Isolation and enzymatic characterization of lamjapin, the first ribosome-inactivating protein from cryptogamic algal plant ( Laminaria japonica A) Ren-shui Liu 1 , Jia-hua Yang 2 and Wang-Yi Liu 1 1 State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China; 2 Department of Biochemistry, Yantai University, Shandong, China Lamjapin,anoveltypeI ribosome-inactivating protein, has been isolated from kelp (Laminaria japonica A), a marine alga. This protein has been extensively purified through multiple chromatography columns. With a molecular mass of  36 kDa, lamjapin is slightly larger than the other known single-chain ribosome-inactivating proteins from the higher plants. Lamjapin can inhibit protein synthesis in rabbit reticulocyte lysate with an IC 50 of 0.69 n M .Itcan depurinate at multiple sites of RNA in rat ribosome and produce the diagnostic R-fragment and three additional larger fragments after the aniline reaction. Lamjapin can deadenylate specifically at the site A20 of the synthetic oli- goribonucleotide (35-mer) substrate that mimics the sarcin/ ricin domain (SRD) of rat ribosomal 28S RNA. However, it cannot hydrolyze the N-C glycosidic bond of guanosine, cytidine or uridine at the corresponding site of the A20 of three mutant SRD RNAs. Lamjapin exhibits the same base and position requirement as the ribosome-inactivating pro- teins from higher plants. We conclude that lamjapin is an RNA N-glycosidase that belongs to the ribosome-inacti- vating protein family. This study reports for the first time that ribosome-inactivating protein exists in the lower cryp- togamic algal plant. Keywords: kelp; lamjapin; marine alga; ribosome-inacti- vating protein; RNA N-glycosidase. Plant ribosome-inactivating proteins (RIPs) are a group of toxic proteins with RNA N-glycosidase activity that act on the eukaryotic and prokaryotic ribosomes. It was shown that RNA N-glycosidase activity of RIP could remove a specific adenine from a highly conserved loop (the sarcin/ ricin domain; SRD) of the largest RNA in ribosome and thus inhibit the protein synthesis [1,2]. Type I RIPs consist of a single, intact polypeptide chain of about 11–30 kDa. Type II RIPs are composed of two chains linked by a disulfide bond. Type III RIP consists of an amino-terminal domain resembling type I RIP linked to a carboxyl-terminal domain with unknown function [3]. This class of plant toxins has drawn much attention because of their antiviral activity and the potential use as a toxin moiety in immunotoxins for the treatment of several important human diseases such as cancer and AIDS [4–6]. RIPs also have promising application in crop plant biotechnology with the aim of increasing resistance to insects, fungal and viral pathogens [7–9]. In addition, RIPs are a powerful tool to probe the topographic structures of ribosomal RNA and the mechanism of protein synthesis [10]. Moreover, there is no consensus on the physiological function, distribution and the evolutionary links of RIPs. For all these reasons, the search for new RIPs is continuing, and more novel RIPs are being isolated and characterized from terrestrial flowering plants, while no RIP has been isolated from cryptogamic plants so far [11–13]. In this paper, lamjapin is shown to be the first single-chain RIP from kelp, a lower cryptogamic algal plant. This result will help to characterize the function, evolution, as well as the distribution of this class of protein in plant kingdom. MATERIALS AND METHODS Materials The fresh tender leaves of kelp (Laminaria japonica A) were collected in winter at the shore of Yantai in the Shangdong Province of China. CM-Cellulose 52 was purchased from Whatman. Phenyl-Sepharose CL-4B, pI marker, ampho- lyte, the FPLC system, Superose-12 and Mono Q columns were obtained from Pharmacia LKB. L -[ 14 C]leucine was from Amersham. The protein molecular-mass markers were provided by Shanghai Lizhu-dongfeng Biotech. Ultrafiltra- tion