THE J O U R N A L OF BIOLOGICAL CHEMISTRY Vol. 255, No. 14. Issue of July 25, pp. 6947-6953. 19w Prtnted m U.S.A

Gelonin, a New Inhibitor of Protein Synthesis, Nontoxic to Intact Cells ISOLATION, CHARACTERIZATION, AND PREPARATION OF CYTOTOXIC COMPLEXES WITH CONCANAVALIN A*

(Received for publication, November 20, 1979)

Fiorenzo Stirpe,S Sjur Olsnes,§ and Alexander Rh18 From 8 Norsk Hydro's Institute for Cancer Research and the Norwegian Cancer Society, Montebello, Oslo, Norway a n d + Institute of General Pathology, University of Bologna, Bologna, Italy

The protein here named gelonin was extracted from the seeds of Gelonium multiflorum by a buffered phos- phate solution and purified in a single step by chro- matography on a carboxymethyl cellulose column. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and gel filtration on Sephacryl 300 superfine showed that gelonin consists of one poly- peptide chain with a molecular weight of about 28,000 to 30,000. Gelonin bound to a column containing im- mobilized concanavalin A and could be eluted with a- methylmannoside. Pretreatment of gelonin with jack- bean mannosidase prevented this binding, indicating that it is a glycoprotein containing terminal mannose residues. The protein proved t o be extremely stable, as measured by its biological activity, toward treatment with sodium dodecyl sulfate, urea, acid, base, and heat.

Gelonin strongly inhibited protein synthesis in a re- ticulocyte lysate. It inactivated the 60 S ribosomal sub- unit with no effect on the 40 S ribosomal subunit. A t a concentration of 100 pg/ml, gelonin only slightly in- hibited protein synthesis in intact HeLa cells and it gave no microscopically visible cytopathogenic effect. When the inhibitor was linked by a disulfide bridge to concanavalin A, the complex gave 50% inhibition of cellular protein synthesis at a concentration of about 1 pg/ml, corresponding to about 0.2 pg/ml of gelonin.

The results show that gelonin is a single chain protein which acts in a cell-free system like the A chains of abrin, ricin, and modeccin and suggest that it lacks the ability to bind to the cell surface and to enter intact cells. When coupled through a disulfide bond to a pro- tein capable of binding to cells, it is rendered toxic to intact cells.

Studies during recent years have shown that several bac- terial and plant cytotoxins consist of two functionally different domains. The one moiety is involved in the binding of the toxins to receptors on the cell surface, whereas the other somehow enters the cytoplasmic phase and acts by inhibiting protein synthesis. Thus, the A fragments of diphtheria toxin and Pseudomonas aeruginosa A toxin are enzymes capable of inactivating elongation factor 2 by ADP ribosylation (I), whereas the A chains of abrin, ricin, and modeccin irreversibly damage the 60 S subunit of eukaryotic ribosomes by an

* This work was supported by a European Molecular Biology Organization short tem.fellowship (to F. S.) and by Consiglio Na- zionale delle Ricerche, Rome, within the Progetto finalizzato "Con- troll0 della crescita neoplastica." The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

enzymatic mechanism, the nature of which has not been elucidated (2-4). All these toxins are extremely potent, and recent data indicate that a cell is killed when a single toxin molecule (or its A chain) enters the cytoplasmic phase (5, 6).

Recently, it has been found that many plants contain pro- teins that in ceU-free systems inactivate eukaryotic ribosomes, apparently in the same way as the A chains of abrin, ricin, and modeccin, but have little or no effect on intact cells. Such proteins have been purified from the roots of Phy to lacca americana (7, 8), from wheat germ (9), from the seeds of Momordica charantia (IO), and they have been partially purified from the seeds of Croton tiglium and Jatropha curcas (11, 12).

In screening studies, extracts from several other seeds strongly inhibited protein synthesis in a cell-free system, but lacked toxic effect on intact cells (13, 14). One of the most potent extracts was obtained from the seeds of Gelonium multiflorum (Euphorbiaceae). In the present study, we have isolated from such seeds a single chain protein, gelonin, with properties similar to those of the A chains of abrin, ricin, and modeccin. When gelonin was linked to concanavalin A. the complex formed was toxic to intact cells.

EXPERIMENTAL PROCEDURES

Materials-Seeds of G. multiflorum, grown in India, were pur- chased from F. G. Celo, Zweibriicken, West Germany. Jackbean a- mannosidase, concanavalin A, trypsin, chymotrypsin, pepsin, and protease type VI, from Streptomyces griseus, were obtained from Sigma Chemical Co. Abrin A chain was prepared as described earlier (15). Sepharose-concanavalin A and markers for molecular weight determinations were obtained from Pharmacia Fine Chemicals, Upps- ala, Sweden. Carboxymethyl cellulose (CM52) was purchased from Whatman Ltd., Maidstone, U. K. The protein iodination reagents were from Bio-Rad Laboratories, Richmond, Ca.

