Human lens thioltransferase: cloning, purification, and function.
OBJECTIVE
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.
Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, 134 VBS, Lincoln, NE 68583-0905, USA.
F
F
Qiao
K
K
Xing
A
A
Liu
N
N
Ehlers
N
N
Raghavachari
M F
MF
Lou
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D054477:Glutaredoxins; D005979:Glutathione Peroxidase / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007460:Iodoacetamide / Q000494:pharmacology; D007525:Isoelectric Focusing; D007908:Lens, Crystalline / Q000187:drug effects / Q000201:enzymology*; D008969:Molecular Sequence Data; D023282:Nuclease Protection Assays; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D011490:Protein Disulfide Reductase (Glutathione); D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D017931:DNA Primers; D054477:Glutaredoxins; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D005979:Glutathione Peroxidase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011490:Protein Disulfide Reductase (Glutathione); D005982:Glutathione Transferase; D007460:Iodoacetamide