Abstract
Human thioredoxin (Trx) catalyzes intracellular disulfide reductions but has also co-cytokine activity with interleukins after leaderless secretion. A recombinant truncated form of thioredoxin with the 80 N-terminal residues (Trx80) was purified to homogeneity. We discovered that Trx80 by itself is a potent mitogenic cytokine stimulating growth of resting human peripheral blood mononuclear cells. No effect was seen by Trx, but Trx80 at 50-100 nm induced cell proliferation of human peripheral blood mononuclear cells in serum-free synthetic medium, measured as [(3)H]thymidine incorporation after 72 h, with a maximum effect being comparable with that of 5 units/ml of interleukin-2. Trx80 lacked redox activity, but CD spectra suggested a secondary structure similar to Trx. Reduced Trx80 had an M(r) of 25,000, indicating that it is a dimer in solution. We also developed two different sandwich enzyme-linked immunosorbent assays that distinguish between full-length Trx and Trx80 and determined plasma levels of Trx and Trx80 in blood donors. The levels of Trx80 varied from 2 to 175 ng/ml; in comparison levels of Trx varied from 16 to 55 ng/ml without correlation to Trx80. In conclusion, the naturally occurring Trx80 is a novel mitogenic cytokine for normal resting human blood mononuclear cells.
Highlights
Thioredoxins (12 kDa) are ubiquitous enzymes that catalyze thiol-disulfide exchange reactions via two Cys residues in the conserved active site sequence -Trp-Cys-Gly-Pro-Cys- [1,2,3]
The standard deviation of the seven samples were divided with the mean to get the intra-assay coefficient of variation (CV). b Plasma sample from two different donors were measured in duplicates four times in different ELISA plates
The standard deviation of the four samples were divided with the mean to get the inter-assay coefficient of variation (CV). c Placenta extracts were measured for Trx content with sandwich
Summary
TrxR, thioredoxin reductase; Trx, thioredoxin; Trx, truncated thioredoxin containing amino acids 1 to 80 in full-length thioredoxin; DTT, dithiothreitol; TE, 50 mM Tris-HCl, 1 mM EDTA pH 7.5; PBMC, peripheral blood mononuclear cells; LPS, lipopolysaccaride; PBS, phosphate-buffered saline; ELISA, enzymelinked immunosorbent assay; IL, interleukin; HIV, human immunodeficiency virus. An extracellular truncated form of Trx, consisting of the 80 – 84 N-terminal residues of Trx, is known to be secreted from monocytes and CD4ϩ T cells [18, 23,24,25]. This truncated protein, Trx, exhibits eosinophilic cytotoxicity promoting effects and is increased in patients with severe schistosomiasis (26 –28). By using monoclonal antibodies to Trx and Trx, endogenous Trx was localized on the plasma membrane of the monocyte and macrophage cell lines THP-1 and U937, whereas Trx was found on the cell surface of many different cells [29] Both Trx and Trx have been reported to be secreted from cytotrophoblasts [30]. We report the discovery of a unique mitogenic cytokine activity of Trx and have developed methods to measure the protein in plasma samples
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