Saporin and ricin A chain follow different intracellular routes to enter the cytosol of intoxicated cells Riccardo Vago 1, *, Catherine J. Marsden 2, *, J. Michael Lord 2 , Rodolfo Ippoliti 3 , David J. Flavell 4 , Sopsamorn-U Flavell 4 , Aldo Ceriotti 5 and M. Serena Fabbrini 1,5 1 Dibit-S Raffaele Scientific Institute, Milan, Italy 2 Department of Biological Sciences, University of Warwick, Coventry, UK 3 Dipartimento di Biologia di Base ed Applicata, Universita ` degli Studi di L’Aquila, Italy 4 The Simon Flavell Leukaemia Research Unit, University Department of Pathology, Southampton, UK 5 Istituto di Biologia e Biotecnologia Agraria, CNR, Milan, Italy Protein toxins whose substrates are located within the cytosol of mammalian cells must be able to cross an intracellular membrane in order to exert their biologi- cal activity. Following initial internalization, these tox- ins must travel intracellularly to reach their molecular targets [1]. Some bacterial toxins such Pseudomonas ae- ruginosa Exotoxin A (PEA) carry a KDEL-like signal for retrieval to the endoplasmic reticulum (ER) [2,3]. KDEL receptors, normally cycling between the Golgi complex and the ER, can retrieve escaped ER-resident proteins that carry KDEL ⁄ REDL (single amino acid letter code) at their C-termini. In the ER, the presence of a higher pH allows detachment of the retrieved protein from the KDEL receptors [4]. The REDLK Keywords anticancer therapy; bacterial toxins; intracellular trafficking; KDEL retrieval sequence; plant ribosome-inactivating proteins Correspondence M. S. Fabbrini, CNR, via Bassini 15, 20133 Milan, Italy Fax: +39 223 699 411 Tel: +39 223 699 444 E-mail: fabbrini@ibba.cnr.it *Riccardo Vago and Catherine J. Marsden contributed equally to this work. (Received 18 May 2005, revised 11 July 2005, accepted 9 August 2005) doi:10.1111/j.1742-4658.2005.04908.x Several protein toxins, such as the potent plant toxin ricin, enter mamma- lian cells by endocytosis and undergo retrograde transport via the Golgi complex to reach the endoplasmic reticulum (ER). In this compartment the catalytic moieties exploit the ER-associated degradation (ERAD) pathway to reach their cytosolic targets. Bacterial toxins such as cholera toxin or Pseudomonas exotoxin A carry KDEL or KDEL-like C-terminal tetrapep- tides for efficient delivery to the ER. Chimeric toxins containing monomer- ic plant ribosome-inactivating proteins linked to various targeting moieties are highly cytotoxic, but it remains unclear how these molecules travel within the target cell to reach cytosolic ribosomes. We investigated the intracellular pathways of saporin, a monomeric plant ribosome-inactivating protein that can enter cells by receptor-mediated endocytosis. Saporin toxi- city was not affected by treatment with Brefeldin A or chloroquine, indica- ting that this toxin follows a Golgi-independent pathway to the cytosol and does not require a low pH for membrane translocation. In intoxicated Vero or HeLa cells, ricin but not saporin could be clearly visualized in the Golgi complex using immunofluorescence. The saporin signal was not evi- dent in the Golgi, but was found to partially overlap with that of a late endosome ⁄ lysosome marker. Consistently, the toxicities of saporin or sapo- rin-based targeted chimeric polypeptides were not enhanced by the addition of ER retrieval sequences. Thus, the intracellular movement of saporin differs from that followed by ricin and other protein toxins that rely on Golgi-mediated retrograde transport to reach their retrotranslocation site. Abbreviations ATF, amino-terminal fragment of urokinase; BFA, Brefeldin A; DT, diphtheria toxin; ER, endoplasmic reticulum; ERAD, ER-associated degradation; huPAR, human urokinase receptor; LRP, LDL-receptor related protein; PEA, Pseudomonas aeruginosa Exotoxin A; RIP, ribosome-inactivating protein; RTA, ricin A chain; SAP, saporin. FEBS Journal 272 (2005) 4983–4995 ª 2005 FEBS 4983 sequence found at the C-terminus of PEA is essential for the cytotoxicity of the endocytosed toxin, allowing PEA to reach its site of action [2,5]. This implies that PEA may interact with the KDEL receptor in order to traffic from the Golgi to the ER. The plant ribosome-inactivating protein ricin also enters the endocytic pathway and travels backwards from the Golgi complex to the ER where it is thought to parasitize the ER-associated degradation (ERAD) pathway [1,7–10] that normally disposes misfolded or unassembled proteins to the cytosol for proteasomal degradation [6]. Although ricin does not contain a KDEL-like C-terminal sequence, addition of this ER retrieval signal greatly enhances the toxicity of both a reconstituted AB holotoxin and the A chain alone [10–12]. Thus, the catalytic domains of different bacterial and plant protein toxins, including ricin [8], PEA [2,3] cholera toxin and Shiga toxin, can exploit the ERAD pathways [1,4,14] to reach their targets in the cytosol [15]. Here, most of them can irreversibly inactivate protein synthesis [1,13] causing apoptotic cell death [16,17]. PEA ADP-ribosylates elongation factor 2 [2,13,14,20], whereas Shiga and ricin A chain act by specifically depurinating 28S ribosomal RNA [1,3,7,9]. Saporin is a monomeric plant polypeptide that shows the same N-glycosidase activity as the ricin A chain. Different isoforms can be found in seeds and leaves of the soapwort Saponaria officinalis and some have been expressed in Escherichia coli and characterized biochemically [18,19]. The catalytic subunits of protein toxins are used to construct toxic chimeras selectively directed against tumor or meta- static cells via specific targeting domains [20]. One such recombinant chimera, preATF–SAP, targets transformed cells expressing the human urokinase receptor (huPAR) and contains the amino-terminal- fragment (ATF) of human prourokinase