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

Abstract

Highlights: •A glycaemia-reducing lupin seed protein is internalized by HepG2 cells. •The protein accumulates in the cytosol in an intact form. •The internalized protein is multiply phosphorylated. -- 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 waymore » to the understanding of the lupin γ-Conglutin insulin-mimetic activity.« less

Authors:
 [1];  [1]; ;  [2];  [1]; ;  [3]; ; ;  [4];  [1]
  1. Department of Food, Environmental and Nutritional Sciences (DeFENS), Section of Chemistry and Biomolecular Sciences, Università degli Studi di Milano (UNIMI) (Italy)
  2. Department of Agricultural and Environmental Sciences, UNIMI (Italy)
  3. Department of Veterinary Science and Public Health, UNIMI (Italy)
  4. Department of Pharmacological and Biomolecular Sciences, UNIMI (Italy)
Publication Date:
OSTI Identifier:
22239699
Resource Type:
Journal Article
Journal Name:
Biochemical and Biophysical Research Communications
Additional Journal Information:
Journal Volume: 437; Journal Issue: 4; Other Information: Copyright (c) 2013 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0006-291X
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AMINO ACIDS; ANTIBODIES; ELECTROPHORESIS; GLUCOSE; INSULIN; PHOSPHORYLATION; TRANSMISSION ELECTRON MICROSCOPY; TRYPSIN

Citation Formats

Capraro, Jessica, Magni, Chiara, Faoro, Franco, Maffi, Dario, Scarafoni, Alessio, Tedeschi, Gabriella, Maffioli, Elisa, Parolari, Anna, Manzoni, Cristina, Lovati, Maria Rosa, and Duranti, Marcello. Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells. United States: N. p., 2013. Web. doi:10.1016/J.BBRC.2013.07.026.
Capraro, Jessica, Magni, Chiara, Faoro, Franco, Maffi, Dario, Scarafoni, Alessio, Tedeschi, Gabriella, Maffioli, Elisa, Parolari, Anna, Manzoni, Cristina, Lovati, Maria Rosa, & Duranti, Marcello. Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells. United States. https://doi.org/10.1016/J.BBRC.2013.07.026
Capraro, Jessica, Magni, Chiara, Faoro, Franco, Maffi, Dario, Scarafoni, Alessio, Tedeschi, Gabriella, Maffioli, Elisa, Parolari, Anna, Manzoni, Cristina, Lovati, Maria Rosa, and Duranti, Marcello. 2013. "Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells". United States. https://doi.org/10.1016/J.BBRC.2013.07.026.
@article{osti_22239699,
title = {Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells},
author = {Capraro, Jessica and Magni, Chiara and Faoro, Franco and Maffi, Dario and Scarafoni, Alessio and Tedeschi, Gabriella and Maffioli, Elisa and Parolari, Anna and Manzoni, Cristina and Lovati, Maria Rosa and Duranti, Marcello},
abstractNote = {Highlights: •A glycaemia-reducing lupin seed protein is internalized by HepG2 cells. •The protein accumulates in the cytosol in an intact form. •The internalized protein is multiply phosphorylated. -- 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.},
doi = {10.1016/J.BBRC.2013.07.026},
url = {https://www.osti.gov/biblio/22239699}, journal = {Biochemical and Biophysical Research Communications},
issn = {0006-291X},
number = 4,
volume = 437,
place = {United States},
year = {Fri Aug 09 00:00:00 EDT 2013},
month = {Fri Aug 09 00:00:00 EDT 2013}
}