Truncated Thioredoxin Is a Mitogenic Cytokine for Resting Human Peripheral Blood Mononuclear Cells and Is Present in Human Plasma
2000; Elsevier BV; Volume: 275; Issue: 48 Linguagem: Inglês
10.1074/jbc.m001012200
ISSN1083-351X
AutoresKlas Pekkari, Ramanathan Gurunath, Elias S.J. Arnér, Arne Holmgren,
Tópico(s)Heat shock proteins research
ResumoHuman 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 [3H]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 anM 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. 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 [3H]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 anM 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. thioredoxin reductase thioredoxin truncated thioredoxin containing amino acids 1 to 80 in full-length thioredoxin dithiothreitol 50 mm Tris-HCl, 1 mm EDTA pH 7.5 peripheral blood mononuclear cells lipopolysaccaride phosphate-buffered saline enzyme-linked immunosorbent assay interleukin human immunodeficiency virus 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- (1Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 2Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar, 3Powis G. Gasdaska J.R. Baker A. Adv. Pharmacol. 1997; 38: 329-359Crossref PubMed Scopus (114) Google Scholar). The three-dimensional structure of thioredoxins (the thioredoxin fold) is characterized by a central five-stranded sheet surrounded by four α-helices (4Qin J. Clore G.M. Gronenborn A. Structure. 1996; 2: 503-521Abstract Full Text Full Text PDF Scopus (154) Google Scholar, 5Weichsel A. Gasdaska J.R. Powis G. Montfort W.R. Structure. 1996; 4: 735-751Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 6Martin J.L. Structure. 1995; 3: 245-250Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar, 7Eklund H. Gleason F.K. Holmgren A. Proteins Struct. Funct. Genet. 1991; 11: 13-28Crossref PubMed Scopus (329) Google Scholar). The active site disulfide in the oxidized Trx is reduced to a dithiol by NADPH and TrxR1 (8Holmgren A. J. Biol. Chem. 1979; 254: 9113-9119Abstract Full Text PDF PubMed Google Scholar, 9Ren X. Björnstedt M. Shen B. Ericson M.L. Holmgren A. Biochemistry. 1993; 32: 9701-9708Crossref PubMed Scopus (138) Google Scholar). Human T-lymphotropic virus-1 transformed human T cells produce a factor previously named adult T cell leukemia-derived factor, which is identical to human Trx (10Wakasugi N. Tagaya Y. Wakasugi H. Mitsui A. Maeda M. Yodoi J. Tursz T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8282-8286Crossref PubMed Scopus (258) Google Scholar). Trx is also secreted from several other cell types including activated normal B lymphocytes, B cell lines from B-type chronic lymphocytic leukemia, liver cells, fibroblasts, and T lymphocytes (11Rubartelli A. Bajetto A. Allavena G. Wollman E. Sitia R. J. Biol. Chem. 1992; 267: 24161-24164Abstract Full Text PDF PubMed Google Scholar, 12Ericson M.L. Hörling J. Wendel H.V. Holmgren A. Rosén A. Lymphokine Cytokine Res. 1992; 11: 201-207PubMed Google Scholar). Among its regulatory functions, Trx is involved in redox control of DNA binding of transcription factors like NF-κB and AP-1 (13Hayashi T. Ueno Y. Okamoto T. J. Biol. 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Nilsson K. Holmgren A. Int. Immunol. 1995; 7: 625-633Crossref PubMed Scopus (90) Google Scholar, 19Nakamura H. Masutani H. Tagaya Y. Yamauchi A. Inamoto T. Nanbu Y. Fujii S. Ozawa K. Yodoi J. Cancer. 