NOVEL HUMAN OCULAR GLUTATHIONE S-TRANSFERASES WITH HIGH-ACTIVITY TOWARD 4-HYDROXYNONENAL

Purpose. To study the distribution and expression of glutathione S-tra nsferase isozymes involved in detoxification of endogenously generated toxic products of lipid peroxidation, namely, 4-hydroxynonenal (4-HNE ) in human lens, retina, cornea, iris, ciliary body and to study their kinetic and structural properties. Methods. The authors have previous ly cloned and sequenced cDNA of mouse mGSTA4-4, which shows high activ ity towards 4-HNE. They have expressed it in Escherichia coli and have raised antibodies against the recombinant mGSTA4-4. In the present st udy, these antibodies were used in Western blot analysis and immunoaff inity chromatography to study the expression and to purify the human o rtholog(s) of mGSTA4-4 from ocular tissues. Results. Western blot anal yses of human ocular tissues indicated that a glutathione S-transferas es (GST) isozyme immunologically similar to mGSTA4-4 was expressed in cornea, retina, and iris and ciliary body, but not in lens. This isozy me designated as hGST 5.8 was purified to homogeneity from human retin a, cornea, and iris and ciliary body by immunoabsorption on immobilize d antibodies against mGSTA4-4. The human ortholog of mGSTA4-4, designa ted as hGST 5.8 purified from all these tissues and pI value of 5.8, s ubunit Mr value of 25 k and blocked N-terminal. Amino acid sequences o f CNBr fragments of hGST 5.8 isozymes of human ocular tissues showed a high degree of primary structure homologies with the corresponding re gions of mGSTA4-4. There were noticeable differences in the amino acid sequences of hGST 5.8 of cornea, retina, and iris and ciliary body, s uggesting the presence of several closely related hGST 5.8 subunits in the ocular tissues. This heterogeneity was due to tissue-specific exp ression rather than simple allelic polymorphism. The hGST 5.8 had abou t sixfold to eightfold higher activity toward 4-hydroxynonenal than 1- chloro-2,4-dinitrobenzene, or CDNB. The catalytic efficiency (Kcat/Km) of ocular hGST 5.8 for 4-HNE was about 100-fold higher than those for the alpha, mu, or pi classes of GST. In addition, hGST 5.8 expressed glutathione peroxidase activity toward phospholipid hydroperoxides and GSH-conjugating activity toward 9,10-epoxy stearic acid. Conclusions. The results indicate that hGST 5.8 isozyme(s) distinct from the alpha , mu, and pi classes of GSTs, are differentially expressed in human oc ular tissues and may pray an important role in protective mechanisms a gainst endogenous toxicants generated during lipid peroxidation.