Biochimica et Biophysica Acta, 1087 (1990) 293-302 293 Elsevier

BBAEXP 92182

Purification and properties of new ribosome-inactivating proteins with R N A N-glycosidase activity

Andrea Bolognesi 1, Luigi Barbieri 1, Ada Abbondanza 1, Anna Ida Falasca 2, Domenica Carnicelli 1, Maria Giulia Battelli 1 and Fiorenzo Stirpe 1

1 Dipartimento di Patologia Sperimentale and 2 Dipartimento di Biochimica dell' Universitd, di Bologna, Bologna (Italy)

Key words: Asparin, 1 and 2; Bryodin-L; Colocin, 1 and 2; Lychnin; Mapalmin; PAP-R; Ribosome-inactivating protein; RNA N-glycosidase

Ribosome-inactivating proteins (RIPs) similar to those already known (Stirpe & Barbieri (1986) FEBS l~tt. 195, 1-8) were purified from the seeds of Asparagus officinalis (two proteins, asparin 1 and 2), of CitruUus colocynthis (two proteins, colocin 1 and 2), of Lychnis chalcedonica (lychnin) and of Manihot palmata (mapalmin), from the roots of Phytolacca americana (pokeweed antivirai protein from roots, PAP-R) and from the leaves of Bryonia dioica Coryodin-L). The two latter proteins can be considered as isoforms, respectively, of previously purified PAP, from the leaves of P. americana, and of bryodin-R, from the roots of B. dioica. All proteins have an M r at approx. 30000, and an alkaline isoelectric point. Bryodin-L, colocins, lychnin and mapalmin are glycoproteins. All RIPs inhibit protein synthesis by a rabbit reticulocyte lysate and phenylalanine polymerization by isolated ribosomes and alter rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912).

Introduction

Ribosome-inactivating proteins from plants (RIPs, reviews in Refs. 1-4) catalytically inactivate the 60S ribosomal subunits of eukaryotic ribosomes. They can be either single-chain proteins (RIPs type 1) or two- chain proteins (RIPs type 2), in which one chain has the enzymic activity and the other one has the properties of a galactose-specific lectin [1]. RIPs type 1 are more frequent, being present in several parts of many and possibly all plants, including seeds, roots, leaves and latices [5], sometimes in more than one form, possibly isoforms. RIPs have an unusual N-glycosidase activity, and cleave the N-glycosidic bond of adenine 4324 of 28S rRNA [6-8] introducing a lesion that renders RNA cleavable by aniline at a site adjacent to the cleavage induced by a-sarcin. This makes ribosomes unable to bind the elongation factors 1 [9] or 2 [10,11], with consequent arrest of protein synthesis. In spite of the

Abbreviations: RIP(s), ribosome-inactivating protein(s); PAP, poke- weed antiviral protein; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; ICso , concentration causing 50% inhibition; SPDP, N-succinimidyl-3-(2-pyridylthio)propionate; 2-IT, 2-iminothiolane.

Correspondence: F. Stirpe, Dipartimento di Patologia Sperimentale, Via San Giacomo 14, 1-40126 Bologna, Italy.

numerous similarities in their structure and of the iden- tical enzymic activity, RIPs have different effects on ribosomes from different organisms (from plants, proto- zoa, insects and other metazoa) [12-17].

All RIPs tested inhibited the infectivity of plant and animal viruses. Actually the first identified RIP type 1, namely the pokeweed antiviral protein (PAP), was ini- tially purified as an anti-viral protein (reviewed in Ref. 18). This property of RIPs was attributed to easier penetration into virally infected cells with consequent inactivation of their ribosomes and arrest of viral multi- plication. Recently, however, a RIP type 1, tri- chosanthin, was reported to inhibit replication of hu- man immunodeficiency virus through a mechanism ap- parently independent of the effect on ribosomes [19].

Interest in RIPs is growing since they are used as components of ' immunotoxins', hybrid molecules con- sisting of a toxic moiety linked to an antibody [20]. Hopefully immunotoxins will be useful to eliminate harmful cells, neoplastic, immunocompetent and para- sitic cells being considered as possible targets. The availability of several RIPs is essential to solve some problems arising in the preparation of immunotoxins: (i) not all RIPs are effective on target cells; (ii) some RIPs become more toxic, once bound to antibodies; and (iii) some RIPs are inactivated during the conjuga- tion procedure. Further, the in vivo administration of immunotoxins, being RIPs heterologous proteins, would rise an immune reaction neutralizing their action. This

0167-4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

294

could be circumvented with conjugates prepared with several RIPs, immunologically distinct from each other.

The availability of other RIPs would also be useful to increase our knowledge of the distribution and of the antiviral and other properties of these proteins, and of their effects on different ribosomes, which in turn could lead to a better understanding of ribosomal structure and function.

We describe here the purification and some proper- ties of new RIPs that we propose to call: asparin 1 and 2, from the seeds of Asparagus officinalis (asparagus), bryodin-L, from the leaves of Bryonia dioica (white bryony), probably an isoform of bryodin-R (from roots, perviously called bryodin [21]), colocin 1 and 2, from the seeds of Citrullus colocynthis, lychnin, from the seeds of Lychnis chalcedonica, mapalmin, from the seeds of Manihot palmata, and PAP-R, from the roots of Phytolacca americana, probably an isoform of PAP [18]. Some of these RIPs had been identified previously [22], but their purification in convenient amounts is now possible since a large-scale procedure was developed [23] that allows to purify RIPs even from materials where they are present at a relatively low concentration.