membranes and centricons were purchased from Amicon. T7 RNA polymerase, was obtained from Pro- mega. [a- 32 P]UTP was from New England Nuclear. Oligo- deoxynucleotides were synthesized at the Shanghai GenecoreÒ Biotechnology Company. All the other chemi- cals and reagents were of analytical grade. Isolation and purification of lamjapin from kelp The fresh tender leaves of kelp were freeze-dried and powdered in liquid nitrogen. Thirty grams of the finely ground material were extracted in 1000 mL of buffer A Correspondence to W Y. Liu, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China. Fax: + 86 21 64348357, E-mail: liuwy@sunm.shcnc.ac.cn Abbreviations: RIP, ribosome-inactivating proteins; SRD, sarcin/ricin domain. (Received 28 May 2002, revised 22 July 2002, accepted 2 August 2002) Eur. J. Biochem. 269, 4746–4752 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03165.x (50 m M Tris/HCl, pH 9.2, 0.2 M NaCl, 10 m M ascorbic acid) by gentle stirring at 8 °C overnight. The homogenate was centrifuged (8000 g,4°C) for 30 min, then the supernatant was decanted and filtered through four layers of gauze. The filtrate was adjusted to pH 8.5 with 1 M HCl and solid ammonium sulfate was added to 1 M . This fluid was used as the crude extract from which lamjapin was purified by the following four steps of column chromato- graphy. Phenyl-Sepharose CL-4B. The crude extract was applied to the phenyl-Sepharose CL-4B column (10 · 4cm)pre- equilibrated with buffer B [50 m M Tris/HCl, pH 8.5, 1 M (NH 4 ) 2 SO 4 and 0.2 M NaCl]. After being washed with the buffer B until the A 280 fell below 0.1, the column was eluted with buffer C [50 m M Tris/HCl, pH 8.5, 0.4 M (NH 4 ) 2 SO 4 ] and buffer D [50 m M Tris/HCl, pH 8.5, 0.1 M (NH 4 ) 2 SO 4 ] sequentially; the protein in peak 2 was collected. CM-Cellulose 52. The protein solution collected from peak 2 of the phenyl-Sepharose CL-4B column was dialyzed exhaustively against buffer E (5 m M phosphate buffer, pH 6.0) and then loaded on a CM-Cellulose column (10 · 2.4 cm) preequilibrated with the same buffer. After loading the protein, the column was washed with 60 mL of buffer E and then eluted with buffer E containing 0.15 M sodium chloride. The protein in peak 1 was collected. Superose-12 FPLC. After dialysis against distilled water, the proteins in peak 1 from the CM-cellulose column were lyophilized and dissolved in buffer F (50 m M phosphate buffer, pH 7.2, 0.15 M NaCl). The protein was then loaded on a Superose12 HR (30 cm · 10 mm) column preequili- brated with buffer F and eluted the column with buffer F. Three peaks appeared and the protein in peak 2 was collected for further purification. Mono Q FPLC. The protein solution in peak 2 with RIP activity from the Superose-12 column was desalted and adjusted to 10 m M ethanolamine, pH 9.2 by repeated concentration in an Amicon concentrator equipped with a PM10 membrane. Then the protein solution was loaded on toaFPLCMonoQHR5/5column(50· 1.6 mm) preequilibrated with the buffer G (10 m M ethanolamine, pH 9.2). A linear gradient elution with 40 mL of NaCl solution (0–1 M ) in buffer G was performed. The purified RIP in the peak 2 was collected and was named as lamjapin. Protein synthesis in the cell-free system Rabbit reticulocyte lysate was prepared and the protein synthesis in rabbit reticulocyte lysate was performed according to the methods of Sambrook et al. [14]. Various amounts of lamjapin (0.25–8 ng) were added to the 50 lL of reaction buffer to measure their inhibition of protein synthesis. Activity of lamjapin on rat ribosomes Rat liver ribosomes were isolated as described by Spedding [15]. One point five A 260 units of ribosomes (27 pmol) were incubated with 20 or 30 ng of lamjapin in 100 lLofbuffer H(25m M Tris/HCl, pH 7.6, 25 m M KCl, 5 m M MgCl 2 )at 37 °C for 15 min. After adding 10 lL of 10% SDS solution to the reaction mixture in an ice-bath, ribosomal RNAs were extracted with phenol/chloroform and precipitated by ethanol. After acidic aniline treatment at 60 °C for 10 min, ribosomal RNAs were separated on 8 M urea-denatured polyacrylamide gel (3.5%) for 1 h or on 8 M urea-denatured polyacrylamide gel (4.5%) for 3 h. The RNA fragments were stained by methylene blue. Fluorescence assay of the adenine released by lamjapin Adenine was quantitated by the fluorimetric method of Zamboni et al.