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Susceptibility of insect cells and ribosomes to ricin
Comp. Biochem. Physiol. Vol. 96B,No. 3, pp. 543-548, 1990
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SUSCEPTIBILITY OF INSECT CELLS A N D RIBOSOMES TO RICIN J. E. MARUNIAK,*S. E. FIESLERt and P. M. McGuIREt *Department of Entomology and tDepartment of Biochemistry and Molecular Biology, University of Florida, 0711 1FAS, Gainesville, FL 32611-0711, USA (Received 5 December 1989)
Abstract--1. The sensitivity of insect cells to whole ricin and its catalytic subunit has been examined. Evidence is presented that although cells from Trichoplusia ni and Spodopterafrugiperda are resistant to ricin, cell-free protein synthesis extracts are inhibited by ricin A chain and that the isolated insect ribosomes are sensitive to the N-glycosidase depurination activity of the A chain. 2. These results are consistent with the model that, as with other sensitive eukaroytic ribosomes, insect ribosomes appear to have a consensus sequence in the ribosomal RNA on the surface of the ribosome which is sensitive to this N-glycosidase.
INTRODUCTION Ricin is a cytotoxic glycoprotein produced in the seeds of the castorbean plant, Ricinus communis (Olsnes and Pihl, 1976; 1982b). It is one of a large number of plant-derived proteins which inhibit eukaroytic protein synthesis (Gasperi-Campagni et al., 1985). Some, including ricin, abrin, and modeccin, are extremely toxic to animals and to cells grown in culture. Others, among them pokeweed antiviral protein (PAP) and gelonin, are not very toxic to animals, but inhibit protein synthesis in cell-free systems. Although the ricin A chain gene has been expressed in bacteria, the recombinant product is only marginally soluble, probably largely because it lacks carbohydrate. Since there is abundant evidence for the glycosylation of heteroiogous recombinant products in the baculovirus-expression system, subcloning into this vector could greatly improve the ability to manipulate the ricin A chain gene. The insect baculovirus expression system has been publicised as a "super-vector" for the production of recombinant proteins (Wright, 1986). A recent review (Luckow and Summers, 1988) described the expression of a diverse collection of foreign genes from bacteria, viruses, plants, and humans. The expressed polypeptides can be antigenic, immunogenic, catalytic, enzymatic, secreted, glycosylated or phosphorylated. The mol. wts of the polypeptides range from 160,000 to 15,500. An impressive feature of the baculovirus expression vectors is the amount of polypeptide produced. Late (24-27 hr) after infection of Spodoptera frugiperda insect cell cultures with the recombinant virus, the foreign polypeptide can reach levels of expression of from 1 to greater than 500 mg/l. The observation that many of these gene products are secreted from the cells into the cell culture supernatant solution demonstrates that they could easily be purified from contaminating proteins. The baculovirus expression system has been shown to be able to process polypeptides similar in
complexity to the ricin molecule which is composed of two polypeptide chains, covalently linked by a disulfide bond and processed from a precursor molecule by post-translational excision of leader and joining peptides (Lamb et al., 1985). The A and B chains may be separated by reduction and ionexchange chromatography (Olnes and Pihl, 1973) and individually are relatively non-toxic to intact cells (Jansen et al., 1982). The B chain is responsible for binding the toxin to the cell surface and for its internalization (Olnes et al., 1974, 1976). The A chain, once released from the B chain within the cell, inhibits cytoplasmic protein synthesis (Lin et al., 1971); one internalized ricin molecule can kill a cell (Olnes and Pihl, 1982a). This property makes ricin an attractive reagent for the design of immunotoxins. In many respects, the ricin A chain serves as a prototype of work with other toxins. The ricin A chain is composed of 267 amino acids and has a mol. wt of ca 30,000. The A chain (Piatak et al., 1988; O'Hare et al., 1987) and the B chain (Chang et al., 1987) have been cloned into other expression vectors. A genomic clone coding for the entire ricin precursor has been constructed (Hailing et al., 1985); as is the case with other lectins (Vodkin et al., 1983; Hoffman, 1984), the gene lacks introns. There are two N-glycosylation sites on the native A chain at residues 10 and 236 containing one residue of xylose and one of fucose in addition to mannose and N-acetylglucosamine (Foxwell et al., 1985). The cDNA for ricin A chain has been cloned, sequenced, and inserted into several bacterial expression systems under control of inducible promoters (Piatak et al., 1988). This has permitted production of large amounts of recombinant ricin A chain (rRTA). Though it lacks the carbohydrate associated with native ricin A, rRTA is as active as the native molecule in inhibiting protein synthesis in vitro. The cloning/expression system has also allowed production of a number of rRTA mutants in a range of sizes and with a range of inhibitory activities (Bradley et al., 1989).