1992; 69: 2091-2097Crossref PubMed Scopus (148) Google Scholar, 20Biguet C. Wakasugi N. Mishal Z. Holmgren A. Chouaib S. Tursz T. Wakasugi H. J. Biol. Chem. 1994; 269: 28865-28870Abstract Full Text PDF PubMed Google Scholar) and prevents apoptosis via direct binding interaction of the reduced form with apoptosis signal-regulating kinase 1 (21Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2092) Google Scholar). Chemokine activity of Trx has also recently been discovered in vivo and in vitro, and this function is not mediated via known chemokine receptors (22Bertini R. Howard O.M.Z. Dong H. Oppenheim J.J. Bizarri C. Sergi R. Caselli G. Pagliei S. et al.J. Exp. Med. 1999; 189: 1783-1789Crossref PubMed Scopus (290) Google Scholar). An active site mutant with the cysteine residues substituted to serines is not active as a chemokine, suggesting that the redox function of Trx is required for its chemokine activity (22Bertini R. Howard O.M.Z. Dong H. Oppenheim J.J. Bizarri C. Sergi R. Caselli G. Pagliei S. et al.J. Exp. Med. 1999; 189: 1783-1789Crossref PubMed Scopus (290) Google Scholar). An extracellular truncated form of Trx, consisting of the 80–84 N-terminal residues of Trx, is known to be secreted from monocytes and CD4+ T cells (18Rosén A. Lundman P. Carlsson M. Bhavani K. Srinivasa B.R. Kjellström G. Nilsson K. Holmgren A. Int. Immunol. 1995; 7: 625-633Crossref PubMed Scopus (90) Google Scholar, 23Silberstein D.S. Dessein A.J. Elsas P.P. Fontaine B. David J.R. J. Immunol. 1987; 138: 3042-3050PubMed Google Scholar, 24Silberstein D.S. Ali M.H. Baker S.L. David J.R. J. Immunol. 1989; 143: 979-983PubMed Google Scholar, 25Balcewicz S.M. Wollman E.E. Gorti R. Silberstein D.S. J. Immunol. 1991; 147: 2170-2174PubMed Google Scholar). This truncated protein, Trx80, exhibits eosinophilic cytotoxicity promoting effects and is increased in patients with severe schistosomiasis (26Dessein A.J. Lenzi H.L. Bina J.C. Carvalho E.M. Weiser W.Y. Andrade Z.A. David J.R. Cell Immunol. 1984; 85: 100-113Crossref PubMed Scopus (40) Google Scholar, 27Lenzi H.L. Mednis A.D. Dessein A.J. Cell Immunol. 1985; 94: 333-346Crossref PubMed Scopus (36) Google Scholar, 28Silberstein D.S. McDonough S. Minkoff M.S. Balcewicz Sablinska M.K. J. Biol. Chem. 1993; 268: 9138-9142Abstract Full Text PDF PubMed Google Scholar). By using monoclonal antibodies to Trx and Trx80, endogenous Trx80 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 (29Sahaf B. Söderberg A. Spyrou G. Barral A.M. Pekkari K. Holmgren A. Rosén A. Exp. Cell Res. 1997; 236: 181-192Crossref PubMed Scopus (77) Google Scholar). Both Trx and Trx80 have been reported to be secreted from cytotrophoblasts (30Di Trapani G. Perkins A. Clarke F. Mol. Hum. Repr. 1998; 4: 369-375Crossref PubMed Scopus (36) Google Scholar). It was also reported that Trx80 as a fusion protein increased HIV production in HIV-infected macrophages, whereas Trx showed inhibitory effects, and when Trx was added to HIV-infected macrophages cleavage of the protein to a truncated form was seen (31Newman G.W. Balcewicz-Sablinska M.K. Guarnaccia J.R. Remold H.G. Silberstein D.S. J. Exp. Med. 1995; 180: 359-363Crossref Scopus (50) Google Scholar), although these results have not been confirmed. In this study we have prepared large quantities of recombinant homogenous Trx80 and characterized the molecule. We also report the discovery of a unique mitogenic cytokine activity of Trx80 and have developed methods to measure the protein in plasma samples. AIM V cell culture medium andl-glutamine were purchased from Life Technologies, Inc.; primers were from Scandinavian Gene Synthesis AB; isopropyl thiogalactoside was from Saveen Biotech AB; NcoI was from Promega; Ficoll-Paque, Sephadex G-50 and G-75, and RPC 18 column were from Amersham Pharmacia Biotech; polymyxin B sulfate and agarose-linked polymyxin B sulfate were from Sigma; 5-[3H]thymidine was from Amersham Pharmacia Biotech; Spin-X filters from Corning Costar Corporation; penicillin/streptomycin was from Bio Whittaker Europe; and alkaline phosphate-linked streptavidin was from Mabtech AB. Recombinant Trx80 was constructed on the rationale that the lysine-rich region of amino acids 80–84 in full-length thioredoxin (KKGQK) could be a favorable site for a cleavage event to take place. Two primers were synthesized (5′-ATT CTA AGG AAA ACC ATG GTG AAA CAG-3′ and 5′-CAC CCA CCC ATG GTC ACT ACT TAA AAA ACT GG-3′) introducing an NcoI cleavage site at the ATG and an NcoI site and stop codon at the C terminus. A polymerase chain reaction was run with pACA/Trx (9Ren X. Björnstedt M. Shen B. Ericson M.L. Holmgren A. Biochemistry. 