Ricin A Chain Can Transport Unfolded Dihydrofolate Reductase into the Cytosol
1997; Elsevier BV; Volume: 272; Issue: 35 Linguagem: Inglês
10.1074/jbc.272.35.22097
ISSN1083-351X
AutoresBruno Beaumelle, Marie‐Pierre Taupiac, Janet M. Lord, Lynne M. Roberts,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoRicin is a heterodimeric protein toxin. The ricin A chain is able to cross the membrane of intracellular compartments to reach the cytosol where it catalytically inactivates protein synthesis. It is linked via a disulfide bond to the B chain, a galactose-specific lectin, which allows ricin binding at the cell surface and endocytosis. To examine the potential of ricin A to carry proteins into the cytosol and the requirement for unfolding of the passenger protein, we connected mouse dihydrofolate reductase (DHFR) to ricin A by gene fusion via a spacer peptide. DHFR-ricin A expressed inEscherichia coli displayed the biological activities of the parent proteins and associated quantitatively with ricin B to form DHFR-ricin. The resulting toxin was highly cytotoxic to cells (4–8-fold less than recombinant ricin). DHFR-ricin cytotoxicity was inhibited by methotrexate, a DHFR inhibitor stabilizing DHFR-ricin A in a folded conformation. The DHFR moiety of DHFR ricin bound to the plasma membrane. Although methotrexate prevented this binding, it did not significantly affect DHFR-ricin endocytosis, which proceeded via ricin B chain. Intoxication kinetics data and a cell-free translocation assay demonstrated that protection of cells from DHFR-ricin cytotoxicity resulted from a selective inhibition by methotrexate of DHFR-ricin A translocation. We conclude that ricin A is a potential carrier of proteins to the cytosol, provided that the passenger protein is able to unfold for transmembrane transport. Ricin is a heterodimeric protein toxin. The ricin A chain is able to cross the membrane of intracellular compartments to reach the cytosol where it catalytically inactivates protein synthesis. It is linked via a disulfide bond to the B chain, a galactose-specific lectin, which allows ricin binding at the cell surface and endocytosis. To examine the potential of ricin A to carry proteins into the cytosol and the requirement for unfolding of the passenger protein, we connected mouse dihydrofolate reductase (DHFR) to ricin A by gene fusion via a spacer peptide. DHFR-ricin A expressed inEscherichia coli displayed the biological activities of the parent proteins and associated quantitatively with ricin B to form DHFR-ricin. The resulting toxin was highly cytotoxic to cells (4–8-fold less than recombinant ricin). DHFR-ricin cytotoxicity was inhibited by methotrexate, a DHFR inhibitor stabilizing DHFR-ricin A in a folded conformation. The DHFR moiety of DHFR ricin bound to the plasma membrane. Although methotrexate prevented this binding, it did not significantly affect DHFR-ricin endocytosis, which proceeded via ricin B chain. Intoxication kinetics data and a cell-free translocation assay demonstrated that protection of cells from DHFR-ricin cytotoxicity resulted from a selective inhibition by methotrexate of DHFR-ricin A translocation. We conclude that ricin A is a potential carrier of proteins to the cytosol, provided that the passenger protein is able to unfold for transmembrane transport. Toxins such as ricin and diphtheria toxin (DT), 1The abbreviations used are: DT, diphtheria toxin; DTA, diphtheria toxin A chain; CTL, cytotoxic T lymphocyte; DHFR, dihydrofolate reductase; MTX, methotrexate; RTA, ricin A chain; rRTA, recombinant RTA; RTB, ricin B chain; Pipes, 1,4-piperazinediethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; PBS, phosphate buffered saline. which have intracellular targets, are able to cross