FASEB J. Thermo Fisher Scientific
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by FABBRINI, M. S.
Right arrow Articles by CERIOTTI, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by FABBRINI, M. S.
Right arrow Articles by CERIOTTI, A.
(The FASEB Journal. 2000;14:391-398.)
© 2000 FASEB

Cytosolic immunization allows the expression of preATF-saporin chimeric toxin in eukaryotic cells

M. SERENA FABBRINI1, DANIELA CARPANI, MARCO R. SORIA and ALDO CERIOTTI*

Department of Biological and Technological Research-Dibit, San Raffaele Scientific Institute, 20132 Milano, Italy; and
* Istituto Biosintesi Vegetali, Consiglio Nazionale delle Ricerche, 20133 Milano, Italy

1Correspondence: Dibit-Department of Biological and Technological Research, San Raffaele Scientific Institute, via Olgettina 58, 20132 Milano, Italia. E-mail: fabbrini.serena{at}hsr.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this work, we have devised an intracellular immunization strategy for the expression in high amounts of ATF-saporin, a targeted chimeric toxin constituted by the ATF receptor binding domain of human urokinase and the plant ribosome-inactivating protein saporin, which has been shown to be highly cytotoxic to target cells. This strategy may allow the production of highly toxic secretory proteins in eukaryotic cells, avoiding cell suicide caused by autointoxication. The procedure consists of equipping host cells with cytosolic neutralizing antibodies directed toward the toxic domain of the heterologous polypeptide. We show that this intracellular immunization is essential for the synthesis of correctly folded, biologically active ATF-SAP in the high amounts needed to investigate its in vivo anti-metastatic potential. Such a strategy should be generally useful for the production of toxic molecules of therapeutic value whose folding and maturation require transit through the eukaryotic secretory pathway. Fabbrini, M. S., Carpani, D., Soria, M. R., Ceriotti, A. Cytosolic immunization allows the expression of preATF-saporin chimeric toxin in eukaryotic cells.


Key Words: ribosome-inactivating proteins • urokinase receptor • urokinase amino-terminal fragment • intracellular immunization • anti-cancer therapy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PLANT AND BACTERIAL toxins have been widely used for producing tumor-directed chimeras in which a targeting moiety is linked to a catalytic, toxic domain (1) . Several kinds of such targeted toxic chimeric molecules have been generated, but in many cases coupling between the toxin and the cell binding domain has been obtained in the test tube, using cross-linking agents (2 3 4 5) . The large-scale production of these chimeras could be better achieved via expression of recombinant immunotoxins (6) or other fusion proteins in appropriate hosts.

Most targeting domains (such as growth factors, hormones, or antibody fragments) used for the construction of chimeric toxins derive from secretory proteins. Thus, recombinant fusion proteins should be ideally expressed in eukaryotic cells, where they can be directed to the endoplasmic reticulum (ER). In this compartment, newly synthesized polypeptides encounter the appropriate set of molecular chaperones and enzymes that assist polypeptide folding and catalyze various cotranslational and posttranslational modifications (7) . These modifications often are needed for the polypeptides to acquire the correct conformation and, hence, full biological activity.

Recently, we have produced in Escherichia coli a recombinant chimeric toxin (ATF-SAP) (8) that has potential as an anti-metastatic polypeptide and consists of the receptor binding domain of human urokinase (uPA) fused to saporin (SAP), a single-chain plant ribosome-inactivating protein (RIP) that specifically depurinates a universally conserved adenine residue of 28S/26S/25S ribosomal RNA (9) . The human uPA receptor (uPAR) is overexpressed in several metastatic tumor cells (10) and is a strong prognostic marker in some human tumors. This GPI-linked receptor focalizes uPA proteolytic activity, particularly at the leading edge of migrating cells, and is a potential target for anti-cancer therapies since it behaves as an activatable cell surface chemokine capable of triggering signaling in a variety of cell types (11) . Recently, it has been demonstrated that cooperation between uPA/uPAR and metalloproteinase MMP-9 is required for breaching of vascular wall for intravasation and consequently for metastasis spread (12) . uPAR binds in a species-specific fashion the uPA precursor (Pro-uPA), catalytically active uPA, and its derived amino-terminal fragment (ATF). In the ATF-SAP chimera, the saporin domain also mediates internalization of the chimeric toxin by binding to the low density, lipoprotein-related receptor protein (LRP) (13 , 8) . ATF-SAP is therefore efficiently endocytosed and highly cytotoxic toward target cells. After internalization, the RIP domain must translocate to the cytosol to depurinate 28S rRNA. This irreversibly blocks protein synthesis and leads to cell death.

When ATF-SAP was expressed in prokaryotic hosts, only a minor proportion of correctly folded, active protein was recovered in the soluble cytosolic fraction (8) . Since soluble, active saporin can be produced in E. coli (14) , this was presumably due to inefficient folding of the ATF domain. ATF contains six disulfide bonds essential for generating the complex and characteristic architecture of the kringle and growth factor-like subdomains and whose formation is required for receptor binding activity. Attempts to favor disulfide bond formation by targeting ATF-saporin to the periplasmic space were unsuccessful, and using a bacterial strain able to support disulfide bond formation did not result in better yields of correctly folded ATF-SAP (M. S. Fabbrini, unpublished results). On the other hand, the use of eukaryotic hosts for ATF-SAP expression would be hampered by cytotoxic effects against the host cells (15 , 16) .