1993; 32: 9701-9708Crossref PubMed Scopus (138) Google Scholar) as template using 27 cycles of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min. A product of approximately 240 base pairs was purified.Escherichia coli BL-21 (DE3) cells transformed with the pET 3d-Trx80 plasmid were grown in LB medium containing 50 μg/ml ampicillin at 37 °C to an optical density of 0.5 at 595 nm and then induced with 0.5 mm isopropyl thiogalactoside. The induced cells were harvested by centrifugation after 4 h of induction. The bacterial pellet was dissolved in 5 ml of TE buffer with 1 mm phenylmethylsulfonyl fluoride for 1 gram of bacterial pellet and lyzed using a combination of lysozyme and sonication. Then 10 mm DTT, 2 mm MgCl2, and 50 μg/ml DNase1 were added, and the cell lysate was centrifuged at 13,000 rpm for 30 min. Trx80 was found to be present in inclusion bodies. The pellet fraction was therefore washed with 0.5 murea, incubated on ice for 15 min, centrifuged at 13,000 rpm for 30 min, and subsequently dissolved in 5 m urea. After this incubation the protein was soluble and was upon centrifugation at 13,000 rpm for 30 min dialyzed extensively against TE buffer. Subsequently the protein was applied to a DE52 column pre-equilibrated with TE buffer and was eluted with a linear NaCl gradient. The protein was eluted around 125 mm NaCl and apparently pure when analyzed on a silver-stained 20% homogenous SDS-polyacrylamide gel. Nevertheless it was reduced by excess DTT and applied to a reverse-phase column (RPC 18, fast protein liquid chromatography) pre-equilibrated with 0.1% NH4HCO3 and eluted with 0.1% NH4HCO3 and 50% acetonitrile. To concentrate the protein, the acetonitrile was removed by evaporation, and the buffer was changed to TE by repeated additions using a Diaflo equipment and a 3-kDa cut-off membrane. This pure Trx80 was then aliquoted and stored at −20 °C. The protein was fully characterized as pure Trx80 using N-terminal sequencing and electrospray ionization mass spectrometry (Protein Analysis Center, Karolinska Institutet). Trx, calf thymus TrxR, and Trx84 were purified as described previously (9Ren X. Björnstedt M. Shen B. Ericson M.L. Holmgren A. Biochemistry. 1993; 32: 9701-9708Crossref PubMed Scopus (138) Google Scholar, 29Sahaf B. Söderberg A. Spyrou G. Barral A.M. Pekkari K. Holmgren A. Rosén A. Exp. Cell Res. 1997; 236: 181-192Crossref PubMed Scopus (77) Google Scholar, 32Luthman M. Holmgren A. Biochemistry. 1982; 21: 6628-6633Crossref PubMed Scopus (510) Google Scholar). A Coomassie-stained 20% homogenous SDS-polyacrylamide gel with Trx80 and Trx using the Amersham Pharmacia Biotech phastgel system is shown in Fig. 1. A theoretical molar extinction coefficient for Trx80 was calculated to 8383 m−1 cm−1 (33Aitken A. Learmonth M. Walker J.M. The Protein Protocols Handbook. Humana Press Inc., Totowa, NJ1996: 3-6Crossref Google Scholar). An experimental molar extinction coefficient was determined by dissolving 5 mg of dry protein powder in 10 mm Tris, 1 mm EDTA, pH 7.5, and A 280 nm was recorded after appropriate dilutions. This gave an experimental value of 8250 m−1 cm−1. This was used to calculate concentrations of pure Trx80. Concentrations for Trx was measured at 280 nm and calculated with molar extinction coefficient 8250 m−1 cm−1 (9Ren X. Björnstedt M. Shen B. Ericson M.L. Holmgren A. Biochemistry. 