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

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

La Plata

Congreso; XXXVII Reunión Anual de la Sociedad Argentina de Biofísica; 2008

Sociedad Argentina de Biofísica

Ovorubin (OR) is the major egg protein from the apple snail (Pomacea canaliculata) penvitellin fluid (PVF). It plays essential roles in embryo development, including transport arid protection of camtenoids, protease inhibition, photoprotection, storage, and nourishment. It is a lipo-glycn-carotenoprotein comple!x with an apparent molecular mas of 300 kDa composed of three subunits of ca. 28, 32, and 35 kDa. OR has a pinkish-red colour, dueto the cofactor astaxanthin (3,3´-hydroxy-@p-caroterie-4,4´dione), a potent ar.tior:idant, that protects the embryos from photoxidative damage. In this work, we focussed on the protease inhibitor rde of OR and correlated this function with its structural features. Partial amino acid sequence of chemically deglymsilated OR by mass spectrometty showed a high degree of homology with a member of the Kunitz iiihibitor family. This Kunitz motif, provided the first evidence of the nature of the inhibitor. Studies of the physical interactions bebeen 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 ils trypsin inhibition capacity, though longexposure 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 pl-I=2 to render it sensible to protease digestion. Finally, studies of OR resistance in a simulated gastrointestinal digestiori showed that retained its tiypsin inhibition capacity. cbatiy indicating thephysiological implication of these findings. These led us to suggest a novel role for OR duiing P canaliculata embryogenesis as an antinutritive protein rather than simply preventing bacteria1 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 come sort of punishment if they ingest the prey (eggs).