G1E Antioxidant Transcripts

Selected genes annotated as "antioxidant" in the G1EDb are shown in Table. These include numerous enzymes previously recognized to function in erythrocytes ¹. Among the most strongly upregulated were glutathione peroxidase (Gpx) 4 and glutathione-S-transferase (GST) theta 2 (Gstt2) (Table), which are distinguished by their ability to directly reduce phospholipid and cholesterol hydroperoxides ^2-4. Because oxidative damage to membranes is a major determinant of erythrocyte senescence ⁵, scavenging of lipid hydroperoxides may be especially important; Gpx4 and Gstt2 potentially fulfill this role.

Gstt2 and D-dopachrome tautomerase (Ddt) transcripts exhibited similar patterns of induction (Table). Both of the proteins are abundant in mature erythrocytes ^6,7 and the corresponding genes are tightly clustered in both mouse and human genomes. In humans, these genes share a bidirectional promoter indicating that they are co-regulated ⁸, which implies common function. Ddt catalyzes conversion of the artificial quinone substrate D-dopachrome to 5, 6-dihydroxyindole ⁹. Quinones are prototypical glutathione depletion agents that propagate deleterious free radical reactions ¹⁰. By detoxifying naturally occurring quinones, Ddt may function to preserve glutathione, a critical substrate for Gstt2 and numerous other antioxidant enzymes ¹¹.

Our data suggest that multiple mechanisms to generate and maintain erythrocyte glutathione exist. For example, the hexose monophosphate (HMP) shunt pathway is the primary mechanism for regenerating glutathione to its reduced form. Two enzymes that recycle sugars to this pathway, transketolase (Tkt) and ribose 5-phosphate isomerase A (Rpia) were induced. In addition, glyoxalase II (Glo2), an enzyme that generates glutathione from glycolytic pathway intermediates, independent of the HMP shunt, was also induced.

Numerous members of the peroxiredoxin (Prdx) family of antioxidant enzymes were present in G1E-ER4 cells. Prdx1 and Prdx2 were expressed at high level and induced, consistent with demonstrated essential requirements in erythrocytes ^12,13. Induction of Prdx 3 and Prdx 6 indicates potential unique and/or overlapping roles for these additional family members ¹⁴. It is believed that thioredoxin acts as the major electron donor for antioxidant reactions catalyzed by peroxiredoxins ¹⁵. Transcripts corresponding to thioredoxin 1 and 2 (Txn1, Txn2) were detected, but declined during the time course. However, a related protein, thioredoxin domain containing protein Txndc1, was upregulated and may, therefore, be a functional thioredoxin in erythrocytes.

Glutathione peroxidases and thioredoxin reductases incorporate selenocysteine residues, which play a role in redox sensing and catalysis ¹⁶. Interestingly, the selenium binding protein (Selenbp1), whose function is unknown, was strongly upregulated throughout the time course. Its ortholog was reported to be the most abundant non-heme protein in a subterranean mole rat ¹⁷, an animal which exists at low oxygen tensions, presumably under conditions of increased oxidative stress. Selenbp1 may play a central role in protecting red cells from oxidant-mediated damage by participating in the production of selenocysteine-containing antioxidants.

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