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It is i m p o r t a n t , then, to determine the sensitivity of insect cells a n d ribosomes to ricin a n d its catalytic subunit, not only for its i n h e r e n t interest, but also to establish the feasibility o f exploiting this eukaroytic expression vector for p r o d u c t i o n o f r e c o m b i n a n t toxins. The work reported here d e m o n s t r a t e s t h a t a l t h o u g h insect cells are resistant to ricin, cell-free protein synthesis extracts are inhibited by ricin A chain a n d t h a t isolated insect ribosomes are sensitive to the N-glycosidase d e p u r i n a t i o n activity of the A chain. MATERIALS AND METHODS
Materials Native ricin toxin, its A chain, and human placental RNase inhibitor were purchased from Sigma Chemical Company, St. Louis, MO. Rabbit reticulocyte lysate was purchased as a kit from Promega Biotec Corporation, Madison, WI. Insect cell culture The cell lines TN368 (Trichoplusia ni, cabbage looper) and SF9 (Spodoptera frugiperda, fall armyworm) were cultured in TCI00 medium with 10% FBS (Maruniak et al., 1984). Experiments were performed in three separate 25 cm I flasks with TN368 cells seeded at 8 x 105 cells/5 mls and SF9 cells seeded at 2 × 106 cells/5 mls. The stock solutions of ricin A chain or whole ricin were diluted into TC100 medium, and either 25/~g ricin A or 0.5 ~g, 5 gg or 50 #g of whole ricin were added to flasks containing either cell line in 5 mls medium. Controls consisted of no addition to cells, or the buffer in which the whole ricin is shipped (0.01 M phosphate buffer, 0.15M NaC1 with 0.1% NaAzide) diluted in medium. When diluted into the cell medium this resulted in the concentration of NaAzide being 0.00036%, 0.0036% and 0.036% in control flasks without ricin. Due to the extreme hazards of exposure to ricin, the NaAzide was not removed. At 1,2 and 3 days after treatment, 0.5 ml of Trypan Blue (Gibco) was added to each set of three flasks and sealed to count viable and non-viable cells in a fixed field of the microscope. Preparation of insect cell-free extracts Approximately l08 Spodoptera frugiperda cells were pelleted and washed in phosphate buffered saline (PBS, Dulbecco and Vogt, 1954) without Mg +2 or Ca +2 and resuspended in 1.5vol of lysis buffer (0.02M Hepes pH 7.6, 0.01 M KAc, 0.005 M DTT) on ice. Following 30 strokes with a dounce homogenizer, a 1/9 volume of a solution containing 0.1M Hepes pH7.6, 1.5MKAc, 0.02 M MgAc 2, 0.05 M DTT was added and mixed 5 times with the homogenizer. Unbroken cells and nuclei were removed by centrifugation at 1800g for 6 rain. The supernatant solution was then centrifuged at 4000g for 10min and the S-4 supernatant fraction for subsequent translations stored at - 7 0 ° C (Swerdel and Fallon, 1989). Translation assays and polyacrylamide gel analyses Reaction mixtures were assembled by combining a mixture containing 35 #1 of Spodoptera frugiperda cell lysate prepared as described above, enriched in amino acids (minus methionine) to 20#M, 1 #g creatine phosphokinase, 4 # g creatine phosphate, 0.55 #g GTP, 15 U of human placental RNase inhibitor, and 15pCi of 35S-methionine (spec. act. = 800 Ci/mmol from NEN). To five of the six reaction mixtures the translation inhibitors cycloheximide or ricin A chain were added to give the following final amounts: 2 m M or 20mM cycloheximide, 50ng, 100ng, or 500ng ricin A chain. These reaction mixtures were adjusted to 50#1 with water and
incubated at 30°C for 1 hr. An aliquot containing 25 #1 of each sample was added to 1 ml of IN NaOH. Following incubation at 37°C for 10 min, 4 ml of a 25% trichloroacetic acid (TCA) solution containing 2% casamino acids at 0 '~ C was added to each sample and incubated at 0°C for 30 min. The samples were filtered through Whatman glass filter discs, washed with 10ml of 10% TCA and 5 ml of acetone, and radioactivity determined in 10 ml of Ecoscint scintillation fluid. An equal volume of 2X Laemmli disruption buffer was added to the remaining 25/~1 of each sample. Each was boiled for 2 min and applied to a 10% polyacrylamide gel containing 0.5% SDS. Following electrophoresis the gel was stained, photographed and then dried onto filter paper for autoradiography.