[16].OneA 260 (18 pmol) of rat liver ribosomes was incubated with 0.64 lgoflamjapinin 100 lL of buffer H at 37 °C for 0–40 min. After incubation, ribosomal RNA was precipitated with 2 vol. of ethanol. The ethanol-soluble fractions were diluted to 1 mL with water, and then 0.4 mL of 0.14 M chloroacetaldehyde containing 0.1 M sodium acetate (pH 5.1) was added to each sample. The samples were incubated at 85 °Cfor1h. After cooling to room temperature, fluorescence was measured at an excitation wavelength of 280 nm and an emission wavelength of 400 nm. Adenine at concentrations ranging from 1 to 1200 pmol was used as a standard for calculation of the amount of adenine in the samples. Assay for the deadenylation of SRD RNA by lamjapin RNA N-glycosidase activity of lamjapin was assayed according to the method of Endo et al. [17] with a slight modification. The radiolabeled wild type SRD RNA and its three mutant SRD RNAs (G20, C20 and U20 instead of A20) were prepared by in vitro transcription using T7 RNA polymerase and synthetic DNA oligomers as template. The radiolabeled SRD RNA and its three mutants were incubated, respectively, with 1 l M lamjapin in 20 lLof buffer I (30 m M sodium citrate, pH 5.0, 1 m M MgCl 2 )at 35 °C for 90 min and then treated with acid aniline. The RNA fragments produced by lamjapin and aniline treat- ment were extracted with phenol/chloroform and precipi- tated by ethanol. After separating the RNA fragments by electrophoresis on 20% polyacryamide gel containing 8 M urea, gels were dried and exposed to X-ray film. Assay of the release of adenine from the SRD RNA by lamjapin The preparation of the oligoribonucletides was as described above except that the synthesis was with [2,8- 3 H] ATP instead of ATP. The assay conditions were also the same. The reaction was carried out in the same way except that, after incubation, 10 lL of a solution of 0.2 M NaCl containing 100 lL of carrier tRNA and 40 lL of ethanol was added to the sample and kept at °C for 60 min. The mixture was centrifuged (15 000 g for 20 min) and the radioactivity in a portion of the supernatant was determined in a liquid scintillation counter. Other analytical methods SDS/PAGE analysis was carried out on 12% SDS-poly- acryamide gels by the method of Laemmli [18] and protein bands were stained with silver according to the method of Ó FEBS 2002 Lamjapin, a novel type I RIP from marine alga (Eur. J. Biochem. 269) 4747 Ansorge [19]. The pI was measured by isoelectric focusing on a polyacrylamide gel in the pH range 3–10 and the proteins were stained with Coomassie Brilliant Blue by the method of Neuhoff et al. [20]. The pI was determined by calculating the linear regression of the marker proteins vs. the migration distances. The protein concentration was determined by the method of Bradford [21]. RESULTS Isolation and purification of lamjapin In a search for RIP from marine plants, we found that the crude kelp extract exhibited the RNA N-glycosidase activity towards rat ribosome, but some inhibitory sub- stances in the extract could inactivate this enzyme activity. To overcome this problem, the phenyl-Sepharose CL-4B column chromatography was chosen as the first step and it separated efficiently the target proteins from the inhibitory substances (Table 1). Proteins of peak 2 from the phenyl-Sepharose CL-4B were then subjected to a general protocol of RIP purification. As Fig. 1 shown, the CM-cellulose 52 column retained the most of the RIP activity and also separated the lamjapin