Materials and Methods

Materials Plant materials were obtained from the following

sources: A. officinalis seeds were purchased from Con- sorzio Agrario Provinciale, Bologna; Citrullus coloc- ynthis seeds were grown in the Giardino officinale of the Azienda Regionale delle Foreste (Casola Valsenio, RA, Italy), from seeds obtained from the Botanic Garden of the University of Padua, Italy; Lychnis chalcedonica seeds were purchased from Florsilva (Idice, BO, Italy); Bryonia dioica leaves and Manihot palmata seeds were from the Botanic Garden of the University of Bologna; Phytolacca americana roots were from a private garden.

L-[14C]Leucine and L-[14C]phenylalanine were from Amersham International (Bucks, U.K.). Cell culture medium and supplements were as in previous work [21]. SPDP and materials for chromatography, including calibrating substances, were from Pharmacia AB, (Up- psala, Sweden). 2-IT, poly(U) and tRNA were from Sigma Chemical (St. Louis, MO, U.S.A.). Phenyl- alanine-tRNA was prepared as described by Hultin & N~islund [24]. Electrophoresis markers were from LKB (Uppsala, Sweden). All other chemicals were as in pre- vious work [25]. PAP and PAP II were a gift of Dr. J.D. Irvin, PAP-S was prepared essentially following the procedure described by Barbieri et al. [23], and were further purified on Blue-Sepharose as described for PAP-C [26].

Purification of ribosome-inactivating proteins The purification of mapalmin, colocin 1 and 2 and

lychnin was carried out following the same small-scale procedure (25-50 g of starting material).

Seeds of Citrullus colocynthis, Lychnis chalcedonica and Manihot palmata, the latter previously ground in a mortar, were homogenized with an Ultraturrax appara- tus in 8 volumes of cold PBS (0.14 M sodium chloride/5 mM sodium phosphate buffer (pH 7.5)). The slurries were extracted overnight at 4 °C with magnetic stirring, filtered through cheesecloth and clarified by centrifuga- tion at 3 0 0 0 0 X g for 30 min at 4 °C. These crude extracts were adjusted to pH 4.0 with glacial acetic acid and centrifuged as above. The supernatants were ap- plied to an S-Sepharose column (20 x 2 cm) equi- librated with 20 mM sodium acetate (pH 4.5). The column was washed with 1 vol. of the equifibration buffer and then extensively with 5 mM sodium phos- phate buffer (pH 6.7). Bound proteins were eluted with 1 M sodium chloride in the same buffer; active fractions were pooled, dialysed exaustively against 5 mM sodium phosphate buffer (pH 6.7) at 4 ° C and centrifuged as above to remove precipitated material. The dialysed extracts were applied to a CM-Sepharose Fast Flow column (25 x 1.6 cm) equilibrated with the same buffer, but for colocins preparation that was performed at higher ionic strength (see Fig. 1). The column was washed with the equilibration buffer and eluted with a linear gradient (500 + 500 ml) of sodium chloride, as specified in the legend to Fig. 1. Fractions inhibiting protein synthesis were pooled, dialysed extensively against water at 4 o C, and freeze-dried.

Colocins were further purified by gel filtration on a Sephacryl S-200 column (90 x 5 cm) in PBS.

Asparin 1 and 2, bryodin-L and PAP-R were purified essentially following the large-scale procedure described by Barbieri et al. [23].

Asparagus officinalis seeds (1 kg) were homogenized with an Ultraturrax apparatus in 8 volumes of PBS. Bryonia dioica leaves (12.5 kg) were ground in a mortar with liquid nitrogen and homogenized with an Ultratur- rax apparatus in 4 volumes of PBS. Phytolacca americana roots (2.7 kg) were peeled, cut in cubes, and the juice was extracted with the aid of a kitchen centri- fuge. The residual pulp was homogenized with an Ultra- turrax apparatus in 3 volumes of PBS. The slurries were extracted overnight at 4 ° C with magnetic stirring and filtered through cheesecloth. The crude extracts (but for bryodin-L preparation) and the root juice were centri- fuged at 10000 × g for 45 min at 4°C. The super- natants of the juice and of the extracts of Phytolacca americana roots were combined.

The supernatants of the crude extracts were adjusted to pH 4.0 with glacial acetic acid, centrifuged as above and the precipitates were discarded. The supernatant from A. officinalis was then vacuum filtered on What-

man No. 2 paper. The acidified extracts were applied to an S-Sepharose column (12 x 10 cm) equilibrated with 20 mM sodium acetate (pH 4.5). The column was extensively washed with 5 mM sodium phosphate buffer (pH 7.0) (pH 6.85 for asparins preparation) and bound proteins were eluted with 1 M sodium chloride in the same buffer. The active fractions were pooled and pre- cipitated by the addition of ammonium sulphate to saturation at 4 ° C. The precipitated material was re- covered by centrifugation as above, the pellet was dis- solved in 5 mM sodium phosphate buffer (pH 7).

Further purification procedure of asparins was dif- ferent from that of bryodin-L and PAP-R.