Ribosome preparation and depurination assay Rabbit reticulocyte ribosomes were prepared from the commercial lysate and insect ribosomes from the S-4 lysate described above by centrifugation for 15 min at 135,000 g. Each pellet was resuspended in PBS. Mixtures containing approximately 10 pmoles of ribosomes were incubated for 1 hr at 30°C with 10 pmoles of ricin A chain, extracted three times with phenol (McGuire et al., 1976), and the aqueous layer treated with lysine at a final concentration of 0.4 M for 2 hr at 45°C (Philippsen et al., 1968). Samples of RNA were separated on a 3.5% polyacrylamide gel containing 7 M urea (Eller et al., 1985). The gel was stained with ethidium bromide and photographed using u.v. transillumination. RESULTS AND DISCUSSION
Resistance o f insect tissue culture cells to ricin To discover whether or not insect cells are sensitive to the toxic effects of ricin, experiments were performed in triplicate with TN368 (cabbage looper) and SF9 (fall a r m y w o r m ) cells with ricin A chain or whole ricin. The ricin A chain at 5 # g / m l had little effect on the growth rate or viability of TN368 cells except at day 2, on which there was some decrease in total cells (Figs 1, 2). Ricin A chain slightly stimulated growth of SF9 cells a n d h a d no effect o n viability. W h o l e toxin at 0.1 # g a n d 1 # g / m l h a d little effect on the growth or viability of TN368 cells. A t 10/~g per ml, cell g r o w t h decreased a n d non-viable cells increased from 7.5% at day 1 to 15.9% at day 3, but this appears to be due to the N a Azide in the whole ricin solution, since the buffer control produced the same p a t t e r n from 7.1% at day 1 to 24.4% at day 3. The SF9 cell growth was stimulated by 0.5 ~ g a n d 1 p g / m l of whole ricin. The SF9 cells also appeared to be affected by only the highest c o n c e n t r a t i o n of N a A z i d e in the ricin, since they did n o t grow at 10 #g/ml, but grew a n d remained viable at the other c o n c e n t r a t i o n s o f ricin. These results suggest t h a t the insect cells are resistant to this ricin p r e p a r a t i o n at c o n c e n t r a t i o n s which affected H e L a cells (1/~g/ml, d a t a not shown).
Susceptibility o f insect ribosomes to ricin A chain A l t h o u g h these results suggest that insect cells are resistant to ricin, the m e c h a n i s m of resistance could be at any of several levels. F o r example, some media c o m p o n e n t s specific to the insect culture system m a y be binding, sequestering a n d / o r degrading the toxin thus inactivating it. Alternatively, the cells m a y be resistant because they lack a receptor a n d / o r endocytotic system compatible with binding or internal-
Insect cells, ribosomes and ricin
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DAY Fig. 1. Growth of insect cells TN368 in TCI00 medium (5 ml/25 cm2flask) containing ricin A, whole ricin or buffer control. Viable cells were counted and then 0.5 ml Trypan Blue was added to count non-viable cells. Values represent average counts in a fixed field for three separate flasks at each time. ization. Finally, the ribosomes themselves may be resistant to ricin activity. Although the ribosomes of many cell types have been found to be sensitive to ricin (Olnes and Pihl, 1976), there is precedent for resistance to its catalytic activity. E. coli ribosomes are clearly resistant to 100/~g/ml (Lugnier et al., 1976), since large amounts of recombinant ricin can be produced in this expression system (Piatak et al., 1988). Furthermore, ribosomes from Tetrahymena are resistant to ricin A up to 2000/~g/ml (Wilde et al., 1979). Although for fungi there is a report to the contrary, mitochondrial ribosomes are resistant at 20/~g/ml ricin in yeast and 12/~g/ml in Neurospora crassa mitochondria (Lugnier et al., 1976), while rat liver mitochondrial ribosomes may also be resistant at 10 to 128#g/ml (Greco et al., 1974). Finally, wheat germ ribosomes are insensitive to 5000-fold (50/~g/ml) the concentration that inhibits mammalian ribosomes (Cawley et al., 1977).
Clearly it would be of interest to ask whether or not insect ribosomes themselves are resistant to the catalytic activity of ricin. To test the sensitivity directly, cellular lysates of Spodoptera frugiperda were prepared as described previously (Swerdel and Fallon, 1989; and as detailed in the Materials and Methods section). It was found that this iysate, without further treatment, was well suited to the inhibition experiments, since even in the absence of exogenous mRNA, approximately 6 x 104 precipitable counts/ min 35S-methionine were found after a 60 min incubation at 30°C. Using this amount of incorporation to represent the control translation activity, the amount of incorporation decreased by 40% and 82%, when either 2 mM or 20 mM cycloheximide was addcd, respectively. The result with ricin A chain was even more striking; when the reaction mixture contained 50, 100 or 500ng of ricin A chain, an average of 88%
SF 9 V I A B L E C E L L S 600 500
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J. E. MARUNlAK et al. IN VITRO TRANSLATION
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AUTORADIOGRAPH 6 7 100 NG 500 NG Ricin Ricin
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Fig. 3. Polyacrylamide get analyses of in vitro translation products. Fig. 3A shows a Coomassie Blue staining pattern of a 10% polyacrylamide gel in which lane 1 contains marker proteins: phosphorylase B (mol. wt 97,400); bovine serum albumin (mol. wt 66,200); ovalbumin (tool. wt 42,699); carbonic anhydrase (mol. wt 31,000); soybean trypsin inhibitor (mol. wt 21,500); and lysozyme (mol. wt 14,400). Lane 2 is the lysate containing no inhibitors. Lanes 3 and 4 contain samples incubated with 2 mM and 20 mM cycloheximide, respectively. Lanes 5-7 of Fig. 3A contain samples with 50, 100, and 500 ng, respectively, of ricin A chain. Figure 3B is the autoradiograph following 10 days exposure of this gel. decrease in activity resulted, regardless of the amount of added ricin A chain. These results are illustrated (Fig. 3) in an autoradiograph of these translation products, in the absence or presence of either cycloheximide or ricin A chain, following electrophoresis of an aliquot of the reaction mixtures on a 10% denaturing polyacrylamide gel. Athough the absolute amount of ~SS-methionine incorporation varied among the three experiments in which the inhibition of ricin A chain was tested, the results consistently demonstrated that greater than 85% inhibition of the control lysate's incorporation occurred at the level of 50 ng of ricin A chain per reaction. Clearly these results suggest that although intact cells appear to be resistant to ricin at a level toxic to HeLa cells, cell-free Spodoptera protein synthesis is sensitive to its catalytic component. To confirm and extend these results, insect ribosomes from the same lysate used in translation analysis were tested for sensitivity to the N-glycosidase activity of ricin A chain. Ten pmoles of either S p o d o p t e r a or rabbit reticulocyte ribosomes were incubated with buffer or equimolar amounts of ricin A chain for 1 hr at 30°C. Following phenol extraction, the RNA from each sample was treated with
