Abstract
Intestinal fructose uptake is mainly mediated by glucose transporter 5 (GLUT5/SLC2A5). Its closest relative, GLUT7, is also expressed in the intestine but does not transport fructose. For rat Glut5, a change of glutamine to glutamic acid at codon 166 (p.Q166E) has been reported to alter the substrate-binding specificity by shifting Glut5-mediated transport from fructose to glucose. Using chimeric proteins of GLUT5 and GLUT7, here we identified amino acid residues of GLUT5 that define its substrate specificity. The proteins were expressed in NIH-3T3 fibroblasts, and their activities were determined by fructose radiotracer flux. We divided the human GLUT5 sequence into 26 fragments and then replaced each fragment with the corresponding region in GLUT7. All fragments that yielded reduced fructose uptake were analyzed further by assessing the role of individual amino acid residues. Various positions in the first extracellular loop, in the fifth, seventh, eighth, ninth, and tenth transmembrane domains (TMDs), and in the regions between the ninth and tenth TMDs and tenth and 11th TMDs were identified as being important for proper fructose uptake. Although the p.Q167E change did not render the human protein into a glucose transporter, molecular dynamics simulations revealed a drastic change in the dynamics and a movement of the intracellular loop connecting the sixth and seventh TMDs, which covers the exit of the ligand. Finally, we generated a GLUT7-GLUT5 chimera consisting of the N-terminal part of GLUT7 and the C-terminal part of GLUT5. Although this chimera was inactive, we demonstrate fructose transport after introduction of four amino acids derived from GLUT5.
Highlights
Intestinal fructose uptake is mainly mediated by glucose transporter 5 (GLUT5/SLC2A5)
The p.Q167E change did not render the human protein into a glucose transporter, molecular dynamics simulations revealed a drastic change in the dynamics and a movement of the intracellular loop connecting the sixth and seventh transmembrane domains (TMDs), which covers the exit of the ligand
Analysis of GLUT5–GLUT3 chimeras revealed that the region between the N terminus and the first intracellular loop and the sequence including the third extracellular loop to the 11th TMD are important for fructose transport (8)
Summary
We constructed GLUT5–GLUT7 chimeras by dividing the complete protein sequence of GLUT5 into 26 fragments that we consecutively replaced with homologous domains of GLUT7. Amino acid changes that showed drastically reduced fructose uptake in GLUT5–GLUT7 chimeras were exchanged in chimera G7-220-G5-B (p.L42V, p.E173Q, and p.I177T). As anticipated by the RMSD and root mean square fluctuation calculations, in p.Q167E, the intracellular (IC) loop between the TMD moves dramatically during the simulation This movement leads to a complete change of the loop position and resembles that of a lid covering the exit of the substrate into the cytosol (Fig. 5). This observation raised our curiosity about the residues that drastically reduce fructose transport These amino acids are located closer to the central cavity, embracing TMD 5, 7, 8, and 11 and loops between TMD 1 and 2 and 10 and 11, they are still asymmetrically distributed, involving only one side of the protein (Fig. 6B, depicted in red).
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