fused to the mature sequence of the ricin-related single-chain ribosome-inactivating protein saporin (SAP) [19]. To allow correct folding of the ATF cell-binding domain, which is essential for binding to huPAR [21] and contains six disulfide bridges forming a kringle and a growth factor-like domain, we expressed a secretory version of the ATF–SAP chi- mera in Xenopus laevis oocytes. Endogenously syn- thesized preATF–SAP was highly cytotoxic to host Xenopus laevis oocytes, but the oocytes could be pro- tected from autointoxication by injecting neutralizing antisaporin antibodies into the cytosol [22]. The mechanism(s) underlying this cytotoxicity remains unclear but these results clearly show that some pre- ATF–SAP polypeptides reached the oocyte cytosol. These observations raised the possibility that saporin may also use ER dislocon channels to enter this compartment. We investigated the pathway followed by saporin in exogenously intoxicated cells. Overall, our results strongly indicate that, in spite of the structural similar- ities with ricin A chain, saporin and derived chimeras follow a different intracellular transport route(s). Results Vero and HeLa cells were treated with drugs known to interfere with ricin holotoxin intracellular delivery and thus cytotoxic activity. The fungal inhibitor Brefel- din A (BFA) causes Golgi complex disassembly, pro- tecting cells against both ricin and PEA intoxication [23,24]. Furthermore, proteasomal inhibition prevents the cytosolic degradation of catalytic A chains of ricin following ER-to-cytosol transport, an effect exacerba- ted in the case of a mutant (ricin-6K) with increased lysine content [25]. Saporin is a lysine-rich protein and proteasomal inhibitors would sensitize target cells if the dislocation mechanism was similar to the one used by ricin. Ricin cytotoxicity was sensibly decreased by BFA treatment, as expected [23], whereas it was slightly increased by the proteasome inhibitor (Table 1). In contrast, neither drug significantly affected saporin- mediated toxicity (Table 1). In a second set of experi- ments, HeLa cells were challenged with different concentrations of saporin, ricin or RTAKDEL, either in the absence or presence of BFA (Table 1). BFA treatment led to a dramatic increase in the ID 50 sof ricin and RTAKDEL but did not have any effect on saporin ID 50 . The proteasomal inhibitor clasto-lacta- cystin-b-lactone sensitizes HeLa cells toward the action of mutated ricin-6K [25], as expected, but its effect on ricin and saporin toxicity was only a two- to threefold sensitization (Table 1). Thus, neither transport via the Golgi to the ER nor dislocation as an unfolded poly- peptide appears to contribute to the productive intoxi- cation route followed by saporin. Intracellular tracing of a fluorescinated saporin in both Vero cells (Fig. 1A) and HeLa cells (not shown) revealed the presence of saporin in punctuate struc- tures after exposure to an excess of the toxin. Although we cannot exclude that some fluid-phase uptake may also have occurred in these conditions, at these time points, we did not observe any colocaliza- tion of saporin with early endosome markers (anti- EEA1), although the late endosomal marker Lamp2 was shown to partially overlap with CY3 saporin fluorescence. Furthermore, unlike for ricin (Fig. 1B), Saporin trafficking in intoxicated mammalian cells R. Vago et al. 4984 FEBS Journal 272 (2005) 4983–4995 ª 2005 FEBS no Golgi localization of fluorescent saporin could be detected using anti-(Golgin 97) serum. This finding is fully consistent with BFA being ineffective in blocking saporin toxicity (Table 1). Toxins exploiting the ERAD pathway have a low lysine content to avoid ubiquitination upon disloca- tion to the cytosol [26]. A paradigm of a second class of toxins with normal lysine content is the diphtheria toxin (DT) that, while transiting in acidic endosomes, undergoes a conformational change triggering forma- tion of a pore through which the catalytic chain escapes into the cytosol and inactivates protein syn- thesis [27,28]. Therefore, in both Vero and HeLa cells, we analyzed the effects of chloroquine, a lysosomal caotropic drug that raises the pH in acidic compart- ments and almost abolished DT toxicity, but could not affect saporin-mediated cytotoxicity (Table 1). Bafylomycin A1, an inhibitor of the H + ATPase pump was able to protect cells from DT intoxication, but again did not affect saporin-mediated cytotoxicity (data not shown). The low cytotoxic activity of saporin in HeLa cells, with ID 50 in the micromolar range after 6 h of expo- sure (Table 1), prompted us to verify that toxicity was due to a genuine depurinating capability of the plant toxin over the endogenous ribosomes. RNA was iso- lated from cells treated with graded saporin concen- trations, as indicated, and either treated or not treated with acetic aniline. Figure 2 shows that saporin is indeed able to reach and inactivate HeLa ribosomes, as shown by the diagnostic aniline fragment vizualized in the denaturing agarose gels. We predicted, based on these observations, that appending an ER recycling signal such as KDEL to the C-terminus of saporin would not affect its cytotoxicity. We therefore com- pared the killing activities of SAPKDEL with SAPwt, independent of any targeting domain, and investigated whether the cytotoxicity of saporin would be potenti- ated by a KDEL motif, as previously shown for both PEA [2] and ricin A chain [12]. Both SAPwt and SAPKDEL (Fig. 3A) were expressed in bacteria and purified to homogeneity as described previously [18] (data not shown), and recombinant saporin-KDEL was specifically immunoprecipitated by monoclonal anti-KDEL serum before assaying its biological activit- ies (Fig. 3B). In Table 2, the in vitro activities of recombinant ricin and saporin polypeptides