1993; 32: 9701-9708Crossref PubMed Scopus (138) Google Scholar). Free SH groups were determined at A 412 nm using 1 mm 5,5′-dithio-bis(2-nitrobenzoic acid) and a molar extinction coefficient of 13 600 m−1cm−1 (9Ren X. Björnstedt M. Shen B. Ericson M.L. Holmgren A. Biochemistry. 1993; 32: 9701-9708Crossref PubMed Scopus (138) Google Scholar). CD spectra of Trx80 was recorded at 25 °C in TE buffer and was compared with the CD spectra of full-length Trx. The CD spectra analyses were performed in a cell path length of 0.2 mm on an Aviv spectrophotometer. Gel filtration experiments were carried out on Sephadex G-50 and G-75 columns. The Sephadex columns were pre-equilibrated with TE buffer containing 1 mm DTT. Both Trx and Trx80 were reduced with 10 mm DTT prior to applying the proteins on the columns. The Sephadex G-75 column had a volume of 131 ml, the sample volume was 2.6 ml, and the flow rate was 5 ml/cm2/h. The concentrations of protein ranged from 1.3 to 2.6 mg/ml. The NADPH consumption assay was carried out at 20 °C in a total volume of 500 μl with 200 μmNADPH in TE buffer. The reaction cuvette contained 10 μmoxidized Trx80 with three free SH groups, and the reaction was initiated by adding 10 μg of calf thymus TrxR (32Luthman M. Holmgren A. Biochemistry. 1982; 21: 6628-6633Crossref PubMed Scopus (510) Google Scholar) to both the reference and the reaction cuvette, and theA 340 nm was recorded. This was followed by addition of 1 μm Trx to both the reference and the reaction cuvette, and NADPH consumption was followed at 340 nm using a Zeiss PM Q3 spectrophotometer equipped with an automatic zero control of the reference cuvette and a recorder. Human PBMC were prepared by standard Ficoll-Paque centrifugation of heparinized blood from healthy donors (Blood Bank, Karolinska Hospital). The cells at a concentration of 1 × 106 cells/ml were suspended in serum-free AIM V medium supplemented with 2 mml-glutamine, penicillin (100 units/ml), and streptomycin (100 μg/ml) for all cell experiments except the experiment where the increase in cell number was investigated. In this experiment PBMC were at a concentration of 0.5 × 106 cells/ml in RPMI medium supplemented with 10% fetal calf serum, 2 mml-glutamine, penicillin (100 units/ml), and streptomycin (100 μg/ml). Before addition to cell cultures, Trx and Trx80 were reduced with 10 mm of DTT in 37 °C for 60 min. To remove any possible contamination with endotoxin, the protein solutions were passed over a column with polymyxin B-linked agarose according to manufacturer's recommendation. Subsequently DTT was removed by chromatography at +4 °C over a PD 10 column pre-equilibrated with degassed ice-cold PBS. The number of free SH groups was determined, and all Trx and Trx80 used in this study had at least 2.8 free SH groups per molecule. The protein solutions were finally, prior to cell culture experiments, filtered with spin-X filters to ensure sterility. All [3H]thymidine assays were performed in 96-well flat bottom plates at 1 × 106 cells/ml in a final volume of 200 μl with all cultures incubated in triplicate at 37 °C and 5% CO2 in humidified cell incubators. For determination of [3H]thymidine uptake, each well was pulsed with 0.5 μCi of 5-[3H]thymidine for 9 h prior to harvesting onto fiberglass filters using a multi-channel cell harvester (Tomtec, Wallac, Sweden). Filters were counted with a MicroBeta Plus Scintillation counter (Tomtec, Wallac). All data reported are mean cpm of triplicate cultures. Catabolism of [3H]thymidine to [3H]dihydrothymine was determined as described (34Bodycote J. Wolff S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4749-4753Crossref PubMed Scopus (50) Google Scholar, 35Arnér E.S.J. Valentin A. Eriksson S. J. Biol. Chem. 1992; 267: 10968-10975Abstract Full Text PDF PubMed Google Scholar, 36Arnér E.S.J. Eriksson S. Anal. Biochem. 