Asparins-containing solution was dialysed against water at 4 ° C, then clarified by centrifugation as above. The supernatant was adjusted to 5 mM phosphate buffer (pH 6.85) and applied to a Sephacryl S-200 column (85 x 10 cm) in the same buffer. Active fractions were pooled and chromatographed on a CM-Sepharose Fast Flow column (30 x 5 cm) equilibrated with 5 mM sodium phosphate buffer (pH 6.85). During this ion exchange chromatography, the pH is critical to obtain the separation of asparin 1 and 2. The column was washed with the equilibration buffer and eluted with a linear gradient (10 + 10 1) of sodium chloride, as speci- fied in the legend to the Fig. 1. Two active peaks were collected, dialysed extensively against water, adjusted to 10 mM Tris-HC1 (pH 8.0) and each of them was chro- matographed through a column of Red-Sepharose (13 x 1.6 cm) equilibrated and washed in the same buffer. Purified asparins were eluted with a 0-0.25 M sodium chloride gradient in the same buffer (500 + 500 ml), dialysed against water and freeze-dried. Asparin 2 was separated from a low molecular weight contaminant by gel filtration on Sephacryl S-200 (90 x 5 cm) equi- librated and eluted in PBS.

After concentration with saturated ammonium sulphate, bryodin-L and PAP-R containing solutions were clarified by centrifugation as above and chromato- graphed through a Sephacryl S-200 (Sephadex G-50 for PAP-R preparation) (85 x 10 cm) in 5 mM sodium phosphate buffer (pH 7.0). Active fraction were pooled and absorbed to a CM-Sepharose Fast Flow column (30 x 5 cm) equilibrated and washed with the same buffer. The column was eluted with a linear gradient (10 + 10 1) of sodium chloride, as specified in the legend to the Fig. 1. The active peaks were adjusted to pH 4.0 with glacial acetic acid and concentrated by chromatog- raphy on a CM-Sepharose column (5 x 5 cm) equi- librated with 20 mM sodium acetate buffer (pH 4.5). The column was washed with the equilibration buffer and proteins were eluted with 1 M sodium chloride/5 mM sodium phosphate buffer (pH 7.0) dialysed exten- sively against water at 4 °C and freeze-dried. The freeze dried proteins were dissolved in 10 mM Tris-HC1 (pH 8.0) and applied to a Blue-Sepharose column (25 x 1.6

295

cm) equilibrated in the same buffer. The column was washed with the equilibration buffer and absorbed pro- teins were eluted with a linear 0-0.2 M sodium chloride gradient in the same buffer (400 + 400 ml), dialysed against water and freeze dried.

Physico-chemical determinations M, values were determined by electrophoresis and by

gel filtration. The polyacrylamide-gel-electrophoresis method of Laemmli [27] was used with the following markers: cytochrome c (5//, 12 300), myoglobin (17200), carbonic anydrase (30 000), ovalbumin (45 000), bovine serum albumin (66 250) and ovotransferrin (76-78 000). Gel filtration was through a column (95 cm x 1.6 cm) of Sephacryl S-200, equilibrated with 0.3 M-sodium chlo- r ide/20 mM sodium phosphate buffer (pH 7.5) and calibrated with ribonuclease A (M, 13 700), chymotryp- sinogen A (25 000), ovalbumin (43000), bovine serum albumin (67000) and dextran blue (2-106).

Isoelectric point and the composition in amino acid, amino sugar and neutral sugar of the protein were determined as described by Falasca et al. [28].

Aminoterminal sequence was determined by the method described by Lappi et al. [29].

Protein was determined spectrophotometrically [30] or by the method of Lowry et al. [31], with bovine serum albumin as a standard.

Modification of ribosomal RNA The effect of RIPs on rRNA was analysed as de-

scribed by Endo et al. [6]. Samples (0.01 ml) of re- ticulocyte lysate were incubated in the presence of 1 /~g/ml RIPs for 20 min at 37°C. The reaction was arrested by addition of SDS to 0.5% final concentration. RNA was extracted with phenol and precipitated with ethanol. For both analysis and purification of frag- ments, RNA was subjected to electrophoresis on 5% acrylamide gels and gels were stained with silver as described previously [8] (details in the legend to Fig. 2).

Preparation of sulfhydryl-modified proteins RIPs were modified with SPDP or with 2-IT, two

reagents used to conjugate the components of im- munotoxins, as described in previous work [25]. The number of sulfhydryl groups introduced per molecule of protein was determined as in [32] for SPDP and as in [33] for 2-IT.

Cell cultures The cells used, all of human origin, were: primary

cultures of trophoblasts and fibroblasts, and the cell lines HeLa, TG (oviduct carcinoma), NB 100 (neuro- blastoma), JAR and BEWO (choriocarcinoma). Cells were maintained as monolayer cultures in RPMI 1640 medium supplemented with antibiotics and 10% foetal calf serum, in a humidified atmosphere containing 5%

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TABLE I

Purification of ribosome-inactivating proteins

The purification procedures are described in the text. Cell-free protein synthesis was determined with a rabbit reticulocytes lysate as de- scribed [22]. The ion exchange chromatographies are shown in Fig. 1. 1 unit of activity is defined as the amount of protein necessary to inhibit protein synthesis by 50% in 1 ml of rabbit reticulocyte lysate reaction mixture.

