Ovorubin as a protease inhibitor in Pomacea canaliculata eggs. Implications in the biochemical defenses of snail embryos.

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

La Plata

Congreso; XXXVII Congreso Soc. Argentina de Biofísica; 2008

SAB

Ovorubin (OR) is the major egg protein from the apple snail (Pomacea canaliculata) perivitellin fluid (PVF). It plays essential roles in embryo development, including transport and protection of carotenoids, protease inhibition, photoprotection, storage, and nourishment. It is a lipo-glyco-carotenoprotein complex with an apparent molecular mass of 300 kDa composed of three subunits of ca. 28, 32, and 35 kDa. OR has a pinkish-red colour, due to the cofactor astaxanthin (3,3L-hydroxy-ƒÀ-ƒÀ-carotene-4,4L-dione), a potent antioxidant, that protects the embryos from photoxidative damage. In this work, we focussed on the protease inhibitor role of OR and correlated this function with its structural features. Partial amino acid sequence of chemically-deglycosilated OR by mass spectrometry showed a high degree of homology with a member of the Kunitz inhibitor family. This Kunitz motif, provided the first evidence of the nature of the inhibitor. Studies of the physical interactions between trypsin and OR by cross-linking and Western blot assays gave further evidence of the interaction of OR inhibitor with serine proteases. Boiling OR at 100‹C for 40 min did not alter its trypsin inhibition capacity, though long exposure to acidic pH values (pH=2) greatly diminished its inhibitory properties. Both results could be explained by an analysis of the structural perturbations of OR by small-angle X-Ray scattering (SAXS) that showed that the particle shape was not significantly affected by temperature and experienced a loss of structure at acidic pH. In vitro pepsin digestion indicated that OR is resistant to this protease action for 2h. It was necessary to pre-incubate OR for 48h at pH=2 to render it sensible to protease digestion. Finally, studies of OR resistance in a simulated gastrointestinal digestion showed that retained its trypsin inhibition capacity, clearly indicating the physiological implication of these findings. These led us to suggest a novel role for OR during P. canaliculata embryogenesis as an antinutritive protein rather than simply preventing bacterial invasions as was previously suggested. This hypothesis is supported by the bright and conspicuous eggs, a warning signal very much used in nature to deter predators advertising of some sort of punishment if they ingest the prey (eggs).