Cloning and expression of a cDNA for human thioredoxin.

Thioredoxin is the best representative enzyme of a group of proteins, widely distributed and possessing dithiol-disulfide oxidoreductase activity. We have constructed a cDNA library from messenger RNAs isolated from a lymphoblastoid B cell line (Epstein-Barr virus-immortalized normal human lymphocytes). Screening of this library with synthetic oligonucleotide probes, constructed from the NH2-terminal amino acid sequence of a protein produced by this line, allowed us to identify a full-length cDNA clone coding for human thioredoxin. The open reading frame (315 nucleotides long) codes for a protein of 104 amino acids (excluding the initial methionine). This protein possesses the highly conserved enzymatic active site common to plant and bacterial thioredoxins: Trp-Cys-Gly-Pro-Cys (amino acids 30-34). These data provide for the first time the complete primary sequence of a thioredoxin of mammalian origin. Recombinant human thioredoxin, expressed in Escherichia coli, possesses a dithiol-reducing enzymatic activity as assayed on mammalian and plant substrates. It is able to reduce the interchain disulfide bridges of murine pentameric IgM and porcin insulin and also to activate vegetal NADP-malate dehydrogenase. Studies of human thioredoxin mRNA expression and regulation in immunocompetent cells of human origin indicate that the protein is weakly expressed in resting lymphocytes and monocytes, but the level of human thioredoxin mRNA transcription is quite important in activated monocytes and established dividing human cell lines.

Thioredoxin is the best representative enzyme of a group of proteins, widely distributed and possessing dithiol-disulfide oxidoreductase activity. We have constructed a cDNA library from messenger RNAs isolated from a lymphoblastoid B cell line (Epstein-Barr virus-immortalized normal human lymphocytes). Screening of this library with synthetic oligonucleotide probes, constructed from the NHz-terminal amino acid sequence of a protein produced by this line, allowed us to identify a full-length cDNA clone coding for human thioredoxin. The open reading frame (315 nucleotides long) codes for a protein of 104 amino acids (excluding the initial methionine), This protein possesses the highly conserved enzymatic active site common to plant and bacterial thioredoxins: Trp-Cys-Gly-Pro-Cys (amino acids 30-34). These data provide for the first time the complete primary sequence of a thioredoxin of mammalian origin.
Recombinant human thioredoxin, expressed in Escherichia coli, possesses a dithiol-reducing enzymatic activity as assayed on mammalian and plant substrates. It is able to reduce the interchain disulfide bridges of murine pentameric IgM and porcin insulin and also to activate vegetal NADP-malate dehydrogenase.
Studies of human thioredoxin mRNA expression and regulation in immunocompetent cells of human origin indicate that the protein is weakly expressed in resting lymphocytes and monocytes, but the level of human thioredoxin mRNA transcription is quite important in activated monocytes and established dividing human cell lines.
Thioredoxin is an oxidoreductase enzyme of 12,000 daltons, containing a dithiol disulfide active site (1,2). It is ubiquitous and found in many organisms, from plants and bacteria to mammalians. It has been identified originally in Escherichia coli as an hydrogen donor for ribonucleotide reductase and deoxyribonucleotide synthesis (1). In higher organisms, the thioredoxin system (which includes NADPH as a proton donor, thioredoxin reductase, and thioredoxin) seems to parla Recherche Medicale Grant CRE 873006, Association Recherche * This work was supported by Institut National de la Sante et de sur le Cancer, Ministere de la Recherche et de I'Enseignement Superieur, and SAN-OFI. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accession numbeds) J04026.
ticipate as a general dithiol-disulfide oxidoreductase in the cells. Multiple i n vitro substrates for thioredoxin have been identified so far, such as ribonuclease, choriogonadotropins, proteolytic enzymes (3), coagulation factors (4), glucocorticoid receptor (5), and insulin (6,7). The reduction of the latter is used as a classical activity test. If thioredoxins have been extensively studied biochemically and functionally, the only cDNA sequence reported yet is from Anabaena 7119 (8).
During the course of studies focused on activation of immune cells via "hormones" called interleukins, we have reported in a previous paper the biochemical purification of a novel protein (9) produced by an EBV' human lymphoblastoid B cell line, named 3B6 (10). This 3B6 protein has been purified biochemically to homogeneity, and a 21-NHs-terminal amino acid sequence has been obtained. In this paper, using synthetic oligonucleotide probes constructed from the protein sequence, we report the successful cloning of a fulllength cDNA coding for the previously purified protein. We also present evidence indicating that this new molecule is a human thioredoxin based upon protein homologies and biological activity of the recombinant protein.
No immunological role for thioredoxin has been identified so far, However, our results demonstrate a regulation of human thioredoxin messenger RNA expression in various immunocompetent cells, such as lymphocytes and monocytes, and in several cell lines of human origin.

