Defensive proteins in Pomacea: Hemagglutinating activity of PsSC, the major egg perivitellin of P. scalaris

ITUARTE,S.; DREON, M.S.; HERAS, H.

Santiago de Compostela

Congreso; Physiomar 12; 2012

Universidad de Santiago de Compostela

Pomacea snails (apple snails) lay colored egg clutches outside the water. Aerial oviposition was a key event in the Ampullariidae evolution, that required physiological innovations as eggs are exposed for weeks to sunlight, high temperature, desiccation and predators. Apple snail egg-proteins, perivitellins, were only studied in two species: P. canaliculata (Lamarck, 1822) and P. scalaris. The major perivitellin of P. canaliculata, ovorubin, is a carotene-glycoprotein which plays multiple roles including nutrition, photo protection, antioxidant stabilization, andidigestive defense and warning signaling. In P. scalaris, a carotenoid binding glycoprotein, PsSC, which shares several structural and functional features with ovorubin, was recently isolated and biochemically characterized. Many glycan-binding proteins, like lectins, have been isolated from mollusks, where they function as recognition and immune molecules. Here we report the hemagglutinatig activity of PsSC and characterize this activity in the context of the structural stability of the protein. The protein showed high agglutinating activity against rabbit red blood cells, not altered by the presence of divalent cations. Hemaglutination was strongly inhibited by glucosamine, galactosamine and GalNAc. In contrast with ovorubin, PsSC did not inhibit trypsin, although both perivitellins share a resistance to pepsin and trypsin digestion. Temperature and pH stability, analyzed by fluorescence and Vis spectroscopy and by small angle X-ray scattering (SAXS), indicated that the native protein complex was stable up to 60°C; in coincidence with a loss of hemaglutinating activity at this temperature. While PsSC did not show structural perturbations between pH 2.0 and 10.0, a loss in its biological activity was observed below pH 4.0 and above pH 8.0. These results suggest that PsSC, like ovorubin, would be involved in egg defenses against predation being indigestible by potential predators (antinutritive role). Structural and functional properties of PsSC are similar to plant storage proteins that play a dual role to nourish embryos and as defensive lectins against predators PsSC has the potential of becoming a biotechnologically useful lectin, due to its wide pH and temperature stability range. This is the first report of a protease-resistant glycan binding egg protein in a Pomacea snail, a genus where novel egg-defense systems were recently described.