Structure-function relationships of ovorubin, a protease inhibitor from the eggs of Pomacea canaliculata

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

Buzios, Brasil

Congreso; VII Iberoamerican Congress of Biological Physics; 2009

Sociedad Argentina de Biofísica

Ovorubin (OR) is the major protein from the apple snail (Pomacea canaliculata) egg 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, a potent antioxidant, that protects the embryos from photoxidative damage. In this work, we focussed on the primary structural features of OR and analyzed their functional implications. Partial amino acid sequence of chemically-deglycosilated OR obtained 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 inhibition which was further analyzed. Studies of the physical interactions between trypsin and OR by cross-linking and Western blot assays and small angle X-ray scattering (SAXS) gave further evidence of the interaction between OR and serine proteases. Moreover, SAXS results showed a 1:1 complex between OR and trypsin. Given the high resistance of OR to a range of 4-12. the interaccion OR-trypsin at different pH was also studied. Functional studies of simulated gastrointestinal digestion showed that OR retained its trypsin inhibition capacity, clearly indicating the physiological implication of these findings. Moreover, no antibacterial activity could be detected on native OR As a whole, results suggest a novel role for OR during P. canaliculata embryogenesis as an antinutritive protein rather than simply preventing bacterial invasions as was previously thought.