INVESTIGATIONS ON STRUCTURAL STABILITY, BIOLOGICAL ACTIVITY AND METABOLIC FATE OF GAMMA-CONGLUTIN, A BLOOD GLUCOSE-LOWERING LUPIN SEED PROTEIN

gamma-Conglutin is a glycoprotein representing about 5% of lupin seed proteins. gamma-Conglutin is oligomeric at neutral pH, while at acidic pH it dissociates into monomers of 50 kDa. Each monomer is composed of two disulphide linked subunits of 17 and 29 kDa, originating from the endogenous proteolysis of the pro-polypeptide. Other 5 interchain disulphide bridges are also present. gamma-Conglutin sequence does not match any canonical legume storage proteins sequence; this protein is not degraded during seed germination and it was found to be extracellularly secreted during germination. These findings suggest a functional role for gamma-conglutin in the seed other than a mere storage one. gamma-Conglutin shows a great similarity (64%) with the basic globulin (BG7S) of soybean seed, which has been attributed an in vitro kinase activity and a binding activity towards some small regulatory proteins, such as a 4 kDa plant hormone-like peptide and mammalian insulin. -Conglutin shows resistance to in vitro proteolysis unless it was previously fully denatured. It can bind various divalent ions, such as Zn++ and Ni++, and protein such as acid phosphatase and insulin with a Kd of 10-5 M. Finally, gamma-conglutin was found to decrease glycemia in glucose overloading experiments on rats. Therefore, it seemed worth to investigate the molecular bases of this biological activity, as well as the properties related to gamma-conglutin metabolic fate and interaction with target cells. Indeed, aim of this work was to unravel the structural bases of gamma-conglutin assembly and stability to proteolysis and to develop suitable models to monitor its pathway after assumption and the cellular mechanisms involved in gamma-conglutin stimulation. By using dynamic light scattering, we investigated the assembly state of gamma-conglutin at different pH conditions. The results indicated a transition from monomer to oligomer, likely hexamer, via the transient formation of a dimer while pH was raised from 4.5 to neutral values. A model of these pH transitions based on the role of charge attraction/repulsions between acidic and basic amino acid residues in driving gamma-conglutin aggregation state was proposed. By using far- and near-UV circular dichroism, and intrinsic/ANS fluorescence analyses, the conformational changes as a function of pH were measured. All results suggested that the protein secondary and tertiary structures were unmodified by pH change above 3.5, while for lower values the structural collapse of the protein took place. Nevertheless, indirect evidence obtained with a set of proteolytic enzymes showed that gamma-conglutin became susceptible to proteolysis at pH values lower than 4.25, thus confirming its overall stability at neutral to slightly acidic pHs and suggesting also that a partial opening of the protein, without loss of overall three dimensional conformation, already occurred. Although γ-conglutin 3D structure is not available, it can be argued that its native conformation, which is stabilized also by a number of intrachain disulphide bonds, is extremely compact, especially if compared to the canonical seed storage proteins. The survival of intact gamma-conglutin into the intestinal lumen as well as its presence in the blood of suitable animal models were not proved with our assays in this work. However, the transit of gamma-conglutin through the intestinal barrier, by using in vitro and ex vivo models, namely Caco-2 cell monolayers and intestinal everted sacs, was demonstrated. Both experimental approaches showed that the transit through a transcellular way was possible for the intact gamma-conglutin, though limited to a small percentage of the applied protein. Although we were not able to detect gamma-conglutin in the sera samples of animals fed gamma-conglutin, the hypothesis of an at least partial preservation of the protein into the gastro-intestinal tract is also consistent with the observed immunogenic activity as measured in orally-treated mice in a parallel study. Eventually, we showed that the full length protein in its native conformation was capable to activate the insulin-signaling pathway in miocyte models in an insulin-like pattern, despite the dramatic structural differences between the two proteins. Moreover, the cellular absorption of gamma-conglutin by miocytes was shown by using a 2D electrophoretic approach. This finding, obtained with a cellular model not specifically involved in protein transport, confirmed the transcellular transport for gamma-conglutin observed with Caco2 cells. Very recent unpublished data reporting the results of the first human trial with healthy human subjects confirmed gamma-conglutin biological activity. Therefore, the present set of data is relevant to design further approaches aimed at exploiting lupin gamma-conglutin as a preventing and therapeutic agent in the cases of impaired glucose tolerance and possibly pre-diabetic and diabetic conditions.