Protein IC50 Total Yield (g) (ng/ml) activity (%)

(10 -6 • units)

Asparagus officinalis (1 kg of seeds)

Crude extract Acidified extract

S-Sepharose Sephacryl S-200 CM-Sepharose (asparin 1)

(asparin 2) Red-Sepharose I (asparin 1) Red-Sepharose II (asparin 2) Sephacryl S-200 (asparin 2)

Bryonia dioica (12.5 kg of fresh leaves)

Crude extract acidified S-Sepharose Sephacryl S-200 CM-Sepharose Blue-Sepharose (bryodin-L)

Citrullus colocynthis (50 g of seeds)

Crude extract Acidified extract S-Sepharose CM-Sepharose (colocin 1)

(colocin 2) Sephacryl S-200 1 (colocin 1) Sephacryl S-200 II (colocin 2)

Lychnis chalcedonica (25 g of seeds)

Crude extract Acidified extract S-Sepharose CM-Sepharose (lychnin)

Manihot palmata (25 g of seeds)

Crude extract Acidified extract S-Sepharose CM-Sepharose (mapalmin)

Phytolacca americana (2.7 kg of roots)

Crude extract Acidified extract S-Sepharose Sephadex G-50 CM-Sepharose Blue-Sepharose (PAP-R)

23.2 127 184 (100) 13.6 61.4 221 120

7.55 28.3 266 145 2.04 14.4 142 77.2 0.159 10.1 15.7 8.5 0.229 4.59 49.9 27.2 0.054 8.09 6.67 3.6 0.081 5.48 14.8 8.0 0.049 4.46 11.0 6.0

294 1726 170 (100) 1.74 12.3 141 83.2 0.411 8.13 50.6 29.7 0.034 3.97 8.69 5.1 0.021 2.76 7.61 4.5

0.908 123 7.35 (100) 0.627 32.1 19.6 266 0.129 9.33 13.8 187 0.007 1.20 5.83 79.4 0.006 1.32 4.55 61.8 0.005 1.20 4.17 56.7 0.004 1.26 3.17 43.2

0.826 56.6 14.6 (100) 0.783 23.3 33.7 231 0.323 23.0 14.1 96.6 0.040 4.54 8.70 59.6

0.412 57.0 7.21 (100) 0.384 57.0 6.72 93.2 0.103 18.5 5.44 75.5 0.004 1.49 2.52 35.0

39.6 54.8 723 (100) 33.7 44.8 752 104

not determined 1.79 3.00 597 82.5 0.327 2.50 131 18.1 0.192 1.50 128 17.7

297

CO 2, at 3 7 ° C . Subcul tu res were ob t a ined by t ryps in t r ea tmen t of conf luent cultures.

Effect on protein synthesis Cell-free p ro te in synthesis was measu red as descr ibed

[22] wi th a r abb i t re t iculocytes lysa te [34], or as po ly (U) -d i rec ted pheny la l an ine po lymer i za t i on with r ibosomes pur i f ied f rom a r abb i t re t iculocytes lysate [35]. The effect of RIPs on p ro te in synthesis by cells was evalua ted f rom the i nco rpo ra t i on of [3H]leucine, af ter a 2 h pulse wi th 0.1 # C i / m l , as descr ibed previ- ously [21].

The IC50 (concen t ra t ion giving 50% inhib i t ion) was ca lcu la ted by l inear- regress ion analysis .

Toxicity to mice and histological preparation The toxici ty of the pur i f ied p ro te in was eva lua ted in

Swiss female mice weighing 2 7 - 3 2 g as descr ibed previ-

ously [21]. LDs0 were ca lcu la ted at 48 h of po i son ing by the l inear regression method . F r o m dead animals , sam- ples of the fol lowing organs were removed, f ixed in

3.7% fo rma ldehyde in sal ine and e m b e d d e d in paraf f in : heart , intest ine, k idney, liver, lung, pancreas and spleen.

Sect ions were cut and s ta ined with hematoxy l in -eos in for l ight microscopy.

Results

Purification of ribosome-inactivating proteins The genera l p rocedure desc r ibed by Barbier i et al.

[23] was fol lowed with some modi f i ca t ions dur ing the p r epa ra t i on of c rude extracts in o rde r to a d a p t the me thod to di f ferent s ta r t ing mater ia ls . The behav iou r of var ious RIPs dur ing pur i f i ca t ion was very similar . Some difference was no ted dur ing ion-exchange ch roma tog ra -

phy, in that each R I P e lu ted at a d i f ferent ionic strength. Fu r the rmore , the pur i f i ca t ion of a spa r in 1 and 2 re- qui red a di f ferent pH, as shown in Fig. 1.

Bryod in -L and P A P - R were fur ther pur i f ied on Blue-Sepharose as descr ibed for P A P - C [26]. This proce- dure was not effective for aspar ins pur i f ica t ion , which was achieved by c h r o m a t o g r a p h y on the more acid resin Red-Sepharose , fol lowed (for a spa r in 2 only) b y gel f i l t ra t ion on Sephacry l S-200. Pur i f ied p ro te ins were homogeneous at S D S - P A G E (Fig. 2).

The p ro te in - syn the t i c inh ib i to ry act ivi ty at the ma in pur i f ica t ion steps and the yield of the pur i f ied RIPs are given in Table I. The highest yield of R I P was o b t a i n e d f rom the seeds of Lychnis chalcedonica. As obse rved prev ious ly with o ther extracts , in some cases the inhibi - tory act ivi ty recovered af ter some s t e p s of pur i f i ca t ion was higher than the or ig inal act ivi ty, poss ib ly due to removal of an inh ib i to r or to an ' a c t i va t i on ' of the RIP.

298

Mr(k) 7 6 - 7 8 - - .

