Proteome analysis to study the effects of lupin gamma-conglutin on cellular metabolism in cultured C2C12 and HepG2 cells

gamma-Conglutin is a tetrameric glycoprotein representing about 5% of lupin seed proteins. Each monomer is made up of two disulphide-linked subunits of 17 and 29 kDa (1). The protein was found to decrease glycaemia in glucose overload trials carried out on animal models and healthy human subjects (2, 3). The translocation of the intact protein through the intestinal barrier in in vitro and ex vivo experiments has been demonstrated (4). \uf067-Conglutin showed insulin-mimetic activity and was able to increase glucose consumption by myocites and HepG2 cells (5, 6). This experimental set of data leads to consider \uf067-conglutin as a possible nutraceutical molecule for the prevention of diabetes II-related diseases. In order to contribute to unveiling the molecular mechanisms at the basis of its effects on cell metabolism, two different cell lines, namely myoblastic C2C12 and HepG2, were incubated with \uf067-conglutin for 4 and 6 hours, respectively. After accurate cell washing, the proteomes were analyzed by 2D electrophoresis, Western blot and in-gel fluorescence stain (Phos Tag) for phosphoprotein detection. The results showed that the protein was able to enter the cells, where it was detected in intact form. No polypeptides with Mr lower than those of the full size subunits were evidenced. Interestingly, a polypeptide of 29 kDa subunit reacting with specific \uf067-conglutin antibodies, but not with Phos Tag dye, appeared with a different pI in HepG2 cells. The nature of such modifications and possible changes in the phosphorylation pattern of the cells are still under investigation. (1) Magni et al (2007) Phytochem 68(7), 997. (2) Magni et al (2004) J Nutr Biochem 15, 646. (3) Bertoglio et al (2011) Fitoterapia 82(7), 933. (4) Capraro et al (2011) Food Chem 125, 1279. (5) Terruzzi et al (2011) Nutr Metab Cardiovasc Dis 21(3), 197. (6) Lovati et al (2012) Br J Nutr, 107(1), 67