Interaction of cibacron blue F3GA and polynucleotides with ricin A-chain, 60 S ribosomal subunit-inactivating protein

Biochimica et Biophysica Acta 914 (1987) 177-184

177

Elsevier BBA 32889

Interaction of Cibacron blue F3GA and polynucleotides with ricin A-chain, 60 S ribosomal subunit-inactivating protein Keiichi Watanabe and Gunki Funatsu Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka (Japan) (Received 26 January 1987)

Key words: Ricin A-chain; Cibacron blue F3GA; Dye-protein interaction; Polynucleotide binding site; Ribosome inactivation

Cibacron blue F3GA, a sulfonated polyaromatic blue dye, inhibited the ability of ricin A-chain to inactivate ribosomes. Difference-spectroscopic study revealed that the dye bound to the A-chain ( K d --0.72 pM), producing a difference spectrum with a single maximum at 688 nm and two minima at 585 and 628 nm. Such a significant difference spectrum was not observed in the presence of ricin B-chain or intact ricin, neither of which can inactivate ribosomes. Modification of arginine residues in the A-chain with phenylglyoxal showed a correlation between the loss of inhibitory activity on protein synthesis and the loss of difference absorbance produced by the dye-A-chain interaction. Both losses occurred significantly at an early stage of the modification. Furthermore, the dye protected the A-chain against a loss of its inhibitory activity resulting from the modification of arginine residues. These results suggest that the same arginine residues participate both in the interaction with the dye and in the inactivation of ribosomes. Based on these data, the dye appears to interact with the active site of the A-chain. Addition of several polynucleotides, namely rRNA, tRNA, poly(U) and DNA, to the dye-A-chain complex resulted in a marked displacement of the dye, whereas mono- and dinucleotides had little or no effect on the dye-A-chain interaction. These findings indicate the possible existence of a polynucleotide binding site in the active site of the A-chain. A combination of these and other results suggests that the A-chain recognizes and acts on some part of RNA of the 60 S ribosomal subunit.

Introduction Ricin, a toxic lectin from seeds of the castor bean (Ricinus communis), consists of two different polypeptide chains (A- and B-chains) linked by a single disulfide bond [1,2]. The B-chain binds

Abbreviation: Buffer A, 20 mM Tris-HC1 buffer (pH 7.5)/5 mM magnesium acetate/100 mM NH4C1/1 mM dithiothreitol. Correspondence: G. Funatsu, Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka 812, Japan.

galactose-containing receptors onto the cell surface [3] allowing the toxin to enter cells, whereas the A-chain enzymaticaUy inactivates 60 S ribosomal subunits [4-6], resulting in an inhibition of elongation factors-dependent functions of ribosomes [5,7-9]. Although the A-chain has been recently reported to exhibit ribonulcease activity with naked ribosomal RNA [10], rRNA-cleavage has not been detected when intact ribisomes are treated with the A-chain [10-12]. Thus, the molecular mechanism for ribosome inactivation is still not clear. The A-chain is a glycoprotein consisting of 265 amino-acid residues, and its amino-acid sequence has been determined in our laboratory

0167-4838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

178

[13]. Our chemical modification studies have demonstrated that the arginine residues of the A-chain are essential for the inactivation of ribosomes [14]. Cibacron blue F3GA, a sulfonated polyaromatic blue dye, can be used for probing nucleotide binding sites in many proteins (for example, Refs. 15-17). The A-chain has been reported to interact with this dye [18]. This interaction, however, has not been analyzed in detail, nor has a relationship between the interaction with the dye and the inactivation of ribosomes been established. Here we present experiments which suggest that Cibacron blue F3GA and polynucleotides, including rRNA, interact with the active site of the A-chain, and that the same arginine residues participate both in the interaction with the dye and in the inactivation of ribosomes. Materials and Methods

