Structure-Function Study of Human Glucose Transporter 9 (hSLC2A9)

• Author / Creator

  Long, Wentong

• Abnormal serum uric acid (urate) is found related to many medical complications, such as hypertension, gout and kidney stones. Both Single Nucleotide Polymorphism and genome wide association studies demonstrated that the glucose transporter 9 (hSLC2A9) is highly associated with abnormal human plasma urate levels, indicating that hSLC2A9 has an essential physiological role in urate regulation in the human body. Human SLC2A9 is certainly a hexose and urate transporter. However, investigations are still being undertaken to try to reveal the mechanisms of how hSLC2A9 transports hexoses and urate. As a result, this thesis explores the possible pathway of urate translocation mediated by hSLC2A9. We applied site-directed mutagenesis, followed by functional studies with radiolabelled fluxes and electrophysiological measurements to screen several possible groups of residues predicted by our homology hSLC2A9 computer model, which might be involved in urate transport. We hypothesized that the hydrophobic residues I335 and W110, cysteine residues C181, C297, C301, C398, C451, and C459, transmembrane helix 7 (H7), and two residues, Y298 and N429, predicted to be in the binding sites are important for hSLC2A9 urate transport. The results indicate that the I335V mutant transports urate similarly to the wild type hSLC2A9; however, I335 is necessary for urate/fructose trans-acceleration exchange to occur. In addition, tryptophan 110 (W110) is a critical site for urate transport. Two homology computer models of hSLC2A9 and hSLC2A5 reveal that I335 (or the homologous I296 in hSLC2A5) is a key component for protein conformational changes when the protein translocates substrates and W110 is a critical site that could directly interact with urate during transport. Our results for the cysteine residues in hSLC2A9 demonstrated that C181 and C398 are important for urate translocation while C297 and C451 are not required. Mutation of either C301 or C459 significantly reduced urate transport. Combining the computer model analysis, we suggest that C181 is the residue with which pCMBS reacts; C301 and C459 could plausibly have an intermolecular disulfide interaction, which serves as a central core for the protein structure, thus, also critical for urate transport. Analysis of the chimæric proteins, hSLC2A9(7)5 and hSLC2A5(7)9 indicate that helix H7 is necessary for urate transport in hSLC2A9b, but not sufficient to allow hSLC2A5(7)9 to gain urate transport. In silico docking and functional studies allow us to postulate that N429 is a key residue that could form part of the urate binding.

• Subjects / Keywords

  • Urate, urate transport
  • Glucose transporter
  • HSLC2A9
  • Structure-function

• Graduation date

  Fall 2015

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3W08WX19

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.