In this work, we describe an intracellular immunization strategy that allows us to produce biologically active ATF-SAP in a eukaryotic cell. We show that the synthesis of a secretory version of ATF-SAP is highly toxic in Xenopus oocytes, but that the presence of anti-SAP neutralizing antibodies in the cytosol is sufficient to protect oocyte ribosomes from inactivation, still allowing the majority of the synthesized polypeptides to be efficiently secreted in a biologically active form. Cytosolic neutralizing antibodies would be indeed expected to interact with any toxic molecule mislocalized to the cytosol but not with the bulk of ATF-SAP polypeptides that enter the endomembrane system for secretion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of a preATF-SAP expression plasmid
Pro-uPA human cDNA (17) was amplified using Taq DNA polymerase (Cetus, Perkin Elmer, Norwalk, Conn.) with 5'-GCGGTCGACACCATGAGA-3' (forward) and 5'GGCGTCATGAGTTTTCCATCT-3' (reverse) oligonucleotides to generate a DNA fragment encoding the amino-terminal sequence of human Pro-uPA (including the signal peptide) and bearing a SalI restriction enzyme site (italic). This polymerase chain reaction fragment was purified, digested with SalI-ScaI, and ligated to a ScaI-EcoRI-purified DNA fragment deriving from pET-11D-ATF-SAP-3 expression plasmid, which encodes part of the ATF sequence (starting from the ScaI restriction site) fused to the entire mature saporin-3 amino acid sequence (8) . The resulting ligation product encoding full-length preATF-SAP DNA was first subcloned into pBluescript II SK- and sequenced before insertion as a blunt-ended, purified SalI-EcoRI fragment into BglII/blunted pSP64T vector (18) , thus generating pSP64TpAS.

In vitro transcription
pSP64TpAS, pSP6ßPHSLwt (encoding the plant storage protein phaseolin) (19) , and a SP6 transcription construct encoding human L-ferritin (20) (kindly provided by Gaetano Cairo, C.N.R., Milano) were linearized with the appropriate restriction enzymes and used as templates for the production of synthetic mRNAs (21) . In vitro transcription with SP6 RNA polymerase (Boehringer Mannheim, Mannheim, Germany) was performed in the presence of 0.5 mM m7G(5')ppp(5')G cap analog (Amersham Pharmacia Biotech, Little Chalfont, U.K.). mRNAs were resuspended in water to give a final concentration of 0.5–1 mg/ml and stored at -80°C. Ethidium bromide-stained mRNA was quantitated by visual comparison with appropriate standards in agarose/formaldehyde gels (22) .

Antibody preparations
A salt cut of a goat antiserum raised against native seed-extracted SAP was kindly provided by Doug A. Lappi (Advanced Targeting Systems, Del Mar, Calif.). A salt cut of a nonimmune goat serum was also prepared. Immunoglobulins (Igs) were purified from the salted-out sera using HiTrap protein G affinity columns (Amersham Pharmacia Biotech) following the manufacturer’s instructions. After elution, Igs were dialyzed against phosphate-buffered saline (PBS; 20 mM sodium phosphate buffer pH 7.4, 137 mM NaCl) and total protein concentration was determined with a Bio-Rad protein assay kit, using gamma globulin as standard.

In vitro translation assays
The neutralizing activity of goat anti-SAP immunoglobulins was assayed as follows: 50 ng of brome mosaic virus (BMV) RNA were translated in nuclease-treated rabbit reticulocyte lysate (Promega, Madison, Wis.) supplemented with L-[4,5-3H] leucine (4.4–7.0 TBq/mmol, Amersham Pharmacia Biotech) using 0.15 unit/µl RNasin (Promega) in 12.5 µl final samples together with either 1 µl goat nonimmune (13.8 mg/ml) or anti-SAP (13.3 mg/ml) Igs in the presence or absence of 400 pM native seed-extracted SAP. The same conditions were used to assess the effect of goat nonimmune or anti-SAP neutralizing Igs on the translation of 50 ng preATF-SAP mRNA. At the end of the translation period (1 h) the lysates were chilled on ice, brought to 0.1 mg/ml RNase A, and incubated at room temperature for 15 min. Samples were denatured in the presence of 6% sodium dodecyl sulfate (SDS) and 50 mM dithiothreitol and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and fluorography.

RIP activity of secretory ATF-SAP was assayed by measuring inhibition of BMV RNA translation in reticulocyte lysates (14) .

Oocyte preparation, microinjection, labeling, and homogenation
Manually dissected oocytes were prepared, maintained in modified Barths’ saline (MBS), pulse-labeled with L-[35S]methionine and L-[35S]cysteine (Pro-mix, 37 TBq/mmol Amersham Pharmacia Biotech), and homogenized as described (23) , except that the homogenation buffer (40 µl/oocyte) was supplemented with Complete (Boehringer Mannheim) mixture of protease inhibitors. For microinjections, a stock of ~13 mg/ml goat anti-SAP or nonimmune Igs in PBS/RNasin inhibitor (Promega; 1 unit/µl) was freshly prepared and carefully mixed with either water or the mRNA solution. Injections (~40 nl/oocyte) were performed using an IM-1 injector with timer (Narishige). For pulse-chase experiments, the medium (5 µl/oocyte) was collected at the end of the pulse-labeling period and the oocytes were washed with MBS. Some oocytes were immediately frozen and stored at -20°C whereas others were transferred to fresh medium containing 10 mM unlabeled methionine and cysteine and 6% MBS-dialyzed fetal calf serum (Life Technologies, Inc./BRL, Grand Island, N.Y.). In some experiments 10 µM monensin (Sigma, St. Louis, Mo.) was included in the chase medium (24) . The concentration of secretory ATF-SAP in the oocyte incubation medium was estimated by an enzyme-linked immunoassay (8) , using a biotinylated monoclonal antibody to human uPA (Monozyme, Denmark) and human recombinant Pro-uPA as standard.