R N A Extraction
Total RNA was extracted from various lymphoid cells using the guanidium/cesium chloride (CsCl) method (11) slightly modified as follows. Briefly, 50 X lo6 cells in 75 +l of saline buffer were lysed in 0.5 ml of 6 M guanidium isothiocyanate, 5 mM citrate, 100 mM 2mercaptoethanol, and 0.5% Sarkosyl NL30 solution. After sonication for 10 s, the cell extract was layered over 0.5 ml of a 5.7 M csC1, 250 mM sodium acetate, 100 mM EDTA solution. It was then ultracentrifuged for 90 min at 20 "C at 350,000 X g in a TL 100.2 rotor (Beckman). The pellet was washed in 30% deionized formamide, 70% ethanol; dried and resuspended in Tris/EDTA/sodium dodecyl sulfate (SDS). It was kept precipitated in ethanol and sodium acetate.

Construction arzd Screening of cDNA Library
Poly(A)+ RNA from the 3B6 cell line was isolated by oligo(dT)-8 Trisacryl M (IBF) chromatography of the total RNA (12). The cDNA library was constructed by the method of Gubler and Hoffman (13) using Amersham's cDNA synthesis system. The EcoRI sites were methylated with EcoRI methylase (New England Biolabs), and EcoRI 'The abbreviations used are: EBV, Epstein-Barr virus; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DTT, dithiothreitol; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. linkers (New England Biolabs) were ligated onto the ends. The cDNAs were digested with EcoRI (Amersham Corp.) and size fractionated by Sephadex G-100 chromatography. The first two-thirds of the eluate were then ligated into EcoRI-digested GTlO and packaged using a Vector Cloning System (Stratagene) according to the manufacturer's instructions. After infecting and plating Escherichia coli c600 Hfl-, lo5 recombinant phages were obtained. After amplification, the screening was performed at lo3 plaques/plate; and the phages were transferred on duplicate sheets of nylon membrane (Hybond N, Amersham Corp.), processed, and prehybridized for 2 h at 42°C in 20% formamide, 6 X SSC (0.15 M NaCl, 0.15 sodium citrate), 5 X Denhardt's solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.01% bovine serum albumin), 10 mM EDTA, and 0.5% SDS + 100 pg/ml salmon sperm DNA. Hybridization was carried out under the same conditions with two synthetic oligonucleotide probes (see "Results") which had been end-labeled with T4 polynucleotide kinase and [3'P] ATP. Washes were performed at 42°C in 6 X SSC, 0.1% SDS for 60 min and in 2 X SSC, 0.1% SDS for another 60 min. After overnight autoradiographic exposure, plaques that gave positive signals were purified by a secondary plating and rescreening. Their DNA was purified (14), and the inserts were sized by Southern blotting.

Synthetic Oligonucleotides
Oligonucleotides were synthesized by the phosphoramidite method using a Biosearch Model 8600 automated DNA synthesizer. After deprotection, they were purified by acrylamide gel electrophoresis.
cDNA Sequencing The inserts were excised and subcloned into M13mp18 and M13mp19 (15). Both strands were sequenced by the dideoxy termination method of Sanger et al. (16) using universal and specific primers to achieve the sequence of the two DNA strands.

Northern Blots
Northern blots were performed with 20 pg of total RNA from various lymphoid cells. After electrophoretic migration on a 1.2% formaldehyde-agarose gel, the RNA was transferred onto a nylon membrane as described by Maniatis et al. (14). The transferred RNA was probed with the 0.4-kilobase pair EcoRI-DraI fragment from the W1 cDNA clone and labeled with [32P]a-dCTP using a multiprime kit (Amersham Corp.). The equal level of transferred RNA was controlled by hybridization with a 0.6 kilobase pair PstI-TaqI fragment of murine 0-actin cDNA inserted in Bluescript plasmid and labeling with [32P]a-UTP and the T3 RNA polymerase using a riboprobe kit (Amersham Corp.). With the same riboprobe kit, a precise RNA size marker was prepared from a mixture of three DNA templates giving three known fragments of RNA with SP6 RNA polymerase and [32P]cr-UTP. Prehybridization and hybridization were carried out at 65°C in 4 X SSC, 0.01 M phosphate buffer (pH 7), 0.4 mg/ml heparin, 0.1% SDS, 0.2% PPi, and 30% formamide. Washes were also performed at 65°C in 6 X SSC, 0.1% SDS; 2 X SSC, 0.1% SDS; and 0.2 X SSC, 0.1% SDS for 1 h time each.