~-[U-~'C]leucine (specific activity, 342 Ci/mol), N-ethyl-P,3-["C]- rnaleimide (specific activity, 2.1 Ci/moI), Na'"1 (13 to 17 mCi/mg of iodine), and [14C]phenylalanyl-tRNA from Escherichia coli (specific activity, 0.192 pCi/mg) were obtained from The Radiochemical Centre, Amersham, England.

Extraction of Gelonin-Shelled seeds of G. multiflorum were ground in a s o r v d OmniMixer with 8 volumes of 0.14 M NaCI, 5 mM sodium phosphate (pH 7.4). The homogenate was left a t 2-4°C on a magnetic stirrer overnight, then further cooled in an ice bath, and finally centrifuged a t 35,000 X g for 20 min a t 0°C. A cloudy yellow supernatant was separated from the sediment and from a floating layer of solidified fat. The supernatant was dialyzed against 5 mM sodium phosphate (pH 6.5). Any precipitate formed during the di- alysis was removed by centrifugation and discarded.

Polyacrylamide G d Electrophoresis-Protein fractions were made up to contain 2% (w/v) sodium dodecyl sulfate, 60 mM Tris-HC1 (pH 6.8), 1OW (w/v) sucrose, and, in some cases, 0.7 M 2-mercaptoethanol. The samples were then incubated a t 56°C for 5 min and layered into slots of polyacrylamide slabs consisting of a 10 to 20% exponential gradient separating gel (0.85 rnm X 16 cm X 16 cm) and a 3% concentrating gel (0.85 mm X 2 cm X 16 cm) with the buffers described

6947

6948 Gelanin, a Protein Synthesis Inhibitor

by Laemmli (16). The gels were run at constant voltage (150 V) until the tracking dye (bromphenol blue) reached the lower edge of the gel. The gels were stained with Coomassie brilliant blue and in some cases dried and exposed to Kodak Royal X-Omat film.

Iodination-Labeling of pure gelonin with 1251 by the lactoperoxi- dase-glucose oxidase method was carried out with the reagents sup- plied from Bio-Rad Laboratories as described by the manufacturer. Gelonin (100 pg in 60 pl of buffer) was labeled with 2 p1 (1.1 mCi) of Na’2sI. After 20 min at room temperature, the protein was separated from free NaIz5I by filtration through a Sephadex G-25 column, equilibrated with 0.14 M NaCI, 5 m~ sodium phosphate (pH 7.4). The labeled product contained 634 cpm/ng.

Cell-free Protein Synthesis-A rabbit reticulocyte lysate, prepared as earlier described (17), was supplemented as described by Pelham and Jackson (18) and then stored in small aliquots in liquid nitrogen. Protein synthesis was measured in 50-pl samples containing 0.5 pCi of [14C]leucine, with or without inhibitor, as described in the legends to figures. Aliquots (10 pl) were removed at the times indicated and poured into tubes containing 1 ml of 0.1 M KOH, and then the

described (17). trichloroacetic acid-precipitable radioactivity was measured as earlier

Ribosomes and Ribosomal Subunits-Ribosomes were isolated from rabbit reticulocyte lysate as described earlier (19). The ribosomal subunits were obtained after incubation of ribosomes with puromycin and GTP and then treated with high salt concentration and separated on a sucrose gradient as described (19). Poly(U)-directed polyphen- ylalanine synthesis with isolated ribosomes and ribosomal subunits was measured as earlier described (20).

Cells-HeLa Sa cells were maintained in monolayer culture in Eagle’s minimum essential medium containing 10% calf serum as earlier described (21). For toxicity tests, cells were seeded into Linbro tissue culture plates with 16-mm troughs (lo” ceUs/well) and then increasing amounts of gelonin were added to the wells. On the next day, the medium was removed and replaced by serum-free medium containing %II of the normal concentration of leucine and 0.1 pCi/ml of [‘4C]leucine, and the incorporation of radioactivity for 2 h was measured as earlier described (22). Human lymphocytes were pre- pared and cultured as described by Godal et al. (23).

Coupling Experiments-Concanavalin A (25 mg) in 1 r d of 0.1 M sodium phosphate, pH 7.5, 0.1 M NaCl was treated with a 10-fold molar excess of N-succinimidyl-3-(2-pyridyldithio)propionate, ob- tained from Pharmacia Fine Chemicals, Uppsala, Sweden, for 30 min at room temperature with occasional stirring (24). Excess reagent was removed by affinity chromatography on a 2-ml column of Sephacryl 300 supertine, equilibrated with 0.14 M NaC1,5 m~ sodium phosphate (pH 7.4). After washing, concanavalin A was eluted with 0.1 M a- methylmannoside in the same buffer. Finally, a-methylmannoside was removed by dialysis.