Immunoprecipitation analysis and gel electrophoresis
An anti-SAP serum against the native, seed-extracted protein was raised in rabbits and precleared overnight at 4°C on a rocker using a 20–25 oocyte equivalent of a total extract of Xenopus laevis oocytes/ml of serum. The serum was cleared by centrifugation at 10,000 x g and the supernatant was used for immunoprecipitations. An anti-phaseolin rabbit serum was a kind gift from Roberto Bollini (C.N.R., Milano). Monoclonal L01 anti-ferritin-L chain was courtesy of Sonia Levi (DIBIT-HSR, Milano). The anti-kringle 5B4 monoclonal antibody (25) was a kind gift from Maria Luisa Nolli (Lepetit-Dow, Gerenzano, Italy). Immunoprecipitations were performed as described previously (26) , except that protein G-Sepharose was used to adsorb Mab 5B4 monoclonal antibody. Immunoprecipitated polypeptides were analyzed by SDS-PAGE (15% acrylamide, 0.075% bisacrylamide) using the system of Laemmli and Favre (27) . Gels were treated for fluorography as described by Bonner and Laskey (28) .

Cell-killing experiments
U937 human monocytic cells and murine fibroblast LB6 clone19 cells (stably expressing human uPAR; ref 29 ) were used to evaluate the cytotoxicity of secretory ATF-SAP. Briefly, exponentially growing U937 cells were cultured and acid-washed as previously described (30) and plated in 96-well plates (Costar, Cambridge, Mass.) at a cell density of 104 cells/well. The cells were incubated for 48 h at 37°C in the presence of serial log dilutions (prepared in tissue culture medium) of recombinant ATF-SAP (8) or of the 48 h conditioned medium of oocytes coinjected with preATF-SAP RNA and goat anti-SAP Igs. Equivalent dilutions of the conditioned medium of immune Ig-injected oocytes were also assayed as control. At the end of the exposure period, the cells were washed with PBS, pulse-labeled for 16 h with 0.5 µCi/well L-[4,5-3H]leucine (4.4–7.0 TBq/mmol, Amersham Pharmacia Biotech), and total incorporation of radioactivity into protein was measured by liquid scintillation counting after harvesting cells on glass fiber filters. Cytotoxicity was calculated by measuring the dose of toxin inhibiting by 50% the incorporation of untreated cells (ID50). Competition of the ATF-SAP-mediated cytotoxicity with human Pro-uPA was performed as follows: LB6 clone19 cells were cultured as described (29) and plated 16 h before the experiment on gelatin-coated 96-well plates at 104 cells in 80 µl. The cells were exposed for 2 h to 5 x 10-9 M secretory, recombinant ATF-SAP, or 10-7 M native seed-extracted saporin either in the absence or presence of 25 x 10-9 M recombinant Pro-uPA. Cells were then treated as described previously (8) .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Neutralizing anti-SAP antibodies allow efficient in vitro and in vivo synthesis of preATF-SAP
As a first step to develop an intracellular immunization strategy allowing expression of preATF-SAP in eukaryotic cells, we wanted to verify whether antibodies raised against saporin could suppress the toxicity of this protein in an in vitro eukaryotic translation system. Figure 1 shows that goat anti-SAP immunoglobulins were effective, allowing translation of BMV RNA even in the presence of a saporin concentration (400 pM) far above the IC50 (5–10 pM) of this protein (14) (Fig. 1 , lane 4). Goat nonimmune Igs were ineffective in protecting reticulocyte ribosomes from saporin activity (Fig. 1 , lane 3). The protective effect of the anti-SAP Ig preparation was also evident when mRNA coding for the ER-directed version of ATF-SAP carrying the Pro-uPA signal peptide (preATF-SAP; see Materials and Methods for details) was translated in the rabbit reticulocyte system. When RIP-encoding mRNA is translated in vitro, the newly synthesized RIP polypeptides enzymatically inactivate the ribosomes, eventually leading to a complete block of translation (31) . The presence of immune Igs in the translation reaction mixture allowed a substantial increase in the synthesis of preATF-SAP polypeptides (Fig. 1 , compare lanes 8 and 9), thus confirming that the Ig immune preparation could specifically neutralize the saporin-3 isoform (14) that was used for the construction of the preATF-SAP chimera.



View larger version (74K):
[in this window]
[in a new window]
  		Figure 1. In vitro neutralizing activity of goat anti-SAP Igs. BMV RNA or preATF-SAP (pAS) mRNA were translated in nuclease-treated rabbit reticulocyte lysates containing ~1 mg/ml anti-SAP immune (i) or nonimmune (ni) goat Igs either in the absence (-) or presence (+) of 400 pM native seed-extracted saporin (Sap-S). At the end of the translation period (1 h), equivalent amounts of lysates were subjected to SDS-PAGE and fluorography. Arrowhead indicates the position of preATF-SAP polypeptides. The position of molecular mass markers (kDa) is indicated on the left.

To test whether anti-SAP neutralizing antibodies could also protect ribosomes in a living eukaryotic cell, secretory preATF-SAP was expressed in Xenopus oocytes, coinjecting synthetic preATF-SAP mRNA with nonimmune or immune Ig preparations. Xenopus ribosomes have been shown to be sensitive to the action of the plant RIP ricin A chain (32) and are therefore expected to be sensitive to SAP inactivation. Oocytes were pulse-labeled, and immunoprecipitated polypeptides were analyzed by SDS-PAGE and fluorography. Figure 2 shows that very low levels of heterologous protein are synthesized when preATF-SAP mRNA is injected together with water or nonimmune Igs (Fig. 2 , lanes 4 and 5), while substantial amounts of protein are recovered after a 2 h pulse if preATF-SAP mRNA is coinjected with the goat anti-SAP neutralizing Igs (Fig. 2 , lane 6). This protective effect is specific for preATF-SAP-expressing oocytes. Indeed, coinjection of anti-SAP neutralizing antibodies does not induce per se an increase in the synthesis of an unrelated plant polypeptide (Fig. 2 , lanes 7–9).



