Department of Food, Environmental and Nutritional Sciences, Section of Chemistry and Biomolecular Sciences, Università degli Studi di Milano, Italy.
Biochem Biophys Res Commun. 2013 Aug 9;437(4):648-52. doi: 10.1016/j.bbrc.2013.07.026. Epub 2013 Jul 17.
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.
羽扇豆γ-伴球蛋白是一种能够降低哺乳动物血糖水平并增加模型细胞葡萄糖摄取的蛋白质。本研究旨在探讨γ-伴球蛋白在进入靶细胞的过程中是否发生内化，并在此过程中发生任何共价变化，以此作为了解其作用机制的第一步。为此，用γ-伴球蛋白处理和未处理的 HepG2 细胞进行共聚焦和透射电子显微镜检查。在不同时间间隔对γ-伴球蛋白进行免疫揭示，其在细胞质内积累。与此同时，细胞裂解物的 2D 电泳和印迹图谱的抗体反应显示，完整的蛋白质亚基存在于处理过的细胞中，而在对照细胞中则没有发现该蛋白质的痕迹。然而，在图谱中还观察到与 γ-伴球蛋白相关的具有异常低 pI 的斑点。这些斑点被切除、胰蛋白酶处理并进行 MS/MS 光谱分析。检测到磷酸化氨基酸的存在。这些发现表明，γ-伴球蛋白以完整形式被 HepG2 细胞内化，并通过多种磷酸化修饰，为理解羽扇豆γ-伴球蛋白的胰岛素模拟活性开辟了道路。