Expression of cDNA in E. coli
The cDNA for human thioredoxin was expressed in E. coli W3llOcI as previously described by Wingfield et al. (17). Briefly, the cDNA was cleaved by restriction endonuclease PstI at codon 16. A synthetic DNA fragment was used to regenerate the first 16 codons of the natural sequence, to add an initiating methionine as codon 1, and to provide an NcoI sticky end. The modified cDNA fragment was transferred into an expression plasmid under the control of the PL promoter and the bacteriophage p ner gene ribosome-binding site. The expression plasmid was introduced into E. coli W3llOc1, a strain carrying the thermolabile repressor temperature-sensitive c1857 gene on its chromosome. Cells were grown at 30°C to mid-log phase, at which point expression was induced by raising the temperature of the culture to 42°C for 2 h.

Purification of Human Thioredoxin
The buffers used in protein purification were as follows. Buffer A: Bacteria (120 g) were resuspended in 220 ml of Buffer A and passed twice through an Amicon French pressure cell operated at 124 megapascals. The suspension was centrifuged at 100,000 X g for 30 min, and the supernatant was recentrifuged at 61,000 X g for 90 min. The supernatant (260 ml) was diluted 3-fold with Buffer B and applied to a column (25 X 5 cm) of DEAE-Sepharose (Pharmacia LKB Biotechnology Inc.) equilibrated with Buffer B. A 3-liter gradient of 0-0.5 M NaCl in Buffer B was applied. Thioredoxin was eluted with about 0.2 M NaC1; fractions were pooled (285 ml) and dialyzed against Buffer C. The dialysate was clarified by centrifugation at 100,000 X g for 30 min and applied to a second DEAE-Sepharose column (25 X 5 cm) equilibrated with Buffer C. A 3-liter gradient of 0-0.25 M NaCl in Buffer C was applied. Thioredoxin eluted at 125-150 mM NaCl; fractions were pooled (500 ml) and titrated to pH 7.5 with 1 M Tris base, and DDT was added to 0.5 mM. The solution was concentrated to 20 ml by ultrafiltration using Diaflo YM-5 membranes (Amicon), filtered with Millex-GV 0.22-pm filter units (Millipore), and applied to a 98 X 5-cm column of Ultrogel AcA54 (Pharmacia LKB Biotechnology Inc.) equilibrated with Buffer D. Thioredoxin-containing fractions were pooled (50 ml), sterile-filtered using Millex-GV 0.22-pm filter units, and stored at -80°C.

Protein Determination
The concentration of purified protein was determined by measuring the absorbance at 280 nm using a Hewlett-Packard 8450 spectrophotometer. A molar absorption coefficient at 280 nm of 7.14 cm-' (based on a M, of 11,700) was determined by qualitative amino acid analysis as outlined by Allen (18). The t value corresponds to A -= 6.10 at 280 nm.

1.0% 1 cm
Amino Acid Analysis Protein samples were hydrolyzed in constant boiling HCl in NOflushed evacuated tubes in the presence of phenol (1 mg/ml) and 100 mM ethanethiol. Hydrolysates were analyzed on a Beckman 6300 amino acid analyzer.

Analytical Separation Methods
Electrofocusing was carried out on thin-layer polyacrylamide gels (Pharmacia LKB Biotechnology Inc., Ampholine PAGE plates, pH 3.5-9.5) according to the manufacturer's instructions. SDS-PAGE was carried out on 15% (w/v) polyacrylamide gels using the buffer system described by Laemmli (19).