particular biological membranes. Both ricin and DT are heterodimeric proteins comprising an A chain bearing the catalytic activity responsible for the arrest of protein synthesis and a B chain enabling the toxin to bind at the plasma membrane (1Lord J.M. Roberts L.M. Robertus J.D. FASEB J. 1994; 8: 201-208Crossref PubMed Scopus (405) Google Scholar, 2Olsnes S. Sandvig K. Frankel A.E. Immunotoxins. Kluwer Academic Publishers, New York1988: 39-73Google Scholar). Whereas DT recognizes a specific receptor (3Naglich J.G. Metherall J.E. Russell D.W. Eidels L. Cell. 1992; 69: 1051-1061Abstract Full Text PDF PubMed Scopus (468) Google Scholar), the lectin activity of ricin B chain (RTB) enables interaction with terminal galactose residues present on both glycoproteins and glycolipids. Ricin is then endocytosed, and electron microscopic studies revealed significant routing to endosomes and to thetrans-Golgi network (2Olsnes S. Sandvig K. Frankel A.E. Immunotoxins. Kluwer Academic Publishers, New York1988: 39-73Google Scholar). Expression of mutant GTPases involved in regulating vesicular transport steps early in the secretory pathway protects cells against ricin toxicity, suggesting that this toxin may be further transported to the endoplasmic reticulum (4Simpson J.C. Dascher C. Roberts L.M. Lord J.M. Balch W.E. J. Biol. Chem. 1995; 270: 20078-20083Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). At some point during ricin intracellular routing, the A chain is translocated across a membrane into the cytosol where protein synthesis is inhibited. This translocation step is rate-limiting for cytotoxicity (5Hudson T.H. Neville Jr., D.M. J. Biol. Chem. 1987; 262: 16484-16494Abstract Full Text PDF PubMed Google Scholar), and a number of potential translocation sites are reported in the literature, namely the endosome (6Beaumelle B. Alami M. Hopkins C.R. J. Biol. Chem. 1993; 268: 23661-23669Abstract Full Text PDF PubMed Google Scholar), the trans-Golgi network/Golgi apparatus (7Sandvig K. Prydz K. Hansen S.H. van Deurs B. J. Cell Biol. 1991; 115: 971-981Crossref PubMed Scopus (135) Google Scholar), or the endoplasmic reticulum (4Simpson J.C. Dascher C. Roberts L.M. Lord J.M. Balch W.E. J. Biol. Chem. 1995; 270: 20078-20083Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Nevertheless, a direct assay for ricin translocation is available only in the endosome system (6Beaumelle B. Alami M. Hopkins C.R. J. Biol. Chem. 1993; 268: 23661-23669Abstract Full Text PDF PubMed Google Scholar). The unfolding requirement for protein translocation has essentially been studied using mouse dihydrofolate reductase (DHFR), which once fused to the appropriate targeting sequence, could be transported across the inner membrane of Escherichia coli (8Arkowitz R.A. Joly J.C. Wickner W. EMBO J. 1993; 12: 243-253Crossref PubMed Scopus (100) Google Scholar), imported into mitochondria (9Eilers M. Schatz G. Nature. 1986; 322: 229-232Crossref Scopus (466) Google Scholar), or into the ER lumen (10Schlenstedt G. Zimmermann M. Zimmermann R. FEBS Lett. 1994; 340: 139-144Crossref PubMed Scopus (7) Google Scholar). Such transport processes are blocked by methotrexate (MTX), a folate analogue that stabilizes the native conformation of DHFR, thereby preventing the unfolding deemed necessary for the membrane translocation of most polypeptides (8Arkowitz R.A. Joly J.C. Wickner W. EMBO J. 1993; 12: 243-253Crossref PubMed Scopus (100) Google Scholar, 9Eilers M. Schatz G. Nature. 1986; 322: 229-232Crossref Scopus (466) Google Scholar, 10Schlenstedt G. Zimmermann M. Zimmermann R. FEBS Lett. 1994; 340: 139-144Crossref PubMed Scopus (7) Google Scholar). However, when DHFR was targeted to the chloroplast (11Guéra A. America T. van Waas M. Weisbeek P.J. Plant Mol. Biol. 1993; 23: 309-324Crossref PubMed Scopus (41) Google Scholar), or the glycosome (12Häusler T. Stierhof Y.