Materials. Yeast tRNA, poly(U) and puromycin dihydrochloride were purchased from Boehringer Mannheim. Calf thymus DNA, creatine kinase (type I) ( A T P : c r e a t i n e N - p h o s photransferase, EC 2.7.3.2) and pyruvate kinase (type II) ( A T P : p y r u v a t e O2-phosphotransferase, EC 2.7.1.40) were from Sigma Chemicals. Phenylglyoxal monohydrate was from Aldrich Chemicals. L-[4,5-3H]Leucine (60 C i / m m o l ) and L-[U14C]phenylalanine (514 m C i / m m o l ) were from Amersham. All other reagents used were of analytical grade. General preparations. Ricin (ricin D) was prepared as described in Ref. 19. A- and B-chains were isolated from reduced ricin as described previously [20,21]. Protein concentration was determined by amino-acid analysis with a JLC-6AH amino-acid analyzer after hydrolysis of protein with 5.7 M HC1 containing 0.05% 2-mercaptoethanol at 107 ° C for 24 h. Cibacron blue F3GA from Ciba-Geigy was further purified by chromatography on a Whatman 3MM paper [22]. The dye concentration was measured spectrophotometrically at 610 nm using an absorption coefficient of 13.6 mM -1 • cm -a [16]. Crude rat liver polysomes, S-100 and p H 5 enzymes were prepared by established methods [23]. S-100 was dialyzed against 20 mM Tris-HC1

buffer (pH 7.5)/100 mM NH4C1/5 mM magnesium acetate/1 mM dithiothreitol (buffer A) and clarified by centrifugation. The 80 S ribosomes were prepared by a procedure involving the puromycin treatment of the crude polysomes [24]. The ribosome concentration was determined by assuming 17 pmol/A260 unit [25]. Anti-A-chain antibody was prepared from the serum of rabbits immunized with glutaraldehydetreated A-chain. The y-globulin fraction, obtained by ammonium sulfate fractionations of the antiserum, was dialyzed against Tris-HC1 buffer (pH 7.5)/0.14 M KC1. 1 /zl of this antibody preparation, which had an absorbance of 35 at 280 nm, was able to neutralize a minimum of 2 #g A-chain, as judged from its ability to completely prevent ribosome inactivation by the A-chain. Rat liver rRNA was extracted with phenol from the ribosomal fraction and fractionated into 28 S, 18 S and 5 S rRNAs by sucrose density-gradient centrifugation [26]. Difference spectroscopy. Difference spectra were recorded at room temperature in cuvettes having 10 mm light paths, using a Hitachi spectrophotometer (Model 200-10). The base-line difference spectrum was measured with 2 ml of a protein solution in the sample cuvette and 2 ml of buffer in the reference cuvette. The difference spectra were recorded after addition of 5 or 10 /~1 increments of a 0.8 mM Cibacron blue F3GA solution to both cuvettes. The ability of several nucleotides to displace the dye from the protein-dye complex was examined by measuring either the difference spectrum or difference absorbance after addition of identical incremental volumes of a concentrated solution of the nucleotides to a sample cuvette containing protein and dye as well as a reference cuvette containing the same concentration of dye only. Modification with phenylglyoxal. Modification of A-chain with phenylglyoxal was performed essentially as described earlier [14]. A-chain (1 m g / ml) was incubated with 5 mM reagent at 22 ° C in a 0.125 M N a H C O 3 solution (pH 8.3) for different times in the dark. Modified arginine residues were determined by amino-acid analysis after HC1 hydrolysis. Poly(phenylalanine) synthesis. Poly(U)-directed poly(phenylalanine) synthesis by rat liver 80 S

179 ribosome was assayed in a 50/~1 reaction mixture consisting of 1 m M A T P / 0 . 4 m M G T P / 1 0 m M phosphoenolpyruvate/0.5 ttg pyruvate kinase/60 nCi [14C]phenylalanine (514 m C i / m m o l ) / 5 /~g poly(U)/0.5 /~g t R N A / 7 . 5 /~1 5% S-100/12.5 /tl p H 5 enzymes/1.7 pmol 80 S ribosomes/1 mM dithiothreitol/9 m M magnesium acetate/70 mM N H 4 C 1 / 8 0 m M K C 1 / 2 0 mM Tris-HC1 (pH 7.5). After incubation at 37 ° C for 20 min, the reaction mixture was plated on a glass-fiber filter (Whatman GFC, 25 mm) and dipped in 10% trichloroacetic acid. The filter was heated in 5% trichloroacetic acid at 90 to 1 0 0 ° C for 20 rain, washed with 5% trichloroacetic acid, and dried. The radioactivity on the filter was measured with a liquid scintillation counter (Aloka LSC-602). Pretreatment of ribosomes with the A-chain was performed in 10 /~1 buffer A containing 1.7 pmol ribosomes and increasing amounts of the A-chain. After incubation at 37 ° C for 10 min, anti-A-chain antibody (1 ttl) was added and poly(phenylalanine) synthesis was assayed with 40/~1 of other components. Cell-free protein synthesis. The cell-free system of protein synthesis was prepared using unfractioanted lysate from rabbit reticulocyte essentially according to Ref. 27 as described previously [28,29]. The increasing amounts of A-chain (in 20 /~1 of 20 m M Tris-HC1 buffer (pH 7.8)) were added directly to 180 #1 of the cell-free system. The assay mixture was incubated at 30 ° C for 60 min. The [3H]leucine radioactivity of the labeled trichloroacetic acid-insoluble fraction from a 30/~1 assay mixture was measured as described earlier [28,29]. The inhibitory activity of the A-chain on protein synthesis was determined based on the amounts required to give 50% inhibition.