are com- pared: RTA and SAP IC 50 values were in the pico- molar range and were essentially the same as their KDEL-extended versions (Table 2). Vero cells are greatly sensitized to RTAKDEL [10–12] and were therefore used to compare the cytotoxic activities of the recombinant polypeptides (Table 2). The KDEL sequence increased the cytotoxicity of RTA almost 20-fold (ID 50 of 1.8 nm for RTA-KDEL vs. 44 nm for RTAwt). In contrast, addition of the KDEL sequence did not potentiate the cytotoxic activity of saporin after 4 h exposure (ID 50 of 5 nm for SAPKDEL vs. 3.7 nm for SAPwt) or even after longer exposure, indi- cating that this effect was independent of the kinetics Table 1. Saporin cytotoxicity is resistant to treatments affecting ricin or diphtheria toxin toxicities. Cell-killing of Vero cells was per- formed as described in the Experimental Procedures. The ID 50 val- ues of plant intact ricin (A + B) and diphtheria toxin (DT) were determined, using these same assays, and found to be around 1.7 and 2.5 p M, respectively. Vero cells were exposed to either 9 nM saporin or 5 pM ricin or 10 pM DT for 4 h in the presence or absence of the Golgi disrupting drug BFA (0.5 lgÆmL )1 ) or a protea- some inhibitor (MG-132, 10 l M)or10lM chloroquine. The data referred to in A, B and C show the percent of relative light units (% RLU) referred to 100% luciferase expression in the untreated samples ± S.E.M. n, number of independent experiments. Where indicated, HeLa cells were pretreated for 15 min at 37 °C with 10 l M BFA, 60 min with 20 lM proteasome inhibitor clasto-lacta- cystin-b-lactone, 60 min with 100 l M chloroquine. Cells were then exposed to the various toxins for the indicated times. Residual pro- tein synthesis was measured by incubating cells at 37 °C for 90 min in the presence of 1 lCi [ 35 S]-methionine in NaCl ⁄ P i . The ID 50 values obtained in the absence (–) or (+) presence of drugs are reported. Cell type Toxin exposures BFA –+ A Vero % RLU (n ¼ 6) 5 p M Ricin 30.4 ± 9.8 89.3 ± 7.1 9n M Saporin 19 ± 5.6 34 ± 12 HeLa ID 50 (6 h) Ricin 3.3 pM >1700pM RTA-KDEL 33.4 nM >1670nM Saporin 2100 nM 1940 nM Cell type Toxin exposures Proteasome inihibitors –+ B Vero % RLU (n ¼ 3) 5 p M Ricin 17.4 ± 3 6.8 ± 1 9n M Saporin 16.8 ± 4.8 20.5 ± 7.3 HeLa ID 50 Ricin-6K (4 h) 2140 pM 33.4 pM Ricin (18 h) 0.199 pM 0.0997 pM Saporin (18 h) 17.6 nM 5.3 nM Cell type Toxin exposures Chloroquine –+ C Vero % RLU (n ¼ 3) 10 p M DT 7.1 ± 0,9 60.5 ± 21.7 9n M Saporin 19.2 ± 4.9 21.1 ± 3.3 HeLa ID 50 (4 h) DT 0.143 nM > 3.17 nM Ricin 9.97 pM 8.30 pM Saporin 2640 nM 2820 nM R. Vago et al. Saporin trafficking in intoxicated mammalian cells FEBS Journal 272 (2005) 4983–4995 ª 2005 FEBS 4985 of intoxication. Neither the human leukemic cell line HSB-2 nor the Burkitt lymphoma Ramo cells showed any potentiation of cytotoxicity by addition of the KDEL sequence to the C-terminus of saporin (data not shown). An intriguing result was the decrease in cytotoxicity of SAPKDEL observed in U937 cells (Table 2). However, recombinant SAPAARL assayed as a control showed same ID 50 as SAPKDEL. We then tested if a targeted saporin chimera such as ATF–SAP, containing six disulfides, present in a kringle and the huPAR-binding growth factor domain [19,21,22] would need to partially unfold and ⁄ or undergo reduction prior to membrane dislo- cation. If these steps occurred in the ER, cytotox- icity could potentially be enhanced by ER-retrieval motifs. Figure 4 summarizes the secretory mutant chimeras that were constructed and expressed in Xenopus oocytes. When the terminal lysine residue of ATFSAPREDLK (here in bold) is removed by extra- cellular carboxypeptidase(s) normally present in cell culture medium [5], does the REDL sequence behave as an active KDEL-like motif. Therefore, this mutant chimera should be initially efficiently secreted by the protected oocytes and, as in the case of PEA, when exposed to the target cell, would be endo- cytically taken up and possibly retrieved to the ER. As a control, we have also expressed preATF– SAPKDEL. Synthetic mRNAs encoding preATF–SAP or the mutants were produced and in an in vitro translation assay, these COOH-extended mutants could inactivate reticulocyte lysate ribosomes (Fig. 4B), as shown for preATF–SAP [22], by blocking their own translation. Indeed, polypeptides translated from preATF–SAPwt Fig. 1. Intracellular distribution of saporin in intoxicated Vero cells. Vero cells were trea- ted with 100 lgÆmL )1 Cy-3-labeled saporin (A) or Cy-3-labeled ricin (B) for 4 h before methanol fixation and immunostaining using the antibodies indicated. The scale bar rep- resents 20 lm. Saporin trafficking in intoxicated mammalian cells R. Vago et al. 4986 FEBS Journal 272 (2005) 4983–4995 ª 2005 FEBS and either preATF–SAPKDEL or preATF–SAP- REDLK cRNAs, but not those translated from con- trol RNA (BMV, compare lanes 4 and 6), were seen only when translated in the presence of anti-saporin neutralizing immune Igs (Fig. 4B lanes 7, 10 and 13, respectively). Thus, as previously shown for SAPK- DEL, these C-terminal amino acid extensions to the ATF–saporin chimera did not affect the in vitro toxic- ity of the chimeras. In pulse-labeled, Ig-protected Xenopus oocytes (Fig. 5A), the newly synthesized polypeptides all showed the expected electrophoretic mobility, those of the mutants being decreased com- pared with the wild-type chimera. At the end of the 24 h chase period, most of the ATFSAPREDLK poly- peptides (lane 8) were, as expected, secreted into the culture medium, as was the wild-type polypeptide (lane 12) [22]. In contrast, KDEL mutant polypeptides were mostly retained within the oocyte (compare lanes 3 and 4). Fewer than 20% of the newly synthesized ATFSAPKDEL polypeptides were found in the oocyte medium after 24 h of chase. Western blot analysis of the 72 h