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, while 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 internalized 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.

Internalisation and multiple phosphorylation of γ-conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells / J. Capraro, C. Magni, F. Faoro, D. Maffi, A. Scarafoni, G. Tedeschi, E. Maffioli, A. Parolari, C. Manzoni, M.R. Lovati, M. Duranti. - In: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. - ISSN 0006-291X. - 437:4(2013 Jul 17), pp. 648-652. [10.1016/j.bbrc.2013.07.026]

Internalisation and multiple phosphorylation of γ-conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells

J. Capraro;C. Magni;F. Faoro;D. Maffi;A. Scarafoni;G. Tedeschi;E. Maffioli;A. Parolari;C. Manzoni;M.R. Lovati;M. Duranti
2013

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, while 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 internalized 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.
2D electrophoresis ; Lupinus albus ; hypoglycaemic protein ; microscopy ; protein phosphorylation
Settore BIO/10 - Biochimica
17-lug-2013
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/223102
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