Exogenous expression of human SGLT1 exhibits aggregations in sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Exogenous expression of human SGLT1 exhibits aggregations in sodium dodecyl sulfate polyacrylamide gel electrophoresis.
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发表时间：
2013-05
影响因子：
2.2
通讯作者：
Wei‐Chien Huang;S. Hsu;Shyh-Jer Huang;Yun-Ju Chen;Yu-Chun Hsiao;Weihua Zhang;I. Fidler;M. Hung
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作者：
Wei‐Chien Huang;S. Hsu;Shyh-Jer Huang;Yun-Ju Chen;Yu-Chun Hsiao;Weihua Zhang;I. Fidler;M. Hung
钠/葡萄糖协同转运蛋白 1 (SGLT1) 主动且能量依赖性地摄取葡萄糖，在包括糖尿病和癌症在内的各种疾病的发展中发挥着关键作用，并被视为这些疾病的有前途的治疗靶点。 EGFR 的蛋白质-蛋白质相互作用已被证明可以调节 SGLT1 的表达和活性。 SGLT1的外源表达是表征其功能的重要方法之一；然而，外源表达的 SGLT1 无法通过蛋白质印迹以其计算的分子量可靠地检测到，这为进一步了解 SGLT1 调控的分子事件造成了障碍。在本研究中，我们证明外源性 SGLT1 在葡萄糖摄取中正常发挥作用，但始终在堆积胶和电泳胶之间的界面附近检测到，而不是在蛋白质印迹分析中在计算的分子量处检测到，这表明过表达的 SGLT1 形成了 SDS 抗性聚集体，该聚集体不能在 SDS-PAGE 上变性和有效分离。 EGFR 的共表达增强了 SGLT1 的葡萄糖摄取活性和蛋白水平。然而，在其羧基端而非氨基端与 Flag 或 HA 标签融合消除了外源 SGLT1 的葡萄糖摄取活性，而不影响其蛋白质水平。此外，SGLT1聚集体的溶解度不受其他去垢剂的影响，但通过抑制o-link糖基化而部分改善。这些发现表明，SGLT1 的外源过度表达可以正常发挥作用，但由于其通过形成 SDS 抗性聚集体而产生的凝胶迁移行为，因此在其配方重量下可能无法一致地检测到。
Sodium/glucose co-transporter 1 (SGLT1), which actively and energy-dependently uptakes glucose, plays critical roles in the development of various diseases including diabetes mellitus and cancer, and has been viewed as a promising therapeutic target for these diseases. Protein-protein interaction with EGFR has been shown to regulate the expression and activity of SGLT1. Exogenous expression of SGLT1 is one of the essential approaches to characterize its functions; however, exogenously expressed SGLT1 is not firmly detectable by Western blot at its calculated molecular weight, which creates a hurdle for further understanding the molecular events by which SGLT1 is regulated. In this study, we demonstrated that exogenous SGLT1 functions in glucose-uptake normally but is consistently detected near the interface between stacking gel and running gel rather than at the calculated molecular weight in Western blot analysis, suggesting that the overexpressed SGLT1 forms SDS-resistant aggregates, which cannot be denatured and effectively separated on SDS-PAGE. Co-expression of EGFR enhances both the glucose-uptake activity and protein level of the SGLT1. However, fusion with Flag or HA tag at its carboxy- but not its amino-terminus abolished the glucose-uptake activity of exogenous SGLT1 without affecting its protein level. Furthermore, the solubility of SGLT1 aggregates was not affected by other detergents but was partially improved by inhibition of o-link glycosylation. These findings suggested exogenous overexpression of SGLT1 can function normally but may not be consistently detectable at its formula weight due to its gel-shift behavior by forming the SDS-resistant aggregates.