oocyte incubation media with anti-(ATF krin- gle domain) (Fig. 5B) and anti-SAP sera (Fig. 5C, lower panel) showed that only the full-length poly- peptides were secreted by the protected oocytes, whereas blotting with a monoclonal anti-KDEL serum (Fig. 5C, upper panel) indicated that an intact KDEL sequence was still present in the corresponding KDEL- secreted chimera. The specific cytotoxicity of the chimeric proteins was evaluated using standard cell-killing experiments. U937 cells express both human uPAR and endocytic recep- tors belonging to the LDL-related receptor family (LRP) that are required for the efficient targeting and internalization of these toxic chimeras [19,33]. The cytotoxic activity of the seed-extracted saporin in U937 cells was almost three orders of magnitude lower than that of the huPAR-targeted chimera [19,33] (Fig. 6, compare ID 50 of SAP [35 nm] with that of ATF–SAPwt [0.04 nm]). However, both mutant chime- ras were slightly less active than the wild-type chimera with REDL- and KDEL-extended versions showing an ID 50 of 0.1 and 0.2 nm, respectively. This is consistent with the decrease in cytotoxicity of SAPKDEL com- pared with SAPwt observed in U937 monocytes (Table 2). Appending a KDEL sequence enhanced both RTA [12] and PEA cytotoxicity [29]. The finding that ER-retrieval sequences did not enhance the cytotoxici- ty of either saporin or the ATF–SAP chimera was, indeed, expected and confirms our initial observations that the intracellular transport of saporin bypasses the Golgi complex. Fig. 2. Saporin cytotoxicity is a direct result of ribosome modifica- tion. HeLa cells were treated with increasing concentrations of saporin for 18 h. To ensure that the N-glycosidase activity seen was entirely due to depurination of ribosomes during the saporin exposures for the cytotoxicity assay, ribosomes were isolated by denaturing all proteins upon lysis. After lysis of the cells, RNA was isolated and aniline treated before running on denaturing agarose gels. The arrow indicates the aniline band, which is diagnostic of N-glycosidase activity. The spike sample received the highest con- centration of saporin, added just prior to cell lysis, and a control sample received no saporin (–). A B Fig. 3. Schematic representation of the DNA constructs expressed in E. coli and purifed from bacteria lysates. (A) Mature saporin (SAPwt) (black and white bars) or mature saporin with a C-terminal KDEL (SAPKDEL) or AARL (single amino acid letter code) were expressed in BL21 (De3) pLys E. coli and recombinant toxins puri- fied to homogeneity; RIP: ribosome-inactivating catalytic domain. (B) SAPKDEL is immunoprecipitated by monoclonal anti-KDEL sera. Immunoprecipitates were recovered on protein G–Sepharose beads and polypeptides transferred on nitrocellulose were revealed with an antisaporin serum and detected by enhanced-chemiolumines- cence; H: heavy chains of the Igs. Molecular mass markers are shown in the right. The arrow points to the position of SAPKDEL (lane 3). Saporin wt or the mock (–) induced lysates gave no signal (lanes1 and 2). R. Vago et al. Saporin trafficking in intoxicated mammalian cells FEBS Journal 272 (2005) 4983–4995 ª 2005 FEBS 4987 Discussion The therapeutic use of saporin-based immunotoxins [30,31] prompted us to investigate whether saporin would follow the same route of entry into the cytosol as the related plant toxin ricin [10–12] or the bacterial toxin PEA [2,29]. This would imply that saporin-mediated cytotoxicity should be increased by introducing KDEL- like sequences at the C-terminus of this molecule. Cell-surface binding of saporin is mediated, at least in part, by members of the LDL-related family of receptors [18,19,32,33] and LRP-minus MEF cells show a 10-fold decrease in saporin sensitivity (our unpub- lished results). LRP mediates internalization of the ATF–saporin chimera through clathrin-coated pits [19,33] and the binding and internalization of another type I RIP, trichosanthin [34], and PEA [3,13]. Thus, saporin is able to use the same internalization receptor as PEA bacterial toxin. However, when Vero or HeLa cells were treated with BFA although Golgi disassem- bly clearly impaired ricin cytotoxicity, it did not signifi- cantly affect saporin-mediated toxicity. We therefore concluded that the Golgi complex is not a major intra- cellular compartment for productive trafficking of sap- orin. When we investigated the intracellular route of a human prourokinase–saporin TRITC conjugate [33], the fluorescence of the saporin chimera did not overlap either with a fluorescinated ricin holotoxin or with the Golgi marker NBD-ceramide. Toxins that use ERAD pathways [1,7–10,14], such as ricin, PEA [13] and chol- era toxin [1,10,14,35], must avoid proteasomal degrada- tion to exert their toxic action [15,35] and their paucity in lysine (but not in DT retrotranslocating from a dif- ferent compartment) [27,28] helps avoid ubiquitination and subsequent proteasome degradation [26]. Cholera toxin essentially avoids ubiquitination [35] and, in Table 2. Saporin with a KDEL C-terminal extension has similar in vitro and Vero cell-killing activity as the wild-type saporin. (A) Saporin RIP activities were compared using the cell-free system reticulocyte lysate (in vitro) or by intoxicating Vero cells. The con- centration inhibiting 50% of BMV RNA translation in vitro was measured (IC 50 ) in replicated samples and reported with the stand- ard deviations (SD). Vero cells were exposed for 4 h to serial log dilutions of each toxin before luciferase reporter transfection (Experimental Procedures). Relative light units (RLU) were quanti- fied in each sample in a luminometer and the dose of toxin that inhibits reporter expression by 50% over the untreated controls (ID 50 ) was calculated with the SEM of at least two independent experiments, each