Thioredoxin Assay
Three assays were performed to evaluate the dithiol-disulfide oxidoreductase activity of recombinant human thioredoxin.
ZgM Reduction-IgM was prepared from ascitic fluid of mice injected intraperitoneally with an IgM antibody-secreting murine hybridoma and purified by 50% ammonium sulfate precipitation, followed by gel filtration chromatography on a Pharmacia LKB Biotechnology, Inc. S300 column. Thioredoxin (1 mM) was reduced by 100 mM DTT for 1 h 20°C and then microdialyzed for 1 h on a Millipore filter (0.025-pm pore size) against 80 mM HEPES, 10 mM EDTA buffer.
Using the same buffer, 2 pl of 1.7 p M IgM and 0.1, 1, and 5 p1 of a 1 mM solution of reduced thioredoxin were mixed for overnight reaction at 20°C. SDS-PAGE analysis for protein size determination was performed under nonreducing conditions using a 2-13.5% acrylamide gradient and silver nitrate staining as previously reported by Laemmli (19) and Merril et al. (20).
DTT-dependent Activation of Corn Leaf NADP-Malate Dehydrogenase-Corn leaf NADP-malate dehydrogenase (21) and spinach thioredoxin were purified as described (22). The activation medium of NADP-malate dehydrogenase contained 3 p~ Tris-HC1 (pH 7.9), 0.3 p~ DTT, 0.15 nM NADP-malate dehydrogenase, and either thioredoxin m or human thioredoxin as indicated. After a 30 min incubation at 20"C, a 10 pl aliquot was used to determine the activity of NADP-malate dehydrogenase, followed by changes in absorbance at 340 nm as described earlier.
Catalytic Activity of Human Thioredoxin on DTT-dependent Insulin Reduction (6)"Comparison was made between recombinant human thioredoxin and commercial E. coli thioredoxin (IMCO). In a total volume of 1 ml, the reaction medium contained 100 p~ phosphate buffer (pH 7), 0.13 p M porcin insulin (Novo Pharmaceutical Industry, Paris, France), and thioredoxin as indicated. Each reaction was initiated by adding 0.5 pM DTT to the cuvette, and absorbance otides downstream from this TAA codon. An EcoRI-DraI at 650 nm was immediately recorded at 30°C. restriction fragment from clone W1 was used to probe a Northern blot of the original 3B6 RNA (Fig. 3). One single band was observed, indicating that a single species of mRNA RESULTS Isolation of cDNA Clones of 550 base pairs is present in these cells.
A XGTlO library constructed from the poly(A)+ RNA of Analvsis of Deduced Amino Acid Seauence 3B6 lymphoid cells was screened with two synthetic oligonucleotide probes deduced from the NH2-terminal amino acid sequence of the 3B6 protein (Fig. 1). Among 48,000 clones screened, seven hybridized with both probes. The DNA of two of them (W1 and W2) was purified and shown to contain 0.6kilobase pair inserts. Both clones have an identical sequence which is shown in Fig. 2. This sequence is 500 base pairs long excluding the poly(A) tail and has a single reading frame. The first ATG codon located at nucleotide 64 from the 5'-end is followed by a 312-nucleotide-long open reading frame ending with an in-frame termination codon TAA at position 379 and is therefore able to code for a 104-amino acid protein. The classical polyadenylation site, AATAAA, is located 102 nucle-Comparison of the deduced amino acid sequence with the sequence information available from the biochemical analysis of the 3B6 protein showed that the previously determined NHz-terminal 21 amino acids were located within this reading frame, immediately after the first methionine codon of the cDNA clone (Fig. 2). The theoretical M, of 11,680 and the PI of 4.88 were in good agreement with the experimental values previously found for the 3B6 protein (9). No potential glycosylation sites were detected. The molecule contained 5 cysteine residues. Computer search against the National Biomedical Research Foundation DNA sequence data bank revealed no homology with any known gene, but comparison at the

5'
First oligonucleotide   amino acid level showed a 30% homology with thioredoxin, a protein well characterized in plants and bacteria. The homology was absolute over a highly conserved five amino acid sequence, Trp-Cys-Gly-Pro-Cys, referred to as the active site characteristic of all thioredoxins previously described. A few other conserved amino acids were found apart from the active site: a shorter region of homology is found upstream, Val-Asp-Phe, and an alanine and downstream a proline. No significant homology with vegetal thioredoxin from Anabaena 7119 (8) and spinach chloroplast (3) or bacterial thioredoxin from Corynebacterium nephridii (23) and E. coli (24) could be determined elsewhere in the molecule. In contrast, many homologies were noticed with a partial amino acid sequence of rat hepatoma thioredoxin (25) at the NH2 terminus (Fig.  4).

