McIver, J., Grady, G. F., and Keusch, G. T. (1975) J. Infect. Dis. 3. Keusch, G. T., and Jacewicz, ... Mathews, M. B. (1977) FEBS Lett. 15,201-204. 18. Vazquez, D.
THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256. No. 16. Issue of August 25. pp. 8739-8744, 1981 Prrnted in U.S.A.
The Cytotoxic Activity of Shigella Toxin EVIDENCE FOR CATALYTIC INACTIVATION OF THE 60 S RIBOSOMAL SUBUNIT* (Received for publication, January 22,
1981)
Richard Reisbig, Sjur Olsnes, and Kristin Eiklid From Norsk Hydro’s Institute forCancer Research and theNorwegian Cancer Society, Montebello, Oslo 3, Norway
The cytotoxic test system for Shigella shigae toxin in the preceding paper (9).Partially purified toxin is the pH 11was improved and used to study the stability of the extracted toxin (IO). A chain and AI fragment were isolated by SDS’ electrophoresis as described in the preceding paper (9). Molar contoxin to various pH values, temperature, and chemicals. centrations of toxin and thesubunits were determined using molecular Inhibition of protein synthesis is the first demonstra- weights of 65,000 for whole toxin, 30,500 for the A chain, and 27,500 ble effect in cells treated with Shigella toxin. This in- for the A1 fragment. hibition appears to beatthe level of peptidechain Cell Culture and In Vivo Measurements-HeLa S.3 cells were elongation. maintained in monolayer cultures in Dulbecco’s modifiedEagle’s An inhibitory effect on cell-free protein synthesis is Minimum Essential Medium with 10% fetal calf serum as previously exhibited by toxin pretreated first with trypsin and described (11). I n vivo toxicity tests were done in 24-well tissue culture trays (5 then with dithiothreitol and 8 M ureaor 1% sodium dodecyl sulfate. Ribosomes treated with toxin or its Al X IO4 cells/well) by adding increasing amounts of toxin to the wells. fragment had lost most of their ability to polymerize The next day, the medium was removed and replaced by serum-free with 21 mM HEPES (pH7.7),instead of bicarbonate and no [14C]phenylalaninein a poly(U)-dependent cell-free sys- medium unlabeled leucine. Fifty nCi of [I4C]leucine (342mCi/mol) was added tem. Salt-washed ribosomes in simple buffered soluto each well and the incorporation of radioactivity during 1 h was tions were inactivated at a rate of at least 40 ribosomes/ measured as previously described (11). (min) (A, fragment). Addition of antitoxin immediatelyProtein Synthesis in Rabbit Reticulocyte Lysate-Rabbit reticustopped further inactivation, but it did not reactivate locyte lysate was prepared by the method of Lingrel (12),supplemented as described by Pelham and Jackson (13),and then stored in the inactivated ribosomes. small aliquots in liquid nitrogen. Protein synthesis was measured in 60 S ribosomalsubunitsfromtoxin-treatedribosomes had a marked reduction in ability to support 50-pJ samples containing 0.5 pCi of [’4C]leucine (342 mCi/mol) with polyphenylanine synthesis,whereas 40 S subunits from and without toxin as described in the legends to thefigures. Aliquots were transferred to tubes containing 1 ml of 100 mM KOH and the toxin-treated ribosomes retained their activity. acid-precipitable radioactivity was measured as preToxin-treated ribosomes retained their ability to in- trichloroacetic viously described (14). corporate [3H]puromycin into growing peptide chains, Polymerization of Phenylalanine-The incubation mixture conindicating that the peptide bond formation is not the sisted of the indicated amounts of ribosomes or ribosomal subunits in function inhibited. 100 p1of buffer (50 mM Tris-HC1 (pH 7.4), m 60 M KCl, 4 mM MgCI2,
Shigella toxin is a potent inhibitor of protein synthesis in cells (1-7) and in cell-free systems (8).In the preceding paper (9),it was shown that Shigellatoxin consists of two different kinds of polypeptide chains. The heaviest chain, the A chain, is responsible for the inhibition of protein synthesis in vitro. This chain contains a trypsin-sensitive region. After cleavage with trypsin, a smaller fragment, A*, remains bound to the larger fragment, AI, by a disulfide linkage. The six to seven shorter chains (the B chains) appear to be bound to the A chain primarily via the AP fragment. In this investigation, we have studied the in vitro and in vivo activity of the toxin and its A chain. Confiiing the results of Brown et al. (8)we have found that thetoxic activity on cells occurs at the level of inhibition of protein synthesis. We have extended their results by identifying the intracellular target for the toxin and we present evidence that it acts enzymatically.