Gelonin (0.5 mg) was mixed with a trace of ‘251-labeled gelonin (los cpm) and dialyzed against 0.1 M sodium phosphate (pH 7.5), 0.1 M NaCI. Then a 10-fold molar excess of SPDP’ was added and the mixture was incubated for 30 min at room temperature. (Control experiments showed that even treatment with a 25-fold molar excess of SPDP did not impair the ability of gelonin to inhibit cell-free protein synthesis, whereas some reduction in activity was observed after treatment with a 100-fold molar excess of SPDP.) Then, suffi- cient acetic acid was added to reduce the pH to 4.5, dithiothreitol was added to a final concentration of 50 m ~ , and the mixture was incubated for 20 min at room temperature. Excess reducing agent was removed by gel filtration on Sephadex G-25 (medium), equilibrated with 0.1 M sodium phosphate (pH 7.5), 0.1 M NaC1. Finally, the gelonin thus treated was mixed with approximately equimolar amounts of concanavalin A, pretreated with SPDP as described above, and the mixture was incubated at room temperature overnight.

On the next day, the mixture was submitted to centrifugation at 300,000 X g for 18 h in 5 to 20% (w/v) sucrose gradients containing 0.1 M a-methylmannoside in 0.14 M NaCI, 5 m~ sodium phosphate, pH 7.4. Fractions (0.25 ml each) were collected by puncturing the bottom of the tubes, the radioactivity in each fraction was measured to localize gelonin, and the absorbance at 280 nm was measured to localize concanavalin A. Analysis of the different fractions by poly- acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and subsequent autoradiography of the gel showed that the radioactive material sedimenting in front of the main peak of material absorbing at 280 nm, moved more slowly than gelonin alone, indicat-

’ The abbreviation used is: SPDP, N-succinimidyl-3-(2-pyridyldi- thio)propionate.

ing that it consisted of complexes with concanavalin A. Other Methods-Free thiol groups were determined by measuring

the binding of N-ethyl[’4C]maleimide as earlier described (25). Pro- tein was determined spectrophotometrically (26). I4C radioactivity was measured in a Beckman LS-330 liquid scintillation spectrometer and Iz5I radioactivity in an Intertechnique gamma spectrometer.

RESULTS

Purification of Gelonin-Seeds of G. multiftorum were extracted as described under “Experimental Procedures,” and the dialyzed extract was applied to a CM52 column equili- brated with 5 mM sodium phosphate (pH 6.5). The adsorbed material was eluted with a linear 0 to 0.3 M NaCl gradient in the same buffer. The results are shown in Fig. 1. The different fractions were tested for their ability to inhibit protein syn- thesis in a cell-free system from rabbit reticulocytes. Most of the inhibitory activity became adsorbed to the column and was eluted corresponding to Peak IV.

Polyacrylamide gel electrophoresis of the material in the different fractions showed that the material in Peak IV con- sisted of one polypeptide chain with molecular weight of about 30,000 (Fig. 2, A and B). It is clear from Fig. 2 that this protein, which we named gelonin, is the most abundant one in the extract. The yield was 4 to 5 mg of pure gelonin from 3.5 g of seeds. Approximately 40% of the inhibitory activity found in the dialyzed extract could be recovered in pure gelonin.

Characterization of Gelonin-To test whether the native molecule consists of one or more copies of this chain, it was Ntered through a column of Sephacryl 300 superbe which had been calibrated with proteins of known molecular weights. It was found (Fig. 3) that gelonin was eluted at a position corresponding to a molecular weight of about 28,000. The different fractions were also tested in a cell-free protein syn- thesizing system. As shown in Fig. 3, there was good corre- spondence between the elution pattern of the protein and the ability to inhibit cell-free protein synthesis. The results indi- cate that gelonin consists of only one copy of the peptide chain. A small shoulder at the leading edge of the peak probably corresponds to dimers of the protein.

P

U-

u-

n 40 SO M 100 120 250

- 0.3

-0.2

- 0 1

Fraction number

FIG. 1. Purification of gelonin by chromatography on car- boxymethyl cellulose. Seed extracts (20 d, containing 100 mg of protein), dialyzed against 5 mM sodium phosphate (pH 6.5). was applied to a CM52 column (20 X 1.5 cm) equilibrated with the same buffer. The column was washed and then bound material was eluted with a 400-ml0 to 0.3 M linear NaCl gradient in the same buffer at a rate of 25 ml/h at room temperature. Fractions (1.4 ml each) were collected and the absorbance at 280 nm was measured. From some of the fractions, 5-pl aliquots were taken and added to a cell-free system as described under “Experimental Procedures.” The incorporation of [‘4C]leucine was measured after 10 min. The fractions indicated with the bur were collected as pure gelonin.

Gelonin, a Protein Synthesis Inhibitor 6949

3

A B C D E FIG. 2. Polyacrylamide gel electrophoresis of crude and

pure gelonin in the presence of sodium dodecyl sulfate. Aliquots ( 5 0 pl) from fractions in Fig. 1 were submitted to polyacrylamide gel electrophoresis as described under "Experimental Procedures." A, fraction 127; B, fraction 129; C, crude gelonin; D, molecular weight markers: phosphorylase B (94,000). bovine serum albumin (67,000), ovalbumin (43,0001, carbonic anhydrase (30,000). trypsin inhibitor (20,000). a-lactalbumin (14,000); E , abrin A chain.