Recombinant Protein Purification
Recombinant human thioredoxin was expressed in E. coli W3llOcI (see "Experimental Procedures") as a soluble protein accounting for 15% of the total proteins present in induced cells (Fig. 5, lane A). The protein was purified with an overall yield of 40%, representing a recovery of 6  estimated from the amino acid composition. Amino acid analysis indicated excellent correlation between the experimental and the theoretical amino acid compositions (data not shown). Sulfhydryl analysis by modification with dithiothreitol and iodoacetamide followed by amino acid analysis indicated the presence of 5 cysteine residues/molecule as predicted by the cDNA sequence.

Biological Characterization
Since the major function of thioredoxins is a dithiol-disulfide oxidoreductase enzymatic activity, we tried at first to reduce an immunological substrate and next to confirm the thioredoxin specificity by classical tests.
IgM Reduction-We thought that pentameric IgM, which contains 26 interchain disulfide bridges, might constitute a potential substrate for thioredoxin. Three different doses of reduced recombinant human thioredoxin were mixed with a constant concentration of IgM and incubated overnight at room temperature (see "Experimental Procedures"). The products resulting from the reaction were analyzed by SDS-PAGE under nonreducing conditions (Fig. 6). With the highest dose of recombinant human thioredoxin (600-fold molar excess of recombinant human thioredoxin/IgM, i.e., 23-fold excess of disulfide bridges), four major bands were identified corresponding to 70 kDa (heavy chain IgM, p), 25 kDa (light chain IgM = L), 27 kDa (dimeric human thioredoxin), and 13 kDa (monomeric human thioredoxin). A minor band a t 95 kDa (pL) was also seen (Fig. 6, lane D). With intermediate doses of recombinant human thioredoxin (5-fold excess of human thioredoxin/IgM disulfide bridges), the pentameric IgM a t 950 kDa and heavy chain dimers of IgM at 140 kDa (pz) were predominant (Fig. 6, lane E ) . The four major bands seen with the highest dose were not so intense. With the lowest dose of recombinant human thioredoxin (a 0.5 ratio of human thioredoxin/IgM disulfide bridges), the pentarneric IgM was left intact (Fig. 6, lane F ) .
DTT-linked Activation of NADP-Malate Dehydrogeme-A common and specific assay to test the thioredoxin activity was then used. A dose-dependent curve of human and spinach thioredoxin m on the DTT-dependent activation of corn leaf NADP-malate dehydrogenase is shown in Fig. 7. Although a t equivalent concentrations, recombinant human thioredoxin was less efficient than spinach thioredoxin m, it was able nevertheless to activate strongly NADP-malate dehydrogenase a t higher concentrations (80 PM). At low concentrations of recombinant human thioredoxin (1-2 p~)  was observed in the time course of NADP-malate dehydrogenase activation (data not shown). The apparent K , values computed from these reactions were 70 and 4 PM for human thioredoxin and spinach thioredoxins, respectively.
Thioredoxin-catalyzed Reduction of Insulin by Dithiothreitol-In order to confirm the specificity of our recombinant protein, we examined the reduction of insulin, which is a classical assay for thioredoxins. For this purpose, we compared the activities of E. coli thioredoxin with recombinant human thioredoxin. The reduction of insulin disulfide bridges by DTT at pH 7.0 was determined with different concentrations of thioredoxins. As seen in Fig. 8, both thioredoxins displayed the same activity: the rates of insulin reduction were 1.7 AAw/rnin/mg of thioredoxin.

Humun Thwredoxin mRNA Expression and Regulation in Immunocompetent Cells
The expression of human thioredoxin mRNA in various cells of lymphoid or related origin was studied by Northern blot analysis using two different probes; an EcoRI-DraI fragment of clone W1 or the synthetic oligonucleotide EW2. As shown in Fig. 9, human thioredoxin mRNA was present in all human T and B cell lines, as well as in the monocytic and erythroid lines tested. However, whereas it was high in the EBV 3B6 and 721 lines, it was lower in the Burkitt lines Daudi and Raji, indicating a different expression level in the different cell lines. The same Northern blot hybridized with a @-actin riboprobe revealed the same level of mRNA in all the cells tested (data not shown).
We next investigated the human thioredoxin mRNA expression level, during activation in vitro by various stimulating agents, in lymphocytes and monocytes (Fig. 10). The steady level of mRNA in these freshly isolated cells was quite low. Mitogenic stimulation for 24 h by phytohemagglutinin of peripheral blood lymphocytes increased the expression of human thioredoxin mRNA. The activation of monocytes by bacterial lipopolysaccharide or phorbol myristate acetate, a known activator of protein kinase C, increased the level of human thioredoxin mRNA from 1 to 18 h of stimulation. Phorbol myristate acetate-activated 3B6 cells also showed an increased level of human thioredoxin expression.
Finally, as seen in Fig. 11, the human thioredoxin probes (both the cDNA and the oligonucleotide probes) did not hybridize with RNA extracted from murine lymphoid lines (EL4 and CTL L2). In contrast, the gibbon ape lymphoid line MLA 144 contained a mRNA species of equivalent size which positively hybridized with the human thioredoxin probes.