1993; 210: 102-105Crossref PubMed Scopus (4) Google Scholar) and was found to be negligible in all treatments, showing that [3H]thymidine uptake thereby was a trustworthy measure of DNA replication (34Bodycote J. Wolff S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4749-4753Crossref PubMed Scopus (50) Google Scholar, 35Arnér E.S.J. Valentin A. Eriksson S. J. Biol. Chem. 1992; 267: 10968-10975Abstract Full Text PDF PubMed Google Scholar, 36Arnér E.S.J. Eriksson S. Anal. Biochem. 1993; 210: 102-105Crossref PubMed Scopus (4) Google Scholar). As controls to show that the cell growth effect seen was caused by Trx80 and not from any contaminant, two sets of experiments were performed. First, pure polyclonal goat anti-Trx80 antibodies were kept at 0.83 mg/ml in 0.2 m citrate buffer, pH 6.5, and coupled to cyanogen bromide-activated Sepharose 4B, to give a 1.5-ml column with 8 nmol of coupled antibody. Over this column 1.5 ml of Trx80 solution with 1.5 nmol of Trx80 was passed. After that 1.5 ml of PBS was added to column, and the flow through was collected; this was sterile-filtered and added to cells. In a second control experiment, the endotoxin inhibitor, polymyxin B sulfate, was added to cell cultures treated with Trx80 or lipopolysaccaride, and the [3H]thymidine incorporation was determined. Cell death was measured by trypan blue exclusion and did not exceed 10% in any experiment. The cell experiments where optical density of cell cultures were used to determine the cell number was performed as described earlier (37Kravtsov V.D. Eur. J. Cancer. 1994; 30A: 1564-1570Abstract Full Text PDF PubMed Scopus (14) Google Scholar). Briefly, 200 μl of cell suspension was seeded in 96-well plates with 3 μm DTT. 2.5 μg/ml phytohemoglutine A, 1 μm Trx80, or 1 μm Trx84 were added in triplicate to the cell suspension. Cells were grown for 6 days at 37 °C and 5% CO2 in humidified cell incubators, and the optical density at 650 nm was measured during the incubation time using a micro plate reader (Thermomax, Molecular Devices). Data were analyzed using the accompanying SOFTmax version 2.31 software. As a blank only medium and DTT was used, and as a control only 3 μm DTT was used. The final concentration of DTT was 3 μm in all cultures. Specific monoclonal mouse anti-Trx (clone 2G11) and anti-Trx80 (clone 7D11) antibodies were obtained as described previously (29Sahaf B. Söderberg A. Spyrou G. Barral A.M. Pekkari K. Holmgren A. Rosén A. Exp. Cell Res. 1997; 236: 181-192Crossref PubMed Scopus (77) Google Scholar). Standard samples of Trx and Trx80 were aliquoted at 100 μg/ml in PBS with 0.5% bovine serum albumin and kept at −70 °C. Each aliquot was discarded after being thawed once. 96-well plates were coated with 50 μl of 10 μg/ml anti-Trx or anti-Trx80 antibody in PBS and incubated at +4 °C overnight. Subsequently the mixture was discarded, and 200 μl of incubation buffer (0.5% bovine serum albumin, 0.05% Tween 20, 0.02% NaN3 in PBS) was added and incubated 2 h in room temperature to block unspecific protein binding sites. Plates were washed four times with washing buffer (0.05% Tween 20 in 0.9% saline). Standard dilutions of Trx or Trx80, and samples were prepared in incubation buffer, and 50 μl of standards or samples were added in duplicates and incubated at +4 °C overnight. Thereafter plates were washed four times with washing buffer and biotinylated goat anti-Trx polyclonal antibody was added at 2 μg/ml in 50 μl and incubated 2 h in room temperature. Subsequently plates were washed four times with washing buffer, and 50 μl of alkaline phosphatase-conjugated streptavidin diluted 1:1000 in incubation buffer was added and incubated 1 h in room