View larger version (95K):
[in this window]
[in a new window]
  		Figure 2. Neutralizing anti-SAP Igs can protect Xenopus oocytes from self-intoxication. Oocytes were injected with preATF-SAP (ATF-SAP, 500 ng/µl) or phaseolin (PHSL, 100 ng/µl) synthetic mRNAs either alone or together with goat nonimmune (ni, 6.7 mg/ml) or immune Igs (i, 6.5 mg/ml). Control oocytes (lanes 1–3) were either left uninjected or were injected with nonimmune or immune Igs alone. After overnight incubation at 19°C, oocytes were pulse-labeled for 2 h, lysates corresponding to two oocytes were immunoprecipitated with anti-SAP and anti-PHSL rabbit sera, and polypeptides were analyzed by SDS-PAGE and fluorography. The position of molecular mass markers (kDa) is indicated on the left.

We next wanted to determine whether injection of the immune Igs was efficiently restoring the protein biosynthetic ability of the oocyte. For this purpose, mRNAs coding for both ER-targeted (phaseolin, PHSL) and cytosolic (human ferritin L chain) polypeptides were injected either alone or together with preATF-SAP mRNA (Fig. 3 ). The two control mRNAs were selected to take into account the possibility that membrane-bound and free polysomes recruit ribosomal subunits from two distinct populations in Xenopus oocytes (33) . In oocytes injected with the three mRNAs (lane 4), the synthesis of both PHSL and ferritin L chain was greatly reduced with respect to control oocytes injected with PHSL and ferritin L chain mRNAs alone (lane 5). This effect was due to saporin cytotoxicity and not to a competition for the protein synthesis machinery, since coinjection of the three mRNAs together with immune Igs (lane 2), but not with nonimmune Igs (lane 3), led to a complete recovery of PHSL and ferritin L chain expression. This was accompanied by a large increase in the synthesis of preATF-SAP. Thus, expression of ER-targeted preATF-SAP is highly toxic to Xenopus oocytes, impairing the synthesis of both secretory and nonsecretory proteins. We conclude that under the conditions used in our experiments, oocyte ribosomes can be efficiently protected by injecting cytosolic anti-SAP neutralizing antibodies.



View larger version (49K):
[in this window]
[in a new window]
  		Figure 3. Neutralizing anti-SAP Igs can fully restore the oocyte biosynthetic capability. RNAs coding for PHSL (33 ng/µl) and cytosolic ferritin L chain (133 ng/µl) were injected either alone or together with preATF-SAP (ATF-SAP) mRNA (250 ng/µl). These mRNAs were coinjected with either water (-), immune (i) (3,25 mg/ml), or nonimmune (ni) Igs (3,35 mg/ml). After overnight incubation, oocytes were pulse-labeled for 2 h and lysates were immunoprecipitated with an anti-ferritin L chain monoclonal antibody together with anti-PHSL and anti-SAP rabbit sera. Proteins were resolved by SDS-PAGE and visualized by fluorography. Dot: ferritin L chain; triangle ATF-SAP; asterisks: singly and fully glycosylated PHSL polypeptides. The position of molecular mass markers (kDa) is indicated on the left.

ATF-SAP is secreted by Xenopus oocytes
Both pro-urokinase (34) and saporin are naturally transported along the exocytic pathway (35 , 36) . We therefore investigated whether the chimera between these two proteins could be secreted by the oocytes. Figure 4 shows that this is indeed the case. Transport and processing of preATF-SAP were investigated in oocytes protected by neutralizing Igs and subjected to a pulse-labeling period, followed by a chase. Some oocytes were chased in the presence of monensin, a drug affecting Golgi trafficking in various cells types (37) , including Xenopus laevis oocytes (24) . The majority of the newly synthesized polypeptides (Fig. 4A , lane 1) were secreted into the medium during the chase period (Fig. 4A , lanes 4 and 6). However, a fraction of the newly-synthesized polypeptides was proteolytically processed to yield a 32 kDa product, which was fully retained within the oocyte (Fig. 4A , lanes 3 and 5). The conformational 5B4 monoclonal antibody recognizing the kringle subdomain of ATF could immunoprecipitate (although less efficiently than the rabbit anti-SAP serum) only the full-size ATF-SAP polypeptide. Conversely, it did not recognize the 32 kDa processing product, indicating this should correspond to the saporin moiety of the chimera (Fig. 4B ).



View larger version (40K):
[in this window]
[in a new window]
  		Figure 4. Intracellular processing and secretion of ATF-SAP polypeptides. A) oocytes coinjected with preATF-SAP mRNA (250 ng/µl) and goat anti-SAP Igs (6.5 mg/ml) were pulse-labeled for 2 h (0 h chase) and further chased for 24 or 48 h. Monensin (10 µM) was included in the chase medium of some of the oocytes subjected to a 24 h chase. At the end of the pulse and chase period, the media were collected and the oocytes washed with MBS. Equivalent amounts of oocyte lysates (o) and incubation media (m) were immunoprecipitated with rabbit anti-SAP serum and proteins were analyzed by SDS-PAGE and fluorography. B) Oocytes were injected preATF-SAP mRNA (250 ng/µl) and nonimmune Igs (6.7 mg/ml). After overnight incubation, oocytes were pulse-labeled for 2 h and then chased for 24 or 48 h. Identical aliquots of the oocyte lysates were immunoprecipitated with rabbit anti-SAP serum (lanes 1–2) or with Mab 5B4, which recognizes the human ATF kringle domain (lanes 3, 4). Proteins were separated by SDS-PAGE and visualized by fluorography. The arrows indicate the intracellular 32 kDa processing product(s). The position of molecular mass markers (kDa) is indicated on the left.