-D. Wirtz E. Clayton C. J. Cell Biol. 1996; 132: 311-324Crossref PubMed Scopus (76) Google Scholar), import was not affected by MTX or its analogue, aminopterin. In the former case at least, this is due to a potent unfolding activity associated with the chloroplast surface (13Walker D. Chaddock A.M. Chaddock J.A. Roberts L.M. Lord J.M. Robinson C. J. Biol. Chem. 1996; 271: 4082-4085Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Mutant DT A chain (DTA) and ricin A chain (RTA) with introduced disulfides were either not or poorly translocated (14Falnes P. Choe S. Madshus I.H. Wilson B.A. Olsnes S. J. Biol. Chem. 1994; 269: 8402-8407Abstract Full Text PDF PubMed Google Scholar, 15Argent R.H. Roberts L.M. Wales R. Robertus J.D. Lord J.M. J. Biol. Chem. 1994; 269: 26705-26710Abstract Full Text PDF PubMed Google Scholar), indicating that toxin A chains must also unfold to cross membranes. Bacterial toxins such as Pseudomonas exotoxin A (16Donnely J.J. Ulmer J.B. Hawe L.A. Friedman A. Shi X.-P. Leander K.R. Shiver J.W. Oliff A.I. Martinez D. Montgomery D. Liu M.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3530-3534Crossref PubMed Scopus (77) Google Scholar) and DTA (17Wieldocha A. Madshus I.H. Mach H. Middaugh C.R. Olsnes S. EMBO J. 1992; 11: 4835-4842Crossref PubMed Scopus (84) Google Scholar, 18Klingenberg O. Olsnes S. Biochem. J. 1996; 313: 647-653Crossref PubMed Scopus (35) Google Scholar) can transport foreign proteins into the cytosol. This transfer also requires unfolding of the passenger protein as shown for DTA with both acidic fibroblast growth factor (17Wieldocha A. Madshus I.H. Mach H. Middaugh C.R. Olsnes S. EMBO J. 1992; 11: 4835-4842Crossref PubMed Scopus (84) Google Scholar) and DHFR (18Klingenberg O. Olsnes S. Biochem. J. 1996; 313: 647-653Crossref PubMed Scopus (35) Google Scholar), using heparin and MTX, respectively, to stabilize the folded structure of the passenger protein and arrest translocation. Nothing is known concerning the potential of RTA to deliver foreign proteins to the cytosol. Ultimately, however, enzymatically inactive ricin, like DT, is a potential carrier of antigenic peptides (19Yewdell J.W. Bennick J.R. Cell. 1990; 62: 203-206Abstract Full Text PDF PubMed Scopus (203) Google Scholar). This is of special interest since an initial step in the presentation of antigens by the major histocompatibility complex class I molecules requires peptide processing in the cytosol of antigen-presenting cells (20Germain R.N. Cell. 1994; 76: 287-299Abstract Full Text PDF PubMed Scopus (1272) Google Scholar). Ricin may thus be used for cytosolic delivery of such peptides or even of whole proteins for subsequent processing, cell surface presentation and induction of a protective cytotoxic T lymphocyte (CTL) response (16Donnely J.J. Ulmer J.B. Hawe L.A. Friedman A. Shi X.-P. Leander K.R. Shiver J.W. Oliff A.I. Martinez D. Montgomery D. Liu M.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3530-3534Crossref PubMed Scopus (77) Google Scholar, 19Yewdell J.W. Bennick J.R. Cell. 1990; 62: 203-206Abstract Full Text PDF PubMed Scopus (203) Google Scholar). In this study, we genetically fused DHFR to RTA and demonstrated the ability of RTA to introduce fused DHFR into the cytosol. This import was blocked by MTX, indicating that DHFR must unfold in order for it to translocate with RTA. This finding has obvious implications for the use of RTA as a carrier of cellular epitopes. All enzymes needed for DNA manipulations were obtained from Life Technologies, Inc. or Pharmacia. Taq DNA polymerase for polymerase chain reaction, plasmid miniprep purification system, DNA silver sequencing