temperature. Plates were then washed six times in washing buffer and 50 μl of 1 mg/ml p-nitrophenyl phosphate dissolved in 10% diethanolamine, 0.02% NaN3, 0.5 mmMgCl2, pH 9.8, was added to each well, and absorbance was recorded at 405 nm by a microplate reader (Thermomax, Molecular Devices). Data were analyzed using the accompanying SOFTmax version 2.31 software. Blood was collected anonymously by vein puncture in heparinized tubes from blood donors (n = 12) at Karolinska Hospital. Samples were subsequently centrifuged at 2000 × g for 20 min at room temperature and plasma was collected and kept at −20 °C. Degree of hemolysis in the plasma samples was determined using a commercial plasma hemoglobin kit from Sigma following the manufacturer's instructions. The hemoglobin levels in these samples varied from 29 to 67 μg/ml, corresponding to 0.02–0.05% hemolysis, assuming a normal total blood hemoglobin level of 140 g/liter. The contribution of released intracellular full-length Trx because of this degree of hemolysis to the total determined extracellular levels given in Table III was then calculated following the method of Nakamuraet al. (38Nakamura H. De Rosa S. Roederer M. Anderson M.T. Dubs J.G. Yodoi J. Holmgren A. Herzenberg L.A. Herzenberg L.A. Int. Immunol. 1996; 8: 603-611Crossref PubMed Scopus (179) Google Scholar) and found to be 2.7–6.3 ng/ml. Crude extracts of human placenta tissue were prepared by homogenization in 2 ml of TE buffer/gram of fresh placenta tissue using a Polytron tissue disruptor. The supernatant after centrifugation at 10,000 × g for 10 min at +4 °C was used for the Trx determinations.Table IIIPlasma levels of thioredoxin and Trx80 in healthy individualsSubjectTrx 3-aMeasured with sandwich ELISA for Trx, presented as mean of duplicate values with standard deviations.Trx80 3-bMeasured with sandwich ELISA for Trx80, presented as mean of duplicate values with standard deviations. About 5% (3–8%) of the measured Trx levels contributes to the given Trx80 levels because of the cross-reactivity of the Trx80 ELISA as described under "Experimental Procedures," resulting in a corrected Trx80 range of 1–171 ng/ml.ng/mlng/ml129 ± 66 ± 5226 ± 339 ± 3322 ± 122 ± 3432 ± 358 ± 6538 ± 6116 ± 8626 ± 112 ± 1753 ± 2174 ± 49816 ± 312 ± 1931 ± 253 ± 161016 ± 216 ± 81147 ± 717 ± 21225 ± 22 ± 13-a Measured with sandwich ELISA for Trx, presented as mean of duplicate values with standard deviations.3-b Measured with sandwich ELISA for Trx80, presented as mean of duplicate values with standard deviations. About 5% (3–8%) of the measured Trx levels contributes to the given Trx80 levels because of the cross-reactivity of the Trx80 ELISA as described under "Experimental Procedures," resulting in a corrected Trx80 range of 1–171 ng/ml. Open table in a new tab The novel procedure to produce Trx80 developed in this study yielded pure protein (Fig. 1 A) free from LPS and suitable for testing in cell cultures. Because it was produced as inclusion bodies and refolded from 5 m urea, we established that the refolding had succeeded. Therefore, CD spectra of Trx80 and Trx were recorded and compared; Fig. 1 B shows that Trx and Trx80 had similar spectra with characteristic minima at 208–212 and 221–223 nm. The mean residue ellipticities of these signals were, however, lower for Trx80, which also showed a small negative band around 187 nm, whereas Trx only showed a positive shoulder at the same wavelength. The loss of the C-terminal 25 residues corresponds to loss of one helix (residues 93–105 of Trx) and one strand (residues 84–92 of Trx) (5Weichsel A. Gasdaska J.R. Powis G. Montfort W.R. Structure. 