Efficiency of Selenocysteine Incorporation in Human Thioredoxin Reductase

Abstract

Thioredoxin reductase (TR) is a flavoenzyme, containing one selenocysteine (Sec) residue at the penultimate carboxyl-terminus, that catalyzes the NADPH-dependent reduction of oxidized thioredoxin. Sec is encoded by the UGA stop codon in the open reading frame of the mRNA, and the conserved stem-loop structure in 3'-untranslated regions functions as the determinant of Sec incorporation instead of termination of translation. The efficiency of Sec incorporation in Sec-containing enzymes in physiological or selenium (Se)-deficient condition remains unclear. To clarify this, we have developed monoclonal antibodies to human TR, and established a sandwich enzyme-linked immunosorbent assay to determine TR protein content. We observed that the specific activity of cytosolic TR in NCI-H441 cells increased with increasing concentrations of Se in a serum-free medium. The specific activity of TR purified from each cytosol was essentially equal to the calculated specific activity of each cytosolic TR. The Se content of TR increased with increasing concentration of Se in the medium, from 0.32 mol/mol of TR subunit (no SE) to 0.98 mol/mol of TR subunit (500 nM Se), and was directly correlated with the specific activity of TR. When calculated from the cytosolic TR specific activity of human peripheral mononuclear cell, the theoretical efficiency of Sec incorporation in physiological conditions is assumed to be 87%.

Cell Death by SecTRAPs: Thioredoxin Reductase as a Prooxidant Killer of Cells

“…To our knowledge, no studies have yet been published demonstrating endogenous production of the truncated enzyme in mammalian tissues. Evidence for production of a truncated TrxR1 species was reported for NCI-H441 cells [71], while in other liver or kidney-derived cell lines clear evidence for such production at selenium depletion could not be seen [72]. The toxicity of SecTRAPs to A549 and HeLa cells could possibly explain prior difficulties encountered in attempts to establish a stable TrxR1-overexpressing cell line.…”

A Comparative Study on the Hydroperoxide and Thiol Specificity of the Glutathione Peroxidase Family and Selenoprotein P

“…PHGPx was purified from human placental cytosol as described previously (21) with a slight modification. TRX reductase was purified from human placenta as described previously (22) with some modifications (23).…”

The molecular biology of selenocysteine

“…For example, testicular GPX4 (142), plasma SelP (129) and the ubiquitous TrxR1 (143) are produced in high quantities. In fact, the calculated Sec incorporation efficiency for endogenous TrxR1 is 87% in human blood cells (144). Indeed, more studies are needed to clarify the discrepancy between the tissue samples and the in vitro systems.…”