Abstract
Human GLUT5 is a fructose-specific transporter in the glucose transporter family (GLUT, SLC2 gene family). Its substrate-specificity and tissue-specific expression make it a promising target for treatment of diabetes, metabolic syndrome and cancer, but few GLUT5 inhibitors are known. To identify and characterize potential GLUT5 ligands, we developed a whole-cell system based on a yeast strain deficient in fructose uptake, in which GLUT5 transport activity is associated with cell growth in fructose-based media or assayed by fructose uptake in whole cells. The former method is convenient for high-throughput screening of potential GLUT5 inhibitors and activators, while the latter enables detailed kinetic characterization of identified GLUT5 ligands. We show that functional expression of GLUT5 in yeast requires mutations at specific positions of the transporter sequence. The mutated proteins exhibit kinetic properties similar to the wild-type transporter and are inhibited by established GLUT5 inhibitors N-[4-(methylsulfonyl)-2-nitrophenyl]-1,3-benzodioxol-5-amine (MSNBA) and (−)-epicatechin-gallate (ECG). Thus, this system has the potential to greatly accelerate the discovery of compounds that modulate the fructose transport activity of GLUT5.
Highlights
Most glucose transporters (GLUTs), members of the SLC2 family, facilitate the passive diffusion of glucose and related monosaccharides in mammalian cells
To generate plasmids for GLUT5 expression in yeast we used a construct previously codon-optimized for expression in insect cells and encoding a GLUT5 protein truncated for the first seven amino acids at the N-terminus
In addition to GLUT5tr ORFs, a variant, in which the codons for the initial seven amino acids were re-introduced into GLUT5tr by an oligonucleotide, was amplified
Summary
Most glucose transporters (GLUTs), members of the SLC2 family, facilitate the passive diffusion of glucose and related monosaccharides in mammalian cells. Inhibitor effect on GLUT5 fructose transport in the hxt[0] strain, as indicated by inhibitor IC50 value, is similar to that determined for GLUT5 inhibition in the human breast cancer cells MCF-7 (in the case of MSNBA) and in GLUT5-expressing Xenopus laevis oocytes (in the case of ECG). Both MSNBA and ECG decrease cell growth of GLUT5-expressing hxt[0] strain in a dose-dependent manner. Subsequent optimization of GLUT5 ligands as selective and potent in vivo chemical probes will allow for advancement of our understanding of GLUT5 biology, its role in human disease, and its pharmaceutical control
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