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Fig. 1. Effect of Cibacron blue F3GA on inactivation of ribosome by ricin A-chain. Rat liver 80 S ribosomes(1.7 pmol) were incubated at 37°C for 10 rain in 10 #l of buffer A with various concentrations of the A-chain in the absence (O) or presence (e) of 10 laM dye, and the A-chain was neutralizedby addition of anti-A-chain antibody. The ribosomes were then assayed for their ability to synthesize poly(phenylalanine)as described in Materials and Methods. Each assay was done in duplicate, and the averagevalues are plotted as percentagesof the value obtained with untreated ribosomes (4030 cpm). The activity in the absence of poly(U) was 59 cpm. The poly(phenylalanine) synthesis was not affected by the dye at the concentration used.

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Effect of Cibacron blue F3GA on inactioation of ribosomes by ricin A-chain The data in Fig. 1 show that the ability of ricin A-chain to inactivate ribosome is significantly reduced by addition of Cibacron blue F3GA. In the presence of 10 # M dye about 5-times the concentration of A-chain was required to give 50% inhibition of poly(phenylalanine) synthesis as in the absence of the dye. This finding confirms the

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Fig. 2. Difference spectra of Cibacron blue F3GA in the presence of ricin A-chain. The sample cuvette contained 10 /~M A-chain, while both the sample and reference cuvettes contained: (1), no dye (base line); (2), 3.98/tM dye; (3), 7.92 /~M dye; (4), 11.8 #M dye; (5), 15.7btM dye; and (b), 19.5/~M dye. The solvent was 20 mM Tris-HC1buffer (pH 7.5)/1 mM dithiothreitol.

180

previous report of an interaction of the dye with ricin A-chain [18], and further suggests that the dye binds at or near the active site of A-chain.

Difference spectroscopic analysis of the dye-A-chain interaction Cibacron blue F3GA in 20 mM Tris-HC1 buffer (pH 7.5) showed a spectral absorption maximum at 610 nm as reported earlier [16]. Addition of the A-chain caused a red shift in the absorption spectrum of the dye, producing a difference spectrum having a single maximum at 688 nm and two minima at 585 and 628 nm (Fig. 2). The difference absorbance at 688 n m (AA688) increased in a hyperbolic manner with increasing dye concentrations (Fig. 3), indicating the formation of a dyeA-chain complex. The concentrations of bound

dye were determined spectrophotometrically at 688 nm, using a difference absorption coefficient of 1.7 mM -1- cm -1 which was obtained from AA688 of the dye saturated with the A-chain. The double-reciprocal plot of the concentration of bound dye versus that of free dye gave a straight line (inset in Fig. 3). From the intercepts, the dissociation constant for the dye-A-chain binding and the number of bound dye molecules per Achain were calculated to be 0.72 #M and 1.4, respectively. In contrast, addition of ricin B-chain or intact ricin, neither of which can inactivate ribosomes [3], did not produce a significant difference spectrum of the dye (Fig. 3).

