Phosphorylation upon uptake of gamma-conglutin, a lupin seed protein able to lower glycaemia in animals and humans, by HepG2 cells

gamma-Conglutin, a lupin seed glycoprotein, is capable of positively influencing glucose uptake by various model cells and reducing glycaemia in animal models and human subjects. In particular, gamma-conglutin can activate in differentiating myocyte different signaling pathways that closely resembled those of insulin. A recent study aimed at identifying the metabolic fate of the protein, showed that it was transcytosed through a CaCo2 cell monolayer and crossed an ex-vivo intestinal barrier in an intact form. Beside, the protein displays strong resistance to endogenous and exogenous proteolytic enzymes. These findings encouraged us to further study the molecular mechanisms of action of this bioactive protein, which are still not completely elucidated. Aim of the present work was to study how gamma-conglutin interact with the target cells and show if the protein undergoes any covalent change during the process. 2D-electrophoresis of the gamma-conglutin treated cell lysates and antibody revelation of the blotted maps showed the presence of the intact protein inside the cells. Moreover, spots with an unexpectedly low pI, not present in the natural protein, were detected. These spots were submitted to MS/MS spectrometric analysis. Phosphorylated amino acids were evidenced in some gamma-conglutin peptides. To unveil gamma-conglutin cellular fate, HepG2 cells treated with the lupin protein were submitted to confocal microscopy and transmission electron microscopy (TEM). gamma-Conglutin was found accumulated inside the cells and spread as protein aggregates into the cytoplasm. Microvilli were involved in trapping the protein at the beginning of the process. A prerequisite for any biological activity is the interaction with or entry into the target cells. These findings, by showing that gamma-conglutin is uptaked by HepG2 cells in an intact form and is modified by multiple phosphorylation, open the way to new studies for the understanding of the described gamma-conglutin insulin-mimetic activity.