Both secretion and processing of preATF-SAP polypeptides are sensitive to monensin treatment (Fig. 4A , lanes 7 and 8), indicating that ATF-SAP secretion is Golgi mediated and suggesting that some of the preATF-SAP polypeptides were diverted from exocytosis for delivery to a hydrolytic post-Golgi compartment. By subcellular fractionation we found that these polypeptides are indeed membrane surrounded (M. S. Fabbrini, unpublished observations).

Using a specific immunoassay (8) , we evaluated that each oocyte coinjected with preATF-SAP mRNA and neutralizing Igs secreted into the medium up to 0,2 µg of recombinant protein during the first 64 h after injection. The cell-free RIP activity of secretory ATF-SAP was evaluated in reticulocyte lysates and its IC50 was ~15 pM, similar to that of native SAP (14) .

Secretory ATF-SAP is specifically cytotoxic to target cells expressing human uPAR and LRP
The secretory behavior of ATF-SAP raised the possibility that the observed cytotoxic effect of preATF-SAP expression could be due to a reuptake of the toxic polypeptide from the oocyte incubation medium. However, when uninjected oocytes were exposed to the 48 h conditioned medium of oocytes coinjected with preATF-SAP mRNA and immune Igs, no inhibition of incorporation of radiolabeled amino acids was observed (data not shown).

The uPA-SAP/Pro-uPA-SAP chemical conjugates and the ATF-SAP recombinant chimera produced in E. coli have previously been shown to intoxicate human U937 monocytic cells, which express both the human uPAR and LRP (38 , 8) . To evaluate the specific cell killing activity of secretory ATF-SAP, human U937 monocytic cells were exposed for 48 h either to the chimera secreted by Xenopus oocytes or (for comparison) to ATF-SAP purified from E. coli (Fig. 5A ). Cells were then pulse labeled with [3H]leucine and incorporation into total protein was measured. Both recombinant polypeptides showed an ID50 of ~10-10 M, indicating that secretory ATF-SAP has the expected specific biological activity (8) .



View larger version (28K):
[in this window]
[in a new window]
  		Figure 5. Oocyte-synthesized ATF-SAP is specifically cytotoxic to target cells. A) The cytotoxicities of recombinant ATF-SAP (filled diamond), of the medium conditioned by immune Ig-injected oocytes (filled circle), and of oocytes secreting ATF-SAP (empty square) were compared on U937 cells. Data are expressed as percent incorporation of the untreated cells and represent means and SE of two experiments, each performed in quadruplicate. Upper x axis: oocyte media fold dilution. Lower x axis concentration of oocyte and E. coli-synthesized ATF-SAP. B) Specificity of ATF-SAP binding. Human uPAR expressing LB6 clone 19 cells were exposed 2 h to 5 x 10-9 M recombinant (black), secretory (hatched) ATF-SAP, or 10-7 M native saporin (empty) either in the absence (-) or presence (+) of 25 x 10-9 M human pro-uPA. At the end of the exposure, cells were acid-washed, incubated 16 h at 37°C, then pulse-labeled 4 h with tritiated leucine. Data are expressed as percent protein synthesis of the untreated cells and represent means and SE of two experiments, each performed in quadruplicate.

Human recombinant Pro-uPA has previously been shown to compete ATF-SAP cytotoxicity on LB6 clone19 murine cells, which are stably transfected with the human urokinase receptor gene and express high amounts of the heterologous receptor (29) . Pro-uPA is also able to bind to LRP, but has a much greater affinity for the human uPAR and therefore can be used to discriminate between the two receptors (39) . To investigate whether binding to human uPAR is indeed mediating the uptake of secretory ATF-SAP, LB6 clone 19 murine cells were exposed for 2 h to E. coli-synthesized or secretory ATF-SAP, either in the absence or presence of human pro-uPA (Fig. 5B ). The effect of human Pro-uPA on the cytotoxicity of native saporin (which is endocytosed via LRP; see ref 13 ) was also evaluated. Pro-uPA competed to a similar extent the cytotoxicity of both oocyte and E. coli-synthesized ATF-SAP, but was unable to affect SAP-mediated cytotoxicity. The level of recovery in the presence of a fivefold molar excess of Pro-uPA is fully consistent with previously reported data on Pro-uPA competition of ATF-SAP-mediated cytotoxicity (8) .

We conclude that ATF-SAP secreted by Ig-protected oocytes contains functional human ATF and saporin domains and is endowed with the expected biological activity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this work, we propose an intracellular immunization strategy for the eukaryotic expression of secretory ATF-saporin. The ability to protect eukaryotic cells from autointoxication should allow to overcome some of the problems encountered when producing chimeras whose folding is strictly dependent on the presence of an appropriate intracellular environment or of cotranslational/posttranslational modifications not supported by bacterial hosts. In addition, novel approaches indicate that secretory chimeric toxins can potentiate the anti-tumor activity of lymphokine-activated killer cells (40) . Expression of secretory toxic polypeptides has been reported for fusions between single-chain antibodies and PE40, a truncated form of Pseudomonas aeruginosa exotoxin A (PE) (40 , 41) . However, fusions to PE presumably need to be proteolytically activated along the endocytic pathway before becoming translocation competent, and thus cytotoxic (6) . This peculiarity may be crucial for the host-safe expression of these particular fusions. Another fusion protein containing diphtheria toxin (DT) catalytic and membrane translocation domains fused to human granulocyte-macrophage colony stimulation factor (GMCSF) was successfully produced in the cytosol of baculovirus infected insect cells (42) . DT is also normally synthesized as a precursor, and only the free catalytic A fragment appears to be enzymatically active (43) . Presumably, the cytosolically synthesized DT-GMCSF represents an inactive, and thus nontoxic, precursor.