kit, and reticulocyte lysates were from Promega. Most of the chemicals were obtained from Sigma. Pure RTB (without any detectable RTA) was purchased from Inland Biologicals, Austin, TX, whereas recombinant RTA (rRTA) was provided by Zeneca (UK). Western blotting detection kit and radiochemicals were from Amersham. AnEcoRI-PstI DHFR fragment was obtained by polymerase chain reaction using the full-length coding sequence of the mouse DHFR in pDS5/3 vector (21Stuber D. Ibrahimi I. Cutler D. Dobberstein B. Bujard H. EMBO J. 1984; 3: 3143-3148Crossref PubMed Scopus (143) Google Scholar) as a template. The sense primer (CTAAG↓AATTCATGGTTCGACCATTG) was used to introduce anEcoRI site (↓) immediately upstream of the initiation codon. The antisense primer (CTTACTGCA↓GGTCTTTCTTCTCGTA) provided aPstI cleavage site (↓) immediately before the natural stop codon. The full-length RTA-coding sequence (22O'Hare M. Roberts L.M. Thorpe P.E. Watson G.J. Prior B. Lord J.M. FEBS Lett. 1987; 216: 73-78Crossref PubMed Scopus (76) Google Scholar) in pKK 223.3 (Pharmacia) was used as a template to prepare by polymerase chain reaction an PstI-HindIII RTA fragment. The sense primer (GATCTCTGCA↓GATATTCCCCAAACAA) provided a PstI site (↓) before the second codon of RTA and the antisense primer (TTACCA↓AGCTTTCAAAACTGTGACGA) provided a HindIII site (↓) after the stop codon. Polymerase chain reaction products were gel-isolated after restriction and ligated in a stepwise manner into pKK 223.3. Preliminary experiments showed that the fusion protein prepared without any spacer was inactive in several assays for biological or biochemical activities (data not shown). A double-stranded oligonucleotide containing an internal BamHI site and coding for a spacer peptide (His-Ala-Ser-Thr-Pro-Glu-Pro-Asp-Pro-Val) was thus inserted using thePstI site. This peptide linker is similar to the flexible hinge region of a monoclonal antibody (23Brinkman U. Buchner J. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3075-3079Crossref PubMed Scopus (95) Google Scholar). Purified plasmids were restricted with PstI before transformation of E. coli TG2. Clones were screened for acquisition of theBamHI restriction site, and DNA sequencing enabled the assessment of its orientation. The resulting protein is denoted DHFR-RTA. A 1.5-liter culture of E. coli TG2 containing the DHFR-RTA plasmid was grown at 30 °C. Expression was induced at anA595 ∼ 0.4 using 1 mmisopropylthiogalactoside. After 2 h at 30 °C, E. coli lysates were prepared by sonication, clarified by centrifugation for 30 min at 20,000 × g (22O'Hare M. Roberts L.M. Thorpe P.E. Watson G.J. Prior B. Lord J.M. FEBS Lett. 1987; 216: 73-78Crossref PubMed Scopus (76) Google Scholar), and dialyzed against 20 mm potassium phosphate, pH 7.4, 1 mm EDTA, 0.1 mm phenylmethylsulfonyl fluoride, 0.5 mm dithiothreitol (buffer A) before loading (at 0.2 ml/min) onto a 5-ml MTX-agarose column (Sigma). After washing with 45 ml of buffer A and 60 ml of buffer A supplemented with 1 mKCl, DHFR-RTA was eluted with 1 mm MTX in buffer A and stored sterile at −80 °C, after adding 15% glycerol. The final yield of purified protein was greater than 10 mg/liter of culture. DHFR activity was measured as described previously (24Pastore E.J. Plante L.T. Kisliuk R.L. Methods Enzymol. 1974; 34: 281-288Crossref PubMed Scopus (22) Google Scholar). The ability of RTA to inhibit protein synthesis was assayed using rabbit reticulocyte lysates (25Pelham H.R. Jackson R.J. Eur. J. Biochem. 