It has been suggested that a reduced pKa in the first cysteine (Cys32 in human thioredoxin) of the active-site sequence is important for modulation of the redox potential in thioredoxin. A hydrogen bond between the sulfhydryls of Cys32 and Cys35 may reduce the pKa of Cys32 and this pKa depression probably results in increased nucleophilicity of the Cys32 thiolate group. This nucleophilicity, in tum, is thought to be necessary for the role of thioredoxin in disulfide-bond reduction. The physiological role, if any, of thioredoxin dimer formation remains unknown. It is possible that dimerization may provide a mechanism for regulation of the protein, or a means of sensing oxidative stress.

N-glycosylation is critical to the function of monoclonal antibodies (mAbs) and distinguishes various systems used for their production. We expressed human mAbs in the small aquatic plant Lemna minor, which offers several advantages for manufacturing therapeutic proteins free of zoonotic pathogens. Glycosylation of a mAb against human CD30 was optimized by co-expressing the heavy and light chains of the mAb with an RNA interference construct targeting expression of the endogenous alpha-1,3-fucosyltransferase and beta-1,2-xylosyltransferase genes. The resultant mAbs contained a single major N-glycan species without detectable plant-specific N-glycans and had better antibody-dependent cell-mediated cytotoxicity and effector cell receptor binding activities than mAbs expressed in cultured Chinese hamster ovary (CHO) cells.

E. coli thioredoxin reductase has been cloned and sequenced [21,22] and its biochemical and physical properties extensively studied [23,24]. Eukaryotic thioredoxin reductases have so far been only cloned from Penicillium chrysogenum [25], Saccharomyces cerevisiae [26], and Arabidopsis thaliana [27] and they show 44-50% sequence identity to the bacterial enzyme. We now report the cloning and sequencing of a putative thioredoxin reductase from human placenta.

In a study of juvenile hormone-regulated gene expression, we isolated an anonymous cDNA representing a message that was strongly suppressed by juvenile hormone in the fat body of the cockroach Blaberus discoidais. (= conalbumin). These are generally at low levels in circulation but are prominent in secreted fluids such as milk, tears, and egg white, where by avid binding of free iron they act as bacteriostats. Finally, membrane-anchored melanotransferrin (antigen p97), though found at trace amounts in several tissues, is dramatically overexpressed in melanomas, where it may support rapid cellular proliferation.Complete sequences for several vertebrate transferrins are known (4-12). They share >35% amino acid positional identity with one another. Within each transferrin there is significant sequence identity between the N-and C-terminal halves, a feature suggestive of an ancestral intragenic duplication that has been confirmed by structural gene analysis (13). In harmony with their internal homology and bilobed tertiary structure, all of the vertebrate transferrins appropriately analyzed (with exception of melanotransferrin; ref. 14) have the capacity to bind two ferric ions per protein molecule. Crystallographic studies (15, 16) suggest that the two cleft lobes of the transferrins have identical sets of residues that serve as ligands to iron. Positions ofcysteine residues (34 average per polypeptide) are highly conserved.Although iron transport in vertebrates has been investigated intensively over recent decades, the subject has received little attention in studies of invertebrate animals. Circulating iron-binding proteins have been described from a tunicate (17), a crab (18), and a spider (19), but to date the only invertebrate transferrin that has been characterized and reported as a primary structure is from the sphinx moth, Manduca sexta (20,21). The insect glycoprotein (77 kDa, a molecular mass consistent with the transferrin superfamily) has significant (25-30%) global sequence identity with the vertebrate transferrins, with most notable similarity around the N-terminal iron-binding sites and in the positioning of cysteine residues. Its CD spectrum is highly reminiscent of human transferrin. M. sexta transferrin circulates and donates iron (20), indicating a primary role in iron transport. These features suggest that, despite having the capacity to bind only one ferric ion instead of two (reflected by absence of a complete iron-binding motif in the C-terminal half), the moth transferrin is evolutionarily related and functionally similar to the transferrins of vertebrates. The fat body (the main source of hemolymph proteins in insects) is a site of transferrin synthesis in M. sexta. This paper represents a dovetail of our interests in iron metabolism and hormonal control of gene expression in insects. It originated with isolation of an anonymous cDNA to a cockroach (Blaberus discoidalis) fat body mRNA that was, in the adult female, markedly suppressed by the sesquiterpenoid juvenile hormone (H),...

