Seco Autnoma de Cincias da Sade

Mestrado em Biomedicina Molecular

2015/2016

Exogenous expression of human SGLT1 exhibits

aggregations in dodecyl sulfate polyacrilamide gel
electrophoresis
The metabolism is an important way to maintain the celular viability and
the homeostase of an organism. Some normal cells produce, by oxidative
phosphorilation, among 38 ATPs for each molecule of glucose. In other hand
a few types of cancer cells produce only 2 ATP per glucose. This
phenomenum is descripte as Warburg effect. On this cancer cells the GLUT1
surface protein responsible for higher glucose uptake is overexpressed as
well the SGLT1, an active energy-dependente sodium/glucose cotransporter.
The role of this cotransporter allows to prevent autophagic cell death and
its correlated with EGFR expression. So, EGFR facilitates glucose transport
into cells by associating with and stabilizing a sodium/glucose cotransporter
(SGLT1).
A among quantity of protein can be detected by SDS Page, a technique of
protein separation according to their molecular weight. SDS could be
performed in one of two conditions: in reduction condition or in nonreduction condition (maintain of the tertiary structure of protein). But some
proteins, in particular glycosilated transmembrane and sulfitoyled protein,
N- and O-linked glycosylation, have an high tendency to form SDS-resistant
aggregates wich exhibit retarded SDS-Page mobility in non-reducing gels.
However, it remains unclear whether exogenously overexpressed SGLT1
forms SDS-resistant aggregates like other transmembrane proteins.
The main purpose of this study is to evaluate the aggregation of SGLT1
protein in dodecyl sulfate polyacrilamide gel electrophoresis in order to
study the SGLT1 protein and her main purpose on cancer. This basic
research will allow development cancer targets for new therapeutics. First,
the authors used a cellular line, HEK293, wich was maintaing in optimal
conditions. Then they generate an anti-SGLT1 antibody and a flag-tagged
SGLT1. Afther, the authors performed a transiente transfection to test the
interation between SGLT1 and EGFR. Cells were cotransfected with SGLT1
and EGFR for 24 hours, and then harvested and subjected to Western Blot
(WB) and immunoprecipitation. For knockdown of SGLT1, cells were
cotransfected with or without SGLT1 siRNA. The total RNA was isolated from
transfected HEK293 cells according manufactures protocol and was subject
to reverse transcription (RT) and polymerase chain reaction (PCR). PCR
primer sequences for SGLT1 are 5-TTCCACATCTTCCGAGATCC-3 and 5GGACGACACAGGCAATTTTT-3. The proteins was separated by 8% SDS and
then, after transfered and blocked with 5% nonfat milk, they were detected
by WB analysis (anti-SLGT1 antibody and secondary antibody).
Immunoprecipation was also realized and analysed by WB. The activity of
SGLT1 was determined by measuring the uptake of MDG, a specific
substrate for SGLT1. Prior to MDG uptake assays, cells were pre-treated
with or without 100 M phloridzin, an SGLT1-specific inhibitor.

Beatriz Martins, Daniela Sousa, Mariana Marques