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..,-17.2 - - - 1 2 . 3

a b Fig. 2. Polyacryamide-gel electrophoresis of ribosome-inactivating proteins. Electrophoresis was performed as described in the text. Staining was with Coomassie blue. Migration was from top to bottom. The gel was loaded with 3-10 jag of each protein, but lychnin that was overloaded to show the purity, as an example. (a) Lane 1, asparin 1; lane 2, asparin 2; lane 3, bryodin-L; lane 4, PAP-R. (b) Lane 1, lychnin; lane 2, mapalmin;

lane 3, colocin 1; lane 4, colocin 2.

Properties of purified ribosome-inactivating proteins The main physico-chemical characteristics of the

purified RIPs are summarized in Table II. Like previ- ously purified RIPs, they have M r in the 30 000 region and an alkaline pI. Bryodin-L, colocins, lychnin and mapalmin are giycoproteins, whereas asparins and PAP- R do not contain sugars.

The amino acid composition of RIPs purified from the same material is obviously very similar, whereas there are differences amongst protein from different sources. Bryodin-L, colocins 1 and 2, and mapalmin do not contain cyst(e)ine.

The NHz-terminal amino acid sequence of PAP-R shows complete identity with those of PAP and of PAP-C, and several homologies with that of dode- candrin, a RIP from Phytolacca dodecandra, a plant taxonomically close to Phytolacca americana, and more limited homologies with PAP-II and with PAP-S (Table III).

Biological properties All RIPs inhibited protein synthesis by a rabbit

reticulocyte lysate, colocin 1, mapalmin and PAP-R being the most active proteins (Table IV). In all cases the ICs0's were well below nM, and similar to those previously reported for other RIPs. The inhibitory ef- fect was somewhat less marked on ribosomes purified from the same reticulocyte lysate.

The purified proteins inhibited also protein synthesis by whole cells, but only at concentrations some thou- sand-fold higher than those affecting cell-free systems. There was a significant variation in the effect on the various cells examined, NB 100, a neuroblastoma-de-

rived cell line, being the most sensitive, and HeLa cells the most resistant.

All proteins damaged rRNA in the same manner as ricin and other RIPs, as is indicated by the fragment that can be obtained by aniline treatment of rRNA extracted from ribosomes incubated with the RIPs (Fig. 3).

The toxicity of RIPs to mice is shown in Table V. The LDso'S were in the average range of toxicity as compared with other RIPs type 1, except those of PAP-R and asparin 1 which were, respectively, in the higher and lower range. Histological examination of the organs of mice killed by lethal doses of the purified RIPs showed mainly necrotic alterations in the liver as observed with other RIPs.

In view of the possible use of the newly identified RIPs for the preparation of immunotoxins, their resis- tance to modification with SPDP or 2-iminothiolane was examined. No loss of activity was observed after modification of PAP-R with SPDP. With all other RIPs a variable loss of activity, more marked in the case of lychnin, was observed. Unexpectedly colocin 1 was in- activated more by 2-iminothiolane than by SPDP, at difference with what observed in other cases (Table VI).

D i s c u s s i o n

Described above are eight additions to the list of RIPs already known. The proteins purified with present experiments are RIPs, in that they inhibit protein synthesis by inactivating ribosomes in a catalytic manner and modify rRNA in an apparently identical manner as

TABLE II

Physico-chemical properties of ribosome-inactivating proteins

Experimental conditions are described in the text.

299

Asparin 1 Asparin 2 Bryodin-L Colocin 1 Colocin 2 Lychnin Mapalmin PAP-R

M R (gel filtration) 29 700 (SDS-PAGE) 30 500

pI 8.7 1~ 10 A1 ¢m.280

Total neutral sugar content (%) traces

Fuc

Glc Man Xyl

28100 27 300 20 400 19 500 20 000 26 900 25 000 29 800 28 800 26 300 26 300 26 600 32 300 29 800

9.2 > 9.5 > 9.5 > 9.5 > 9.5 > 9.5 > 9.5 10 8 6.3 7 7.3 7.1 8

traces 2.72 0.40 1.59 0.31 5.99 traces

Amino acid composition (mol /mol )

Sugar composition (mol /mol ) "

1.52 0 0 0 2.26 0.43 0.65 2.58 0.51 5.83 2.52 0 0 0 3.46 0.63 0 0 0 0.96

a,b

Lys 15.3 14.9 10.8 His 3.4 2.9 1.0 Arg 14.4 14.8 11.0 Asx 26.5 27.2 25.5 Thr 13.9 14.2 17.4 Ser 11.0 11.3 24.4 Glx 24.3 23.2 18.9 Pro 14.9 15.4 7.2 Gly 15.0 15.2 11.4 Ala 19.5 18.8 24.1 1 / 2 Cysc 3.3 1.3 0 Val 16.7 16.9 14.4 Met 4.1 1.1 2.2 Ile 11.4 11.5 15.4 Leu 26.3 26.6 24.5 Tyr 9.8 9.8 11.7 Phe 6.3 6.3 7.4 Trp n.d. n.d. n.d.