The cytotoxic effect we observed in oocytes expressing preATF-SAP contrasts with results obtained with PE-40 and DT-based chimeras. However, in the case of the plant single-chain saporin, one major difference is that presumably no activation step to become catalytically active is required. Expression of cytosolic saporin and even of a preprosaporin precursor polypeptide was already extremely cytotoxic to the Xenopus oocytes (M. S. Fabbrini, unpublished observations). In principle, cotranslational segregation in the ER of the preATF-SAP nascent polypeptides could be expected to protect cytosolic ribosomes from saporin inactivation. However, even when targeted to this compartment, RIPs are still toxic to heterologous cells, either because a small amount of the toxin is initially mislocalized in the cytosol or because RIPs are able to retrotranslocate out of the ER back into the cytosol (44 , 16) .

After internalization, the plant RIP ricin travels in a retrograde fashion along the exocytic pathway, finally reaching the ER where retrotranslocation of the catalytic A chain may occur through the same channel that mediates cotranslational insertion of secretory proteins into this compartment (44 , 45) . The actual site of retrotranslocation of SAP or SAP-containing conjugates has not been yet clarified. However, it might be possible that during secretion, secretory ATF-SAP crosses the same compartment(s) from which the endocytosed protein translocates to the cytosol when exogenously supplied to target cells. Although it still remains to be established how endogenously expressed preATF-SAP reaches the cytosol, our results indicate that the problem of self-intoxication can be solved by providing the host cell with the appropriate neutralizing activity.

Eukaryotic expression systems present some distinctive advantages with respect to the prokaryotic ones. Solubilization and refolding of the bacterial recombinant product are often required, and contamination with bacterial endotoxin or other undesired products is a common concern (42) . For example, ATF-SAP purified form E. coli lysates was heavily contaminated by chymotryptic-like, saporin-containing cleavage products similar to those retained in the oocytes (8) . Conversely, notwithstanding the presence of intracellular processing product(s) (see Fig. 4A, B ), only full-sized ATF-SAP polypeptides are secreted in the incubation medium. The strategy described in this paper was devised to solve the folding problems encountered during the E. coli expression of ATF-saporin, with the final goal of producing it in a biologically active form in high amounts and easily purifiable. Although Xenopus oocytes are a system that allow us to produce sufficient amounts of toxic chimera to investigate the anti-cancer potential of this recombinant molecule in tumor models in animals, cell lines stably expressing the anti-toxin neutralizing activity will be required for a large-scale production. Several studies have shown that antibody fragments maintain biological activity when cytosolically expressed in various eukaryotic cells (46 , 47) . These results indicate that constitutive expression of neutralizing anti-RIP antibody fragments in eukaryotic cells is a readily achievable goal. It should be noted that only a minor fraction of the Igs microinjected into the oocytes would be constituted by anti-SAP antibodies. In addition, only some of these Igs are expected to possess neutralizing activity. Thus, the actual concentration of neutralizing antibodies required to control self-intoxication might be quite low. Being the vast majority of ATF-SAP polypeptides transported along the secretory pathway, the neutralizing Igs only have to take care of the minute fraction of endogenously synthesized polypeptides that escapes segregation into the endomembrane system.

The intracellular immunization strategy described should be generally applicable, and so several other hybrid toxic molecules could be developed to be used for different pharmacological or cellular targets. The appropriate neutralizing activities might be selected after the screening of phage-displayed, single-chain antibody libraries. Stable cell line(s) can then be used as ‘immunized’ host cells for production of the hybrid molecule(s). Thus, generation of potentiated killer cell lines together with the production of bulk amounts of secretory chimeras for anti-cancer preclinical and clinical studies or for saporin-mediated immunolesioning (4) are some of the potential applications that could take advantage of this intracellular immunization strategy.


   ACKNOWLEDGMENTS
 
This work is dedicated to the memory of G. Nitti. We thank L. Benatti, F. Blasi, N. Borgese, J. M. Lord, L. Monaco, and E. Pedrazzini for critical reading of the manuscript, L. Spanò for useful suggestions, and R. Bollini, G. Cairo, U. Cavallaro, D. A. Lappi, S. Levi, M. L. Nolli, M. Mazzanti, and S. Toma for kindly providing reagents. Supported by P. F. Biotecnologie and ACRO of the Consiglio Nazionale delle Ricerche.