1996; 4: 735-751Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). The CD spectra of Trx80 found here clearly established that truncated Trx80 has a substantial secondary structure and are compatible with loss of the C-terminal helix strand motif. When reduced Trx80 was applied to a G-50 Sephadex gel filtration column and chromatographed under reducing conditions, the protein eluted with the void volume and ahead of Trx (data not shown). However, Trx80 eluted at approximately 60% of the column volume on a calibrated Sephadex G-75 column, showing an apparent M r of 25,000 or double the size of Trx, which has a molecular weight of 12,000 (2Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar) (Fig. 2). The anomalous elution of Trx80 is compatible of a dimer in solution. Previous results showed that Trx80 as a fusion protein, in contrast to Trx, did not catalyze the reduction of insulin disulfides by DTT (28Silberstein D.S. McDonough S. Minkoff M.S. Balcewicz Sablinska M.K. J. Biol. Chem. 1993; 268: 9138-9142Abstract Full Text PDF PubMed Google Scholar). Our Trx80 was not a substrate for TrxR (Fig. 3). It also did not act as an inhibitor for reduction of oxidized Trx by TrxR (data not shown). We also tried to complement Trx80 with the C-terminal peptide of 24 residues of full-length Trx to examine whether this could restore any redox activity by noncovalent reconstitution (39Slaby I. Holmgren A. J. Biol. Chem. 1975; 250: 1340-1347Abstract Full Text PDF PubMed Google Scholar). However, this was not the case (data not shown). Trx80 did not have any protein-disulfide isomerase-like activity in refolding of RNase when tested in an assay previously used to assess such activity (data not shown) (40Lundström J. Krause G. Holmgren A. J. Biol. Chem. 1992; 267: 9052-9407Google Scholar). When Trx was added to a mixture containing TrxR, NADPH, and oxidized Trx80 with three free SH groups, Trx80 was reduced by Trx (Fig. 3). This showed that Trx and Trx80 interact and that Trx can reduce disulfides in oxidized Trx80, whereas Trx80 itself lacks redox activity in all systems analyzed above. Knowing that Trx80 was homogeneous and devoid of detectable disulfide oxidoreductase activity, we wanted to analyze cytokine-like effects of the protein, and we chose normal human PBMC for this analysis. PBMC proliferation in RPMI medium with 10% fetal calf serum measured by optical density showed that 2.5 μg/ml phytohemaglutine A increased the cell number by 60% after 4 days in culture compared with 3 μm DTT alone. Trx80 at 1 μm indeed showed a stimulation of cell proliferation, with a significantly better effect than that of Trx84 (35% compared with 20% increase in cell number), whereas Trx alone showed no effect compared with DTT alone (data not shown). This finding made us continue with Trx80 for further analyses, and we switched to a serum-free synthetic AIM V medium with no fetal calf serum added, because Trx is known to be present in fetal calf serum (41Holmgren A. Luthman M. Biochemistry. 1978; 17: 4071-4077Crossref PubMed Scopus (123) Google Scholar). We confirmed the increase in cell number by an increase in [3H]thymidine incorporation after stimulation of PBMC with Trx80 using concentrations as low as 10 nm. The maximum thymidine incorporation was seen after 72 and 96 h of treatment. With 100 nm Trx80 there was a 10–15-fold increase in thymidine incorporation as compared with untreated cells. The effect of Trx80 increased dose-dependently up to 50–100 nm, after which the effect on cell proliferation was the same or even lower at higher Trx80. The stimulation of thymidine incorporation by 100 nm Trx80 was at the same level as that given by 5 units/ml of IL-2. In great contrast, Trx lacked PBMC-stimulating effects at concentrations up to 1 μm. These results are summarized in Fig.4. There were no synergy effects between IL-2 and Trx80/Trx or between Trx80 and Trx in stimulat
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