from the other impurities. The proteins obtained from the CM-cellulose 52 column were resolved into three peaks by Superose-12 FPLC; peak 2 with RIP activity was further fractionated by Mono Q FPLC from which the pure lamjapin was eluted in peak 2 with  0.25 M NaCl. Approximately 30 lg of pure lamjapin could be obtained from 30 g of dry kelp powder. The low yield of lamjapin was due to its low abundance in the total proteins and also to the low efficiency of extraction. Physical properties of lamjapin Lamjapin obtained from the Mono Q column migrated as a single band characterized by both 12% SDS/PAGE and isoelectric focusing gel electrophoresis, indicating that it is a homogeneous protein. Furthermore, it appeared as a single band by SDS/PAGE in the presence or absence of dithiothreitol, demonstrating that it is a type I RIP com- posed of a single peptide chain without an intradisulfide bond (Fig. 2). Lamjapin has an apparent molecular mass of  36 000 Da, which is a little larger than the average molecular mass ( 30 000 Da) of other known single-chain RIPs from the higher plant. Like other type I RIPs, lamjapin is a basic protein with a pI of 8.4, as determined by isoelectrophoresis (Fig. 2). Inhibition of protein synthesis in cell-free system by lamjapin As shown in Fig. 3A, lamjapin inhibited protein synthesis in the cell-free system of rabbit reticulocyte. The protein synthesis decreased gradually with the increment of lamja- pin in the reaction mixture. The IC 50 (the concentration of RIP causing 50% inhibition of translation) of lamjapin is about 0.69 n M , a very low value for an inhibitor of protein Table 1. Purification procedures of lamjapin from 30 g dry powder of L. japonica A. One unit of specific activity is defined as the amount of protein necessary to inhibit protein synthesis by 50% in 50 lL of reaction mixture including rabbit reticulocyte lysate. Procedures Total protein (mg) Total activity (10 5 U) Specific activity (10 5 UÆmg )1 ) Yield (%) Crude extract 160.00 6.25 0.04 100 Phenyl-Sepharose CL-4B a 32.00 9.48 0.29 152 CM-52 cellulose 2.32 6.01 2.59 96 FPLC Superose-12 0.78 4.38 5.61 70 FPLC Mono-Q 0.03 2.42 80.67 39 a This step can separate lamjapin from the inhibitory substances in the crude extract. As a result, the total activity is larger than that of the crude extract. Fig. 1. Purification of lamjapin from L. japonica A by column chro- matography. (A) Phenyl-Sepharose CL-4B; (B) CM-cellulose; (C) Superose-12 FPLC and (D) Mono Q. The experimental procedures are described in Material and methods. The solid lines represent the elu- tion curves; dash line represents the NaCl gradient. Each fraction was assayed for the RNA N-glycosidase activity to rat ribosome. Those fractions that contained the major activity inhibiting the protein syn- thesis in rabbit reticulocyte lysate were pooled and subjected to further purification. 4748 R s. Liu et al. (Eur. J. Biochem. 269) Ó FEBS 2002 biosynthesis, and still in the range of the IC 50 (0.03–4 n M )of type I RIPs. The IC 90 (the concentration of RIP causing 90% inhibition of translation) of lamjapin in the cell-free system of rabbit reticulocyte is  5.56 n M . RNA N-glycosidase activity of lamjapin to rat ribosomes As shown in Fig. 3B, the RNA N-glycosidase activity of lamjapin is compared with that of cinnamomin, a type II RIP purified in our laboratory. At a molar ratio of lamjapin/ribosome of 1 : 48, 20 ng of lamjapin could induce rat liver ribosome to produce the ricin/sarcin fragment (R-fragment) after aniline treatment. The R-frag- ment did not appear if the ribosome was incubated with only lamjapin without aniline treatment. Therefore, the R-fragment was not caused by the RNase contamination in the purified sample. This demonstrated that lamjapin has an RNA N-glycosidase activity like other RIPs from higher plants. Lamjapin acts on ribosomal RNAs at multiple sites Besides the predominant R-fragment