   FOOTNOTES
 
Received for publication January 29, 1999. Revised for publication June 24, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Fitzgerald, D. (1996) Why toxins! Semin. Cancer Biol 7,87-95
  2. Pai, L. H., Wittes, R., Setser, A., Willingham, M. C., Pastan, I. (1996) Treatment of advanced solid tumors with immunotoxin LMB-1: an antibody linked to Pseudomonas exotoxin. Nature Med 2,350-353[Medline]
  3. Laske, D. W., Youle, R. J., Oldfield, E. H. (1997) Tumor regression with regional distribution of the targeted toxin TF-CRM107 in patients with malignant brain tumors. Nature Med 3,1362-1368[Medline]
  4. Mantyh, P. W., Rogers, S. D., Honore, P., Allen, B. J., Ghilardi, J. R., Li, J., Daughters, R. S., Lappi, D. A., Wiley, R. G., Simone, D. A. (1997) Inhibition of hyperalgesia by ablation of lamina I spinal neurons expressing the substance P receptor. Science 278,275-279[Abstract/Free Full Text]
  5. Flavell, D. J. (1998) Saporin immunotoxins. Curr. Top. Microbiol. Immunol. 234,57-61[Medline]
  6. Pastan, I. (1997) Targeted therapy of cancer with recombinant immunotoxins. Biochim. Biophys. Acta 1333,C1-C6[Medline]
  7. Leitzgen, K., Haas, I. G. (1998) Protein maturation in the endoplasmic reticulum. Chemtracts-Biochem. Mol. Biol. 11,423-445
  8. Fabbrini, M. S., Carpani, D., Bello-Rivero, I., Soria, M. R. (1997) The amino-terminal fragment of human urokinase directs a recombinant chimeric toxin to target cells: internalization is toxin mediated. FASEB J 11,1169-1176[Abstract]
  9. Endo, Y., Tsurugi, K., Lambert, J. M. (1988) The site of action of six different ribosome-inactivating proteins from plants on eukaryotic ribosomes: the RNA N-glycosidase activity of the proteins. Biochem. Biophys. Res. Commun. 150,1032-1036[Medline]
  10. Andreasen, P. A., Kjøller, L., Christensen, L., Duffy, M. J. (1997) The urokinase-type plasminogen activator system in cancer metastasis: a review. Int. J. Cancer 72,1-22[Medline]
  11. Blasi, F., Stoppelli, M. P. (1998) Proteases and cancer invasion: from belief to certainty. Biochim. Biophys. Acta 1423,R35-R44
  12. Kim, J., Yu, K., Ossowski, L. (1998) Requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay. Cell 94,353-362[Medline]
  13. Cavallaro, U., Nykjær, A., Nielsen, M., Soria, M. R. (1995) {alpha}2-Macroglobulin receptor mediates binding and cytotoxicity of plant ribosome-inactivating proteins. Eur. J. Biochem. 232,165-171[Medline]
  14. Fabbrini, M. S., Rappocciolo, E., Carpani, D., Solinas, M., Valsasina, B., Breme, U., Cavallaro, U., Nykjaer, A., Rovida, E., Legname, G., Soria, M. R. (1997) Characterization of a saporin isoform with lower ribosome-inhibiting activity. Biochem. J. 322,719-727
  15. Hur, Y., Hwang, D.-J., Zoubenko, O., Coetzer, C., Uckun, F. M., Tumer, N. E. (1995) Isolation and characterization of pokeweed antiviral protein mutations in Saccharomices cerevisiae: identification of residues important for toxicity. Proc. Natl. Acad. Sci. USA 92,8448-8452[Abstract/Free Full Text]
  16. Frigerio, L., Vitale, A., Lord, J. M., Ceriotti, A., Roberts, L. M. (1998) Free ricin A chain, proricin and native toxin have different cellular fates when expressed in tobacco protoplasts. J. Biol. Chem. 273,14194-14199[Abstract/Free Full Text]
  17. Brandazza, A., Lee, E., Ferrera, M., Tillman, U., Sarmientos, P., Westphal, H. (1991) Use of the urokinase-type plasminogen activator gene as a general tool to monitor expression in transgenic animals: study of the tissue-specificity of the murine whey acidic protein expression signals. J. Biotechnol. 20,201-212[Medline]
  18. Krieg, P. A., Melton, D. A. (1984) Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res 12,7057-7070[Abstract/Free Full Text]
  19. Ceriotti, A., Pedrazzini, E., Fabbrini, M. S., Zoppè, M., Bollini, R., Vitale, A. (1991) Expression of wild-type and mutated vacuolar storage protein phaseolin in Xenopus oocytes reveals relationships between assembly and intracellular transport. Eur. J. Biochem. 202,959-968[Medline]
  20. Santoro, C., Marone, M., Ferrone, M., Costanzo, M., Colombo, M., Minganti, C., Cortese, R., Silengo, L. (1986) Cloning of the gene coding for human L apoferritin. Nucleic Acids Res 14,2863-2876[Abstract/Free Full Text]
  21. Ceriotti, A., Pedrazzini, E., de Silvetris, M., Vitale, A. (1995) Galbraith, D. W. Bourque, D. P. Bohnert, H. J. eds. Methods in Cell Biology Vol. 50B, chap. 21,295-308 Academic Press San Diego. [Medline]
  22. Sambrook, T., Fritsch, E. F., Maniatis, T. (1989) Nolan, C. eds. Molecular Cloning. A Laboratory Manual Cold Spring Harbor Laboratory Press Cold Spring Harbor, N.Y..
  23. Ceriotti, A., Colman, A. (1995) mRNA Translation in Xenopus Oocytes. Tymms, M. J. eds. Methods in Molecular Biology Vol. 37,151-178 Humana Press Totowa, New Jersey.
  24. Fabbrini, M. S., Vitale, A., Pedrazzini, E., Nitti, G., Zamai, M., Tamburin, M., Caiolfa, V. R., Patrono, C., Benatti, L. (1993) In vivo expression of mutant preproendothelins: hierarchy of processing events but no strict requirement of Trp-Val at the processing site. Proc. Natl. Acad. Sci. USA 90,3923-3927[Abstract/Free Full Text]
  25. Corti, A., Sarubbi, E., Nolli, M. L., Zanni, A., Galimberti, M., Parenti, F., Cassani, G. (1989) Epitope mapping of the anti-urokinase monoclonal antibody 5B4 by isolated domains of urokinase. Thromb. Haemost. 62,934-939[Medline]
