Villagrán M et al. (2023), Identification of Structural Determinants of th...

XB-ART-59635

Molecules 2023 Jan 05;282:. doi: 10.3390/molecules28020521.

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Identification of Structural Determinants of the Transport of the Dehydroascorbic Acid Mediated by Glucose Transport GLUT1.

Villagrán M , Burgos CF , Rivas CI , Mardones L .

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GLUT1 is a facilitative glucose transporter that can transport oxidized vitamin C (i.e., dehydroascorbic acid) and complements the action of reduced vitamin C transporters. To identify the residues involved in human GLUT1's transport of dehydroascorbic acid, we performed docking studies in the 5 Å grid of the glucose-binding cavity of GLUT1. The interactions of the bicyclic hemiacetal form of dehydroascorbic acid with GLUT1 through hydrogen bonds with the -OH group of C3 and C5 were less favorable than the interactions with the sugars transported by GLUT1. The eight most relevant residues in such interactions (i.e., F26, Q161, I164, Q282, Y292, and W412) were mutated to alanine to perform functional studies for dehydroascorbic acid and the glucose analog, 2-deoxiglucose, in Xenopus laevis oocytes. All the mutants decreased the uptake of both substrates to less than 50%. The partial effect of the N317A mutant in transporting dehydroascorbic acid was associated with a 30% decrease in the Vmax compared to the wildtype GLUT1. The results show that both substrates share the eight residues studied in GLUT1, albeit with a differential contribution of N317. Our work, combining docking with functional studies, marks the first to identify structural determinants of oxidized vitamin C's transport via GLUT1.

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Species referenced: Xenopus laevis
Genes referenced: grap2 slc2a1
GO keywords: glucose transmembrane transporter activity [+]

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	Graphical Abstract
	Figure 1. Schematic general model for members of the GLUT family. The conserved signature sequences appear in boxes, conserved residues in white circles, and high conserved residues in gray circles. The N-linked glycosylation site of Class I is indicated in green. The schema is based on the new GLUT1 crystal structure [24].
	Figure 2. Interactions between GLUT1 and the different substrates as predicted by protein-ligand docking. (A) Position of the 5 Å grid interaction (purple box) used for docking simulations with the amino acids that are part of the sugar-binding sites indicated in blue and red; (B) Docking XP scores for the seven molecules capable of forming a stable complex with GLUT1 (i.e., glucose-6-phosphate, D-galactose, D-glucose, D-glucosamine, dehydroascorbic acid, bicyclic dehydroascorbic acid, and ascorbic acid); (C) Theoretical ∆Gbind values for the seven molecules analyzed with data expressed in kcal/mol and estimated using the MM-GBSA method; (D) Structure of the seven molecules analyzed in this study. G-6-P: glucose-6-phosphate; Galac: D-galactose; Glc: D-glucose; GlcN: D-glucosamine; DHA (bicyclic): dehydroascobic acid bicyclic form; DHA: dehydroascorbic acid; AA: ascorbic acid.
	Figure 3. Comparative analysis of the DHA(bi) and glucose-binding mode of GLUT1 in the 5 Å grid. Representative complexes of GLUT1 with (A) DHA(bi) and (B) glucose are shown. The docking simulations were performed in a 5 Å grid of the GLUT1 junction pocket. Amino acid residues that interact with each molecule are indicated; the arrows indicate specific interactions between each substrate and the amino acid residues in GLUT1. Light dotted arrows indicate H-bond interactions with amino acid side chains. Negatively charged residues appear in red, hydrophobic residues in green, and uncharged polar residues in blue.
	Figure 4. Initial rates for DOG and DHA transport mediated by GLUT1 mutants in Xenopus laevis oocytes. Uptake was carried out at 2 min intervals using DOG (0.5 mM) and DHA (0.1 mM), and the slopes of the time-curse plots were graphed. The maximum transport time was 10 min for DOG and 15 min for DHA. The X. laevis oocytes were injected with 40 ng of hGLUT1 cRNA or its mutants or if in the control, an equal volume of water. Three experiments were carried out in triplicates, counting four oocytes per sample. The data are expressed as mean ± SD with respect to the WT (6.30 ± 0.68 pmoles/oocyte × min for DOG and 6.30 ± 0.60 pmoles/oocyte × min). DHA: dehydroascorbic acid, DOG: 2-deoxyglucose, WT: wildtype GLUT1, Vo: initial rate. * p > 0.01 with respect to the control substrate and # p < 0.05 DHA transport with respect to DOG transport by the same mutant.
	Figure 5. Effects of N317A on DHA transport on GLUT1. (A) DHA saturation curves of GLUT1 wildtype and N317A mutant; (B) Eadie-Hofstee transformation of the data shown in (A); (C) Effect of N317 in response to glucose. DHA’s transport in mutant N317A was performed in the presence of 30–50 mM glucose. (D) Effect of N317 in response to galactose and glucosamine. DHA’s transport in mutant N317A was performed in the presence of 10–30 mM galactose and galactosamine DHA. The data are expressed as mean ± SD of three experiments performed in triplicates. In (C,D), the results are presented as a percentage of the wildtype or mutant in the absence of the hexose explored, and 0.1 mM DHA was used. Xenopus laevis oocytes were injected with 40 ng of hGLUT1 or N317A mutant cRNA or if in the control, with equal volume of water. All the experiments were done at 8 min. For competition assays, hexoses were added simultaneously with DHA. DHA: dehydroascorbic acid, DOG: 2-deoxyglucose, Vo: initial rate, V: velocity, S: substrate, WT: wildtype GLUT1, Galac: galactose, GlcN: glucosamine. * p > 0.01.
	Figure 6. Amino acid sequence alignment of DHA transporters. Transmembrane domains are highlighted in yellow based on the new GLUT1 crystal structure [20,24]. The GLUT1 residues in our study and the equivalent residues among the other members of the GLUT family that transport DHA are marked in blue. Alignment was performed using Clustal 0 (1.2.4).

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Ancey, Glucose transporters in cancer - from tumor cells to the tumor microenvironment. 2018, Pubmed