1.2 mM spermidine, 9 mM 2-mercaptoethanol) containing 10 pgof poly(U), 500 p~ GTP, 10 plof pH 5 supernatant, and 5 nCi of [14C]phenylalanyl-tRNA(115 mCi/mmol) or 92 nCi of [3H]phenylalanyl-tRNA (7 Ci/mmoI). After incubation for 15 min a t 37 “C, the heat-stable, acid-precipitable radioactivity was measured as previously described (15). Ribosomes and Ribosomal Subunits-Rabbit reticulocyte and rat liver ribosomes were prepared as described earlier (14).The ribosomes were washed once with 500 mMKC1 or 500 mM NH,Cl, resuspended in 50 mM Tris-HC1 (pH 7.4), m 25M KCI, 5 mMMgC12, and 6 mM 2mercaptoethanol, and stored on ice. They were used within 5 days of preparation. Ribosomal subunits were prepared by incubating ribosomes or reticulocyte lysate with 100 p~ GTP, 1 mM puromycin, and 300 mMKC1 for 30 min a t 37 “C. The subunits were separated by sucrose gradient centrifugation as described (14). Purification ofAntibodies against ShigellaToxin-Anti-ShigeLla toxin serum was obtained by immunizing rabbits with partially purified toxin (IO) as described (5). The IgG fraction was purified as described by Brandtzaeg (16)except that 10 mM Na-phosphate (pH 7.6) rather than Tris-HC1 was used. It was further purified by gel filtration on a column of Sephacryl-300 Superfine in 0.14 M NaCl, 5 mM Na-phosphate (pH 7.4). RESULTS
EXPERIMENTAL PROCEDURES
Toxin-Unless otherwise mentioned, the Shigella toxin was isolated from the pH 11extract (IO)of Shigella shigae60 R as described
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Activity of Shigella Toxin on Cells in Culture Test System for Cytotoxicity-To assay the toxic effect of Shigella toxin in vivo we have measured its ability to inhibit The abbreviations used are: SDS, sodium dodecyl sulfate; HEPES, 4-(2-hydroxyethyl)-I-piperazineethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride.
8739
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Cytotoxic Activityof Shigella Toxin
protein synthesis in HeLa Sacells. This is the most sensitive TABLE I cell line thus far tested (5).As previously observedby Keusch Effect ofphysical and chemical treatments on the toxic activity of Shigella toxin to intact cells and Jacewicz ( 3 ) the dose-response curve for Shigella toxin is peculiar in the sense that protein synthesis declines gradually Time Tempera- Toxic activity Addition ture on cells" when the toxin concentration is increased over several powers rnin "C 5 % control of10. This suggested that the HeLa S3 cells consisted of a 100 heterogeneous population of cells with different sensitivities. None 30 50 100 To test this, we randomly selected six different clones and None None 5 65 10 determined the cytotoxic activity of Shigella toxin on each. None 5 100 0 As shown in Fig. 1, the clones differed widely in sensitivity. 6 M urea 25 120 100 The most sensitive clone was used for all further studies. No 8 M urea 60 25 32 60 25 100 segregation of resistant cells wasdetected upon recloningafter 12 m~ dithiothreitol Trypsin (5 pg/ml) 60 37 100 1 month. 8 M urea + 12 n m dithiothrei60 25 20 When the cells were grownin the presence of newborn calf to1 serum, no toxic effect was observed even at high concentra- Trypsin + 8 M urea + 12 mM 60 25 16 tions of Shigella toxin (Fig. 1) in agreement with the data of dithiothreitol' Vicari et al. (1). The highest sensitivity of HeLa Ss cells to 6 M guanidine HCl 25 120 0.5 0.2% 2-mercaptoethanol 60 25 100 Shigella toxin was obtained with 10%fetal calf serum. 20 180 100 Stability of Shigella Toxin-Shigella toxin could be frozen pH 11 60 25 100 and thawed many times without loss of activity. Incubation at PH 3 SDS (0.01%) 60 20 100 50 "C for 30 min did not reduce the activity, whereas incuba- SDS (0.1%) 60 20 1 tion at 65 "C for 5 min resulted in a 90% reduction in toxic SDS (1%) 15 20 0.2 activity. Boiling for 5 min completely removed the toxic Trypsin + 1%SDS + 12 mM 60 25