0.4 M lysine for 2 hr at 45"C. If depurination occurred, the site should be sensitive to amine-catalyzed hydrolysis (Philippsen et al., 1968). Figure 4 demonstrates that a fragment of RNA is produced only in ricin A chain-treated samples. In addition, an endogenous fragment of slightly higher tool. wt appears in untreated as well as treated reticulocyte ribosome samples, as was reported earlier for rat (Endo et al., 1987), but not for yeast (Bradley et al., 1987), ribosomes. Furthermore, as in the yeast system, not only is an endogenous fragment absent in the untreated insect ribosome sample, but the size of the fragment produced by ricin A chain is smaller than that generated in the mammalian systems (Endo et al., 1987). The isolated ricin A chain catalytically inactivates the 50S subunit of the ribosome at a rate of 1500/rain and with a Km of 0.1~3,2/IM (Olsnes et al., 1975). Recent evidence (Endo et al., 1987; Bradley et al., 1987; Endo and Tsurugi, 1987) has demonstrated that ricin A chain depurinates yeast 26S and rat liver 26S ribosomal RNA at a single specific site. Prior work which provided the primary structure of the rRNA sequence which is sensitive to ricin A chain (Endo et al., 1987) as well as experiments which elucidated
547
Insect cells, ribosomes and ricin
RNA DEPURINATION ASSAY 1
2
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Fig. 4. Depurination assay. Rabbit reticulocyte or Spodoptera ribosomes (10 pmoles/reaction) were treated with phosphate buffered saline (lanes 3 and 6) or with equimolar amounts of ricin A chain (lanes 4 and 7, respectively), for 1 hr at 30°C, followed by phenol extraction, treatment with 0.4 M lysine for 2 hr at 45°C, and electrophoresis on a 3.5% polyacrylamide gel containing 7 M urea. Lanes 1 and 9 contain marker 16S plus 23S rRNA and 5S rRNA, respectively. The positions of the RNA fragments produced by ricin A chain following treatment of reticulocyte ( > ) and insect ( < ) ribosomes are indicated.
the secondary structure of other eukaroytic ribosomal R N A s (Endo et al., 1987; Veldman et al., 1981) suggest that the site of depurination on the large ribosomal R N A is a highly conserved sequence forming a loop in an area exposed to the surface and accessible to reagents such as kethoxal (Hogan et al., 1984). This domain maps to a region involved in elongation factor-dependent binding of aminoacylt R N A (Veldman et al., 1981), which is consistent with earlier work on the mode of action of ricin (Carrasco et al., 1975; Fernandezopuentes et al., 1976; Cawley et al., 1979). A short report (Piatak et al., 1988) recently described the expression of ricin in insect cells using a baculovirus expression vector. Active ricin was secreted at a concentration of 10-100ng/ml into culture medium of SF9 cells after infection with a recombinant baculovirus. The authors reported that intact SF9 cells were resistant to ricin as we have observed in this report. Their failure to isolate ricin A chain recombinants led the authors to speculate that the insect ribosomes were sensitive to the A chain. We showed by two methods that isolated SF9 ribosomes were sensitive to the A chain. Collectively, these results suggest that while insect cells in culture are resistant to whole ricin as well as its catalytic A chain subunit, Spodoptera frugiperda ribosomes are sensitive to the N-glycosidase activity of ricin A chain, which not only inhibits translation in cell-free extracts primed with endogenous m R N A , but also depurinates the insect r R N A . Thus insect ribosomes, like other sensitive eukaryotic ribosomes, appear to have a consensus sequence conserved on their surface. As in yeast, the depurination site appears to be
considerably closer to the 3' end of the r R N A than in mammalian systems, since a smaller fragment is produced. It would be of interest to determine the mechanism of insect cell resistance. Ricin labeled with a fluorescent probe could be used to determine if the toxin binds to the surface of insect cells. Experiments using radio-labeled whole ricin could also be used to determine if the molecule is transported into insect cells or if it is bound but not transported. Since it appears that whole ricin A is processed in the endoplasmic reticulum as suggested (Piatak et al., 1988) to obtain active A chain, this opens the possibility to manipulate the ricin A gene and still obtain expression of a recombinant product with the baculovirus expression system. Acknowledgements--This research was supported by the Division of Sponsored Research and the Interdisciplinary Center for Biotechnology Research at the University of Florida. Florida Experiment Station Journal Series number R-00155. Note added in proof--Additional experiments were done with dialyzed ricin (1 #g/ml) in SF9 cell cultures. No inhibition of cell growth was noted at day 1 and 2 with a slight decrease at day 3. REFERENCES
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SUSCEPTIBILITY OF INSECT CELLS A N D RIBOSOMES TO RICIN J. E. MARUNIAK,*S. E. FIESLERt and P. M. McGuIREt *Department of Entomology and tDepartment of Biochemistry and Molecular Biology, University of Florida, 0711 1FAS, Gainesville, FL 32611-0711, USA (Received 5 December 1989)
Abstract--1. The sensitivity of insect cells to whole ricin and its catalytic subunit has been examined. Evidence is presented that although cells from Trichoplusia ni and Spodopterafrugiperda are resistant to ricin, cell-free protein synthesis extracts are inhibited by ricin A chain and that the isolated insect ribosomes are sensitive to the N-glycosidase depurination activity of the A chain. 2. These results are consistent with the model that, as with other sensitive eukaroytic ribosomes, insect ribosomes appear to have a consensus sequence in the ribosomal RNA on the surface of the ribosome which is sensitive to this N-glycosidase.