Effect of chemical modification of arginine residues on the dye-A-chain interaction Specific modification of arginine residues with phenylglyoxal has been found to cause the inactivation of the A-chain, suggesting the existence of arginine residue(s) in A-chain's active site [14]. Since Cibacron blue F3GA seemed to interact with the active site of A-chain, it was expected

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20 30 Dye (;aM) Fig. 3. Difference spectral titrations of A-chain (II), B-chain (©) and intact ricin (*) with Cibacron blue F3GA. The initial protein concentration was 10 /~M. The values of maximal difference absorbance are plotted. They were obtained at 688 nm for A-chain, at 674 nm for B-chain and at 684 nm for intact ricin. The solvent for A- and B-chains was 20 mM Tris-HC1 buffer (pH 7.5)/1 mM dithiothreitol, and that for intact ricin was the same buffer without dithiothreitol. The inset shows the double-reciprocal plot of the titration of A-chain with the dye.

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Wavelength [nm] Fig. 4. Difference spectra of Cibacron blue F3GA in_.the presence of phenylglyoxal-modified A-chains. Ricin A-chain was modified with phenylglyoxal as described in Materials and Methods. The reaction time and the number of modified arginine residues per A-chain were: (1), zero, zero; (2), 6 min, 2.7; (3), 12 rain, 4.2; and (4), 60 min, 9.4. The sample cuvette contained 8 / t M modified A-chain, while both the sample and reference cuvettes contained 20 /~M dye. The solvent was 20 mM Tris-HCl buffer (pH 7.5)/1 m M dithiothreitol.

181 that modification of arginine residues should also affect the dye-A-chain interaction. In fact, modification of A-chain with phenylglyoxal resulted in a significant decrease in the magnitude of the difference spectrum generated upon binding of the dye to the A-chain (Fig. 4). A blue shift in the position of the difference absorption maximum also accompanied this modification (Fig. 4). These results suggest that arginine residues are involved in the dye-A-chain interaction. A molecule of A-chain contains 20 arginine residues [13]. Fig. 5 shows the decrease in the maximum difference absorbance as a function of the number of modified arginine residues per A-chain, comparing it with the loss of A-chain's inhibitory activity on protein synthesis. The difference absorbance was not lost so much as the activity. In both cases, however, significant changes were observed at an early stage of the modification and almost maximum decreases were achieved when about four arginine residues were modified. This observation indicates a correlation between the loss of the

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difference absorbance and the loss of the activity, and suggests that definite arginine residue(s) which react rapidly with phenylglyoxal may be involved both in the interaction with the dye and in the inactivation of ribosomes. To explore this correlation further, the effect of the dye on the modification of the A-chain with phenylglyoxal was examined. The A-chain (1 m g / mi) was modified with 1 mM phenylglyoxal for 60 min in the absence or presence of 0.32 mM dye. The modified protein was separated from the dye by dialysis against 5 mM Tris-HC1 buffer (pH 7.5) followed by electrodialysis. In the absence of the dye, 3.3 arginine residues per A-chain were modified and the inhibitory activity on protein synthesis decreased to 3.5%. In the presence of the dye, however, 2.1 arginine residues were modified with a retention of fairly high inhibitory activity (24%). Thus, the dye protected one arginine residue in the A-chain from modification by phenylglyoxal, which probably plays an important role in ribosome inactivation.

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Fig. 5. Loss of the difference absorbance generated upon Cibacron blue F3GA-A-chain interaction (O) and loss of A-chain's inhibitory activity on protein synthesis (O) with increasingmodificationof arginineresidues.RicinA-chainwas modified with phenylglyoxalas described in Materials and Methods. The values of maximaldifferenceabsorbance were obtained fromFig. 4. The inhibitoryactivityon protein synthesis was determinedusingrabbit reticulocytelysateas described in Materials and Mehtods. The data of modifiedA-chain are plotted as percentages of the value obtained with unmodified A-chain.

When increasing amounts of unfractionated rRNA were added to the dye-A-chain complex, a progressive decrease in the AA688 was observed (Fig. 6A), indicating the displacmeent of the dye from the dye-A-chain complex. Subsequent increase in the dye concentration resulted in a recovery of the difference absorbance (Fig. 6B). These findings suggest that dye and rRNA compete for the same binding site on the A-chain. Direct addition of rRNA (to a final concentration of 0.3 mg/ml) to the dye solution did not change the absorption spectrum of the dye, indicating that no direct interaction of rRNA with the dye took place in the experiments described above. The molar concentration of rRNA required to displace the dye from the dye-A-chain complex was much lower than the A-chain concentration. From the results in Fig. 6A the molar concentration of rRNA required to cause the 50% loss of AA688 was calculated to be about 1/40th of the A-chain concentration. This result suggests that the A-chain interacts with several sites on rRNA. To examine whether the A-chain binds to a particular rRNA, 28 S, 18 S and 5 S rRNAs were