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Thioredoxins are a group of ca. 12 kDa redox proteins that mediate numerous cytosolic processes in all cells. Human thioredoxin can be exported out of the cell where it has additional functions including the ability to stimulate cell growth. A recent crystal structure determination of human thioredoxin revealed an inactive dimeric form of the protein covalently linked through a disulfide bond involving Cys 73 from each monomer [Weichsel et al. (1996) Structure 4, 735-751]. In the present study, apparent dissociation constants (Kapp) for the noncovalently linked dimers were determined at various pHs using a novel assay in which preformed dimers, but not monomers, were rapidly linked through oxidation (with diamide) of the Cys 73 disulfide bond, and the relative amounts of monomer and dimer were detected by gel filtration. The values obtained were pH-dependent, varying between 6.1 and 166 microM for the pH range of 3.8-8.0, and were consistent with the titration of a single ionizable group having a pKa of 6.5. A similar value was obtained using gel filtration at pH 3.8 (Kapp = 164 microM), and the crystal structure of the diamide-oxidized protein was determined to be nearly identical to that obtained in the absence of diamide. Asp 60 lies in the dimer interface and was found to be responsible for the pH dependence for dimer formation, and therefore must have a pKa elevated by approximately 2.5 pH units. Mutation of Asp 60 to asparagine abolished nearly all of the pH dependence for dimer formation. The crystal structure of the D60N mutant revealed a dimer nearly identical to the wild type, but, surprisingly, it had the Asn 60 side chain rotated out of the dimer interface and replaced with two water molecules. The values obtained for Kapp suggest human thioredoxin may dimerize in vivo and possible roles for such dimers are discussed.

Thioredoxin reductases function in regulating cellular redox and function through their substrate, thioredoxin, in the proper folding of enzymes and redox regulation of transcription factor activity. These enzymes are overexpressed in certain tumors and cancer cells and down-regulated in apoptosis and may play a role in regulating cell growth. Mammalian thioredoxin reductases contain a selenocysteine residue, encoded by a UGA codon, as the penultimate carboxyl-terminal amino acid. This amino acid has been proposed to carry reducing equivalents from the active site to substrates. We report expression of a wild-type thioredoxin reductase selenoenzyme, a cysteine mutant enzyme, and the UGAterminated protein in mammalian cells and overexpression of the cysteine mutant and UGA-terminated proteins in the baculovirus insect cell system. We show that substitution of cysteine for selenocysteine decreases enzyme activity for thioredoxin by 2 orders magnitude, and that termination at the UGA codon abolishes activity. We further demonstrate the presence of a functional selenocysteine insertion sequence element that is highly active but only moderately responsive to selenium supplementation. Finally, we show that thioredoxin reductase mRNA levels are down-regulated by other sequences in the 3-untranslated region, which contains multiple AU-rich instability elements. These sequences are found in a number of cytokine and proto-oncogene mRNAs and have been shown to confer rapid mRNA turnover. Mammalian thioredoxin reductases (TRRs)1 have many diverse cellular functions, ranging from regulation of overall redox balance to generating reducing equivalents for ribonucleotide reductase, which is required for deoxyribonucleotide synthesis. These enzymes exhibit broad substrate specificity, reducing lipid hydroperoxides and lipoic acid, protein disulfide isomerase, and several low molecular weight disulfide substrates in addition to thioredoxin. The substrate thioredoxin functions in the proper folding and redox regulation of enzymes and transcription factors, including nuclear factor B and transcription factor IIIC (1, 2), and in catalyzing electron transport to other reductive enzymes. Expression of both thioredoxin and TRR is elevated in a number of cancer cells and tumors (3, 4). Cells transfected with thioredoxin cDNA show increased tumor growth, and thioredoxin has been shown to contribute to the transformed phenotype of some human cancer cells (5). Finally, expression of TRR is down-regulated in apoptosis (4), and expression of the substrate, thioredoxin, inhibits apoptosis (6). Thus, the thioredoxin redox system is a major contributor to regulating cell growth and death.Selenoenzymes have long been known to play important roles in protection from oxidative stress and the damaging effects of reactive oxygen species. The glutathione peroxidases, the best characterized family of selenoenzymes, were thought to be solely responsible for the role of selenium in this protection. Selenium has also been implicated through nutritional a...

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