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Sci Rep
2017 Jan 24;7:41167. doi: 10.1038/srep41167.
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Identification of Key Residues for Urate Specific Transport in Human Glucose Transporter 9 (hSLC2A9).
Long W
,
Panigrahi R
,
Panwar P
,
Wong K
,
O Neill D
,
Chen XZ
,
Lemieux MJ
,
Cheeseman CI
.
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Human glucose transporter 9 (hSLC2A9) is critical in human urate homeostasis, for which very small deviations can lead to chronic or acute metabolic disorders. Human SLC2A9 is unique in that it transports hexoses as well as the organic anion, urate. This ability is in contrast to other homologous sugar transporters such as glucose transporters 1 and 5 (SLC2A1 &SLC2A5) and the xylose transporter (XylE), despite the fact that these transporters have similar protein structures. Our in silico substrate docking study has revealed that urate and fructose bind within the same binding pocket in hSLC2A9, yet with distinct orientations, and allowed us to identify novel residues for urate binding. Our functional studies confirmed that N429 is a key residue for both urate binding and transport. We have shown that cysteine residues, C181, C301 and C459 in hSLC2A9 are also essential elements for mediating urate transport. Additional data from chimæric protein analysis illustrated that transmembrane helix 7 of hSLC2A9 is necessary for urate transport but not sufficient to allow urate transport to be induced in glucose transporter 5 (hSLC2A5). These data indicate that urate transport in hSLC2A9 involves several structural elements rather than just a unique substrate binding pocket.
Figure 1. List of residues involved in interaction with urate in SLC2A9 and fructose in SLC2A9/SLC2A5.Note: The residues of SLC2A9 and SLC2A5 located at the similar position are color coded. The transmembrane helices to which the residues belong have been noted.
Figure 2. Docking studies of human SLC2A9b with fructose and urate.Panel (A and B) Binding pocket for fructose and urate, respectively. The residues involved in hydrogen bonding interaction (orange dash lines) have been shown in stick (green). The nitrogen and oxygen atoms are shown in blue and red respectively. The nitrogen and oxygen atoms are shown in blue and red respectively. Panel (C and D) Ligplot showing all the residues forming the binding pocket for fructose and urate, respectively. Hydrogen bonding interactions are shown in green dashed lines and the hydrophobically interacting pairs of atom are shown in red lines patterns.
Figure 3. Urate and fructose transport mediated by WT hSLC2A9 and its Y298Q and N429H mutants.Panel (A) Michaelis-Menten curves of 14C urate kinetics of hSLC2A9 WT (▪), Y298Q (○) and N429H (Δ). Panel (B) 14C urate kinetic constants and the standard error of the regression (Sy. X) of the three isoforms (n ≥ 3). Panel (C) Michaelis-Menten curves of urate-induced currents of WT hSLC2A9, Y298Q and N429H mutants. Panel (D) Urate-induced current kinetic constants and the standard error of the regression (Sy. X) of the WT and two mutants (n ≥ 15 oocytes from 3 frogs). Panel (E) Current-voltage curve of 1 mM urate-induced current obtained using a RAMP protocol WT hSLC2A9 (▪), Y298Q (○) and N429H (Δ) mutant expressing oocytes. (n ≥ 15 oocytes from 3 frogs). Panel (F) 14C fructose uptake mediated by WT hSLC2A9 and its mutants. (n ≥ 3, One-way ANOVA, *p < 0.05).
Figure 4. Urate and fructose transport mediated by WT hSLC2A9 and its cysteine mutants.Panel (A) Molecular model of human SLC2A9b with predicted locations of cysteine residues. View from the extracellular side of the outward facing conformation of hSLC2A9b. Panel (B) 14C fructose uptake mediated by WT hSLC2A9 and its cysteine mutants. Bar graphs represent fructose uptake activities, which were corrected for non-specific transport measured in control water injected oocytes from the same batch of oocytes (n ≥ 3, One-way ANOVA, *p < 0.05). Panel (C) Michaelis-Menten curves of 14C urate kinetics of hSLC2A9 WT (◼) and its mutants C297G (⬒), C451S (⬓), C181T (♢), C398A (▴), double mutant C181T/C398A (▿), C301S (), and C459L ().Uptake activity was corrected for non-specific transport measured in control water injected oocytes from the same batch of oocytes. Panel (D) Michaelis-Menten curves of urate-induced currents of WT hSLC2A9 and its cysteine mutants. Panel (E) 14C urate kinetic constants of the 3 isoforms (n ≥ 3). Panel (F) Urate-induced current kinetic constants of the WT and cysteine mutants (n ≥ 15 oocytes from 3 frogs).