INTRODUCTION Ricin is a cytotoxic glycoprotein produced in the seeds of the castorbean plant, Ricinus communis (Olsnes and Pihl, 1976; 1982b). It is one of a large number of plant-derived proteins which inhibit eukaroytic protein synthesis (Gasperi-Campagni et al., 1985). Some, including ricin, abrin, and modeccin, are extremely toxic to animals and to cells grown in culture. Others, among them pokeweed antiviral protein (PAP) and gelonin, are not very toxic to animals, but inhibit protein synthesis in cell-free systems. Although the ricin A chain gene has been expressed in bacteria, the recombinant product is only marginally soluble, probably largely because it lacks carbohydrate. Since there is abundant evidence for the glycosylation of heteroiogous recombinant products in the baculovirus-expression system, subcloning into this vector could greatly improve the ability to manipulate the ricin A chain gene. The insect baculovirus expression system has been publicised as a "super-vector" for the production of recombinant proteins (Wright, 1986). A recent review (Luckow and Summers, 1988) described the expression of a diverse collection of foreign genes from bacteria, viruses, plants, and humans. The expressed polypeptides can be antigenic, immunogenic, catalytic, enzymatic, secreted, glycosylated or phosphorylated. The mol. wts of the polypeptides range from 160,000 to 15,500. An impressive feature of the baculovirus expression vectors is the amount of polypeptide produced. Late (24-27 hr) after infection of Spodoptera frugiperda insect cell cultures with the recombinant virus, the foreign polypeptide can reach levels of expression of from 1 to greater than 500 mg/l. The observation that many of these gene products are secreted from the cells into the cell culture supernatant solution demonstrates that they could easily be purified from contaminating proteins. The baculovirus expression system has been shown to be able to process polypeptides similar in
complexity to the ricin molecule which is composed of two polypeptide chains, covalently linked by a disulfide bond and processed from a precursor molecule by post-translational excision of leader and joining peptides (Lamb et al., 1985). The A and B chains may be separated by reduction and ionexchange chromatography (Olnes and Pihl, 1973) and individually are relatively non-toxic to intact cells (Jansen et al., 1982). The B chain is responsible for binding the toxin to the cell surface and for its internalization (Olnes et al., 1974, 1976). The A chain, once released from the B chain within the cell, inhibits cytoplasmic protein synthesis (Lin et al., 1971); one internalized ricin molecule can kill a cell (Olnes and Pihl, 1982a). This property makes ricin an attractive reagent for the design of immunotoxins. In many respects, the ricin A chain serves as a prototype of work with other toxins. The ricin A chain is composed of 267 amino acids and has a mol. wt of ca 30,000. The A chain (Piatak et al., 1988; O'Hare et al., 1987) and the B chain (Chang et al., 1987) have been cloned into other expression vectors. A genomic clone coding for the entire ricin precursor has been constructed (Hailing et al., 1985); as is the case with other lectins (Vodkin et al., 1983; Hoffman, 1984), the gene lacks introns. There are two N-glycosylation sites on the native A chain at residues 10 and 236 containing one residue of xylose and one of fucose in addition to mannose and N-acetylglucosamine (Foxwell et al., 1985). The cDNA for ricin A chain has been cloned, sequenced, and inserted into several bacterial expression systems under control of inducible promoters (Piatak et al., 1988). This has permitted production of large amounts of recombinant ricin A chain (rRTA). Though it lacks the carbohydrate associated with native ricin A, rRTA is as active as the native molecule in inhibiting protein synthesis in vitro. The cloning/expression system has also allowed production of a number of rRTA mutants in a range of sizes and with a range of inhibitory activities (Bradley et al., 1989).