The extinction coefficient for gelonin at 280 nm was esti- mated from the weight of a sample of gelonin which had been extensively dialyzed against distilled water and then lyophi- lized in a preweighed tube. The value obtained was E!':., = 6.7.

Gelonin exhibits many properties in common with the A chains of abrin, ricin, and modeccin (see below). Since abrin, ricin, and modeccin all are glycoproteins and bind to concan- avalin A, we studied whether gelonin binds to a column of immobilized concanavalin A. It was found (Fig. 4) that most of the labeled material was indeed bound to the column and could be eluted with a-methylmannoside. When the different fractions were tested for their ability to inhibit cell-free protein synthesis, a good correspondence between radioactivity and inhibitory activity was found (not shown). If gelonin had been pretreated with jackbean a-mannosidase, very little radioac- tivity was bound to the concanavalin A column. This indicates that gelonin is a glycoprotein containing terminal mannose residues. Treatment of gelonin with jackbean a-mannosidase did not impair its ability to inhibit cell-free protein synthesis.

Less than 0.1 mol of N-ethyl['4C]maleimide was bound per mol of gelonin, indicating that it contains no free thiol group.

Stability of Gelonin-To test the stability of gelonin its ability to inhibit cell-free protein synthesis after various treat- ments was studied. The activity was unchanged after incuba- tion for 1 h at 37°C and more than 12 h at room temperature. Freezing and thawing for 10 consecutive times, as well as freeze-drying, did not affect the inhibitory activity, and the

activity was only slightly decreased (about 15%) by treatment overnight with 1% sodium dodecyl sulfate or 6 M urea. Gelonin was not affected by treatment overnight with 0.1 M HCl, or 0.1 M NaOH, or by incubating a sample of gelonin (0.35 mg/ ml) overnight at room temperature with an equal amount of trypsin, chymotrypsin, pepsin, or S. griseus protease. Labeling with 1 molecule of iodine/molecule of gelonin did not affect the inhibitory activity. However, it was completely destroyed by boiling for 20 min. Heat-denatured gelonin was completely

n n

Fraction number FIG. 3. Gel filtration of gelonin on a Sephacryl300 superfine

column. Gelonin (2 mg) was mixed with a trace of dextran blue in 0.25 ml of buffer (0.14 M NaCI, 5 r n ~ sodium phosphate, pH 7.4), applied at 20°C to a Sephacryl column (80 X 1.5 cm), and eluted with the same buffer. Fractions (35 drops each) were collected, and the absorbance at 280 and 620 nm was measured. Aliquots of the fractions were tested for their ability to inhibit cell-free protein synthesis as in Fig. 1. The proteins used to calibrate the Sephacryl column were: bovine serum albumin (BSA) (M, = 67,0001, ovalbumin (M, = 43,000), carbonic anhydrase (M, = 30,000), trypsin (M, = 24,000). and lysozyme (M, = 13,000). The aliquots tested in the cell-free system were: A, 1 pl; A, 0.3 pl; X, 0.01 pl.

4 1

Fraction number FIG. 4. Binding of gelonin to a Sepharose-concanavalin A

column. A sample of '2511-labeled gelonin (0.2 pg, 120,000 cpm) in 0.14 M NaCI, 5 mM sodium phosphate (pH 7.5) was treated at 37OC for 30 min with 50 pg/ml of a-mannosidase. A parallel sample was incubated without enzyme. The samples were then applied at room temperature to columns of Sepharose-concanavalin A (2 X 1.5 cm). After washing with buffer, the columns were eluted with 0.1 M a-methylmannoside in the same buffer. The radioactivity in each fraction was measured. 0, gelonin incubated without enzyme; 0, gelonin pretreated with a- mannosidase. The arrow indicates the start of the elution with a- methylmannoside.

6950 Gelonin, a Protein Synthesis Inhibitor

degraded by chymotrypsin and S. griseus protease and also became somewhat sensitive to trypsin and pepsin.

Effect on Protein Synthesis in Cell-free Systems-Gelonin is a very potent inhibitor of protein synthesis in a cell-free system from a rabbit reticulocyte lysate. Thus, as shown in Fig. 5, as little as 0.4 ng/assay exhibited a definite effect. It is clear that gelonin inhibited protein synthesis even more effi- ciently than did abrin A chain. I t can be seen from Fig. 6 that the concentration giving 50% inhibition after 5 min of incu- bation, when the rate of protein synthesis was still linear, was about 12.5 ng/ml.

We have earlier found that the inhibitory effect of abrin, ricin, and modeccin is strongly increased by pretreatment of the toxins with 2-mercaptoethanol. No similar effect was obtained with gelonin (Fig. 6 ) .