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added separately to the dye-A-chain complex under the same conditions as in Fig. 6A. Each r R N A had the same ability to replace the dye from the dye-A-chain complex as judged from the weights required to give 50% decrease of AA688 (not shown). In the same manner, the interactions of A-chain with various kinds of nucleotide were compared. As shown in Fig. 7, addition of yeast tRNA, poly(U) or calf thymus D N A to the dye-A-chain complex displaced the dye to approximately the same extent as rRNA, while mono- and dinucleotides (ATP, GTP, CMP, UMP, N A D ÷, N A D H and N A D P H ) had a much smaller effect on the dye-A-chain complex.

Discussion The present results suggest that Cibacron blue F 3 G A interacts with the active site of ricin A-chain and that the dye can be used for probing the A-chain's active site. This conclusion is based on the following criteria: (a) the dye inhibited the A-chain's ability to inactivate ribosome; (b) the dye bound to the A-chain ( K d = 0.72 /IM), producing a significant difference spectrum, but not to the B-chain nor to the intact ricin, neither of which can inactivate ribosomes [3]; (c) the results of specific modification of arginine residues in the A-chain suggest that definite arginine residue(s) which react rapidly with phenylglycoxal are involved both in the interaction with the dye and in the inactivation of ribosomes. Cibacron blue F 3 G A has the capability to participate in both hydrophobic and ionic interactions with protein molecules. In the case of Achain, the cationic arginine residue may possibly interact with the anionic sulfonate group of the dye. Indeed, the involvement of ionic interaction in the dye-A-chain binding was indicated by the result that in the presence of high salt (1 M NH4C1 ) the amount of the dye required to saturate the A-chain was larger than in the absence of salt (data not shown). H y d r o p h o b i c interaction through the aromatic rings of the dye also seems likely to contribute to the dye-A-chain binding because m a n y continuous sequences of hydrophobic amino acids occur over the A-chain [13]. Cibacron blue F 3 G A has been used for probing

183

nucleotide-binding sites in various proteins [15-17]. Addition of several polynucleotides (rRNA, tRNA, poly(U) and DNA) to the dye-Achain complex resulted in a marked displacement of the dye, while mono- and dinucleotides had little or no effect on the dye-A-chain interaction. The polynucleotides appear to compete with the dye for the same site on the A-chain. These observations indicate the possible existence of a polynucleotide-binding site in the active site of the A-chain. Previous reports on the interaction of the dye with restriction endonucleases [30], polynucleotide phosphorylase [17] and isoleucyl transfer RNA synthetase [31] have also shown the recognition of polynucleotide binding sites on the enzymes by the dye. The A-chain has long been known to catalytically inactivate the eukaryotic 60 S ribosomal subunit without any added cofactors [4-6]. As to the molecular mechanism of the ribosome inactivation, RNAase activity of the A-chain with naked rRNA has been recently reported [10]. After treatment of intact ribosomes with the A-chain, however, attempts to detect the rRNA-cleavage have been unsuccessful [10-12]. Nevertheless, our results, indicating the binding of polynucleotides to the A-chain, suggest that the A-chain recognizes some part of rRNA on the surface of 60 S ribosomal subunits. Since blocking this polynucleotide binding site with the dye Cibacron blue clearly inhibits inactivation of the ribosome by the A-chain, our results are consistent with the proposal that the A-chain acts on ribosomal RNA. Our previous [14] and present data, which demonstrate the involvement of arginine residues in the inactivation of ribosomes, are consistent with a nucleotide-binding role for these residues. We have now localized the functionaly important arginine residues to the Cibacron blue/polynucleotidebinding site, and one may easily imagine that the positively charged arginine residues might interact with negatively charged phosphate groups of rRNA.

Acknowledgement This work was supported in part by a Grant-inAid for Scientific Research from the Ministry of Education, Japan.

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31 Moe, J.G. and Piszkiewicz, D. (1979) Biochemistry 18, 2810-2814