Format

Send to

Choose Destination
J Biol Chem. 2018 Feb 9;293(6):2115-2124. doi: 10.1074/jbc.RA117.001442. Epub 2017 Dec 19.

Identification of essential amino acids for glucose transporter 5 (GLUT5)-mediated fructose transport.

Author information

1
From the Pediatric Nutritional Medicine, Klinikum rechts der Isar (MRI), Else Kröner-Fresenius-Zentrum für Ernährungsmedizin.
2
Department of Biosciences and Center for Integrated Protein Science Munich, and.
3
Nutritional Physiology, Technische Universität München, 85354 Freising, Germany.
4
Department of Biosciences and Center for Integrated Protein Science Munich, and antes@tum.de.
5
From the Pediatric Nutritional Medicine, Klinikum rechts der Isar (MRI), Else Kröner-Fresenius-Zentrum für Ernährungsmedizin, heiko.witt@tum.de.

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.

KEYWORDS:

GLUT5; GLUT7; fructose; glucose transport; membrane protein; molecular dynamics; protein chimera; transporter

PMID:
29259131
PMCID:
PMC5808771
DOI:
10.1074/jbc.RA117.001442

Supplemental Content

Full text links

Icon for HighWire
Loading ...
Support Center