The A chains of abrin, ricin, and modeccin, as well as curcin, crotin, and the pokeweed inhibitor, all exert their effect by inactivating the 60 S ribosomal subunit (2-4, 7, 12). Since washed ribosomes from gelonin-treated rabbit reticulocyte lysate had a strongly reduced activity in a cell-free system (not shown), we tested which of the ribosomal subunits is inactivated by gelonin. Ribosomes were isolated from a rabbit reticulocyte lysate, part of the ribosomes were treated with gelonin, whereas another part was untreated. Ribosomal sub- units were prepared as described under "Experimental Pro- cedures" and a reconstituted cell-free system was prepared. With polyuridylic acid as synthetic messenger it was found (Table 1) that when the 60 S ribosomal subunit had been exposed to gelonin, the incorporation of ['4C]phenylalanine was very low, whereas with 40 S subunits derived from ge- lonin-treated ribosomes essentially the same incorporation was obtained as when both subunits were untreated. It is therefore clear that also gelonin exerts its effect on the 60 S ribosomal subunit.

Experiments with Intact Cells-When gelonin was added I I J

T i m e ( m i n )

FIG. 5. Ability of gelonin and abrin A chain to inhibit protein synthesis in a cell-free system from rabbit reticulocytes. The indicated amounts of gelonin or abrin A chain were added to a cell- free system prepared from a rabbit reticulocyte lysate. The final volume was 80 pl. Aliquots (10 pl) were taken, as indicated, and the alkali-stable, acid-precipitable radioactivity was measured. X, control; 0, 0.4 ng of gelonin; 0, 0.4 ng of abrin A chain; W, 4 ng of gelonin; 0, 4 ng of abrin A chain; A, 40 ng of gelonin; A, 40 ng of abrin A chain.

-

4" 50 100 250 500

Inhibitor added (ng/ml) - . FIG. 6. Inability of 2-mercaptoethanol to alter the inhibitory

effect of gelonin in a cell-free system from rabbit reticulocytes. Gelonin was incubated for 2 h at 37°C with and without 1% 2- mercaptoethanol. Then, increasing amounts of the protein were added to cell-free systems as in Fig. 5 (except that the final volume was 65 pl) and, after incubation a t 28°C for 5 min, 10-p1 aliquots were taken, and the amount of acid-precipitable radioactivity was measured. 0, untreated gelonin; 0, gelonin pretreated with 1%' 2-mercaptoethanol.

TABLE I Ability of untreated and gelonin-treated ribosomal subunits to

polymerize phenylalanine Ribosomes were isolated from 10 ml of rabbit reticulocyte lysate

and resuspended in 1 ml of 50 mM Tris-HC1 (pH 7.4). 60 m~ KCI, 2 mM MgC12, and 9 mM 2-mercaptoethanol. The suspension was divided into two equal parts to one of which was added 40 p g of gelonin. The samples were incubated a t 37°C for 10 min, then treated with puro- mycin and GTP, and ribosomal subunits were isolated (19). Aliquots (0.5 pmol) of each subunit as indicated were incubated as described (20), and the ability to polymerize ['4C]phenylalanine was measured.

IJntreated subunit Ge'onin-treated Radioactivity unit

cpm

40, 60 264 40,60 117

40 60 85 60 40 35 1

to cultures of HeLa S:, cells, very little effect was observed. Thus, only very high concentrations (100 pg/ml) of gelonin induced some reduction (about 20%) of protein synthesis in the cells after incubation with gelonin overnight.

To test whether gelonin is able to bind to cells, we incubated 'L'I-labeled gelonin with HeLa S:, cells for 60 min at 0°C. The cells were sedimented and washed three times with cell culture medidm. No binding above background was found.

A protein synthesis inhibitor from M . charantia was found to be selectively toxic to lymphocytes.2 To test whether ge-

F. Licastro, C. Franceschi, L. Barbieri, and Stirpe, F. (1980) Virchows Arch. B. Cell. Pathol., in press.

Gelonin, a Protein Synthesis Inhibitor 6951

lonin has a similar effect, different amounts of gelonin were added to lymphocytes stimulated with three different mito- gens (Table 11). In cultures stimulated with phytohemagglu- tinin from Phaseolus vulgaris and with pokeweed mitogen, very little inhibition was observed. However, in cultures stim- ulated with concanavalin A, an inhibitory effect was seen a t the highest concentration of gelonin. Since gelonin binds to

TABLE I1 Effect of gelonin on thymidine incorporation by mitogen-stimulated

lymphocytes Human lymphocytes were isolated and 2.5 X IO5 cells/well were

incubated with or without mitogens as described (23). The indicated amounts of gelonin were added to the cultures and after incubation a t 37°C for 48 h, 1.25 pCi of [“Hlthymidine was added. The incorpo- ration of radioactivity into acid-precipitable material during the fol- lowing 18 h was measured.

Mitogen used Gelonin added (pg/ml)

None 0.4 4.0 40 ~ ~~~ ~

cPm None (controls) 1,538 1,985 1,317 1,320 Phytohemagglutinin 87,262 64,140 51,642 62,393 Concanavalin A 32,117 27,471 23,098 7,834 Pokeweed mitogen 16,807 13,449 11,715 12,584

94 K= 67 K-

43 K-

FIG. 7. Polyacrylamide gel electrophoresis of complexes of gelonin and concanavalin A. ‘”I-labeled gelonin and unlabeled concanavalin A were joined by disulfide bridges as described under “Experimental Procedures.” Aliquots ( 5 0 pl) were incubated with sodium dodecyl sulfate with and without 2-mercaptoethanol and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The dried gel was submitted to autoradiog- raphy. A, nonreduced sample; B, sample pretreated with 2-mercap- toethanol. The horizontal bars indicate the migration of the same molecular weight markers as in Fig. 2.

Protein added (Pg/ml)

FIG. 8. Ability of complexes of gelonin and concanavalin A to inhibit protein synthesis in HeLa Sa cells. Gelonin, concana- valin A, and complexes of both were added to HeLa S:, cells growing in Linbro plates (FB 16-24 TC) in serum-free medium containing 1 g/ liter of galactose instead of glucose. After 8 h of incubation at 37°C. 1 g/liter of glucose and 10% calf serum were added and the plates were incubated further overnight. Then, the medium was changed to low leucine medium containing 0.05 pCi of [“C]leucine/ml. and the incorporation of radioactivity into acid-precipitable material during 1 h was measured. 0, complex of gelonin and concanavalin A, the same as shown in Fig. 7A; 0, the same complex, 50 m~ a-methylman- noside added to the medium; A, mixture of SPDP-treated concana- valin A and gelonin with only about 10% of the gelonin present in covalent complexes; A, the same mixture, 50 mM a-methylmannoside added to the medium; X, SPDP-treated concanavalin A alqne.

concanavalin A and may inhibit the binding of the lectin to its receptors on the lymphocytes, it is possible that the inhi- bition seen is not due to a direct effect of gelonin on the cells.

Complexes of Gelonin and Concanavalin A-The above experiments indicated that gelonin fails to intoxicate cells because it is unable to bind to the cell surface. We therefore attempted to bind gelonin through a disulfide bridge to a molecule capable of binding to cell surface receptors. For this purpose, we chose concanavalin A. Concanavalin A and ge- lonin were f i t reacted with N-succinimidyl-3-(2-pyrimidyl- thio)propionate, and free SH groups were then introduced in gelonin by treatment with dithiothreitol as described under “Experimental Procedures.” The proteins were then mixed and after reaction overnight, the complexes formed were separated from unreacted proteins by sucrose gradient cen- trifugation. Since ‘”“I-labeled gelonin was used in this experi- ment, complexes of gelonin with concanavalin A should be revealed by autoradiography after polyacrylamide gel electro- phoresis in the presence of sodium dodecyl sulfate under nonreducing conditions. Since the monomer of concanavalin A has a molecular weight of 27,000, complexes containing one monomer of each protein should move in the gel correspond- ing to a molecular weight of about 57,000. As shown in Fig. 7 A , a major labeled band was indeed found a t this position. Bands with higher molecular weights were also observed, probably corresponding to complexes containing more than one copy of each protein. When the complexes were treated with 2mercaptoethanol in the presence of sodium dodecyl

6952 Gelonin, a Protein Synthesis Inhibitor

sulfate (Fig. 7B), all the radioactivity moved corresponding to a molecular weight of 30,000, i.e. like free gelonin. When the treatment with 2-mercaptoethanol was carried out under non- denaturing conditions, dissociation was observed only in part of the complexes (not shown).

When tested in a cell-free system under conditions as in Fig. 5, gelonin in complex with concanavalin A was considerably less efficient than free gelonin in inhibiting protein synthesis. A considerable increase in activity was found after pretreat- ment of the complex with 2-mercaptoethanol (not shown). It has earlier been observed that treatment of diphtheria toxin, abrin, ricin, and modeccin with 2-mercaptoethanol strongly increased their ability to inhibit protein synthesis in cell-free systems (1-4).

To test the toxicity of the complex, it was added to ceils in culture and the inhibition of protein synthesis was measured. Since glucose and serum proteins present in the medium compete with the cell surface receptors for binding of concan- avalin A, the complex was added to cells in serum-free medium containing galactose instead of glucose, and serum and glucose were added after 8 h of incubation. It was found (Fig. 8) that 50% inhibition of protein synthesis occurred when about 1 pg of complex, containing about 0.2 pg of gelonin, was added per ml of medium. This inhibition was prevented when a-meth- ylmannoside was added to the medium. Since about 30 p,g of SPDP-treated concanavalin A alone was required to give the same inhibition of protein synthesis and since as much as 100 pg/ml of free gelonin inhibited protein synthesis only slightly, the data indicate that concanavalin A, attached to gelonin through a disulfide bridge, sewed as a haptomer that mediated the binding and uptake of the inhibitor by the cells.