13.0 13.1 16.7 11.5 18.0 2.4 2.0 1.3 1.0 1.6 9.1 9.5 10.5 14.1 10.0

22.7 23.2 22.9 27.4 32.3 12.4 12.5 11.9 16.5 17.7 23.7 22.3 17.5 26.1 17.0 15.4 14.9 18.1 24.0 20.7

5.4 5.8 6.1 12.6 11.2 23.4 12.9 12.4 11.5 13.4 16.7 16.5 18.0 17.8 12.8

0 0 2.6 0 3.4 10.8 10.6 12.7 18.6 17.0

1.3 1.0 2.1 1.1 3.3 15.8 15.7 7.5 13.3 15.0 18.5 18.6 21.4 25.9 21.0 12.0 11.6 9.4 12.8 8.8

7.2 7.4 14.1 13.5 7.8 n.d. n.d. n.d. n.d. n.d.

Calculated assuming the M, determined by SDS-gel electrophoresis. b Average values from the hydrolysis at 24, 48 and 72 h. c Determined as cysteic acid and methionine sulphone.

RIPs do. They differ from previously identified RIPs for the amino acid and sugar composition.

Asparins are the first RIPs purified from a plant member of the Liliaceae family. Bryodin-L is a new example of RIP purified from plants belonging to the Cucurbitaceae family, after momordin I (previously

called M. charantia inhibitor [38]), luffin [39], bryodin-R [21], t r ichosantin [40], trichokirin [41] and momorcochin-S [25]. Bryodin-L and PAP-R are from plants (bryony, pokeweed) in other parts of which RIPs had been found. Bryodin-R, PAP, PAP-II and PAP-S are different from bryodin-L and PAP-R, respectively,

TABLE III

Aminoterminal sequences of ribosome-inactivating proteins from Phytolaccaceae

Sequences of PAP, PAP II and PAP-S are from Bjorn et al. [36], dodecandrin from Ready et al. [37] and PAP-R present experiments.

PAP-C V N T I I Y N V G S T T I S K Y A T F L N D L R N E A K D P S PAP T I I Y N V G S T T I S K Y A T F L N D L R N E A K D P S PAP II - ~ I ~ T ~ ' ~ Y ~ - - ~ F L~ -~L R ~ E A ~ K : K L T PAP-S ~ N T I I N L ~ Y A T ElM E S lL X N E A - K D o d e c a n d r i n I V N T I I Y N V B S T T ! SlNIY A T FIR D N IL R N E A - K D P S LI

300

TABLE IV

Protein synthesis inhibition by ribosome-inactivating proteins

Cell-free protein synthesis was measured with a rabbit reticulocytes lysate as described in the legend to Table I and as phenylalanine polymerisation with ribosomes purified from a rabbit reticulocytes lysate as described [35]. The inhibition of protein synthesis by various cells was determined as described [21]. Experimental conditions are described in the text.

Protein synthesis inhibition (IC50)

Asparin 1 Asparin 2 Bryodin-L Colocin 1 Colocin 2 Lychnin Mapalmin PAP-R

Reticulocytes ICs0 (nM)

Lysate 0.27 0.15 0.09 0.04 0.13 0.17 0.05 0.05 Ribosomes 8.8 6.9 1.3

Cells IC50 (/~M)

Trophoblasts - - 0.18 0.32 0.47 0.46 - Fibroblasts > 3.33 2.02 0.80 0.40 0.16 0.67 0.61 0.07

HeLa > 3.33 > 3.33 > 3.33 > 3.33 1.41 > 3.33 > 3.33 > 3.33 TG 0.61 a 0.21 a 0.77 a 0.54 0.25 2.11 1.68 1.07 NB 100 0.18 0.18 0.05 0.01 0.004 0.03 0.03 0.18 JAR > 3.33 > 3.33 > 3.33 0.32 0.14 1.53 1.64 0.50 BEWO - - - 0.23 0.10 0.33 0.08 -

a Mean of two separate experiments.

as indicated by the amino acid composition. At least in the case of PAP-R, however, the differences from other forms of PAP must be small, since the first part of the respective amino acid sequence is identical. Presumably, they are isoforms (isoenzymes) as probably are the other RIPs previously purified from the same source and the colocins and asparins described above.

All RIPs examined are more active on the crude reticulocyte lysate system than on ribosomes purified from the same lysate, consistently with previous ob- servations with other RIPs [22]. It is possible that poly(U)-directed phenylalanine polymerization is less sensitive to RIPs than mRNA-directed globin synthesis. It is also possible that manipulation of ribosomes pro-

gressively brings about a modification, conformational or otherwise, of rRNA that makes it less sensitive to RIPs. It should be remembered that purified rRNA is less sensitive than whole ribosomes to the action of ricin A-chain [7].

Like other RIPs, those described here are much less toxic to whole cells than to cell-free systems. The cyto- toxic effects vary greatly, both from RIP to RIP (by more than 10-fold in the case of NB 100 and TG cell lines and of fibroblasts) and from a cell type to another. The different cytotoxicity of RIPs does not appear related to their sugar content. A "neuroblastoma cell line, human trophoblasts and the chorioncarcinoma-de- rived BEWO cell line showed the highest sensitivity to

TABLE V

Effect of the artificial introduction of reactive groups suitable for the preparation of immunotoxins on the activity of ribosome-inactivating proteins

Ribosome-inactivating proteins were treated with N-succinimidyl 3-(2-pyridyldithio)-propionate, added at the indicated molar ratio, or with 1.25 mM 2-iminothiolane. Activity was measured from the inhibition of proteins synthesis by a rabbit reticulocytes lysate as described in the legend to Table I. Experimental conditions are described in the text.

