report (1) the effect of pH on ovotubin stability, using small angle X-ray scattering (SAXS), electron microscopy (EM), circular dichroism, fluorescence and absorption spectroscopy and (2) pH stability in relation to a potential role of ovorubin in the egg defence against predators evaluating a simulated gastrointestinal digestion. Results showed that the protein has an axial length of 69 Å and a radius of 43 Å, a very large protease inhibitor compared to those of most animals or plants. The quaternary structure was only affected at pH values below 4 showing a reduction in size and loss of tertiary structure. Astaxanthin binding was not affected at pH values between 2 - 12. Taken together these results indicate a high structural stability in a wide pH range, showing a disassembling, but not an unfolding of the oligomer only at low pH values. Considering the high structural stability in a wide pH range and the previously reported protease inhibitor function, we speculated that ovorubin was actively involved in the chemical defence of embryos by limiting the predator’s ability to digest and use essential nutrients from the eggs. To test this hypothesis we subjected ovorubin to a simulated in vitro gastrointestinal digestion (4h with pepsin, pH 2.5, followed by 4 h with trypsin pH 7.5) Electrophoretic and spectrophotometric analysis showed that ovorubin resisted gastrointestinal digestion and retained its serine protease inhibiting activity. In addition, partial aminoacid sequencing showed that ovorubin belongs to the Kunitz-type serine protease inhibitors´ family. We therefore show evidence that ovorubin may withstand the passage through the digestive tract of potential predators affecting trypsin activity, thus rendering the eggs antinutritive to predators. This is a novel function of perivitellins in the chemical defence of embryos.