1976; 67: 247-256Crossref PubMed Scopus (2434) Google Scholar) supplemented with globin mRNA (Life Technologies, Inc.). The two proteins (20 μm each) were mixed in PBS in the presence of 8 mm GSH. After 3 h at room temperature and overnight dialysis at 4 °C against PBS, the mixture was analyzed by nonreducing SDS-PAGE. Recombinant ricin (rRTA-RTB) was prepared using the same protocol. We experienced that 125I labeling of isolated RTA impaired subsequent association with RTB (data not shown). DHFR-RTA, as well as rRTA, used as a control throughout this study, was thus radiolabeled with 125I (26Fraker P.J. Speck J.C. Biochem. Biophys. Res. Commun. 1978; 80: 849-857Crossref PubMed Scopus (3626) Google Scholar) after association with RTB. Transferrin and DHFR-ricin were conjugated with FITC, and ricin was complexed to colloidal gold as described elsewhere (27Beaumelle B. Hopkins C.R. Biochem. J. 1989; 264: 137-149Crossref PubMed Scopus (19) Google Scholar). Endocytosis efficiency of radiolabeled DHFR-RTA-RTB (DHFR-ricin) by mouse BW5147 lymphocytes was measured using recombinant ricin as a control. Washes with 0.1 m lactose were used to displace plasma membrane-bound molecules (6Beaumelle B. Alami M. Hopkins C.R. J. Biol. Chem. 1993; 268: 23661-23669Abstract Full Text PDF PubMed Google Scholar, 28Sandvig K. Olsnes S. Exp. Cell Res. 1979; 121: 15-25Crossref PubMed Scopus (82) Google Scholar). The assay for toxin translocation from purified lymphocyte endosomes is described elsewhere (6Beaumelle B. Alami M. Hopkins C.R. J. Biol. Chem. 1993; 268: 23661-23669Abstract Full Text PDF PubMed Google Scholar). 125I-Transferrin, as a membrane-bound tracer, and 125I-horseradish peroxidase, as a soluble maker, were used as negative controls in all translocation experiments to monitor the integrity of endosomes. BW5147 cells were labeled with DHFR-ricin for 30 min at 37 °C in Dulbecco's modified Eagle's medium containing 0.2 mg/ml bovine serum albumin and 0.15 mg/ml low density lipoproteins/ml, before lactose-scraping to displace membrane-bound ligand, ricin-gold binding, and lysis under hypotonic conditions. Unbroken cells and nuclei were removed by low speed centrifugation, and crude membranes collected by ultracentrifugation. They were then layered on a discontinuous sucrose gradient (40%/30%/20% sucrose). After 2 h at 100,000 ×g, endosomes were obtained from the 30%/20% interface, washed, and finally resuspended in translocation buffer (110 mm KCl, 15 mm MgCl2, 1 mm dithiothreitol, 0.15 mg/ml bovine serum albumin, 20 mm Pipes, pH 7.1, supplemented with ATP except when otherwise indicated). Translocation was assayed for 2 h at 37 °C and was stopped by chilling on ice. The medium was then separated from endosomes by ultracentrifugation (160,000 ×g for 5 min) on a 17% sucrose cushion. Translocated proteins were precipitated with 10% trichloroacetic acid and separated by reducing SDS-PAGE before autoradiography. Quantification was performed by densitometric analysis of films exposed within their linear range of detection. Direct γ counting of sliced gels was occasionally used to follow 125I-rRTA or125I-RTB translocation and gave identical results. When translocation of unlabeled DHFR-ricin and recombinant ricin was examined, Western blots (29Chaddock J.A. Roberts L.M. Protein Eng. 1993; 6: 425-431Crossref PubMed Scopus (46) Google Scholar) of translocated proteins were quantified using a Storm apparatus (Molecular Dynamics). Exponentially growing BW5147 cells were washed, then labeled with DHFR-ricin-FITC and ricin-tetramethylrhodamine isothiocyanate for 30 min at 37 °C in Dulbecco's modified Eagle's medium/bovine serum albumin/low density lipoprotein (see above). After lactose scraping, cells were fixed for 15 min at 2 °C in PBS containing 3.7% paraformaldehyde before quenching for 15 min using 50 mm NH4Cl in PBS. They were then washed with PBS, mounted in PBS supplemented with 2.5% 1,4-diacylbicyclo(2,2,2)octane, and examined under a Leica confocal microscope using a 63× lens