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It is i m p o r t a n t , then, to determine the sensitivity of insect cells a n d ribosomes to ricin a n d its catalytic subunit, not only for its i n h e r e n t interest, but also to establish the feasibility o f exploiting this eukaroytic expression vector for p r o d u c t i o n o f r e c o m b i n a n t toxins. The work reported here d e m o n s t r a t e s t h a t a l t h o u g h insect cells are resistant to ricin, cell-free protein synthesis extracts are inhibited by ricin A chain a n d t h a t isolated insect ribosomes are sensitive to the N-glycosidase d e p u r i n a t i o n activity of the A chain. MATERIALS AND METHODS
Materials Native ricin toxin, its A chain, and human placental RNase inhibitor were purchased from Sigma Chemical Company, St. Louis, MO. Rabbit reticulocyte lysate was purchased as a kit from Promega Biotec Corporation, Madison, WI. Insect cell culture The cell lines TN368 (Trichoplusia ni, cabbage looper) and SF9 (Spodoptera frugiperda, fall armyworm) were cultured in TCI00 medium with 10% FBS (Maruniak et al., 1984). Experiments were performed in three separate 25 cm I flasks with TN368 cells seeded at 8 x 105 cells/5 mls and SF9 cells seeded at 2 × 106 cells/5 mls. The stock solutions of ricin A chain or whole ricin were diluted into TC100 medium, and either 25/~g ricin A or 0.5 ~g, 5 gg or 50 #g of whole ricin were added to flasks containing either cell line in 5 mls medium. Controls consisted of no addition to cells, or the buffer in which the whole ricin is shipped (0.01 M phosphate buffer, 0.15M NaC1 with 0.1% NaAzide) diluted in medium. When diluted into the cell medium this resulted in the concentration of NaAzide being 0.00036%, 0.0036% and 0.036% in control flasks without ricin. Due to the extreme hazards of exposure to ricin, the NaAzide was not removed. At 1,2 and 3 days after treatment, 0.5 ml of Trypan Blue (Gibco) was added to each set of three flasks and sealed to count viable and non-viable cells in a fixed field of the microscope. Preparation of insect cell-free extracts Approximately l08 Spodoptera frugiperda cells were pelleted and washed in phosphate buffered saline (PBS, Dulbecco and Vogt, 1954) without Mg +2 or Ca +2 and resuspended in 1.5vol of lysis buffer (0.02M Hepes pH 7.6, 0.01 M KAc, 0.005 M DTT) on ice. Following 30 strokes with a dounce homogenizer, a 1/9 volume of a solution containing 0.1M Hepes pH7.6, 1.5MKAc, 0.02 M MgAc 2, 0.05 M DTT was added and mixed 5 times with the homogenizer. Unbroken cells and nuclei were removed by centrifugation at 1800g for 6 rain. The supernatant solution was then centrifuged at 4000g for 10min and the S-4 supernatant fraction for subsequent translations stored at - 7 0 ° C (Swerdel and Fallon, 1989). Translation assays and polyacrylamide gel analyses Reaction mixtures were assembled by combining a mixture containing 35 #1 of Spodoptera frugiperda cell lysate prepared as described above, enriched in amino acids (minus methionine) to 20#M, 1 #g creatine phosphokinase, 4 # g creatine phosphate, 0.55 #g GTP, 15 U of human placental RNase inhibitor, and 15pCi of 35S-methionine (spec. act. = 800 Ci/mmol from NEN). To five of the six reaction mixtures the translation inhibitors cycloheximide or ricin A chain were added to give the following final amounts: 2 m M or 20mM cycloheximide, 50ng, 100ng, or 500ng ricin A chain. These reaction mixtures were adjusted to 50#1 with water and
incubated at 30°C for 1 hr. An aliquot containing 25 #1 of each sample was added to 1 ml of IN NaOH. Following incubation at 37°C for 10 min, 4 ml of a 25% trichloroacetic acid (TCA) solution containing 2% casamino acids at 0 '~ C was added to each sample and incubated at 0°C for 30 min. The samples were filtered through Whatman glass filter discs, washed with 10ml of 10% TCA and 5 ml of acetone, and radioactivity determined in 10 ml of Ecoscint scintillation fluid. An equal volume of 2X Laemmli disruption buffer was added to the remaining 25/~1 of each sample. Each was boiled for 2 min and applied to a 10% polyacrylamide gel containing 0.5% SDS. Following electrophoresis the gel was stained, photographed and then dried onto filter paper for autoradiography.