produced by lamjapin from rat ribosomal RNAs, three additional RNA fragments larger than the R-fragment were found when the treated ribosomal RNAs were separated by 8 M urea-denatured polyacrylamide gel (4.5%) electrophoresis for a longer time (3 h). The R-fragment and three larger fragments did not appear if the ribosome was incubated with only lamjapin without aniline treatment. These data demonstrated con- clusively that the emergence of the larger RNA fragments by lamjapin was not artifact caused by the nuclease contaminant. It was also shown that the ratio of fragments Fig. 3. Activity of lamjapin. (A) Effect of lamjapin on protein synthesis in rabbit reticulocyte lysate. The protein synthesis system contained the indicated amount of lamjapin in a final volume of 50 lLofreaction mixture as described in Material and methods. The control value of [ 14 C]leucine incorporated is 22 000 d.p.m. (B). Activity of lamjapin to rat liver ribosomes. Ribosomes were treated with lamjapin and acid aniline. The ribosomal RNAs were extracted and electrophoresed on 3.5% polyacrylamide gel (8 M urea) at 25 mA for 1 h and ribosomal RNAs were visualized with methylene blue. The R-fragment was produced with acid aniline from the treated ribosomes. B indicates ribosomes were treated with only buffer H; C indicates ribosomes were treated with cinnamomin (20 ng), L indicates ribosomes were treated with lamjapin (20 or 30 ng). Fig. 2. Purity of lamjapin identified by SDS/PAGE and isoelectric focusing. (A) SDS/PAGE of the purified lamjapin. M, protein markers. Lane 1, lamjapin (4 lg) without treatment by dithiothreitol; Lane 2, lamjapin (4 lg) treated with dithiothreitol. SDS/PAGE (12%) was performed and the protein bands were silver stained as described in Materials and methods. (B) Isoelectric focusing of the purified lamja- pin. Lane L, lamjapin (4 lg); M, protein markers. Proteins were focused and stained with Coomassie Brilliant Blue. Regression analysis of the migration distance plotted vs. the pI values of the protein markers was used to calculate the pI value of lamjapin. Ó FEBS 2002 Lamjapin, a novel type I RIP from marine alga (Eur. J. Biochem. 269) 4749 a, b, c and the R-fragment was constant (0.2 : 0.3 : 0.1 : 1.0), independent of the amount of lamjapin employed. This indicated that the action of lamjapin on these sites of ribosomal RNA was specific but the sensitivity of these sites to lamjapin was much lower than that of A4324 in the S/R domain. In order to confirm the multiple sites of depurination of lamjapin on the rat ribosomal RNA, the adenine base released from ribosomal RNA by lamjapin were quantita- tively analyzed by the chloroacetaldehyde method. A time course study demonstrated that the release of adenine by lamjapin continued at a linear rate for at least 40 min when RIP and ribosome are presented at 1 : 1 molar ratio. Quantitative analysis revealed that lamjapin could release more than one mole of adenine from each mole of ribosomes in 10 min and even up to 12 mol of adenines in 40 min (Fig. 4B). Base and position specificity of lamjapin in depurination of SRD RNA Synthetic oligoribonucletide (a 35-mer) that mimics the S/R domain of rat ribosomal RNA (SRD RNA) is an useful substrate for studying the mechanism of action of RIP and for analysis of the chemistry of recognition of RNA by RIP [22,23]. As shown in Fig. 5, ricin could deadenylate A20 of SRD RNA, the site corresponding to position A4324 of rat ribosomal RNA, producing two fragments (20-mer and 15-mer). The SRD RNA treated with lamjapin and acidic aniline also released two fragments with the same size as that produced by ricin, while there was no fragment released when the SRD RNA was treated only with lamjapin. The activity of lamjapin on the SRD RNA exhibited the base specificity as demonstrated by the fact that both the transitional and transversionanl mutants (A20 to G20, C20 or U20) were insensitive to