[Frontiers In Bioscience, Landmark, 24, 1024-1036, March 1, 2019]

Characterization of structural requirement for binding of gigantol and aldose reductase

Yong Yang 1,2, Qiaohong Yang1, Juan Yu1, Wencheng Wan1, Xiaoyong Wei1,2

1School of Basic Medical Sciences,Guangzhou University of Chinese Medicine, Guangzhou Guangdong,510006, PR China, 2School of Medicine, Hangzhou Normal University, Hangzhou, Zhejiang 310000, PR China


1. Abstract
2. Introduction
3. Materials and methods
3.1. Material, reagents and instruments
3.2. Animals and animal care
3.3. Effect of gigantol on lens opacity in rats
3.4. Extraction of AR from the rat lens and measurement of the inhibitory activity of gigantol on AR
3.5. Molecular dynamics simulation and molecular docking
3.6. Construction of pET28a vector expressing AR and its mutants
3.7. Expression and purification of AR and the mutants
3.8. Determination of catalytic activity of recombinant AR and its mutants
3.9. Detection of non-covalent bonding of gigantol to AR with cold-spray ionization mass spectrometry (CSI-MS)
3.10. Statistical methods
4. Results
4.1. Protection of gigantol against STZ-induced lens opacity in rats
4.2. Gigantol inhibits AR activity
4.3. Prediction of the binding sites between gigantol and AR by MD
4.4. The interaction trajectory of gigantol and AR
4.5. Identification of the binding sites between gigantol and AR through site-directed mutagenesis
4.6. Determination of non-covalent bonding between gigantol and AR by CSI-MS
5. Discussion
6. Acknowledgments
7. References


We previously reported that gigantol extracted from Caulis Dendrobii has significant therapeutic benefits for the treatment of galactosemic cataracts through its ability to inhibit aldose reductase (AR) activity. In this study, we identified the binding sites and structurally characterized the interaction between gigantol and AR, to understand the mechanism (s) of the effects of gigantol on cataracts. Gigantol was found to be protective against diabetic cataracts (DC) in rats induced by streptozotocin. Molecular docking predicted the binding sites between AR and gigantol to be residues Trp111, His110, Tyr48 and Trp20. Mutation of each of these residues led to a significant reduction in AR activity. Cold-spray ionization mass spectrometry measurements showed that the binding of gigantol to AR is concentration-dependent and that the maximum stoichiometric ratio of non-covalent bonding is 1:24.4. pH and temperature did not influence the interaction. Taken together, we provide further mechanistic evidence of the beneficial effects of gigantol on DC.


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Abbreviations: diabetic cataracts (DC), aldose reductase (AR), aldose reductase inhibitors (ARI), AR,Ala substitution at Trp20 (W20A), AR,Ala substitution at Tyr48 (Y48A), AR,Ala substitution at His110 (H110A), AR,Ala substitution at Trp111 (W111A), phosphate buffer saline (PBS), dimethyl sulfoxide (DMSO), molecular docking (MD), protein data bank (PDB), 50% inhibition of enzyme activity (IC50), transferable intermolecular potential 3 points (TIP3P), wild-type (WT), root mean square deviation (RMSD), cold spray ionization mass spetrometry (CSI-MS), Streptozotocin (STZ), Kanamycin (Kan), Dalton (Da), Nanosecond (ns), reduced nicotinamide adenine dinucleotide phosphate (NADPH), three times a day (TID), complementary DNA (cDNA), polymerase chain reaction (PCR), basic local alignment search tool (BLAST), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Michaelis-Menten constant (Km), maximum velocity (Vax)

Key Words: Binding Sites, Gigantol, Aldose Reductase, Diabetic Cataract, Site-Directed Mutagenesis, Cold-Spray Ionization Mass Spectrometry

Send correspondence to: Xiaoyong Wei, School of Basic Medical Sciences, Guangzhou University of Chinese Medicine; School of Medicine, Hangzhou Normal University. Tel: 8618520498313, Fax: 862039358588, E-mail:jidewowxy@163.com