title: Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells.
abstract: Lupin seed γ-Conglutin is a protein capable of reducing glycaemia in mammalians and increasing glucose uptake by model cells. This work investigated whether γ-Conglutin is internalised into the target cells and undergoes any covalent change during the process, as a first step to understanding its mechanism of action. To this purpose, γ-Conglutin-treated and untreated HepG2 cells were submitted to confocal and transmission electron microscopy. Immune-revelation of γ-Conglutin at various intervals revealed its accumulation inside the cytosol. In parallel, 2D-electrophoresis of the cell lysates and antibody reaction of the blotted maps showed the presence of the protein intact subunits inside the treated cells, whilest no trace of the protein was found in the control cells. However, γ-Conglutin-related spots with an unexpectedly low pI were also observed in the maps. These spots were excised, trypsin-treated and submitted to MS/MS spectrometric analysis. The presence of phosphorylated amino acids was detected. These findings, by showing that γ-Conglutin is internalised by HepG2 cells in an intact form and is modified by multiple phosphorylation, open the way to the understanding of the lupin γ-Conglutin insulin-mimetic activity.
authors.affiliation: Department of Food, Environmental and Nutritional Sciences, Section of Chemistry and Biomolecular Sciences, Università degli Studi di Milano, Italy.
authors.forename: Jessica
authors.initials: J
authors.lastname: Capraro
authors.forename: Chiara
authors.initials: C
authors.lastname: Magni
authors.forename: Franco
authors.initials: F
authors.lastname: Faoro
authors.forename: Dario
authors.initials: D
authors.lastname: Maffi
authors.forename: Alessio
authors.initials: A
authors.lastname: Scarafoni
authors.forename: Gabriella
authors.initials: G
authors.lastname: Tedeschi
authors.forename: Elisa
authors.initials: E
authors.lastname: Maffioli
authors.forename: Anna
authors.initials: A
authors.lastname: Parolari
authors.forename: Cristina
authors.initials: C
authors.lastname: Manzoni
authors.forename: Maria Rosa
authors.initials: MR
authors.lastname: Lovati
authors.forename: Marcello
authors.initials: M
authors.lastname: Duranti
mesh_terms: D000595:Amino Acid Sequence; D056945:Hep G2 Cells; D006801:Humans; D007004:Hypoglycemic Agents / Q000378:metabolism*; D031297:Lupinus / Q000737:chemistry*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010940:Plant Proteins / Q000378:metabolism*; D012639:Seeds / Q000737:chemistry
chemical_list: D007004:Hypoglycemic Agents; D010940:Plant Proteins; C079483:conglutin-gamma protein, Lupinus angustifolius
keywords: 2D electrophoresis; Hypoglycaemic protein; Lupinus albus; Microscopy; Protein phosphorylation