DISCUSSION

The present results indicate that gelonin is a single chain glycoprotein with biological properties similar to those of the A chains of abrin, ricin, and modeccin. Like these, gelonin inactivates the 60 S ribosomal subunits. From the activity of gelonin in a cell-free system from rabbit reticulocytes, it ‘can be estimated that 1 molecule of the toxin inactivates about 200 ribosomes/min, indicating that it acts catalytically. In a cell-free system from rabbit reticulocytes gelonin is even more active than abrin A chain.

Inhibitors similar to gelonin have been found in a variety of plants. The first one described is an antiviral protein (PAP or Phytolacca americana protein) present in the roots of poke- weed (8). This protein (Mr - 27,000) which is non-toxic to cells, may prevent the multiplication of viruses in the cells (27). Like gelonin, it inhibits cell-free protein synthesis by catalytically inactivating the 60 S ribosomal subunit (7). This is also the case with the less well characterized inhibitors crotin and curcin, present in the seeds of C. liglium and J. curcas (11, 12). It is not clear whether the wheat germ and the M . charantia inhibitors act on the 60 S subunit, although in both cases it has been found that the target is on the ribosomes (9, 10).

Several other seed extracts contain a heat-labile inhibitor of cell-free protein synthesis (13,14). Since such inhibitors are found in a variety of unrelated plant species, they may be present in all plants and have an important function. Possibly, by the methods now available, such proteins are only found in those cases where, for one reason or another, they are synthe- sued in exceptionally high amounts.

Probably, the most interesting finding in this paper is that geIonin which is nontoxic to intact cells, acquired toxic activity when linked by a disulfide bridge to concanavalin A. Presum- ably, the lectin serves as a “haptomer” that binds the complex

to cell surface receptors, permitting uptake of gelonin to take place.

In several laboratories, attempts have been made to alter the specificity of diphtheria toxin, abrin, and ricin by replacing the binding part (the B chains) with a protein having a different binding specificity. The goal of these experiments has been to bind the “effectomers,” the A chains, to molecules that may direct the A chains to particular targets in the organism. Thus, diphtheria toxin A fragment has been bound by a disulfide bridge to human placental lactogen (28, 29), concanavalin A (30), Wistaria floribunda lectin (31), Fab fragments of antibodies against L1210 ceUs (32), and, by the aid of a derivative of chlorambucil, to antilymphocyte globu- lins (33). Similarly, ricin A chain has been linked through a disulfide bridge to the /3 chain of human chorionic gonadotro- pin (34, 35) and to concanavalin A (36). In most cases, the hybrid molecules have proved to be toxic to cells having the appropriate receptors, albeit to a much lesser extent than the native toxins. Apart from the fact that the isolated A chains may be diffcult to prepare in sufficient quantities, an inherent difficulty in such experiments is that the purified A chains may contain traces of intact toxins. Since gelonin is an A chain like protein, it appears to be ideal for such studies. It is present in the seeds of G. multiflorum in high concentrations, it is easily isolated, it is exceptionally stable to chemical and physical treatments, and it is nontoxic to cells, unless linked to a haptomer. In mice, no toxic effect was observed after intravenous injection of 1 mg of gelonin/100 g body weight.

The complex of gelonin and concanavalin A here prepared was much less toxic to cells than the toxins abrin, ricin, and modeccin. There may be several explanations for the low toxicity of artificial complexes. First, it is possible that the natural B chains of the toxins have functions other than that of merely binding to cell surface receptors. Probably, the B chains somehow facilitate the entry of the A chain through the plasma membrane. Second, the artificial binding moieties may not bind to those receptors that are most efficient in internalizing the toxins. Possibly, the artificial complexes enter the cells by mechanisms entirely different from those used by the natural toxins. Third, reduction of the interchain disulfide bridge has been shown to be essential for inhibitory effect of the natural toxins on ceU-free protein synthesis. Possibly, the disuKde bridge in the artificial complexes may be less easily split than those in the natural toxins. These questions may be elucidated by binding gelonin to several carrier (haptomer) molecules by different coupling agents and comparing the toxic effect of the complexes on cells in culture. Possibly, similar complexes can be formed with other A chain like inhibitors present in plant material.

An intriguing question is how toxins like abrin, ricin, and modeccin have evolved. These toxins contain in addition to the effectomer a binding or haptomer part which in effect is a lectin. Nontoxic lectins are widely distributed in the plant kingdom. Possibly, abrin, ricin, and modeccin have been as- sembled by the plants from inhibitor molecules and lectin molecules already present for other purposes. The fact that these toxins, which are closely related in structure and func- tion, are found in plants belonging to three different orders is consistent with this possibility.

Acknowledgments-We are indebted to Dr. T. Godal for help with lymphocyte cultures and to Miss Jannikke Ludt for her skiuful technical assistance.