Heterobi- Asparin 1 Asparin 2 Colocin 1 Colocin 2 Lychnin Mapalmin PAP-R

functional - S H a iCsob - S H a ics0b --SH ~ iCsob - S H ~ iCsob -SHa ics0b -SHa ics0b -SHa iCs0b reagent added (mo l / (nM) (mol / (nM) ( tool / (nM) (mol / (nM) (mol / (nM) (mol / (nM) (mol / (nM)

tool) mol) mol) tool) mol) mol) mol)

None - 0.27 - 0.15 - 0.04 - 0.13 - 0.17 - 0.05 - 0.05 SPDP c (1.5:1) 0.56 0.41 0.46 0.19 1.32 0.15 1.53 0.13 0.70 0.31 0.99 0.11 0.99 0.06

(2.0 : 1) 1.25 0.67 0.79 0.40 - - 2.35 0.32 . . . . . . 2-1T d (1.25 mM) 2.04 0.28 1.06 0.26 0.77 0.39 0.75 0.09 1.22 0.44 0.55 0.04 -

a Artificial sulfhydryl groups introduced per molecule of protein. b Concentration of the derivatized protein causing 505[ inhibition of protein synthesis by cell-free system. c N-succinimidyl 3-(2-pyridyldithio)-propionate. d 2-1minothiolane.

TABLE VI

Toxicity to mice of intraperitoneally injected ribosome.inactivating proteins

Experimental conditions are described in the text.

301

Asparin 1 Asparin 2 Bryodin-L Colocin 1 Colocin 2 Lychnin Mapalmin PAP-R

LDso 20.0 10.0 > 10.0 10.7 12.6 9.3 > 8.0 1.2 Maximum dosage

allowing 100% survival 2.5 2.5 - 1.8 4.0 2.0 1.0 < 0.3

RIPs. I t is no tewor thy that RIPs have abor t i fac ien t ac t iv i ty [40] a t t r ibu ted to their toxic i ty to t rophob las t s [42].

1 2 3 4 5 6 - 4" - + - +

Fig. 3. Gel electrophoretic analysis of rRNA from reticulocyte lysates inactivated by ribosome-inactivating proteins. Ribosome-inactivating proteins were present at concentrations of 1 /~g/ml as follows: lanes 1-2, none (controls); lanes 3-4, PAP-S; lanes 5-6, lychnin. Samples in lanes designated + were treated and those in lanes designated - were not treated with aniline, respectively. The arrow indicates the fragment split by aniline from rRNA of RIP-treated ribosomes. The samples were electrophoresed on a 5% acrylamide gel at 1000 V for 2

h, at 45 o C.

Art i f ic ia l d isulf ide groups can be in t roduced in all new RIPs ei ther with S P D P or wi th 2- iminoth io lane , with l i t t le ( lychnin) or no loss of activity. This makes these proteins , and especia l ly the least toxic ones, sui ta- ble for the p r epa ra t i on of immunotox ins . I t is useful to have several RIPs ava i lab le for this purpose , as p o in t ed out in the In t roduc t ion .

Al l together, p resen t and previous results show that RIPs are presen t in several t issues of the same p l a n t as s l ightly di f ferent forms, p r e s u m a b l y isoenzymes, some- t imes in more than one fo rm in the same tissue. I t should be po in t ed out that , in spi te of the same enzymic activity, RIPs m a y differ f rom each o ther in their effects

on r ibosomes f rom di f ferent o rgan i sms (plants [8], p ro- tozoa [14,15], me tazoa [17]) or on whole cells (p resen t results).

A c k n o w l e d g e m e n t s

W e are i ndeb ted to Dr. P. Cenin i for some exper i - ments on cell p ro te in synthesis. W e thank Mr. G. Bugamell i and Mr. L. Magagno l i of the Univers i ty Botanic G a r d e n for the seeds of M. palmata and the Az ienda Regiona le del le Fores te , Reg ione Emil ia - Romagna , for the seeds of C. colocynthis. The work was suppor t ed by grants f rom the Min i s te ro del la Pubb l i ca Is t ruzione, R o m e and f rom the Assoc iaz ione I t a l i ana per la Ricerca sul Cancro , by con t rac t s f rom the Con- siglio Naz iona l e delle Ricerche, Rome, wi th in the Pro- get to F ina l izza to 'B io tecno log ie e Bios t rumentaz ione ' , and f rom the Commiss ion of Eu ropean Communi t i e s ,

Bio technology Ac t ion P r o g r a m m e and by the Pa l lo t t i ' s Legacy for Cancer Research.

R e f e r e n c e s

1 Barbieri, L. and Stirpe, F. (1982) Cancer Surveys 1, 489-520. 2 Jimrnez, A. and Vazquez, D. (1985) Annu. Rev. Microbiol. 39,

649-672. 3 Roberts, W.K. and Selitrennikoff, P.C. (1986) Biosci. Rep. 6,

19-29. 4 Stirpe, F. and Barbieri, L. (1986) FEBS Lett. 195, 1-8. 5 Gasperi-Campani, A., Barbieri, L., Battelli, M.G. and Stirpe, F.

(1985) J. Nat. Prod. 48, 446-454. 6 Endo, Y., Mitsui, K., Motizuki, M. and Tsurugi, K. (1987) J. Biol.

Chem. 262, 5908-5912. 7 Endo, Y. and Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8139

302

8 Stirpe, F., Bailey, S., Miller, S.P. and Bodley, J.W. (1988) Nucleic Acid Res. 16, 1349-1357.

9 Nilsson, L., Asano, K., Svensson, B., Poulsen, F.M. and Nygard, O. (1986) Biochim. Biophys. Acta 868, 62-70.

10 Montanaro, L., Sperti, S. and Stirpe, F. (1973) Biochem. J. 136, 677-683.

11 Obrig, T.G., Irvin, J.D. and Hardesty, B. (1973) Arch. Biochem. Biophys. 155, 278-289.

12 Battelli, M.G., Lorenzorti, E., Stirpe, F., Celia, R. and Parisi, B. (1984) J. Exp. Bot. 35, 882-889.

13 Villemez, C.L., Russel, M.A., Barbieri, L., Stirpe, F. and Robertus, J.D. (1987) in Membrane-mediated Cytotoxicity (Bonavida, B. and Collier, R.J., eds.), pp. 175-182, A.R. Liss, New York.

14 Cenini, P., Battelli, M.G., Bolognesi, A. Stirpe, F. and Villemez, C.L. (1987) Biochem. Biophys. Res. Commun. 148, 521-526.

15 Cenini, P., Bolognesi, A. and Stirpe, F. (1988) J. Protozool. 35, 384-387.

16 Brigotti, M., Rambelfi, F., Zamboni, M., Montanaro, L. and Sperti, S. (1989) Biochem. J. 257, 723-727.

17 Cenini, P., Carnicelli, D. and Stirpe, F. (1990) Comp. Biochem. Physiol. 96B, 581-584.

18 Irvin, J.D. (1983) Pharmacol. Ther. 21,371-387. 19 McGrath, M.S., Hwang, K.M., Caldwell, S.E., Gaston, I., Luk,

K.-C/., Wu, p., Ng. V.L., Crowe, S., Daniels, J., Marsh, J., Deinhart, T., Lekas, P.V., Vennari, J.C., Yeung, H.-W. and Lifson, J.D. (1989) Proc. Natl. Acad. Sci. USA 86, 2844-2848.

20 Frankel, A.E., Houston, L.L. and Fathman, G. (1986) Annu. Rev. Med. 37, 125-142.

21 Stirpe, F., Barbiefi, L., Battelli, M.G., Falasca, A.I., Abbondanza, A., Lorenzoni, E. and Stevens, W.A. (1986) Biochem. J. 240, 659-665.

22 Stirpe, F., Gasperi-Campani, A., Barbieri, L., Falasca, A., Ab- bondanza, A. and Stevens, W.A. (1983) Biochem. J. 216, 617-625.

23 Barbieri, L., Stoppa, C. and Bolognesi, A. (1987) J. Chromatogr. 408, 235-243.

24 Hultin, T. and N~islund, P.H. (1978) Eur. J. Biochem. 88, 143-148. 25 Bolognesi, A., Barbieri, L., Carnicelli, D., Abbondanza, A., Cenini,

P., Falasca, A.I., Dinota, A. and Stirpe, F. (1989) Biochem. Bio- phys. Acta 993, 287-292.

26 Barbiefi, L., Bolognesi, A., Cenini, P., Falasca, A.I., Minghetti, A., Garofano, L., Guicciardi, A., Lappi, D., Miller, S.P. and Stirpe, F. (1989) Biochem. J. 257, 801-807.

27 Laemmli, U.K. (1970) Nature 227, 680-685. 28 Falasca, A., Gasperi-Campani, A., Abbondanza, A., Barbieri, L.

and Stirpe, F. (1982) Biochem. J. 207, 505-509. 29 Lappi, D.A., Esch, F.S., Barbieri, L., Stirpe, F. and Sofia, M.

(1985) Biochem. Biophys. Res. Commun. 129, 934-942. 30 Kalb, V.F., Jr. and Bernlohr, R.W. (1977) Anal Biochem. 82,

362-371. 31 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.

(1951) J. Biol. Chem. 193, 265-275. 31 Carlsson, J., Drevin, D. and Axen, R. (1978) Biochem. J. 173,

727-737. 32 Ellman, G.L. (1959) Arch Biochem. Biophys. 82, 70-77. 34 Allen, E.H. and Schweet, R.S. (1962) J. Biol. Chem. 237, 760-767. 35 Montanaro, L., Sperti, S., Zamboni, M., Denaro, M., Testoni, G.,

Gasperi-Campani, A., and Stirpe, F. (1978) Biochem. J. 176, 265-275.

36 Bjorn, M.J., Larrick, J., Piatak, M. and Robertus, J.D. (1984) Biochim. Biophys. Acta 790, 154-163.

37 Ready, M.P., Adams, R.P. and Robertus, J.D. (1984) Biochim. Biophys. Acta 791, 314-319.

38 Barbieri, L., Zamboni, M., Lorenzoni, E., Montanaro, L., Sperti, S. and Stirpe, F. (1980) Biochem. J. 186, 443-452.

39 Kishida, K., Masuho, Y. and Hara, T. (1983) FEBS Lett. 153, 209-212.

40 Yeung, H.W., Li, W.W., Feng, Z., Barbiefi, L. and Stirpe, F. (1988) Int. J. Peptide Prot. Res. 31,265-268.

41 Casellas, P., Dussossoy, D., Falasca, A.I., Barbieri, L., Guillemot, J.C., Ferrara, P., Bolognesi, A., Cenini, P. and Stirpe, F. (1988) Eur. J. Biochem. 176, 581-588.

42 Tsao, S.W., Yan, K.T. and Yeung, H.W. (1986) Toxicon 24, 831-840.