Biophysical characterization of a perivitellin protein in freshwater snail Pomacea canaliculata.

FRASSA, V.; DREÓN, M.S.; CEOLÍN, M.; HERAS, H.H.

Rosario, Argentina

Congreso; XXXV Reunión anual de la Sociedad Argentina de Biofísica; 2006

Sociedad Argentina de Biofísica

PV2, together with ovorubin are the major high molecular weight lipoglycoproteins presents in the eggs of Pomacea canaliculata. This autochthonous freshwater snail has remarkable economical and health aspects, specially in far east Asia, as a rapidly expanding rice field pest. We have previously characterized Pv2 regarding its lipid, glucid and protein moieties, described the cellular type involved in its synthesis, its tissular distribution, and identified some of its functions such as its contribution to energy metabolism, structural precursors and defenses for the developing embryo. This protein is a very high-density lipoprotein (VHDL) of 400 KDa, 3.75% lipids and 2.5 % carbohydrates. It is composed of two subunits of 67 and 31 kDa. Different structural parameters of Pv2 were studied by Small Angle X-ray Scattering (SAXS) using urea as chaotropic agents (0 to 6M) or temperature ranging from (20 to 80°C) to evaluate the stability and unfolding mechanism. To gain structural insight on the disassembly/denaturing process we analyzed the evolution of the gyration radius obtained from Guinier plots and the shape and position of the Kratky-plots. In order to assay the relevance of the disulfide network on the stability of the oligomer we also performed non-denaturing PAGE and UV-Vis spectroscopy on samples treated with the Ellman´s reagent. From our SAXS results we were able to obtain a low resolution 3D model with a gyration radius of 44 ± 1 Å in the native protein being the first time a structural model of this protein was built. A small but significant decrease of the gyration radius was observed for urea concentrations up to 1.5M indicating the disassembling of the oligomer. From 2M urea on the gyration radius of the protein starts to grow up reaching 66 ± 5 Å at 6M urea well below the value expected for the denatured subunits. Pv2 underwent steep changes between 50 and 60°C as indicated by the shape of Kratky plot.  Pv2 showed a higher sensitivity to thermal and urea denaturalization than observed for the ovorubin. Their complementary roles during embryogenesis (presence of triacylglicerols in Pv2 and an antioxidant and antiprotease activities of ovorubin) are therefore supported by their constrasting structural stability.