Figure 5. Urate and fructose transport mediated by WT hSLC2A9 and its C128V mutant.Panel (A) Michaelis-Menten curves of 14C urate kinetics of hSLC2A9b WT (◼) and C128V (◻).Panel (B) 14C urate kinetic constants and the standard error of the regression (Sy. X) of the 2 isoforms (n = 3). Panel (C) Michaelis-Menten curves of urate-induced currents of WT hSLC2A9b WT and C128V mutant. Panel (D) Urate-induced current kinetic constants and the standard error of the regression (Sy. X) of the WT and C128V mutant (n = 15 oocytes from 3 frogs). Panel (E) Representative pictures of immunohistochemistry and Western blot analysis of protein expression of C128V mutant expressing oocytes. Panel (F) 14C fructose uptake mediated by hSLC2A9b WT and C128V mutant (n = 3).
Figure 6. pCMBS inhibition experiments.Panel (A) pCMBS screening in 14C urate uptake mediated by WT hSLC2A9 and its cysteine mutants. Bar graphs represent 100 μM urate uptake activities before (dark) and after (white) 100 μM pCMBS treatments. Data was corrected for non-specific transport measured in control water injected oocytes from the same batch of oocytes (n ≥ 3, unpaired t-test, *p < 0.05). Panel (B) pCMBS inhibition curves of urate-induced currents of WT hSLC2A9 (◼) expressing and control water injected (○) oocytes (n ≥ 15 oocytes from 3 frogs). Panel (C) pCMBS inhibition curves of urate-induced current of WT hSLC2A9 (◼) and C181T (◊) protein expressing oocytes. Data were corrected with basal currents before the pCMBS treatment. IC50 is the pCMBS concentration for 50% inhibition of the urate-induced current (n ≥ 15 oocytes from 3 frogs). Panel (D and E) Representative trace of urate protecting pCMBS inhibition and control experiment, respectively. Urate-induced current was elicited by perfusing an oocyte expression WT hSLC2A9 (upper trace) or water injected oocyte (lower trace) with 1 mM urate (first urate-induced peak) followed by 1 min 100 μM pCMBS incubation. The oocyte then was washed with STM for at least 1 min to remove both extracellular pCMBS and urate. Finally, the oocyte was perfused with 1 mM urate again (second urate-induced peak). Panel (F) Urate protecting pCMBS inhibition experiments of WT hSLC2A9 and C181T. Bar graphs are data corrected to control (first peak of urate-induced current before pCMBS treatment) currents (dark) currents after oocyte in both 1 mM urate and 100 μM pCMBS (grey), and currents after oocyte in only 100 μM pCMBS (white) (n ≥ 15 oocytes from 3 frogs, One-way ANOVA, *p < 0.05).
Figure 7. Fructose and urate transport mediated by WT hSLC2A9, its chimæra hSLC2A9(7)5 and hSLC2A9(7)5 G297C/S301C.Panel (A) 14C fructose uptake mediated by WT hSLC2A9 and its chimæric mutants. (n ≥ 3, One-way ANOVA, *p < 0.05). Panel (B) Michaelis-Menten curves of 14C urate kinetics of hSLC2A9 WT (◼), its chimærichSLC2A9(7)5 (◻), and hSLC2A9(7)5 G297C/S301C (▴). Panel (C) 14C urate kinetic constants and the standard error of the regression (Sy. X) of the 3 isoforms (n ≥ 3). Panel (D) Michaelis-Menten curves of urate-induced currents of WT hSLC2A9 and its chimæric mutants. Currents were measured by TEVC. Panel E. Urate-induced current kinetic constants and the standard error of the regression (Sy. X) of the WT and its chimæric mutants (n ≥ 15 oocytes from 3 frogs).
Figure 8. Fructose and urate transport mediated by WT hSLC2A5, its chimæra hSLC2A5(7)9 and hSLC2A5(7)9 T171C/A388C/S441C.Panel (A) 14C fructose uptake mediated by WT hSLC2A5 and its chimæric mutants. (n ≥ 3, One-way ANOVA, *p < 0.05). Panel (B) 14C urate uptake time course experiment of hSLC2A9, WT (◼), WT hSLC2A5 (⚪), hSLC2A5(7)9 (▵) and hSLC2A5(7)9 T171C/A388C/S441C (▴).
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