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Am J Transl Res 2013;5(4):441-449
Original Article
Exogenous expression of human SGLT1 exhibits aggregations in
sodium dodecyl sulfate polyacrylamide gel electrophoresis
Wei-Chien Huang, Sheng-Chie Hsu, Shyh-Jer Huang, Yun-Ju Chen, Yu-Chun Hsiao, Weihua Zhang, Isaiah J
Fidler, Mien-Chie Hung
Center for Molecular Medicine, China Medical University Hospital, Taichung 404, Taiwan; Graduate Institute of
Cancer Biology, the Ph.D. program for Cancer Biology and Drug Discovery, China Medical University, Taichung
404, Taiwan; Department of Biotechnology, Asia Univer-sity, Taichung 413, Taiwan; Department of Medical
Research, E-Da Hospital, Kaohsiung 824, Taiwan; Department of Biological Science & Technology, I-Shou
University, Kaohsiung 824, Taiwan; Department of Biology and Biochemistry, University of Houston, Houston, TX,
77204, USA; Department of Cancer Biology, The University of Texas M.D. Anderson Cancer Center, Houston, TX
77030, USA; Graduate School of Biomedical Sciences, The University of Texas Health Science Center, Houston,
TX 77030, USA; Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer
Center, Houston, TX 77030, USA. These authors contributed equally and thus share first-authorship.
Received May 1, 2013; Accepted May 15, 2013; Epub May 24, 2013; Published June 1, 2013
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. (AJTR1305001).
Keywords: Sodium/glucose cotranspoter 1, epidermal growth factor receptor, protein aggregation, glucose
uptake, o-link glycosylation
Address correspondence to: Wei-Chien Huang, Center for Molecular Medicine, China Medical University
Hospital, 9F, No.6, Hsueh-Shih Road, Taichung, 404 Taiwan. Phone: 886-4-22052121 ext 7931; Fax:
886-4-22333496; E-mail: whuang@mail.cmu.edu.tw; Mien-Chie Hung, The University of Texas MD Anderson
Cancer Center, 1515 Holcombe Boulevard Houston, TX 77030. Phone: 713-792-7477; Fax: 713-794-3270;
E-mail: mhung@mdanderson.org
Original Article
Exogenous expression of human SGLT1 exhibits aggregations in
sodium dodecyl sulfate polyacrylamide gel electrophoresis
Wei-Chien Huang, Sheng-Chie Hsu, Shyh-Jer Huang, Yun-Ju Chen, Yu-Chun Hsiao, Weihua Zhang, Isaiah J
Fidler, Mien-Chie Hung
Center for Molecular Medicine, China Medical University Hospital, Taichung 404, Taiwan; Graduate Institute of
Cancer Biology, the Ph.D. program for Cancer Biology and Drug Discovery, China Medical University, Taichung
404, Taiwan; Department of Biotechnology, Asia Univer-sity, Taichung 413, Taiwan; Department of Medical
Research, E-Da Hospital, Kaohsiung 824, Taiwan; Department of Biological Science & Technology, I-Shou
University, Kaohsiung 824, Taiwan; Department of Biology and Biochemistry, University of Houston, Houston, TX,
77204, USA; Department of Cancer Biology, The University of Texas M.D. Anderson Cancer Center, Houston, TX
77030, USA; Graduate School of Biomedical Sciences, The University of Texas Health Science Center, Houston,
TX 77030, USA; Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer
Center, Houston, TX 77030, USA. These authors contributed equally and thus share first-authorship.
Received May 1, 2013; Accepted May 15, 2013; Epub May 24, 2013; Published June 1, 2013
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. (AJTR1305001).
Keywords: Sodium/glucose cotranspoter 1, epidermal growth factor receptor, protein aggregation, glucose
uptake, o-link glycosylation
Address correspondence to: Wei-Chien Huang, Center for Molecular Medicine, China Medical University
Hospital, 9F, No.6, Hsueh-Shih Road, Taichung, 404 Taiwan. Phone: 886-4-22052121 ext 7931; Fax:
886-4-22333496; E-mail: whuang@mail.cmu.edu.tw; Mien-Chie Hung, The University of Texas MD Anderson
Cancer Center, 1515 Holcombe Boulevard Houston, TX 77030. Phone: 713-792-7477; Fax: 713-794-3270;
E-mail: mhung@mdanderson.org
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