and medial optical sections. Bleed through from one channel to the other was negligible. In preliminary experiments transferrin-FITC and an anti-mouse CD45 (clone I3/2) labeled with Cy5 were used as endosomal and plasma membrane markers, respectively (27Beaumelle B. Hopkins C.R. Biochem. J. 1989; 264: 137-149Crossref PubMed Scopus (19) Google Scholar). Cells (18,000 BW5147 in RPMI, 10% fetal calf serum or 6,000 L929 in RPMI, 5% fetal calf serum) were seeded in 96-well plates. Toxin solutions were added immediately (BW5147) or after 2 h at 37 °C to allow cell adherence (L929). After 24 h at 37 °C [35S]methionine (0.25 μCi) was added, and cells were incubated for a further 12–18 h. Precipitation with trichloroacetic acid was then performed either directly (BW5147) or following solubilization with 0.1 nNaOH after aspiration of the medium (L929). Proteins were collected onto glass fiber filters, washed with 5% trichloroacetic acid, then dried for radioactivity determination. Background incorporation was obtained from cells treated with 1 mm cycloheximide. To examine the kinetics of protein synthesis inactivation by recombinant ricin and DHFR-ricin the above protocol was modified. The cell number was increased to 70,000/well (BW5147) or 15,000/well (L929). A pulse incorporation of [35S]methionine (1 h) was performed at various times after the start of intoxication. Radiolabeled toxin (5 nm of125I-DHFR-ricin or 125I-recombinant ricin) was added to BW5147 cells (106/3 ml of RPMI/fetal calf serum) in the presence or absence of 50 nm MTX. After 0–24 h at 37 °C, cells were collected by centrifugation, washed three times with PBS, then lysed in 1 ml of immunoprecipitation buffer (20 mm Tris, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1% Nonidet P-40) (30Shackelford D.A. Trowbridge I.S. J. Biol. Chem. 1986; 261: 8334-8341Abstract Full Text PDF PubMed Google Scholar). After 20 min on ice, insoluble material was removed by centrifugation (13,000 × g for 10 min). This step and the rest of the experiment were performed at 4 °C. The cleared lysate received 2 μl of sheep anti-RTA antibody and was mixed for 1 h on a rotating wheel before adding 7 μl of protein G-agarose (Sigma). After 1 h on the wheel, the immune complexes were recovered by centrifugation (10,000 × g for 3 min), washed twice with immunoprecipitation buffer, then once with PBS, and eluted by boiling in SDS-PAGE reducing sample buffer (30Shackelford D.A. Trowbridge I.S. J. Biol. Chem. 1986; 261: 8334-8341Abstract Full Text PDF PubMed Google Scholar). Gels were exposed to storage phosphor screens that were analyzed using a Storm apparatus. All experiments were done at least in triplicate and repeated twice. Errors are expressed as S.E. Both DHFR (18Klingenberg O. Olsnes S. Biochem. J. 1996; 313: 647-653Crossref PubMed Scopus (35) Google Scholar) and RTA (31O'Hare M. Brown A.N. Hussain K. Gebhard A. Watson G. Roberts L.M. Vitetta E.S. Thorpe P.E. Lord J.M. FEBS Lett. 1990; 273: 200-204Crossref PubMed Scopus (49) Google Scholar) have been fused directly with a targeting protein to prepare biologically active hybrids. To obtain a biologically active DHFR-RTA chimera, we found it necessary to insert a linker peptide including three prolines (23Brinkman U. Buchner J. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3075-3079Crossref PubMed Scopus (95) Google Scholar) between the N terminus of recombinant RTA and the C terminus of DHFR (data not shown). The fusion protein was expressed inE. coli. A major band corresponding to the expected molecular mass of 50 kDa and reacting to both anti-RTA and anti-DHFR sera was observed on the gel of proteins from bacteria where expression was induced (Fig. 1). The DHFR-RTA fusion was purified to homogeneity by affinity chromatography using immobilized MTX (MTX-agarose), as ascertained by protein staining and Western blotting using antisera against both DHFR and RTA (Fig. 2). In particular, cross-reactive material observed in E. coli sonicates using anti-DHFR sera was eliminated by this single chromatographic step (compare lane 4 in Fig. 1 and lane 3 in Fig. 2). A typical yield was greater than 10 mg/liter of culture. Since DHFR-RTA was purified using its affinity for MTX, it is clear that the DHFR portion of the chimera recognizes MTX. This binding is known to stabilize the folded conformation of DHFR (9Eilers M. Schatz G. Nature. 1986; 322: 229-232Crossref Scopus (466) Google Scholar). The high protease resistance of DHFR-RTA, probably due to the well documented resistance of the RTA moiety of the chimera to proteases (13Walker D. Chaddock A.M. Chaddock J.A. Roberts L.M. Lord J.M. Robinson C. J. Biol. Chem. 1996; 271: 4082-4085Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), prevented us from monitoring the folding of the DHFR moiety using protease protection experiments, as conventionally performed (8Arkowitz R.A. Joly J.C. Wickner W. EMBO J. 1993; 12: 243-253Crossref PubMed Scopus (100) Google Scholar, 9Eilers M. Schatz G. Nature. 1986; 322: 229-232Crossref Scopus (466) Google Scholar, 10Schlenstedt G. Zimmermann M. Zimmermann R. FEBS Lett. 1994; 340: 139-144Crossref PubMed Scopus (7) Google Scholar,18Klingenberg O. Olsnes S. Biochem. J. 1996; 313: 647-653Crossref PubMed Scopus (35) Google Scholar). On the other hand, this resistance conferred intracellular stability on the fusion protein (see below). After purification, DHFR-RTA was tested on rabbit reticulocyte lysates for its ability to inactivate protein synthesis. The resulting IC50 (30 ± 10 nm) is similar to that of rRTA (15 ± 7 nm). DHFR-RTA also exhibited a DHFR activity (2.5 108 units/mol), which is virtually identical to the catalytic activity of native mouse DHFR (2.7–2.8 108 units/mol) (11Guéra A. America T. van Waas M. Weisbeek P.J. Plant Mol. Biol. 1993; 23: 309-324Crossref PubMed Scopus (41) Google Scholar). Together these data indicate that connection of DHFR to the N terminus of rRTA did not affect enzymatic activity of either of the parent proteins. Mixing equimolecular amounts of the DHFR-RTA with RTB and 8 mm GSH before dialysis generated homogenous DHFR-RTA-RTB (DHFR-ricin) as seen by nonreducing SDS-PAGE (Fig. 3, lane 6). This shows that the chimera, DHFR-RTA, interacts as efficiently as rRTA with the B chain. Formation of RTB dimers is observed only when the A chain is absent from the mixture (lane 4). Recombinant ricin (rRTA-RTB, lane 1) was generated using the same procedure. This toxin was used as a control throughout the rest of this study. Recombinant ricin migrated slightly faster (lane 1) than native ricin (shown in lane 2) due to the absence of oligosacharides on the A chain. To examine the ability of rRTA to bring DHFR in the cytosol we first investigated intoxication of various cell types by DHFR-ricin. The results of these tests are summarized in Table I. The isolated chains of ricin were essentially nontoxic, whereas DHFR-RTA surprisingly showed moderate cytotoxicity, indicating that fused DHFR somehow promotes more efficient RTA uptake. Nevertheless, since association of DHFR-RTA with RTB to produce DHFR-ricin increased toxicity by 150–650-fold (Table I), DHFR-mediated internalization was not significantly involved in DHFR-ricin cytotoxicity as compared with uptake via the B chain (see below). DHFR-ricin is only 4-fold less toxic than ricin to L929 fibroblasts. It is also highly toxic to BW5147 lymphocytes (8-fold less than ricin).Table ICytotoxic activity of recombinant ricin, DHFR-ricin, and their isolated chains on BW5147 