lamjapin and no fragment appeared from these mutants treated with lamjapin and acid aniline. In the next experiment, SRD RNA and the mutant SRD RNA (A20 to G20) were labeled with [ 3 H]adenine and treated with lamjapin and aniline. The result revealed that lamjapin could release the 3 H-labeled adenine from the wild type SRD RNA (5.3 · 10 4 d.p.m. of 3 H-labeled adenine), while no adenine was released from the A20 mutant SRD RNAs treated with lamjapin. This result showed that only A20 and no other adenines could be released by lamjapin from SRD RNA. The activity of lamjapin to the SRD RNA is absolutely dependent on the preservation of adenine at a proper site. The base- and site-specific RNA N-glycosidase activity of lamjapin is the same as other RIPs from higher plants like ricin A-chain [17]. DISCUSSION Extraction of protein from brow alga is tedious because of its richness in phenolic compounds, pigments and polyan- ionic cell wall consisting of alginates [24]. It was difficult to extract active lamjapin by usual methods of homogenization in the presence of above inhibitory substances. As an alternative method, lyophilized kelp was powdered in liquid nitrogen and then extracted gently at low temperature in the alkaline solution containing ascorbic acid. These conditions could efficiently decrease interaction of proteins with phenolic compounds and alginates, etc. and hence preserved the enzymatic activity of lamjapin. But the efficiency of extraction was still low and the proteins could not be precipitated by ammonium sulfate even up to 85% satura- tion. Poly(ethylene glycol) partition that has been reported to improve the efficiency of protein extraction from other two species of kelp was tried unsuccessfully [25]; this method resulted in the inactivation of lamjapin. Among several methods tested, only the phenyl-Sepharose CL-4B column chromatography could separate efficiently the active lamja- pin from the inhibitory substances in the crude kelp extract. Fig. 4. Multiple sites of action of lamjapin at ribosomal RNAs. (A) Action of lamjapin on ribosomal RNAs. Rat ribosomes were treated with lamjapin and acid aniline. The ribosomal RNAs were extracted and electrophoresed on 4.5% polyacrylamide gel (8 M urea) at 25 mA for a longer time (3 h) and ribosomal RNAs were visualized with methylene blue. a, b, c are three additional larger RNA fragments. (B) Time course of releasing adenine from ribosomes. The experi- mental conditions are described in the Materials and methods. 4750 R s. Liu et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Four methods are commonly used to assess the RNA- N-glycosidase activity of RIPs [26]: (a) quantification of the inhibition of the protein synthesis in cell-free systems, (b) visualization of the RNA fragment produced by aniline cleavage at the site of depurination, (c) measurement of the fluorescent derivative ethenoadenine of released adenine, and (d) detection of the [ 3 H]adenine released. This study showed that lamjapin exhibited strong inhibitory activity to protein synthesis in rabbit reticulocyte lysate. It acted on the rat ribosomal RNA producing the RNA fragments after aniline treatment. In addition, it could release adenine from ribosomal RNA and SRD RNA as revealed by the release of fluorescent derivative ethenoadenine and the [ 3 H]adenine. From these data, we can conclude safely that lamjapin is an RNA N-glycosidase. It belongs to the ribosome-inactivating protein family that were previously found only in higher species of plant kingdom. Intensive studies by the NMR and X-ray demonstrated that SRD RNA possessed a tertiary structure similar to the S/R domain of rat liver ribosomes. It was composed of a stem and a GAGA tetraloop out of which A20 of SRD RNA corresponding to the A4324 of rat ribosomal RNA was flipped [27]. Lamjapin could deadenylate A20 of SRD RNAandreleasedtheRNAfragmentwiththeexactsizeof the R-fragment from the