REFERENCES 1. Pappenheimer, A. M., Jr. (1977) Annu. Rev. Biochem. 46,69-94 2. Olsnes, S., and Pihl, A. (1976) in Receptors and Recognition

Gelonin, a Protein Synthesis Inhibitor 6953

(Cuatrecasas, P., ed) Series B, Vol. 1, pp. 129-173, Chapman and Hall, London

3. Montanaro, L., Sperti, S., Zamboni, M. C., Denaro, M., Testoni, G., Gasperi-Campani, A., and Stirpe, F. (1978) Biochem. J. 176,

4. Olsnes, S., Sandvig, K., Eiklid, K., and Pihl, A. (1978) J. Supramol.

5. Yamaizumi, M., Mekada, E., Uchida, T., and Okada, Y. (1978)

6 . Eiklid, K., Olsnes, S., and Pihl, A. (1980) Exp. Cell Res. 126,321-

7. Obrig, T. G., Irvin, J . D., and Hardesty, B. (1973) Arch. Biochem.

8. Irvin, J . D. (1975) Arch. Biochem. Biophys. 169,522-528 9. Roberts, W. K., and Stewart, T. S. (1979) Biochemistry 18,2615-

10. Barbieri, L., Famboni, M., Lorenzoni, E., Montanaro, L., Sperti,

11. Stirpe, F., Pession-Brizzi, A,, Lorenzoni, E., Strocchi, P., Montan-

12. Sperti, S., Montanaro, L.. Mattioli, A., Testoni, G., and Stirpe, F.

13. Gasperi-Campani, A., Barbieri, L., Lorenzoni, E., and Stirpe, F.

14. Gasperi-Campani, A., Barbieri, L., Morelli, P., and Stirpe, F.

15. Olsnes, S. (1978) Methods Enzymol. 50, 323-330 16. Laemmli, U. K. (1969) Nature (Lond.) 227, 680-685 17. Olsnes, S., and Pihl, A. (1972) FEBS Lett. 20,327-329 18. Pelham, H. R. B., and Jackson, R. J . (1976) Eur. J. Biochem. 67,

19. Benson, S., Olsnes, S., Pihl, A., Skorve, J., and Abraham, A. K.

20. Olsnes, S., and Abraham, A. K. (1979) Eur. J. Biochem. 93,447-

371-379

Struct. 9, 15-25

Cell 15,245-250

326

Biophys. 155, 278-289

2621

S., and Stirpe, F. (1980) Biochem. J. 186,443-452

aro, L., and Sperti, S. (1976) Biochem. J. 156, 1-6

(1976) Biochem. J. 156, 7-13

(1977) FEBS Lett. 76, 173-176

(1980) Biochem. J. 186,439-441

247-256

(1975) Eur. J. Biochem. 59,573-580

452

21. Sandvig, K., Olsnes, S., and Pihl, A. (1976) J. Biol. Chem. 251,

22. Olsnes, S., Haylett, T., and Refsnes, K. (1978) J. Biol. Chem. 253,

23. Godal, T., Henriksen, A., Iversen, J. G., Landaas, T. O., and

24. Carlsson, J., Drevin, H., and Axen, R. (1978) Biochem. J. 173,

25. Olsnes, S., Refsnes, K., Christensen, T. B., and Pihl, A. (1975)

26. Kalb, V. F., Jr., and Bernlohr, R. W. (1977) Anal. Biochem. 82,

27. Wyatt, S. D., and Shepherd, R. J. (1964) Phytopathology 59,

28. Chang, T., and Neville, D. M., Jr. (1977) J. Biol. Chem. 252,

29. Chang, T., Dazord, A., and Neville, D. M., Jr. (1977) J. Biol. Chem. 252,1515-1522

30. Gilliland, D. G., Collier, R. J., Moehring, J. M., and Moehring, T. J. (1978) Proc. Natl. Acad. Sci. U. S. A . 75,5319-5323

31. Uchida, T., Yamaizumi, M., Mekada, E., Okada, Y., Tsuda, M., Kurokawa, T., and Sugino, Y. (1978) J. Biol. Chem. 253,6307- 6310

32. Masuho, Y., Hara, T., and Noguchi, T. (1979) Biochem. Biophys. Res. Commun. 90,320-326

33. Thorpe, P. E., Ross, W. C. J., Cumber, A. J., Hinson, C. A,, Edwards, D. C., and Davies. A. J. S. (1978) Nature 271, 752- 755

34. Oeltmann, T. N., and Heath, E. C. (1979) J . Biol. Chem. 254, 1022-1027

35. Oeltmann, T. N., and Heath, E. C. (1979) J. Biol. Chem. 254, 1028-1032

36. Yamaguchi, T., Kato, R., Beppu, M., Terao, T., Inoue, Y., Ikawa, Y., and Osawa, T. (1979) J. Natl. Cancer. Znst. 62, 1387-1395

3977-3984

5069-5073

Lindmo, T. (1978) Znt. J . Cancer 21,561-569

723-737

Biochim. Biophys. Acta 405, 1-10

362-371

1787-1794

1505-1514