performed four times. Recombinant ricin A chain was used as a control. (B) Comparison of saporin wild-type [19,22], SAPKDEL and SAPAARL killing activities in promyelocytic human U937 cell was carried out essentially as described previously (Experimental Procedures and the legend to Fig. 6), following 48 h exposure to serial dilutions of the toxins and measuring the remain- ing protein synthesis with tritiated leucine incorporation. Mean ID 50 values are reported. Toxin In vitro IC 50 ±SD10 )12 M Vero ID 50 ± SEM 10 )9 M A SAPwt 25 ± 5 3.7 ± 2.3 SAPKDEL 25 ± 14 5 ± 3.5 RTAwt 180 ± 9 44 ± 12 RTAKDEL 150 ± 4 1.8 ± 0.9 Cell type Toxin ID 50 B U937 SAPwt 55 n M SAPKDEL 200 nM SAPAARL 200 nM A B Fig. 4. Neutralizing antisaporin Igs are needed for efficient in vitro translation of the wild-type and COOH-extended ATF–SAPorin chimeras. (A) Schematic representation of the DNA chimeric con- structs expressed in protected Xenopus oocytes. PreATF–SAPorin wild-type (preATF–SAPwt) was obtained by substituting the serine- protease domain of urokinase with the saporin RIP domain and pre- ATF–SAPorin with a C-terminal KDEL (preATF–SAPKDEL) or REDLK (preATF–SAPREDLK) sequence were obtained after introducing synthetic oligonucleotides (see Experimental procedures) SP: signal peptide, ATF: amino-terminal fragment for uPAR cell surface bind- ing. (B) preATF–SAPwt cRNA or those encoding the COOH- mutants (preATF–SAPKDEL or preATF–SAPREDLK) were translated in the presence of tritiated leucine in nuclease-treated rabbit reticu- locyte lysates, supplemented with goat antisaporin immune (i) or nonimmune (ni) Igs or NaCl ⁄ P i (–). BMV RNA was also translated in the same conditions (lanes 4–6), as control. At the end of the trans- lation period (1 h) equivalent amounts of lysates were subjected to a 15% polyacrylamide SDS ⁄ PAGE and fluorography. Saporin trafficking in intoxicated mammalian cells R. Vago et al. 4988 FEBS Journal 272 (2005) 4983–4995 ª 2005 FEBS agreement with this view, the addition of extra lysine residues at selected positions in RTA drastically reduced the cytotoxicity of the holotoxin without affecting its catalytic activity [25]. Saporin is a mono- meric protein whose three-dimensional structure can be superimposed on RTA, despite the fact that their amino acid identity is lower than 30% [36] and that 10% of the amino acids in saporin are lysine residues that also confer an extremely high pI (almost 10) and an unusual stability to this polypeptide [37]. Inhibition of the proteasomes, however, did not lead to a large increase in saporin cytotoxicity, suggesting that this toxin may not use ERAD pathway(s) or may be not subjected to an unfolding step prior to entry into the cytosol, as it has recently been postulated for FGF [38]. That saporin is able to reach the cytosolic compartment was confirmed, because isolated HeLa ribosomes were depurinated in a dose-dependent fashion. Thus, this toxin might well be able to escape through different intracellular compartment(s). Raising the intracellular pH of the endosomal compartment using chloroquine or bafilomycin A1 resulted, as expected, in complete protection from DT. There were, however, no substan- tial differences between the effects on saporin or ricin cytotoxicities. This lack of protection by chloroquine and bafilomycin A1 as well, indicates that whatever the translocation mechanism of saporin is, it is not low-pH dependent, as for DT and would differ also from that of FGF that was shown to be bafilomycin A1 sensitive [39]. Hence, saporin does not appear to possess A B C Fig. 5. (A) KDEL mutant polypeptides are retained by Xenopus oocytes whereas polypeptides carrying REDLK are efficiently secreted. Oocytes were coinjected with preATF–SAPwt cRNA (300 ngÆlL )1 ) or the same amount of synthetic cRNA encoding the COOH-mutants pre- ATF–SAP-KDEL or preATF–SAP-REDLK together with goat neutralizing antisaporin Igs (3.25 lgÆlL )1 ) to protect oocytes from autointoxi- cation. Control oocytes (not shown) were left uninjected. After overnight incubation at 19 °C, oocytes were labeled 2 h with S 35 Promix (0 h chase) and some oocytes were then further chased for 24 h. Equivalent amounts of oocyte lysates (o) and incubation media (m) were immunoprecipitated with rabbit antisaporin serum, and proteins analyzed by 15% polyacrylamide SDS ⁄ PAGE and fluorography. The arrow indicates intracellular polypeptide accumulated in the KDEL mutant. (B) Properly folded, full length polypeptides are secreted by the oocytes. Oocytes were injected as described in Fig. 5A and the unlabeled oocytes incubated at 19 °C for 72 h in the presence of 6% MBS-dialyzed fetal calf serum. Equivalent amounts of wild-type or mutant ATF–SAP polypeptides were subjected to a nonreducing 15% polyacrylamide SDS ⁄ PAGE, and the electroblotted polypeptides were immunodetected using anti-ATF conformational sera, followed by secondary HRP-goat anti-(mouse epitope) Igs and detection by enhanced-chemioluminescence. Conditioned media containing two glycosylated COOH-mutant ATF–SAP chimeras (our unpublished data) were also loaded (lanes 2 and 4), for comparison. Molecular mass markers (kDa) are shown on the right. (C) Secreted ATF–SAPorin (lane 2), ATF–SAP-KDEL (lane 3) or ATF–SAP-REDLK (lane 4) polypeptides were also detected using anti-KDEL sera or rabbit antisaporin. Microsomal membrane preparation (not shown) or SAPKDEL (asterisk) were used as positive controls. The positions of molecular mass markers (kDa) are indicated on the right. Control oocyte media are loaded in lane 1. R. Vago et al. Saporin trafficking in intoxicated mammalian cells FEBS Journal 272 (2005) 4983–4995 ª 2005 FEBS 4989 putative translocation domain(s) and the cytotoxic activity of the ATF–SAPorin chimera was even slightly increased both by chloroquine or bafilomycin A1 treat- ments, suggesting