Human lens thioltransferase: Cloning, purification, and function

PURPOSE. To clone the human lens thioltransferase (TTase) gene and to purif y, characterize and study the possible function of the recombinant human le ns 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 synthe tic substrate hydroxyethyl disulfide. Its function in dethiolating and reac tivating 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 p urified RHLT had a molecular weight of 11.8 kDa and reacted positively with anti-pig liver TTase. It displayed similar structural, functional, and kin etic characteristics to those of TTases from other sources. It was shown th at RHLT effectively regenerated the activities of GST and GPx, after each w as 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-SPD) in H2O2-exposed rabbit lens epithelial c ells. CONCLUSIONS. The human lens TTase gene has been cloned for the first time. Its gene product showed the characteristics which support our speculation t hat TTase may play a major role in maintaining the homeostasis of lens prot ein thiols thus protecting against oxidative stress.