DISCUSSION
Thioredoxin is the best representative enzyme of a group of widely distributed proteins possessing dithiol-disulfide ox-  idoreductase activity (thioredoxin and glutaredoxin systems) (26,27). Although partial amino acid sequences of animal thioredoxins have been reported previously (25,28,29), no cloning of mammalian thioredoxin cDNA or gene has been published yet. We present in this paper the complete amino acid sequence deduced from a cDNA clone of a human thioredoxin. This human enzyme possesses the completely conserved five-amino acid active site. Some surrounding residues which may be of importance for the secondary structure are also conserved. Apart from this region, the protein homology with thioredoxins from various origins is variable. This homology is found to be strong with the incomplete sequences available for rat and rabbit thioredoxins (28,29). In contrast, a weak homology with vegetal (spinach (30)) and bacterial (C. nephridii (23), E. coli (24)) thioredoxins is observed. Interestingly, the human thioredoxin enzymatic activity is similar to the spinach and bacterial thioredoxins, suggesting that the only requirement in the oxidoreductase activity is the active site. At the nucleotide sequence level, however, interspecies mRNA analysis by Northern blot shows a strong cross-hybridization between human and gibbon, but none with mouse. These findings are in support of a nucleotide homology between man and ape. The partial amino acid sequence previously reported (9) has shown that the mature human thioredoxin protein starts with a valine. According to the sequence of the cDNA, this valine follows just a methionine. This implies that the translation product is processed. Several mechanisms are possible. One could be the elimination of the NHz-terminal methionine. This hypothesis is supported by the nucleotide environment of the ATG codon which is in good agreement with Kozak's (31) consensus sequence for an initiation codon. A second possibility could be the existence of an additional NH2-terminal peptide: this peptide could correspond either to a leader peptide allowing the transport of the protein to a specific cellular compartment or to a preprotein possessing or not the biological activity.
The enzymatic oxidoreductase activity of recombinant human thioredoxin produced in E. coli is detected in a wide spectrum of substrates from different origins. Recombinant human thioredoxin can reduce murine IgM disulfide bridges. It displays the same activity as vegetal thioredoxin on plant substrates, although with stoichiometric differences. Finally, it can also catalyze porcin insulin reduction by DTT. It seems therefore that many proteins with disulfide bridges may be potential substrates for thioredoxins. This enzymatic family, with highly conserved active site and molecular weight through evolution, is most probably of major importance for the cells. The principal cellular localization and the major substrates are not really defined. An interesting observation was of the formation, upon oxidation, of human thioredoxin dimers. Indeed immunoblots, using antibodies against a human thioredoxin NH2 terminus synthetic peptide, were positive with crude extracts from lymphoid human cell lines, revealing a monomeric (13 kDa) and a dimeric (26 kDa) band of human thioredoxin (data not shown). This indicates that an equilibrium between monomeric reduced human thioredoxin and dimeric oxidized human thioredoxin may exist physiologically.
Messenger RNA coding for human thioredoxin is detected in most lymphoid and hematopoietic human cell lines tested. This indicates that immortalized cells, which divide actively, have a high level of expression of the human thioredoxin protein. In contrast, resting cells such as lymphocytes or monocytes, freshly isolated from the circulating blood, express human thioredoxin mRNA very weakly. Upon activation, these cells transcribe substantial amounts of human thioredoxin mRNA. These results are in good agreement with previous histo-immunologic studies, using antibodies against rat thioredoxin, which did not detect the presence of the enzyme in resting lymphocytes (32).
The high level of human thioredoxin expression, in dividing lymphocytes, which enabled us to isolate this protein from an EBV B cell line, may suggest some important immunological role for the enzyme. We now have the tools (human genetic probes, recombinant purified protein and antibodies) to examine further these questions in the human.