  26. Galili, G., Altschuler, Y., Ceriotti, A. (1995) Galbraith, D. P. B. Bourque, D. P. ohnert, H. J. eds. Methods in Cell Biology Vol. 50B, chap. 36,497-517 Academic Press San Diego. [Medline]
  27. Laemmli, U. K., Favre, M. (1973) Maturation of the head of bacteriophage T4. I. DNA packaging events. J. Mol. Biol. 80,575-599[Medline]
  28. Bonner, W. M., Laskey, R. A. (1974) A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46,83-88[Medline]
  29. Roldan, A. L., Cubellis, M. V., Masucci, M. T., Behrendt, N., Lund, L. R., Dano, K., Appella, E., Blasi, F. (1990) Cloning and expression of the receptor for human urokinase plasminogen activator; a central molecule in cell surface, plasmin dependent proteolysis. EMBO J 9,467-474[Medline]
  30. Olson, D., Pollanen, J., Hoyer-Hansen, G., Ronne, E., Sakaguchi, K., Wun, T.-C., Appella, E., Dano, K., Blasi, F. (1992) Internalization of the urokinase-plasminogen activator inhibitor type-1 complex is mediated by the urokinase Receptor. J. Biol. Chem. 267,9129-9133[Abstract/Free Full Text]
  31. May, J. M., Hartley, M. R., Roberts, L. M., Krieg, P. A., Osborn, R. W., Lord, J. M. (1989) Ribosome inactivation by ricin A chain: a sensitive method to asses the activity of wild-type and mutant polypeptides. EMBO J 8,301-308[Medline]
  32. Saxena, S. K., O’Brien, A. D., Ackerman, E. J. (1989) Shiga toxin, Shiga-like toxin II variant, and ricin are all single-site RNA N-glycosidases of 28S RNA when microinjected into Xenopus oocytes. J. Biol. Chem. 264,596-601[Abstract/Free Full Text]
  33. Richter, J. D., Smith, L. D. (1981) Differential capacity for translation and lack of competition between mRNAs that segregate to free and membrane bound polysomes. Cell 27,183-191[Medline]
  34. Vassalli, J. D., Dayer, J. M., Wohlwend, A., Belin, D. (1984) Concomitant secretion of prourokinase and of plasminogen activator-specific inhibitor by cultured human monocytes-macrophages. J. Exp. Med. 159,1653-1668[Abstract/Free Full Text]
  35. Benatti, L., Saccardo, M. B., Dani, M., Nitti, G., Sassano, M., Lorenzetti, R., Lappi, D. A., Soria, M. (1989) Nucleotide sequence of cDNA coding for saporin-6, a type-1 ribosome-inactivating protein from Saponaria officinalis. Eur. J. Biochem. 183,465-470[Medline]
  36. Carzaniga, R., Sinclair, L., Fordhamskelton, A. P., Harris, N., Croy, R. R. D. (1994) Cellular and subcellular distribution of saporins, type-1 ribosome-inactivating proteins, in soapwort (Saponaria officinalis L.). Planta 194,461-470
  37. Mollenhauer, H. H., Morre, D. J., Rowe, L. D. (1990) Alteration of intracellular traffic by monensin; mechanism, specificity and relationship to toxicity. Biochim. Biophys. Acta 7,225-246
  38. Cavallaro, U., del Vecchio, A., Lappi, D. A., Soria, M. R. (1993) A conjugate between human urokinase and saporin, a type I ribosome-inactivating protein is selectively cytotoxic in urokinase receptor-expressing cells. J. Biol. Chem. 268,23186-23190[Abstract/Free Full Text]
  39. Nykjaer, A., Kjøller, L., Cohen, R. L., Lawrence, D. A., Garni-Wagner, B. A., Todd, R. F., van Zonneveld, A. J., Gliemann, J., Andreasen, P. A. (1994) Regions involved in binding of uPA inhibitor complex and pro-uPA to LRP. Evidence that the uPAR protects pro-uPA against binding to the endocytic receptor. J. Biol. Chem. 269,25668-25676[Abstract/Free Full Text]
  40. Chen, S.-Y., Yang, A.-G., Chen, J.-D., Kute, T., King, C. R., Collier, J., Cong, Y., Yao, C., Huang, X. F. (1997) Potent antitumor activity of a new class of tumor-specific killer cells. Nature (London) 385,78-80[Medline]
  41. Yang, A. G., Chen, S.-Y. (1997) A new class of antigen-specific killer cells. Nature Biotechnol 15,46-51[Medline]
  42. Williams, M. D., Rostovtsev, A., Narla, R. K., Uckun, F. M. (1998) Production of recombinant DTctGMCSF fusion toxin in a baculovirus expression vector system for biotherapy of GMCSF-receptor positive hematologic malignancies. Prot. Express. Purif. 13,210-221[Medline]
  43. Pappenheimer, A. M. (1977) Diphtheria toxin. Annu. Rev. Biochem. 46,69-94[Medline]
  44. Rapak, A., Pal, O. F., Olsnes, S. (1997) Retrograde transport of mutant ricin to the endoplasmic reticulum with subsequent translocation to cytosol. Proc. Natl. Acad. Sci. USA 94,3783-3788[Abstract/Free Full Text]
  45. Lord, J., Roberts, L. (1998) Toxin entry—retrograde transport through the secretory pathway. J. Cell Biol. 140,733-736[Free Full Text]
  46. Tavladoraki, P., Benvenuto, E., Trinca, S., De Martinis, D., Cattaneo, A., Galeffi, P. (1993) Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack. Nature (London) 366,469-472[Medline]
  47. Biocca, S., Pierandrei-Amaldi, P., Campioni, N., Cattaneo, A. (1994) Intracellular immunization with cytosolic recombinant antibodies. Biotechnology 12,396-399[Medline]



This article has been cited by other articles:


Home page
 FASEB J.Home page
R. IPPOLITI, E. LENDARO, P. A. BENEDETTI, M. R. TORRISI, F. BELLEUDI, D. CARPANI, M. R. SORIA, and M. S. FABBRINI
Endocytosis of a chimera between human pro-urokinase and the plant toxin saporin: an unusual internalization mechanism
FASEB J, July 1, 2000; 14(10): 1335 - 1344.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by FABBRINI, M. S.
Right arrow Articles by CERIOTTI, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by FABBRINI, M. S.
Right arrow Articles by CERIOTTI, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS