Isolation and partial characterization of a new ribosome-inactivating ...

Planta

Planta (1992)186:532-540

9 Springer-Verlag1992

Isolation and partial characterization of a new ribosome-inactivating protein from Petrocoptis glaucifolia (Lag.) Boiss F. Javier Arias 1, M. Angeles Rojo 1, J. Miguel Ferrcras 3, Rosario Iglesias 1 **, Raquel Mufioz 1, Asunci6n Rocher 2, Enrique Mendez 2, Luigi Barbieri 3, and Tomfis Girb~s I * 1 Departamento de Bioquimica, Biologia Molecular y Fisiologia, Facultad de Ciencias, Universidad de Valladolid, E~47005 Valladolid, z Servicio de Endocrinologia, Centro Ram6n y Cajal, Carretera de Colmenar Viejo Km 9,100, E-28034 Madrid, Spain, and 3 Dipartimento di Patologia Sperimentale, Universitfi di Bologna, 1-40126, Bologna, Italy Received 19 February; accepted 29 August 1991

Abstract. Petrocoptis 9laucifolia, a paleoendemic member of the Caryophyllaceae from the North of Spain, was found to contain at least five proteins that inhibit protein synthesis in a rabbit reticulocyte lysate. One of them, for which the name petroglaucin is proposed, was purified to apparent electrophoretic homogeneity by chromatography through S-Sepharose Fast Flow, Sephadex G-75 and CM-Sepharose Fast Flow. The apparent M r of the preparation was 27500. This protein does not contain appreciable glycan chains and displays 45.8% of NHzterminal amino-acid sequence homology with some ribosome-inactivating proteins from Saponaria officinalis, another member o f the Caryophyllaceae. Petroglaucin shows the following functional properties: (i) it strongly inhibits the rabbit-reticulocyte-lysate system and Vicia sativa cell-free extracts, both coded by endogenous messengers, and also inhibits poly(U)-directed polyphenylalanine synthesis by Vicia sativa cell-free extracts and purified rat-liver ribosomes; (ii) it shows much less inhibitory capacity in wheat-germ, Cucumis sativus and rat-liver cell-free systems coded by endogenous messengers; (iii) the inhibitory effects on purified rat-liver ribosomes were irreversible; (vi) it promotes the release of adenine from purified rat-liver ribosomes. The total activity of this translational inhibitor has been found to increase up to 1 l-fold during its purification, indicating that some regulatory factor that normally blocks the translational inhibitory activity of the ribosome-inactivating protein in crude extracts of the plant is removed during purification. Key words: Petrocoptis Protein synthesis inhibitor Ribosome-inactivating protein - Translation (inhibition)

* To whom correspondence should be addressed ** Present address. Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ont., N 9 B3 P4 Canada Abbreviations: DFA=dimethylformamide; PAGE =polyacrylamide gel electrophoresis; poly(U) =polyuridylic acid; RIP = ribosome inactivating protein; SDS = sodium dodecyl sulfate

Introduction Several screening surveys have shows that many plant species contain proteins acting as powerful translational inhibitors and called ribosome-inactivating proteins (Gasperi-Campani et al. 1980; Gasperi-Campani et al. 1985; Ferreras et al. 1989a, b; Merino et al. 1990). The ribosome-inactivating proteins (RIPs) act only on eukaryotic ribosomes and enzymically inactivate the 60S ribosomal subunit (Jimenez and Vfizquez 1985; Roberts and Selitrennikoff 1986; Stirpe and Barbieri 1986). Recently, an N-glycosidase which acts on 28S r R N A to release adenine 4324 has been found to be associated with some of these types of inhibitors (Endo and Tsurugi 1987; Stirpe et al. 1988). The RIPs were classified some years ago as types I (one polypeptide chain) and II (two polypeptide chains) (Barbieri and Stirpe 1982). F r o m a functional point of view, RIPs have different specificities towards ribosomes from different sources such as plants (Harley and Beevers 1982; Batelli et al. 1984; Stirpe et al. 1986; Merino et al. 1990), animals (Brigotti et al. t989) and protozoa (Cenini et al. 1987; Cenini et al. 1988). The biological role played by these proteins in the plant producing them has not yet been elucidated (Roberts and Selitrennikoff 1986; Stirpe and Barbieri 1986). These proteins are broadly distributed throughout the plant species and families so far examined (GasperiCampani et al. 1985; Roberts and Selitrennikoff 1986). Studies o f NHz-terminal amino-acid sequences have shown that different isoforms of RIPs may be produced within the same family of plants (Stirpe and Barbieri 1986; Montecucchi et al. 1989). The interest in RIPs lies not only in their biological role but also in their potential usefulness in the construction of bioconjugates with carriers such as monoclonal antibodies, hormones, or growth factors (Barbieri and Stirpe 1982; Vitetta and Uhr 1985; Frankel et al. 1986; Singh et al. 1989; Koppel 1990), and in their therapeutical use (Frankel et al. 1986; Lord 1987; Till et al. 1988; McGrath et al. 1989; Koppel 1990). These proteins are

F.J. Arias et al.: A new RIP from Petrocoptis i m m u n o g e n i c . T h e r e f o r e , t h e s e a r c h f o r n e w R I P s is justified, taking into account that in the designing of b i o c o n j u g a t e s it is d e s i r a b l e t o h a v e a v a i l a b l e a l t e r n a t i v e t o x i n s t o c i r c u m v e n t t h e i r n e u t r a l i z a t i o n b y i m m u n e responses. In this work, we describe the isolation and some structural and functional properties of a new RIP present in Petrocoptis glaucifolia, a c a r y o p h y l l a c e a e n p l a n t e n d e m ic i n t h e N o r t h o f S p a i n . T h i s n e w R I P a c t s a s a n u n u s u ally powerful and specific inhibitor of protein synthesis only in some cell-free translation systems. A distinct f e a t u r e o f t h i s p r o t e i n is t h a t its t o t a l a c t i v i t y w a s i n c r e a s e d s e v e r a l t i m e s as a r e s u l t o f its p u r i f i c a t i o n .

Material and methods Plant material. Petrocoptis glaucifolia (Lag.) Boiss. Subsp. viscosa (Rothur.) Lainz was collected in the Cantabrian mountains (Spain) in April and stored at - 2 0 ~ C. Seeds of wheat (Triticum aestivum L.), Vicia sativa L., Vicia lens L. and Cucumis sativus L., obtained from commercial sources, were imbibed in sterile tap water and were incubated on layers of moist filter paper at 20 ~ C under sterile conditions in darkness for 3-4 d. Polyuridylic acid(poly(U)), 5'triphosphate nucleotides, creatine phosphokinase, creatin phosphate and dithiothreitol were purchased from Boehringer (Mannheim, FRG). Amino acids and wheat-germ t R N A mixture were from Sigma (St. Louis, Mo., USA). N-Succinimidyl-3-(2-pyridylthio)propionate and 2-iminothiolane were from Fluka (Buchs, Switzerland). Ready-Safe scintillation mixture was from Beckman Instruments (Palo Alto, Cal., USA). ~-[3H]Valine (sp. act. 2.14 TBq - m m o l - 1) and L-[14C]phenylalanine (sp. act. 19 G B q - m m o 1 - 1 ) were purchased from New England Nuclear through Itisa (Madrid, Spain). All reagents and solvents for the sequencer were purchased from Knauer (Berlin, F R G and for the amino-acid analyzer from Beckman Instruments. Chloracetaldehyde was prepared according to M c C a n n et al. (1985). All other reagents used in this work and not specified were of the highest purity available and purchased from E. Merck (Darmstadt, FRG). All chromatographic materials were purchased from PharmaciaLKB (Uppsala, Sweden).

Isolation of the RIP. We followed the procedure described by Barbieri et al. (1987). Freeze-dried plant material (100 g) was extracted overnight under vigorous stirring with 2 1 of 5 m M sodium phosphate (pH 7.2) containing 0.14 M NaC1. The resulting paste was centrifuged at 10000 - g for 45 min at 4 ~ C. Thereafter, the supernatant was removed, avoiding disturbance of the solidified upper fat pad, and filtered through two layers of cheescloth. The resulting fluid was acidified with glacial acetic acid to pH 4 and the solids that appeared were removed by centrifugation at 10000-g for 30 min at 4 ~ C. The resulting supernatant was then filtered through Sephadex G-25 equilibrated with 10 m M sodium acetate (pH 4.5). Thereafter, the clear eluate was applied to an S-Sepharose Fast Flow column (12 cm long, 2.6 cm i.d.) equilibrated with 10 m M sodium acetate (pH 4.5) at a flow rate of 100 ml 9 h-1. The column was washed with 5 m M sodium phosphate (pH 7) at the same rate and the eluate was discarded. The protein retained by the column was eluted with 5 m M sodium phosphate (pH 7) containing 1 M NaC1, at the same flow rate, and aliquots of 4 ml were taken. All the inhibitory protein eluted in the fractions 10-13, and was applied directly to a Sephadex G-75 column (125 cm long, 2.6 cm i.d. previously equilibrated with 5 m M sodium phosphate (pH 7). The column was eluted with the same buffer at a flow rate of 275 ml 9 h 1 The fractions containing inhibitory activity (18-33, 143 ml) were pooled and applied to a CM-Sepharose Fast Flow column (12 cm long, 1.3 cm i.d.) equilibrated with the same buffer. After washing with the same buffer, but this time containing 5 m M NaC1,

533 the column was eluted at a flow rate of 250 ml 9 h - 1 with a 5-400 m M NaC1 linear gradient in the same buffer, and fractions of 4 ml collected. The elution was stopped once 0.3 M NaC1 was reached. The eluate was then assayed for inhibition of protein synthesis and those fractions containing activity were pooled, freeze-dried, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Preparation of cell-free translation systems. The cell-free translation systems were obtained in RNase-free conditions at 0-2 ~ C as follows. In the case of plants, 1-3 g of embryonic axes were dissected by hand, and placed in a precooled @20~ C) mortar and were then ground in the coldroom for 10-12 min. Thereafter, 0.2 ml of extraction buffer containing 25 m M KC1, 20 m M Tris-HC1 (pH 7.8), 9 m M MgC1 z and 5 m M dithiothreitol were added, mixed, and the resulting extract was placed in centrifuge tubes to obtain the 30000 - g supernatant (S-30) as described elsewhere (Merino et al. 1990; Mufioz et al. 1990a). The rat-liver cell-free extracts and the rabbit reticulocyte lysates were obtained by standard procedures as described elsewhere (Pelham and Jackson 1976; Martin et al. 1979). All S-30 systems were filtered through Sephadex G-25 in order to remove low-molecular-weight compounds.

Preparation of purified rat-liver ribosomes. Rat-liver ribosomes were prepared essentially as described by Stahelin and Falvey (1971) in RNase-free conditions. Livers were homogenized in 4 vol. of 0.2 M sucrose containing 20 m M Tris-HC1 (pH 7.8), 10 mMg-acetate, 150 m M KCI, l m M dithiothreitol. The homogenate was centrifuged 10 min at 10000 9g at 2 ~ C. The supernatant was centrifuged for 15 min at 30000 9g at 2 ~ C. The supernatant was diluted 1:1 with cold homogenization buffer containing 2 % (w/v) Triton X-100 and 1% (w/v) sodium deoxicholate, and then centrifuged 3 h at 105000 "g at 2 ~ C. The pellet was resuspended in 24 ml of homogenization buffer containing 0.5 M KC1, and was centrifuged 10 min at 20000. g at 2 ~ C. The supernatant was centrifuged on a cushion of 3 ml of homogenization buffer containing 60% (w/v) sucrose. A 1-mg aliquot of ribosomes was assumed to contain 250 pmol of ribosomes (Montanaro et al. 1978).

Assay of cell-free translation. The assay for protein synthesis coded by either endogenous messenger or poly(U) was performed in RNase-free conditions and contained the components indicated in Table 1. All the cell-free translation systems were optimized for the most important parameters. The concentrations of either S-30 supernatant or ribosomes and postribosomal supernatant required for translations were optimized for each new preparation. After incubation at 30 ~ C for either 20 min, in the case of rabbit reticulocyte lysates, or 30 min in rat liver and plant germs, the reaction was stopped by addition of hot trichloroacetic acid and processed as reported elsewhere (Girbrs et al. 1983). In the cases of rat liver and plant germs, 0.1 ml of 1 m g . ml 1 bovine serum albumin was added to facilitate precipitation (Girbrs et al. 1979). For translation in the S-30 supernatant of V. sativa coded by poly(U), 10 m M MgClz and 80 lag poly(U) were used.

Protein synthesis in isolated rat liver cells. The isolation and purification of rat liver cells was carried out as indicated previously (Girbrs et al. 1983). Incubation and sampling of liver cells, and the assessment of the radioactivity incorporated into protein was carried out as reported elsewhere (Girbrs et al. 1989). Adenine release from petroglaucin-treated ribosomes. Adenine released from treated ribosomes was determined as its etheno derivative and measured by high-performance liquid chromatography (HPLC) as described by Zamboni et al. (1989) essentially following the procedure of M c C a n n et al. (1985). Incubation was in 10 m M NH4CI, 7 m M Mg-acetate, 1 m M dithiothreitol and 20 m M TrisH C1 (pH 7.5). Each sample contained 25 pmol of ribosomes and the appropriate amount of RIP in a final volume of 100 lal. Controls were run without ribosomes and with added adenine.

F.J. Arias et al.: A new RIP from Petrocoptis

534

Table 1. Composition of the cell-free systems. The final volumes were 100 lal for both endogenous messenger-dependent translation and poly(U)-dependent translations Final concentration (mM)

Cell-free translation system Rabbit reticulocyte lysate

Rat-liver endogenous messengers

Rat-liver poly(U)

Plants extracts

NH4C1 Mg-acetate KCI Tris-HC1 (pH 7.8) Dithiothreitol ATP GTP Creatine phosphokinase (mg - ml 1) Creatine phosphate Heroin (laM) Protein amino acids minus L-Val Spermidine Inhibitors of proteases Heparin (mg. m l - 1) Poly(U) (rag. m l - l) S-30 extract or lysate (pl) a Postribosomal supernatant (mg p r o t e i n - m l - 1 ) Ribosomes (pmol 9ml 1) L-[3H]Valine (sp. act. 2146 GBq 9m m o l - l)b L-[14C]Phenylalanine (sp. act. 19 GBq - m m o l - 1)c

100 2.4 10 5 4 1 0.05 8 25 0.1 0.2 10

50 3 180 26 6 4 1 0.05 8 0.1

100 7 20 1 1 0.6 0.003 10 -

25 6 9 28 24 6 4 1 0.05 8 0.1

1

0.01 10

_

-

-

0.8

-

1

0.008 -

0.003 -

20 -0.0003

10 -

0.007 -

a These values currently were optimized for each preparation b The amounts of added radiactivity were: 3.7 kBq, 0.93 kBq and 2.78 kBq for rabbit reticulocyte lysate, rat liver and plant systems, in the order stated c 0.55 kBq added per reaction mixture

Amino-acid analysis. Protein was hydrolyzed with 100 lal of 5.7 M HC1 containing 0.05% (v/v) 2-mercaptoethanol in evacuated and sealed tubes at 110 ~ C for 24, 48 and 72 h as described by Barber et al. (1988). The analysis was performed on a Beckman 6300 mod. analyzer equipped with a CR3A integrator (Shimadzu, Kyoto, Japan). Sequencing procedure. The protein was sequenced as described by Limas et al. (1990) in a new Knauer Protein Sequencer (Model 810) equipped on-line with a Knauer PTH-amino-acid analyzer. The PTH-amino acids were identified and quantified on a reverse-phase HPLC system based upon a C-18 narrow-bore column and gradient elution with 85% 6.5 mM sodium acetate, 15% acetonitrile, adjusted to pH 4.77 as buffer A and 100% acetonitrile as buffer B. Sequences were performed in the absence of polybrene. Polyacrylarnide gel electrophoresis. Analyses by SDS-PAGE were carried out using the discontinous system of Laemmli (1970) with the Mighty-Small II system (8.3 97.4 cm 2) from Hoefer (San Francisco, Cal., USA; Technical Bulletin 134). The proteins used as standards were trypsin inhibitor (M r 20100), alcohol dehydrogenase (M r 37000), lactic dehydrogenase (Mr 35000), glutamate dehydrogenase (M r 54400), bovine serum albumin (Mr 68000). Protein quantification. For the analysis of the protein concentrations in the cell-free translation systems, the method of Lowry et al. (1951) was used. For diluted protein solutions the method of Bradford (1976) was used.

Detection ofglycoprote&s. The presence of glycoproteins was studied using the Glycan Detection Kit from Boehringer. Electrophoresis and staining and all blotting operations were as described in the instructions from the manufacturer.

Results

Isolation and purification of the inhibitor. Petrocopt& glaucifolia is a v e r y scarce, w o o d y species. T h u s , w e c h o s e to to c o l l e c t all t h e p a r t s f r o m t h e a v a i l a b l e p l a n t s . A f t e r acidification of the crude extract, the soluble material was chromatographed on an S-Sepharose Fast Flow c o l u m n to r e m o v e a c i d p r o t e i n s a n d m o s t o f t h e n o n proteinaceous material; basic proteins were then eluted at h i g h i o n i c s t r e n g t h (1 M NaC1). T h e s e p r o t e i n s w e r e t h e n c h r o m a t o g r a p h e d o n S e p h a d e x G - 7 5 . A s s h o w n in F i g . 1, t h e m a t e r i a l a b s o r b i n g at A~8o e l u t e d in t w o p e a k s . T h e p e a k m o v i n g slightly b e h i n d t h e f r o n t c o n tained a very high translational inhibitory activity when a s s a y e d in a r a b b i t r e t i c u l o c y t e lysate ( d i l u t i o n 1:1000). T h e s e c o n d p e a k ( a r o u n d f r a c t i o n 50) C o r r e s p o n d e d to l o w - m o l e c u l a r - w e i g h t m a t e r i a l a n d w a s also i n h i b i t o r y to p r o t e i n synthesis, p r o b a b l y b e c a u s e Of t h e p r e s e n c e o f p i g m e n t s , salts o r o t h e r l o w - m o l e c u l a r - w e i g h t c o m p o u n d s . I n m o r e d i l u t e d e l u a t e s ( 1 : 5 0 0 0 ) o n l y t h e first peak showed translational inhibitory activity. The fract i o n s c o m p r i s i n g the first i n h i b i t o r y p e a k w e r e p o o l e d and chromatographed on a CM-Sepharose Fast Flow column with a NaC1 gradient (0.005-0.4 M). Several p e a k s o f a c t i v i t y i n h i b i t o r y t o p r o t e i n s y n t h e s i s in t h e r e t i c u l o c y t e - l y s a t e s y s t e m w e r e d e t e c t e d (Fig. 2). T h e s e p e a k s w e r e n u m b e r e d f r o m I to V. P e a k I I I p r o v e d to be t h e m o s t a c t i v e a n d c o n t a i n e d a single p o l y p e p t i d e , as j u d g e d b y S D S - P A G E (Fig. 3). W e p r o p o s e n a m i n g this protein petroglaucin. In the presence of 2-mercapt o e t h a n o l , this p r o t e i n m i g r a t e d as a single b a n d w i t h a n

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FRACTION NUMBER Fig.1. Purification of the translational inhibitor(s) from P. glaucifolia by chromatography through Sephadex G-75. Experimental conditions are described in detail in Material and methods. The absorbance at 280 nm (--) was recorded. The eluate was assayed for inhibition of protein synthesis in the rabbit reticulocyte lysate at dilutions of 1 : 1000 (e) and 1 : 5000 (9 The fractions indicated by the horizontal bar were pooled and subjected to chromatography through CM-Sepharose FF

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2. Purification of translational inhibitor(s) from P. glaucifolia by chromatography through CM-Sepharose FF. Experimental conditions are described in detail in Material and methods. (--), absorbance at 280 nm; (..-.), 0.005~).4 M NaCl gradient; (-e--), effect of eluate on protein synthesis carried out by the rabbit reticulocyte lysate at a dilution of 1 : 1000 Fig.

Fig. 3 a - c . Polyacrylamide-gel-electrophoretic profiles of petroglaucin and other well-known RIPs. The SDS-PAGE runs in the presence (a, e) or the absence of 2-mercaptoethanol (b) were conducted in gels of 10% at 20 mA for 60 min. Thereafter, two gels were stained with Coomassie blue (a, b). The third gel was blotted onto an Immobilon membrane (Millipore) and treated for glycan detection with the Glycan Detection Kit from Boehringer (c). Standards were those indicated in Material and methods. Lanes la, lb, petroglaucin; lanes 2a, 2b, PAP-S (pokeweed antiviral protein, from seads) lanes 3a, 3b, gelonin; lanes 4a, 4b, Saporin 6; lanes 5a, 5b, standards; lane lc, 4e, petroglaucin; lane 2c, gelonin; lane 3c, D-32. The arrows on the left ir~dicate the M, of standards in kDa

Mr o f 27500. In the absence o f the reducing agent the protein mobility was slightly increased. This a b n o r m a l b e h a v i o r is shared by the p o k e w e e d antiviral protein f r o m seeds ( P A P - S ) , the R I P f r o m Phytolacca americana (Bjorn et al. 1984), but n o t by gelonin a n d dianthin 30, the R I P s f r o m Gelonium multiflorum (Stirpe et al. 1980) a n d Dianthus caryophyllus (Stirpe et al. 1981), respectively. Petroglaucin p r o v e d not to be a glycoprotein, since as shown in Fig. 3c, it was insensitive to the glycan-detection system. Some other well-characterized R I P s were used as controls (Fig. 3c). The yield o f petroglaucin was rather low by c o m p a r i s o n with that o f other R I P s (Table 2; Stirpe and Barbieri 1986).

A m i n o - a c i d composition and N H z - t e r m i n a l amino-acid sequence ofpetroglaucin. The a m i n o - a c i d c o m p o s i t i o n in a p p r o x i m a t e n u m b e r s o f residues is s h o w n in Table 3. The protein is rich in Asp, Glu, Gly, Ala, Ileu, Leu, Lys and Arg. The first 24 residues o f the a m i n o - t e r m i n a l amino-acid sequence are s h o w n in Fig. 4. W h e n this s e q u e n c e was c o m p a r e d with the N H z - t e r m i n a l sequences o f several isoforms o f saporin, a n o t h e r c a r y o phyllaceaen R I P , 45.8% o f h o m o l o g y with saporins 5a, 5b, 6a and 6b, and 25 % with saporin 1 were found. W e studied the hydrophilicity profile o f the N H z - t e r m i n a l segment according to H o p p a n d W o o d s (1981) a n d c o m p a r e d it with that o f saporin 6 (Lappi et al. 1985). It

536

F.J. Arias et al.: A new RIP from Petroeoptis

Table 2. Purification ofpetrogtaucin. The protein was purified as described in the text. Inhibition of cell-free translation was performed using rabbit reticulocyte lysates as described in Material and methods. One unit (U) is defined as the amount of inhibitory protein which is required to reduce by 50% the translation in a l-ml reaction mixture P. glaucifolia

(100 g of dried material) Crude extract Acidified crude ext. S-Sepharose FF Sephadex G-75 CM-Sepharose FF

Volume (ml)

Protein (mg)

ICs0 (ng-m1-1)

Spec. act. Total act. ( x l 0 - 6 U - m g -~) ( x l 0 6U)

1330 1280 20 143 0.5 a

593 125 20 2.5 0.3"

17 6.2 0.1 0.006 0.8"

0.06 0.16 9.8 163 1.4"

Yield (%)

35 20 200 408 0.42"

(100) 58 571 1165 1.2"

a Values were obtained from peak III of the eluate from CM-Sepharose FF chromatography

Table 3. Amino-acid composition of petroglaucin. Composition is expressed in residues per mole of protein based on an Mr of 27 500 as judged by SDS-PAGE. Each value represents the average from 24, 48 and 72 h hydrolysis Amino acid

No. of residues"

Cys b Asp Thr Ser Glu Pro Gly Ata Val Met lle Leu Tyr Phe Lys His Arg Trp b

26 19 14 24 9 18 24 18 4 15 26 5 9 23 1 14

Values of residues in rounded-off numbers b Trp and Cys were not determined

Table 4. IC5o values of petroglaucin in cell-free translations carried

out by several animal and plant extracts. Experimental details were as described in Material and methods'. The ICso values were obtained from dose-response curves. The incorporation of radiactivity into protein in each cell-free system was as follows: V. lens 61 B q " mg -1 of protein; wheat germ 448 Bq " mg x of proteins; V. sativa 1361 Bq-mg 1 of protein; V. sativa (with poly(U)) 3667 B q " nag-1 of protein; C. sativus 571 B q " nag ~ of protein; rat liver (extracts) 1269 Bq-mg 1 of protein; rat liver (purified ribosomes) 29 350 B q 9 mg ~ of ribosomes; rabbit reticulocyte lysates 72 B q 9 rag- ~ of protein Cell-free translation system

ICso for inhibition of translation (ng 9ml 1)

Vicia lens (S-30) Wheat germ (S 30) Vicia sativa (S 30) Vicia sativa (S 30 suplemented with poly(U)) Cucumis sativus (S 30) Rat liver (S 30) Rat liver (purified ribosomes) b Rabbit reticulocyte lysate

3500 2200 6 16

~

~

800 (29) 1050 (54) 0.5 (0.18) 0.7 (0.025)

a Values in parentheses represent the ICs0 in nmol. 1 1 b Coded by poly(U) and suplemented with a postribosomal supernatant

1 Petroglaucin

(127) a (80) (0.2) (0.6)

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was found that both petroglaucin and saporin 6 have a v e r y s i m i l a r h y d r o p a t h i c c h a r a c t e r in t h e N H 2 - t e r m i n a l stretch (data not shown). The purified translational i n h i b i t o r a c t e d o n r a b b i t r e t i c u l o c y t e l y s a t e at less t h a n a n e q u i m o l e c u l a r r a t i o (the l y s a t e w a s a s s u m e d to c o n Functional character&ation.

t a i n r i b o s o m e s a t a c o n c e n t r a t i o n o f 3.13 n M ; B a r b i e r i et al. 1989). W e f u r t h e r s t u d i e d the i n h i b i t o r y a c t i o n o f p e t r o g l a u c i n o n o t h e r cell-free s y s t e m s o f a n i m a l a n d plant origin coded by endogenous messengers. As shown in T a b l e 4, it i n h i b i t e d p r o t e i n s y n t h e s i s v e r y efficiently in r a b b i t r e t i c u l o c y t e lysates (0.7 n g - m l - 1 ) a n d in t h e cell-free s y s t e m o f Vicia sativa (6 ng 9 m l - t ) . I n c o n t r a s t ,

F.J. Arias et al.: A new RIP from

Petrocoptis

537

Table 5. The poly(U)-directed synthesis of polyphenylalanine by purified rat-liver ribosomes. Incubation I: ribosomes (90 pmol)

Table 6. Effects of petroglaucin on the release of adenine from purified rat-liver ribosomes. Adenine released was measured by

were incubated in the absence ( - , sample 1) or in the presence (+, sample 2) of an equimolecular concentration of petroglaucin for 10 min at 37~ C in a volume of 1 ml with all the components needed for protein synthesis. The reaction was arrested in ice. Aliquots of 2 pmol of ribosomes were removed and the polyphenylalanine synthesized measured as described in Material and methods. Samples 1 and 2 were loaded onto a 5% (w/v) sucrose cushion and ultracentrifuged for 4 h at 195000"9. Incubation II: washed ribosomes from samples I and 2 were resuspended and incubated either in the absence ( - ) or in the presence (+) of equimolar amounts of petroglaucin in conditions of translation for 10 rain at 37~ C. Thereafter, the extent of polyphenylalanine synthesis was assessed as described in Material and methods

high-performance liquid chromatography. Protein-synthesis inhibition was determined by the inhibition of poly(U)-directed phenylalanine polymerization as described in Material and methods. In the absence of RIP, ribosomes (2 pmol) polymerized 5.03 pmol of phenylalanine in 10 min at 37~ C

Petroglaucin added

Phenylalanine polymerized (pmol)

Incubation I

Incubation II

Incubation I

Incubation II

-

-+ -+

5.03

5.89 2.45 2.55 2.19

+

2.33

Molar ratio RIP/ribosomes (pmol of RIP/pmol of ribosomes)

Adenine released (pmol/pmol of ribosomes)

Poly(U)-directed phenylalanine polymerization (%)

0:1 1:10 1:1

0 0.64 0.76

100 69 46.2

100 A

9--, (/) g

its inhibitory activity with cell-free extracts f r o m rat liver, Vicia lens, w h e a t germ, and Cucumis sativus, was small w h e n c o m p a r e d with the other two systems. We also studied the effect o f the toxin on poly(U)-directed polyphenylalanine synthesis in two systems, namely purified rat-liver ribosomes supplemented with a postribosomal s u p e r n a t a n t directed by poly(U), and S-30 f r o m V. sativa directed by added poly(U). As s h o w n also in Table 4, the toxin inhibits in b o t h translation systems. However, in the rat-liver system the effect on polyphenylalanine synthesis was stronger t h a n the effect on protein synthesis c o d e d by e n d o g e n o u s messenger. In m a m malian cell-free systems the sensitivity o f translation to inhibitors was recently reported to be dependent on the nature o f the messenger being used (Mufioz et al. 1990b). In contrast, with V. sativa the results were the same regardless o f the messenger translated. The inhibitory effect o f petroglaucin on poly(U)directed polyphenylalanine synthesis carried o u t by purified rat-liver ribosomes was irreversible. As s h o w n in Table 5, petroglaucin inhibited either when it was present during the assay as well as when it was preincubated with ribosomes, which were then washed by ultracentrifugation t h r o u g h a sucrose cushion before assay. In b o t h cases petroglaucin inhibited by nearly the same extent, as expected for a ribosome-inactivating protein (Stirpe et al. 1986). We also studied the effect o f petroglaucin o n protein synthesis in isolated rat hepatocytes. As with other type-I R I P s (Stirpe a n d Barbieri 1986), the effect was relatively low when c o m p a r e d with the effect on a n y cell-free system, in spite o f the large concentrations o f toxin used. The rate o f radioactivity i n c o r p o r a t e d into proteins was 340 + 39 Bq (mg F W cells)- 1. h - 1 in c o n t r o l cells versus 277 + 22 Bq (mg F W cells)-1, h-~ in cells treated with 6 ktg " m l - 1 o f toxin. Effects o f petroglaucin on the release o f adenine from rat liver ribosomes. As s h o w n in Table 6, the i n c u b a t i o n o f

O 80-

oj

Ill

0

v-

60-

_z

40 -

o e,-

20-

Z

el

o

[]

~z~

]

i 30

60

TEMPERATURE

90 (*C)

Fig. 5. Thermal stability of petroglaucin from P. glauc~olia. Petro-

glaucin (150 ng 9m1-1) was incubated in 20 gl of H20 for 20 min at the indicated temperatures either in the absence (9 or the presence of 50% (v/v) glycerol (1:3) or 250 mM threhalose (A). Thereafter, the toxin (60 ng. ml-1) was assayed for inhibition of protein synthesis in rabbit reticulocyte lysates as indicated in Material and methods. 100% represents 68 Bq 9 -1

purified rat-liver ribosomes with petroglaucin leads to the release o f adenine as described for some other wellk n o w n R I P s ( E n d o and Tsurugi 1987; Z a m b o n i et al. 1989). Even at less than equimolecular ratiO, the toxin leads to the release o f adenine with the c o n c o m i t a n t inactivation o f the ribosomes. Stability ofpetroglaucin. Petroglaucin loses activity when incubated at temperatures over 60 ~ C for several minutes. As illustrated in Fig. 5, the R I P was fully inactivated by incubating at 95 ~ C. Agents, like glycerol or threhalose, that prevent protein d e n a t u r a t i o n by heat (Ferreras et al. 1989b) were only partially effective in protecting petroglaucin (Fig. 5). Threhalose was s o m e w h a t m o r e efficient t h a n glycerol at temperatures higher t h a n 75 ~ C. By contrast, glycerol was the m o s t efficient agent at temperatures lower than 75 ~ C. O w i n g to the lack o f suitable radical g r o u p s in m a n y R I P s these m u s t n o r m a l l y be derivatized with chemical

538 linkers in order to obtain immunotoxins and other bioconjugates (Koppel 1990). We studied the potential damaging effect of two linking agents, N-succinimidyl-3-(2pyridyldithio)propionate and 2-iminothiolane, on the inhibitory action of petroglaucin. Neither of them, up to a concentrations ratio of 18000 molecules of linker to 1 molecule of petroglaucin, affected the inhibitory capacity of the protein (data not shown).

Discussion

The presence of several translational protein inhibitors in extracts from plant species belonging to the Caryophyllaceae has been previously described. Several forms of saporin from Saponaria officinalis have been isolated, but only saporin 6 has been highly purified and partially characterized (Lappi et al. 1985). In this work we have shown that Petrocoptis glaucifolia also contains several proteins with translational inhibitory activity on rabbit reticulocytes lysates. A single-chain protein has been isolated (peak III) that (i) fulfills the major requirements to be considered as an RIP according to data previously obtained using partially purified extracts from P. glaucifolia (Ferreras et al. 1989a), (ii) that irreversibly damages rat-liver ribosomes, and (iii) that shows N-glycosidase activity measurable as release of adenine from ribosomes (Endo and Tsurugi 1987; Stirpe et al. 1988). All this has allowed us to classify this protein as a type-I RIP which we have called petroglaucin. S-Sepharose Fast Flow chromatography yielded a preparation containing nearly sixfold more total activity than the crude extract; this was further increased to nearly 11-fold after chromatography through Sephadex G-75 (Table 2). This observation indicates that the purification process could have removed some highly active inhibitor(s) of the RIP present in both crude and acidified extracts. Some degree of activation (15-20%) by partial purification has been achieved at least in two other cases: during the isolation ofluffin the RIP from Luffa cylindrica Roehm (Kishida et al. 1983) - and of momorcochin - S, the RIP from the seeds of Momordica conchinchinensis (Bolognesi et al. 1989). However, the effect reported here for petroglaucin is more pronounced. This could be explained by a modification in the primary structure due to proteolytic cleavage of precursor forms or, perhaps, deglycosilation. An important question is the comparatively low activity observed in the purified petroglaucin (0.42 9 106 U) as compared with the activity of the five mixed inhibitory peaks eluted from Sephadex G-75 (408- l06 U). There are several possibilities to account for such large differences in activity. One is that purified petroglaucin could be partially inactivated, for example, by freezing/ thawing-dependent precipitation or by oxidation. Another could involve the loss of some cofactor(s) during the last stage of purification. Yet another could be the existence of isoforms of petroglaucin that acted synergically in potentiating ribosome inactivation. We have no evidence supporting any of these possibilities; however, in view of the precautions taken during the

F.J. Arias et al.: A new RIP from Petrocoptis purification procedure of petroglaucin, the last possibility seems to be the most plausible. Petroglaucin shows a fairly good sequence homology with saporin 6 when compared with other RIPs, even those from the same plant species (i.e. momordin I and II isolated from Momordica charantia which display 48 % of NHz-terminal sequence homology; Montecucchi et al. 1989). Similar hydropathic profiles between petroglaucin and saporin 6 were also found (data not shown). Even though there is good homology between RIPs from the same family, it is often difficult to find any substantial homology when RiPs from different families are compared (Montecucchi et al. 1989). Apparently, RIPs inactivate ribosomes in the same way (Stirpe et al. 1988). The lack of sequence homology between RIPs from different families indicates that their mechanism of action is not closely correlated to the primary structure of the N H zterminal end. The plant systems used in our work translated endogenous messengers using homologous protein factors. Those were conditions of maximum coupling; therefore, they should probably detect subtle differences in translation sensitivity to RIPs better than reconstituted plant translation systems like those used by Harley and Beevers (1982) and Batelli et al. (1984). The marked differences in sensitivity to petroglaucin found between the four plant translation systems used by us supports the hypothesis put forward recently that plant ribosomes exhibit conformational and-or structural differences that can be recognized by some RIPs but not by others (Stirpe and Hughes 1989). We have confirmed and extended this proposal also to include mammalian systems for a given RIP (i.e. compare rat liver and rabbit reticulocyte lysates in Table 4). The high sensitivity of the V. sativa cell-free translation system to petroglaucin could be related to the fact that these extracts lack RIP activity (Merino et al. 1990; data not shown). Accordingly, V. sativa ribosomes most probably are not adapted to the intracellular presence of this kind of toxin. The fact that some plant translation systems show a higher degree of sensitivity to petroglaucin than the rat-liver system offers the possibility of using this toxin as a probe for comparative biochemical studies of ribosomes from different organisms, especially plant ribosomes, which in general are not well understood. The potency of petroglaucin on some animal and plant systems, and its chemical stability makes it a good tool for the elaboration of toxin-carrier bioconjugates for human therapy. It may also be potentially useful to transform plants with the gene encoding for this protein in order to protect them against parasitic diseases of plants. Further work will be carried out to isolate isoforms of petroglaucin, to compare their structures and functional properties, and to study their toxicities, either alone or modified, on cells and intact animals, and also to isolate and characterize their genes. The work in Valladolid was supported by grants from CICYT (BIO 88~)705), Junta de Castilla y Le6n and Iberduero S.A.. The work in Bologna was supported by grants from Ministerio della Pubblice Istruzione, Associazione Italiana per la Ricerca sul Cancro, Consig-

F.J. Arias et al.: A new RIP from Petrocoptis lio Nazionale delle Ricerche, within the Progetto finalizzato Biotenologia e biostrumentazione. F.J. Arias and M.A. Rojo hold fellowships from Iberduero S.A.. J.M. Ferreras and R. Iglesias hold postdoctoral fellowships from Ministerio de Educacirn y Ciencia. We thank Professor R. Parrilla (Instituto Gregorio Marafion, Madrid) for his critical reading of the manuscript. The supervision of the English version of the manuscript by N. Skinner is greatly appreciated.

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