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PubMed · 11222536

Human lens thioltransferase: cloning, purification, and function.

F Qiao·K Xing·A Liu·N Ehlers·N Raghavachari·M F Lou
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Abstract

PURPOSE: To clone the human lens thioltransferase (TTase) gene and to purify, characterize and study the possible function of the recombinant human lens thioltransferase (RHLT). METHODS: The human lens TTase gene was cloned by using RT-PCR and verified by sequence and RNase protection assay. TTase overexpressed in Escherichia coli was isolated and purified to homogeneity by column chromatography and identified by Western blot analysis. The activity was assayed with a synthetic substrate hydroxyethyl disulfide. Its function in dethiolating and reactivating other key metabolic enzymes was studied by using pure glutathione S:-transferase (GST) and glutathione peroxidase (GPx) from commercial source and also with the cell extract of rabbit lens epithelial cells preexposed to H2O2. RESULTS: The cloned human lens TTase gene showed identical sequence to the TTase gene from other human tissues. The RNase protection assay displayed a single transcript from the total RNA of human lens epithelial cells. The purified RHLT had a molecular weight of 11.8 kDa and reacted positively with anti-pig liver TTase. It displayed similar structural, functional, and kinetic characteristics to those of TTases from other sources. It was shown that RHLT effectively regenerated the activities of GST and GPx, after each was inactivated by S-thiolation with cystine in vitro. Furthermore, RHLT was able to restore the activity of the oxidatively inactivated glyceraldehyde-3-phosphate dehydrogenase (G-3PD) in H2O2-exposed rabbit lens epithelial cells. CONCLUSIONS: The human lens TTase gene has been cloned for the first time. Its gene product showed the characteristics which support our speculation that TTase may play a major role in maintaining the homeostasis of lens protein thiols thus protecting against oxidative stress.

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Amino Acid SequenceBase SequenceBlotting, WesternCloning, MolecularDNA PrimersElectrophoresis, Polyacrylamide GelEpithelial CellsEscherichia coliGene ExpressionGlutaredoxinsGlutathione PeroxidaseGlutathione TransferaseGlyceraldehyde-3-Phosphate DehydrogenasesHumansHydrogen PeroxideIodoacetamideIsoelectric FocusingLens, CrystallineMolecular Sequence DataNuclease Protection AssaysOxidoreductasesProtein Disulfide Reductase (Glutathione)Recombinant ProteinsReverse Transcriptase Polymerase Chain ReactionTransfectionJournal ArticleResearch Support, U.S. Gov't, P.H.S.

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F Qiao, K Xing, A Liu, N Ehlers, N Raghavachari, M F Lou. 2001. Human lens thioltransferase: cloning, purification, and function.. https://pubmed.ncbi.nlm.nih.gov/11222536/

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