Does glutathione-S-transferase dethiolate lens protein-thiol mixed disulfides? A comparative study with thioltransferase

Protein S-thiolation is a process in which under oxidative stress, vulnerab le sulfhydryl groups of proteins are conjugated to non-protein thiols such as glutathione (GSH) or cysteine resulting in the formation of protein-thio l mixed disulfides, protein-S-S-glutathione (PSSG) and protein-S-S-cysteine (PSSC). This process spontaneously disrupts the redox homeostasis of the c ells, which in turn leads to functional disturbances in the respective tiss ue, In the ocular lens, such modification of proteins may trigger a cascade of events starting with the alteration of protein conformation, protein/en zyme deactivation, protein-S-S-protein aggregation and eventually lens opac ification or cataract. Generally, the first line of defense system in the c ells protects the lens proteins against such damage. Recent studies in our laboratory have shown that in addition to this defense system, lens cells a lso possess a well developed system to repair the oxidative damage to the l ens proteins. We have identified this repair system as thioltransferase (TT ase) and have proved that TTase by its dethiolase activity reverses the pro tein S-thiolation process which returns the oxidatively damaged lens protei ns/enzymes to their original reduced state and restores their physiological functions. We investigated if this repair mechanism was mediated by enzyme s other than TTase, We studied glutathione S-transferase (GST) and report h ere for the first time the cloning, high level expression, and purification of human lens mu and pi isoforms of GST. A comparative study of recombinan t human lens TTase and GST (mu and pi) on their dethiolating abilities usin g lens crystallin-thiol mixed disulfides showed that the lens TTase is 60-7 0% more efficient in the dethiolation/repair process than GST. When TTase a nd GST were tested in conjunction for the dethiolation of thiol mixed disul fides, there was no significant enhancement of dethiolase activity. These f indings suggest that TTase by itself is an efficient enzyme in the dethiola tion/repair process and hence can be considered a crucial system to counter act oxidative stress in the lens. (C) 1999 Academic Press.