Ribosome preparation and depurination assay Rabbit reticulocyte ribosomes were prepared from the commercial lysate and insect ribosomes from the S-4 lysate described above by centrifugation for 15 min at 135,000 g. Each pellet was resuspended in PBS. Mixtures containing approximately 10 pmoles of ribosomes were incubated for 1 hr at 30°C with 10 pmoles of ricin A chain, extracted three times with phenol (McGuire et al., 1976), and the aqueous layer treated with lysine at a final concentration of 0.4 M for 2 hr at 45°C (Philippsen et al., 1968). Samples of RNA were separated on a 3.5% polyacrylamide gel containing 7 M urea (Eller et al., 1985). The gel was stained with ethidium bromide and photographed using u.v. transillumination. RESULTS AND DISCUSSION
Resistance o f insect tissue culture cells to ricin To discover whether or not insect cells are sensitive to the toxic effects of ricin, experiments were performed in triplicate with TN368 (cabbage looper) and SF9 (fall a r m y w o r m ) cells with ricin A chain or whole ricin. The ricin A chain at 5 # g / m l had little effect on the growth rate or viability of TN368 cells except at day 2, on which there was some decrease in total cells (Figs 1, 2). Ricin A chain slightly stimulated growth of SF9 cells a n d h a d no effect o n viability. W h o l e toxin at 0.1 # g a n d 1 # g / m l h a d little effect on the growth or viability of TN368 cells. A t 10/~g per ml, cell g r o w t h decreased a n d non-viable cells increased from 7.5% at day 1 to 15.9% at day 3, but this appears to be due to the N a Azide in the whole ricin solution, since the buffer control produced the same p a t t e r n from 7.1% at day 1 to 24.4% at day 3. The SF9 cell growth was stimulated by 0.5 ~ g a n d 1 p g / m l of whole ricin. The SF9 cells also appeared to be affected by only the highest c o n c e n t r a t i o n of N a A z i d e in the ricin, since they did n o t grow at 10 #g/ml, but grew a n d remained viable at the other c o n c e n t r a t i o n s o f ricin. These results suggest t h a t the insect cells are resistant to this ricin p r e p a r a t i o n at c o n c e n t r a t i o n s which affected H e L a cells (1/~g/ml, d a t a not shown).
Susceptibility o f insect ribosomes to ricin A chain A l t h o u g h these results suggest that insect cells are resistant to ricin, the m e c h a n i s m of resistance could be at any of several levels. F o r example, some media c o m p o n e n t s specific to the insect culture system m a y be binding, sequestering a n d / o r degrading the toxin thus inactivating it. Alternatively, the cells m a y be resistant because they lack a receptor a n d / o r endocytotic system compatible with binding or internal-
Insect cells, ribosomes and ricin
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DAY Fig. 1. Growth of insect cells TN368 in TCI00 medium (5 ml/25 cm2flask) containing ricin A, whole ricin or buffer control. Viable cells were counted and then 0.5 ml Trypan Blue was added to count non-viable cells. Values represent average counts in a fixed field for three separate flasks at each time. ization. Finally, the ribosomes themselves may be resistant to ricin activity. Although the ribosomes of many cell types have been found to be sensitive to ricin (Olnes and Pihl, 1976), there is precedent for resistance to its catalytic activity. E. coli ribosomes are clearly resistant to 100/~g/ml (Lugnier et al., 1976), since large amounts of recombinant ricin can be produced in this expression system (Piatak et al., 1988). Furthermore, ribosomes from Tetrahymena are resistant to ricin A up to 2000/~g/ml (Wilde et al., 1979). Although for fungi there is a report to the contrary, mitochondrial ribosomes are resistant at 20/~g/ml ricin in yeast and 12/~g/ml in Neurospora crassa mitochondria (Lugnier et al., 1976), while rat liver mitochondrial ribosomes may also be resistant at 10 to 128#g/ml (Greco et al., 1974). Finally, wheat germ ribosomes are insensitive to 5000-fold (50/~g/ml) the concentration that inhibits mammalian ribosomes (Cawley et al., 1977).
Clearly it would be of interest to ask whether or not insect ribosomes themselves are resistant to the catalytic activity of ricin. To test the sensitivity directly, cellular lysates of Spodoptera frugiperda were prepared as described previously (Swerdel and Fallon, 1989; and as detailed in the Materials and Methods section). It was found that this iysate, without further treatment, was well suited to the inhibition experiments, since even in the absence of exogenous mRNA, approximately 6 x 104 precipitable counts/ min 35S-methionine were found after a 60 min incubation at 30°C. Using this amount of incorporation to represent the control translation activity, the amount of incorporation decreased by 40% and 82%, when either 2 mM or 20 mM cycloheximide was addcd, respectively. The result with ricin A chain was even more striking; when the reaction mixture contained 50, 100 or 500ng of ricin A chain, an average of 88%
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J. E. MARUNlAK et al. IN VITRO TRANSLATION
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Fig. 3. Polyacrylamide get analyses of in vitro translation products. Fig. 3A shows a Coomassie Blue staining pattern of a 10% polyacrylamide gel in which lane 1 contains marker proteins: phosphorylase B (mol. wt 97,400); bovine serum albumin (mol. wt 66,200); ovalbumin (tool. wt 42,699); carbonic anhydrase (mol. wt 31,000); soybean trypsin inhibitor (mol. wt 21,500); and lysozyme (mol. wt 14,400). Lane 2 is the lysate containing no inhibitors. Lanes 3 and 4 contain samples incubated with 2 mM and 20 mM cycloheximide, respectively. Lanes 5-7 of Fig. 3A contain samples with 50, 100, and 500 ng, respectively, of ricin A chain. Figure 3B is the autoradiograph following 10 days exposure of this gel. decrease in activity resulted, regardless of the amount of added ricin A chain. These results are illustrated (Fig. 3) in an autoradiograph of these translation products, in the absence