title: Exogenous expression of human SGLT1 exhibits aggregations in sodium dodecyl sulfate polyacrylamide gel electrophoresis.
abstract: 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.
authors.affiliation: Center for Molecular Medicine, China Medical University Hospital Taichung 404, Taiwan ; Graduate Institute of Cancer Biology, China Medical University Taichung 404, Taiwan ; the Ph.D. program for Cancer Biology and Drug Discovery, China Medical University Taichung 404, Taiwan ; Department of Biotechnology, Asia University Taichung 413, Taiwan.
authors.forename: Wei-Chien
authors.initials: WC
authors.lastname: Huang
authors.forename: Sheng-Chie
authors.initials: SC
authors.lastname: Hsu
authors.forename: Shyh-Jer
authors.initials: SJ
authors.lastname: Huang
authors.forename: Yun-Ju
authors.initials: YJ
authors.lastname: Chen
authors.forename: Yu-Chun
authors.initials: YC
authors.lastname: Hsiao
authors.forename: Weihua
authors.initials: W
authors.lastname: Zhang
authors.forename: Isaiah J
authors.initials: IJ
authors.lastname: Fidler
authors.forename: Mien-Chie
authors.initials: MC
authors.lastname: Hung
keywords: Sodium/glucose cotranspoter 1; epidermal growth factor receptor; glucose uptake; o-link glycosylation; protein aggregation