lymphocytes and L929 fibroblastsMoleculeIC50BW5147L929Recombinant ricin (rRTA-RTB)16 ± 2 pm10 ± 2 pmRecombinant ricin + MTX15 ± 2 pm10 ± 2 pmRTB, RTA, or rRTA>10 μm>10 μmDHFR-RTA20 ± 5 nm25 ± 5 nmMTX8 ± 5 nm30 ± 5 nmDHFR-Ricin120 ± 10 pm40 ± 3 pmDHFR-Ricin + MTX300 ± 10 pm60 ± 4 pmAssays were performed as described under "Experimental Procedures." When MTX was used (at 10 and 30 nm on BW5147 and L929 cells, respectively) to protect cells against toxins, cytotoxicity was expressed as percentage of [35S]methionine incorporation as compared to that in cells treated with MTX only. Open table in a new tab Assays were performed as described under "Experimental Procedures." When MTX was used (at 10 and 30 nm on BW5147 and L929 cells, respectively) to protect cells against toxins, cytotoxicity was expressed as percentage of [35S]methionine incorporation as compared to that in cells treated with MTX only. To examine the possibility that DHFR-ricin toxicity resulted from processing of DHFR-RTA within cells to generate RTA, which would then be free to translocate to the cytosol, we studied the stability of DHFR-RTA within cells during intoxication by DHFR-ricin. As shown in Fig. 4, 125I-DHFR-RTA is stable upon BW5147 cells intoxication by 5 nm125I-DHFR-ricin, and no processing could be observed even after 24 h of contact. Quantitative analysis of images obtained from storage phosphor screens showed that beyond 24 h less125I-DHFR-RTA was recovered from cells (Fig. 4). This likely arose because more than 92% of cells have by then been killed by 5 nm DHFR-ricin and consequently a proportion of cells are nonpelletable by centrifugation. Identical results have been observed when 125I-rRTA was taken up by cells in the form of free subunit or as125I-recombinant ricin (L.M.R., unpublished). These results strongly indicate that, during intoxication by DHFR-ricin, DHFR-RTA does not break down to generate free rRTA by proteolysis. Together with the toxicity of DHFR-ricin we conclude that rRTA can carry DHFR into the cytosol. To examine whether unfolding of DHFR is required at any stage during intoxication by DHFR-ricin, MTX was used. However, as experienced by others, it was found that MTX alone interfered with the cytotoxicity assay (18Klingenberg O. Olsnes S. Biochem. J. 1996; 313: 647-653Crossref PubMed Scopus (35) Google Scholar), and we could not use concentrations significantly higher than the IC50 (10–30 nm) of MTX. Nevertheless 10 nm MTX was found to slightly protect BW5147 cells (1.5–3-fold) against intoxication by DHFR-ricin but did not protect against recombinant ricin (Table I). Examination of the intoxication curves revealed that, at a DHFR-ricin concentration below its IC50, 5 nm MTX had a strong protective effect (Fig. 5), whereas MTX provided no protective effect when recombinant ricin was added to cells (not shown). We believe that protection by MTX is more efficient at low DHFR-ricin concentration because the ratio [MTX]/[DHFR-RTA] is higher and might ensure the binding of MTX to DHFR even in intracellular vesicles where, as a result of endocytosis, DHFR-RTA is more concentrated compared with MTX. Cell intoxication by toxins is a multistep procedure and before drawing any conclusions from cytotoxicity data it was necessary to determine at which stage MTX was interfering with the DHFR-ricin intoxication process. To study binding and endocytosis, the initial steps of the intoxication process, cells were labeled with ricin-tetramethylrhodamine isothiocyanate and DHFR-ricin-FITC for 30 min at 37 °C before lactose scraping, fixation and examination under a confocal microscope (Fig. 6). Ricin was essentially intracellular, in transferrin-positive endosomes (not s
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