or presence of either cycloheximide or ricin A chain, following electrophoresis of an aliquot of the reaction mixtures on a 10% denaturing polyacrylamide gel. Athough the absolute amount of ~SS-methionine incorporation varied among the three experiments in which the inhibition of ricin A chain was tested, the results consistently demonstrated that greater than 85% inhibition of the control lysate's incorporation occurred at the level of 50 ng of ricin A chain per reaction. Clearly these results suggest that although intact cells appear to be resistant to ricin at a level toxic to HeLa cells, cell-free Spodoptera protein synthesis is sensitive to its catalytic component. To confirm and extend these results, insect ribosomes from the same lysate used in translation analysis were tested for sensitivity to the N-glycosidase activity of ricin A chain. Ten pmoles of either S p o d o p t e r a or rabbit reticulocyte ribosomes were incubated with buffer or equimolar amounts of ricin A chain for 1 hr at 30°C. Following phenol extraction, the RNA from each sample was treated with
0.4 M lysine for 2 hr at 45"C. If depurination occurred, the site should be sensitive to amine-catalyzed hydrolysis (Philippsen et al., 1968). Figure 4 demonstrates that a fragment of RNA is produced only in ricin A chain-treated samples. In addition, an endogenous fragment of slightly higher tool. wt appears in untreated as well as treated reticulocyte ribosome samples, as was reported earlier for rat (Endo et al., 1987), but not for yeast (Bradley et al., 1987), ribosomes. Furthermore, as in the yeast system, not only is an endogenous fragment absent in the untreated insect ribosome sample, but the size of the fragment produced by ricin A chain is smaller than that generated in the mammalian systems (Endo et al., 1987). The isolated ricin A chain catalytically inactivates the 50S subunit of the ribosome at a rate of 1500/rain and with a Km of 0.1~3,2/IM (Olsnes et al., 1975). Recent evidence (Endo et al., 1987; Bradley et al., 1987; Endo and Tsurugi, 1987) has demonstrated that ricin A chain depurinates yeast 26S and rat liver 26S ribosomal RNA at a single specific site. Prior work which provided the primary structure of the rRNA sequence which is sensitive to ricin A chain (Endo et al., 1987) as well as experiments which elucidated
547
Insect cells, ribosomes and ricin
RNA DEPURINATION ASSAY 1
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Fig. 4. Depurination assay. Rabbit reticulocyte or Spodoptera ribosomes (10 pmoles/reaction) were treated with phosphate buffered saline (lanes 3 and 6) or with equimolar amounts of ricin A chain (lanes 4 and 7, respectively), for 1 hr at 30°C, followed by phenol extraction, treatment with 0.4 M lysine for 2 hr at 45°C, and electrophoresis on a 3.5% polyacrylamide gel containing 7 M urea. Lanes 1 and 9 contain marker 16S plus 23S rRNA and 5S rRNA, respectively. The positions of the RNA fragments produced by ricin A chain following treatment of reticulocyte ( > ) and insect ( < ) ribosomes are indicated.
the secondary structure of other eukaroytic ribosomal R N A s (Endo et al., 1987; Veldman et al., 1981) suggest that the site of depurination on the large ribosomal R N A is a highly conserved sequence forming a loop in an area exposed to the surface and accessible to reagents such as kethoxal (Hogan et al., 1984). This domain maps to a region involved in elongation factor-dependent binding of aminoacylt R N A (Veldman et al., 1981), which is consistent with earlier work on the mode of action of ricin (Carrasco et al., 1975; Fernandezopuentes et al., 1976; Cawley et al., 1979). A short report (Piatak et al., 1988) recently described the expression of ricin in insect cells using a baculovirus expression vector. Active ricin was secreted at a concentration of 10-100ng/ml into culture medium of SF9 cells after infection with a recombinant baculovirus. The authors reported that intact SF9 cells were resistant to ricin as we have observed in this report. Their failure to isolate ricin A chain recombinants led the authors to speculate that the insect ribosomes were sensitive to the A chain. We showed by two methods that isolated SF9 ribosomes were sensitive to the A chain. Collectively, these results suggest that while insect cells in culture are resistant to whole ricin as well as its catalytic A chain subunit, Spodoptera frugiperda ribosomes are sensitive to the N-glycosidase activity of ricin A chain, which not only inhibits translation in cell-free extracts primed with endogenous m R N A , but also depurinates the insect r R N A . Thus insect ribosomes, like other sensitive eukaryotic ribosomes, appear to have a consensus sequence conserved on their surface. As in yeast, the depurination site appears to be
considerably closer to the 3' end of the r R N A than in mammalian systems, since a smaller fragment is produced. It would be of interest to determine the mechanism of insect cell resistance. Ricin labeled with a fluorescent probe could be used to determine if the toxin binds to the surface of insect cells. Experiments using radio-labeled whole ricin could also be used to determine if the molecule is transported into insect cells or if it is bound but not transported. Since it appears that whole ricin A is processed in the endoplasmic reticulum as suggested (Piatak et al., 1988) to obtain active A chain, this opens the possibility to manipulate the ricin A gene and still obtain expression of a recombinant product with the baculovirus expression system. Acknowledgements--This research was supported by the Division of Sponsored Research and the Interdisciplinary Center for Biotechnology Research at the University of Florida. Florida Experiment Station Journal Series number R-00155. Note added in proof--Additional experiments were done with dialyzed ricin (1 #g/ml) in SF9 cell cultures. No inhibition of cell growth was noted at day 1 and 2 with a slight decrease at day 3. REFERENCES
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