its passage though a putative proteo- lytic compartment [19]. The fact that we trace saporin passage through late endosomes, because we do see colocalization with a late endosomal marker, is also fully consistent with these previous data. COPI-independent paths do exist to reach the ER, as recently shown for Shiga-like toxin [3,40]. Disrup- tion of COPI retrograde transport or deletion of KDEL in the A2 chain of the cholera toxin could not abolish toxin delivery to the ER [41]. Interaction with the KDEL receptors may not be required to reach the ER, but the KDEL motif might function by retrieving from the Golgi any toxin that escapes by anterograde transport [42,43]. We therefore evaluated the exogen- ous toxicities in target cells, comparing recombinant saporin with SAPKDEL, and exploited a cytosolic immunization strategy [22] to extend this comparison to secreted saporin chimeric polypeptides, carrying sig- nals that confer ER retrieval along the endocytic route. Recombinant SAPKDEL was immunoprecipitated by a monoclonal anti-KDEL serum, indicating that its KDEL sequence remains fully accessible to this anti- body in solution and its RIP activity in reticulocyte lysates was almost superimposable with that of wild-type saporin. Nevertheless, unlike RTA, saporin cytotoxicity was not increased by the addition of a C-terminal KDEL. The KDEL sequence can substitute for the PEA C-terminus increasing PEA toxicity and that of chimeric PEA toxins ending with KDEL [29]. The PEA C-terminal sequence binds to KDEL recep- tors [2] and overexpression of this receptor makes cells more susceptible to the PEA [3]. The terminal lysine residue found in the natural PEA C-terminus is nor- mally removed by extracellular carboxypeptidase(s) present in cell culture medium [5] leaving a REDL sequence which behaves as an active KDEL-like sequence during internalization. We therefore expressed preATF–SAPREDLK mutant polypeptides and found they were, as expected, efficiently secreted by the oocytes, whereas when appended to ATF–sapo- rin the KDEL sequence was recognized by oocytes causing the KDEL-bearing chimera to accumulate in- tracellularly (Fig. 5). This reinforces our assumption that a KDEL sequence appended to the saporin mole- cule should behave as an effective signal if this poly- peptide is able to reach the Golgi complex. The Xenopus oocyte expression system was leaky, allowing recovery of some ATFSAPKDEL polypeptides from the conditioned medium. Blotting with anti-KDEL confirmed the presence of this C-terminal sequence in the secreted polypeptides. Secretion and cell-surface expression of chaperones [44] and proteins carrying KDEL has been already observed in different cell sys- tems, and ATFSAPKDEL secretion by the oocytes might not, therefore, be surprising. C-terminal exten- ded chimeras showed slightly lower activity against U937 cells. A similar difference in activity was also seen when comparing SAPKDEL and SAPAARL with saporin wt (Table 2). Extra C-terminal sequences might interfere with endocytosis, slightly decreasing the efficiency of internalization of KDEL ⁄ REDL sapo- rins in the human promyelocytic cells. However, a 20 amino acid COOH-extended mutant chimera showed an ID 50 of 0.07 nm in U937 cells, closer to that of the wild-type toxin (our unpublished results). Therefore, these data strongly support our assumption that sapo- rin and the derived chimeras do not travel through the Golgi complex to the ER after internalization. Ricin may also bypass the Golgi apparatus, which has been vesiculated by depletion of epsilon-COP, but still reaches the ER [45]. Our data do not completely exclude the possibility that the plant monomeric toxin saporin might also exploit the ER for its retrotranslo- cation. It has recently been postulated that some poly- peptides such as DHFR or GFP may be able to undergo ER dislocation without the need for an unfolding step [46,47]. Our data and the literature Fig. 6. Comparison of the cytotoxicities of ATF–SAPwt and KDEL ⁄ REDLK chimeras in U937 monocytes. Acid-washed cells were exposed 48 h at 37 °C to equivalent serial logarithmic dilu- tions of the secreted ATF–SAP chimeras, wild-type (filled squares) REDLK (middle, filled cones) or KDEL (filled triangles) and seed- extracted saporin (empty triangles). Cells were then pulse-labeled with L - [4,5- 3 H]leucine and radioactivity incorporation measured after harvesting cells onto glass fibre filters. Cytotoxicities were calcula- ted by measuring the dose of toxin that inhibits protein synthesis in treated cells by 50% (dashed line) and compared with untreated control cells exposed to equivalent dilutions of the conditioned medium of goat anti saporin injected oocytes. The dose–response curves are shown with standard deviations. x-axis: percent total leucine incorporation. Saporin trafficking in intoxicated mammalian cells R. Vago et al. 4990 FEBS Journal 272 (2005) 4983–4995 ª 2005 FEBS indicate the existence of multiple intracellular path- way(s) and delivery mechanisms to reach the cytosolic compartment. In addition to the great potential in anti-cancer therapies, saporin should be useful in strat- egies exploiting disarmed toxins as peptide carriers for MHC class I presentation [48–50]. Experimental procedures Cytotoxicity assays using HeLa cells For 4 or 6 h assays HeLa cells were seeded at 1.5 · 10 5 cellsÆmL )1 into 96-well plates and grown over- night at 37 °C. For 18 h assays cells were seeded at 2.5 · 10 5 cellsÆmL )1 into 96-well plates and grown at 37 °C for 8 h. Cells were incubated with 100 lL of media (Dul- becco’s modified Eagle’s medium [DMEM] supplemented with 5% fetal calf serum and 2 mm glutamine) containing increasing concentrations of native purified saporin (Sigma, St Louis, MO, USA) or native purified ricin, recombinant DT, recombinant ricin-6K [25], recombinant ricin A-chain- KDEL (RTA-KDEL) used as controls for the different drug treatments. After the appropriate time of incubation at 37 °C residual protein synthesis was measured by incuba- ting cells at 37 °C for 90 min in the presence of 1 lCi of [ 35 S]-methionine in 50 lL of NaCl ⁄ P i per well. Labeled pro- teins were precipitated by washing with 5% TCA followed by NaCl ⁄ P i and, after the addition of 200 lL of Optiphase ‘SuperMix’ scintillation fluid (Wallac, PerkinElmer-LAS, UK) per well, plates were counted in a MicroBeta 1450 Trilux counter (PerkinElmer-LAS, UK). Experiments using drug treatments were carried out by exactly the same method except that HeLa cells were pretreated for 15 min with 10 lm BFA, 60 min with 20 lm clasto-lactacystin- b-lactone, 60 min with 100 lm chloroquine or 30 min with 500 nm bafilomycin A1 prior to the addition of toxin dilu- tions. In all cases the appropriate drug was maintained at the same concentration in both the toxin dilutions and the labeling mix. RNA extraction and depurination Cells were grown to 80% confluence in 175 cm 2 flasks in DMEM supplemented with 5% fetal calf serum and 2 mm glutamine before incubating for 18 h with increasing con- centrations of saporin. Cells were removed from the plates by treating with 5 mm EDTA for 10 min at 37 °C before pelleting through 30 mL of media for 5 min at 500 g. Cell pellets were resuspended in 1 mL of Trizol (Invitrogen, Carlsbad, CA, USA) and passed through a needle (0.6 · 25 microlance) three times before pelleting at 12 000 g for 10 min at 4 °C. The supernatants were removed and incubated at room temperature for 5 min before adding 0.2 mL chloroform, vortexing briefly and spinning at 12 000 g for 2 min at room temperature. We added 0.5 mL of propan-2-ol to the aqueous layer and after incubation at room temperature for 15 min the samples were spun at 12 000 g for 15 min at 4 °C. The pellets were washed with 1 mL of 75% ethanol prior to vacuum drying and quantitation. Four micrograms of isolated RNA was treated with 20 lL of acetic aniline for 2 min at 60 °C, pre- cipitated using 0.1 vol. of 7 m ammonium acetate and 2.5 vol. of 100% ethanol and pelleted at 12 000 g for 30 min at 4 °C. Pellets were washed with 1 mL of 75% eth- anol prior to vacuum drying and the RNA was resuspend- ed in 20 lL of 60% formamide in 0.1 · TPE (3.6 mm Tris ⁄ HCl pH 8.0, 3 mm sodium dihydrogen phosphate, 0.1 mm EDTA) and electrophoresed on a denaturing form- amide gel (1.2% agarose, 50% formamide, 0.1 · TPE). Labeling with Cy3 Saporin or ricin was labeled with Cy3 using Cy3-reactive dye-pack. Briefly, saporin or ricin in 0.1 m sodium carbon- ate buffer (pH 8.5) was incubated with the dye for 1 h at room temperature and Cy3–saporin was separated from free dye on a PD-10 column (Amersham Pharmacia Biotech Italia, Milan, Italy) before concentrating in microcon cen- trifugal filters (Millipore-Amicon, Madison, WI, USA). A molar ratio between 1.7 and 2.2 mol of Cy3 per mole of saporin was incorporated. The cytotoxicity of the Cy3–sap- orin was assayed and was unchanged as compared to the native saporin, used for the labeling. Saporin uptake and intracellular immunofluorescence Green monkey kidney Vero cells were seeded at 5 · 10 5 cell- sÆmL )1 onto coverslips in 12-well plates and grown over- night at 37 °C in DMEM supplemented with 5% fetal calf serum and 2 mm glutamine. Cy-3-labeled saporin (100 lgÆmL )1 ) or ricin was added to the cells for 1 or 4 h before washing with NaCl ⁄ P i . Cells were fixed and permea- bilized in ice-cold methanol for 4 min prior to immuno- staining. Nonspecific antibody binding was blocked by incubating with 3% BSA in NaCl ⁄ P i for 30 min before incu- bating with the indicated primary antibody followed by the appropriate secondary antibody (Alexafluor, Invitrogen- Molecular Probes, Eugene, OR, USA) each for 1 h. Cover- slips were mounted and viewed by confocal microscopy (Leica Microsystems, Mannheim, Germany). Cytotoxicity experiments Vero cells were used to compare the cytotoxic activities of SAPwt and SAPKDEL to ricin A chain (RTAwt) or to RTAKDEL or to the ricin holotoxin and DT, used as con- trols for the different drug treatments. The cells were plated R. Vago et al. Saporin trafficking in intoxicated mammalian cells FEBS Journal 272 (2005) 4983–4995 ª 2005 FEBS 4991 into 48-well plate at a density of 1.6 · 10 4 cellsÆwell )1 and exposed to serial logarithmic dilutions of each protein toxin, in quadruplicate. At the end of the exposure period (4 or 30 h), cells were washed with NaCl ⁄ P i and transfected with a reporter plasmid encoding firefly cytosolic luciferase under a cytomegalovirus promoter and allowed to express luciferase over the following 18 h. Lysates were read in a luminometer following manufacturer’s instructions (Pro- mega, Milan, Italy). The luciferase activity was quantitated in each sample and results were expressed in relative light units (RLU), as a percentage of that seen in untreated cells. The RLUs per mg of total protein lysate served as an inter- nal control to assess efficiency of transfections. Results are referred to 100% luciferase expression in the untreated con- trol samples and the dose of toxin inhibiting luciferase activity by 50% relative to controls corresponds to the ID 50 . Vero cells are efficiently killed by ricin holotoxin and by DT [27]. In our assays, ricin showed an ID 50 between 1 and 2 pm in agreement with previous published data [8,12]. The ID 50s of RTA, RTAKDEL were compared with those of SAPwt and SAPKDEL. Kinetics of intoxication was analyzed for SAPwt and SAPKDEL toxins and 4 h intoxi- cation period was chosen for the comparison with RTA ⁄ RTAKDEL using the different drug treatments. BFA (Sigma-Aldrich, Milan, Italy), MG-132 proteasome inhib- itor (Calbiochem, San Diego, CA, USA) and chloroquine (Sigma) were first tested at different concentrations to min- imize the inhibition of luciferase expression caused by the drug itself. We observed a high toxicity, in particular when using BFA observing a dose-dependent inhibition of pro- tein synthesis. A final set of experiments in the presence or absence of BFA (0.5 lgÆmL )1 ), MG-132 (10 lm) or chlo- roquine (10 lm) with a fixed amount of toxins for a 4 h total exposure was performed. Each experiment was per- formed in quadruplicate samples and in several independent replicates, as indicated in the results section. Standard cytotoxicity assays were performed testing increasing concentrations of each recombinant toxin and the secreted ATF–SAP wild-type or COOH-mutant polypep- tides in U937 human promyelocytic leukemia cells and for recombinant saporins also in human T-cell leukemia cell line HSB-2 or in the Burkitt lymphoma cell line Ramos, treated as described in Flavell et al. [51]. U937 cells were plated in 96-well plates at a cell density of 2 · 10 4 cellsÆwell )1 and trea- ted as previously described [22]. Cells were typically incuba- ted 48 h at 37 °C in the presence of serial logarithmic dilutions (prepared in tissue culture medium) of the recom- binant polypeptides (ATF–SAP chimeras from the 72 h con- ditioned medium of protected oocytes, see below). At the end of the exposure period, the cells were washed with NaCl ⁄ P i , pulse-labeled for 4 h with 0.5 l CiÆ well )1 l-[4,5- 3 H] leucine (37 TBqÆmmol )1 , Amersham Pharmacia Biotech, Piscata- way, NJ) and total incorporation of radioactivity into pro- tein was measured by liquid scintillation counting after harvesting cells on glass fiber filters. Cytotoxicity was calcula- ted by measuring the dose of toxin inhibiting by 50% incor- poration of untreated cells (ID 50 ). At least two independent experiments were conducted, each in triplicate. Construction of COOH-mutant ATF–SAPorin and saporin expression plasmids Synthetic oligonucleotides were purchased from Genset. pBSpAS, a preATF–SAP-containing vector [22] was muta- genized, inserting a Aat1 ⁄ Stu1 site into the original stop codon. This was achieved by amplifiying ~ 500 bp HpaI– EcoRI DNA fragment with the Pfu thermostable-poly- merase (Stratagene, La Jolla, CA, USA) and the following oligonucleotides: forward Aat1: 5-GAGTTAACCGC CCTTTTCCCAGAGGCCACAA-3OH; (bold, HpaI sequence); reverse Aat1: 5-CGGAATTCGCCTCGTTTGA GGCCTTTGGTT-3OH; (bold, EcoRI sequence). This Aat1 ⁄ stop minus HpaI–EcoRI-restricted DNA fragment was substituted to wild-type ATF–SAP HpaI–EcoRI DNA in pBSpAS yielding the recipient vector pBSpAS- (stop-). Complementary synthetic oligonucleotides with Aat1- and EcoRI-compatible ends were synthesized which enco- ded an in frame KDEL or REDLK COOH amino acid sequence followed by a stop codon. Sense oligonucleotides were phosphorylated with T4-polynucleotide kinase [52] and subsequently annealed with each complementary oligo- nucleotide before ligation into Aat1–EcoRI-digested pBSpAS(stop-). DNA sequencing was performed using the Thermo sequenase (Amersham Pharmacia Biotech), follow- ing manufacturer’s instructions. The ApaI–NotI fragments from preATF–SAP and from each of the confirmed positive clones were purified and ligated into ApaI–NotI digested pSP64TA ⁄ N (courtesy of Giovanna Chimini, CNRS, Mar- seille, France) yielding, respectively, pSP64TA ⁄ N-pAS encoding the pATF–SAP wild-type or the pATF–SAP mutants, those carrying KDEL-like sequences termed pATF–SAPREDLK (REDLK) or pATF–SAPKDEL (KDEL) (Fig. 4). All the COOH-extended mutants also share three extra amino acids (Ala, Ser and Glu) introduced by this cloning strategy. The construct encoding SAP with a KDEL C-terminal extension was obtained by substituting in pet-11d-SAP-3 the BamHI–EcoRI fragment encoding saporin wild-type with an equivalent one derived from BamHI–EcoRI diges- tion of pATF–SAPKDEL, as in Fabbrini et al. [18], giving rise to pet-11d-SAPKDEL. As a control, recombinant SAP with an extended C-terminus encoding SEARRL was also obtained, by annealing complementary synthetic oligonucleo- tides to Aat1–EcoRI-opened pet-11d-SAPKDEL. Expression and purification of the recombinant proteins was performed essentially as described in Fabbrini et al. [18]. Recombinant SAPARRL polypeptides retain the same RIP activity in vitro as saporin wild-type (data not shown). Purified SAPKDEL polypeptides were specifically immunoprecipitated with Saporin trafficking in intoxicated mammalian cells R. Vago et al. 4992 FEBS Journal 272 (2005) 4983–4995 ª 2005 FEBS [...]... RIP activity of each toxin was evaluated by an in vitro inhibition translation assay, using BMV RNA as reporter, as described in Fabbrini et al [18], assaying serial dilutions made in NaCl ⁄ Pi of SAPwt, SAPKDEL, RTAwt, RTAKDEL or of each secretory chimera Data are expressed as the concentration inhibiting the 50% BMV translation (IC50) IC50s of the COOH-extended chimeras were comparable with that of. .. manuscript We are grateful to Serena Camerini (Dibit-HSR, Milan) for the amino acid sequencing of some recombinant protein, to Marco Colombatti for generous gift of reagents and to Lucia Monaco for precious suggestions and continuous support This research was supported by CNR, AIRC, COFIN, University of L’Aquila and Leukaemia Busters grants and work at Warwick was supported by the UK Biotechnology and. .. the secretory pathway J Cell Biol 140, 733–736 Wesche J, Rapak A & Olsnes S (1999) Dependence of ricin toxicity on translocation of the toxin A- chain from the endoplasmic reticulum to the cytosol J Biol Chem 274, 34443–34449 Wales R, Chaddock JA, Roberts LM & Lord JM (1992) Addition of an ER retention signal to the ricin A chain increases the cytotoxicity of the holotoxin Exp Cell Res 203, 1–4 Wales R,... 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