ribosomal RNA. It is likely that lamjapin deadenylates the A4324 of 28S ribosomal RNA and produces this RNA fragment after the acidic aniline treatment. RIPs were originally thought to act exclusively on the specific A4324 of the S/R domain of rat ribosomes. However, several RIPs such as saporin-R2 were found to act on ribosomal RNA at multiple sites [28,29]. In this study, it was found that lamjapin could deadenylate at multiple sites in rat ribosomal RNA and produced three additional RNA fragments in addition to the main R-frag- ment. The ribosomal RNAs are rich in the stem-loop structure that is similar to the S/R domain. Perhaps some adenines of these domains also showed certain sensitivity to lamjapin and saporin-R2. Lamjapin is one of the few ribosome-inactivating proteins acting at multiple sites in ribosomal RNA. Study on the distribution of this class of protein in lower plant is still scarce. Most of plant species examined belong to the class of Angiospermae [30,31]. No RIP has yet been isolated from the class of Gymnospermae and Cryptogamia. Lamjapin is the first single-chain RIP isolated from kelp (L. japonica A) that belongs to the Cryptogamia, the lowest species in the plant kingdom. This first showed that RIPs exist outside of the flowering plants. Our group screened three marine algae and three freshwater algae in Crypto- gamia and the RNA N-glycosidase was only found in kelp (L. japonica A). It is very likely that the distribution of RIPs is sporadic rather than ubiquitous in the plant kingdom as proposed by Van Damme et al. [3]. The existence of lamjapin in L. japonica A demonstrates that such sporadic distribution of RIPs in plant kingdom ranges widely from the lowest plants to the highest plants. ACKNOWLEDGEMENTS This work was supported by one grant of Natural Science Foundation of China (39970163) and one grant of Academia Sinica (KSCX2-02- 04). The authors thank for Dr Zheng Pu for his technical assistance and Dr Lee Zou for his critical reading of this manuscript. REFERENCES 1. Endo. Y., Mitsui, K., Motizuk, M. & Tsurugi, K. (1987) The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins. J. Biol. Chem. 263, 5908–5912. 2. Endo, Y. & Tsurugi, K. (1987) RNA N-glycosidase activity of ricin A-chain: mechanism of action of the toxic lectin ricin on eukaryotic ribosomes. J. Biol. Chem. 262, 8128–8130. 3. Peumans, W.J., Hao, Q. & Van Damme, E.J.M. (2001) Ribosome inactivating protein: More than RNA N-glycosidase? FASEB J. 15, 1493–1510. 4. Tazzari, P.L., Polito, L., Bolognesi, A., Pistillo, M.P., Capanni, P., Palmisano, G.L., Lemoli, R.M., Curti, A., Biancone, L., Camussi, G.,Conte,R.,Ferrara,G.B.&Stirpe,F.(2001)Immunotoxins containing recombinant anti-CTLA-4 single-chain fragment variable antibodies and saporin: in vitro results and in vivo effects in an acute rejection model. J. Immunol. 167, 42222–42229. 5. Klinck,R.,Westhof,E.,Walker,S.,Afashar,M.,Collier,A.& Aboul-Ela, F. (2000) A potential RNA drug target in the hepatitis C virus internal ribosomal entry site. RNA 6, 1423–1431. 6. Munoz,R.,Arias,Y.,Ferreras,J.M.,Jimenez.P.,Rojo,M.A.& Girbes, T. (2001) Sensitivity of cancer cell lines to the novel non- toxic type 2 ribosome-inactivating protein nigrin b. Cancer Lett. 167, 163–169. 7. Girbes, T., Torre, C., Iglesias, R. & Ferreras, J.M. (1996) RIP for viruses. Nature 379, 777–778. 8. Zoubenko, O., Hudak, K. & Tumer, N.E. (2000) A non-toxic pokeweed antiviral protein mutant inhibits pathogen infection via Fig. 5. Depurination of the synthetic SRD RNA by lamjapin. The sus- ceptibility to lamjapin of mutant oligoribonucleotides having single base changes at A20 was evaluated. (A) The substrate was the wild type oligomer; (B) the oligomer has a transition of A20 to G20; (C) it has a transversion of A20 to C20; and (D) it has a transversion of A20 to U20. R, ricin; L, lamjapin. Ó FEBS 2002 Lamjapin, a novel type I RIP from marine alga (Eur. J. Biochem. 269) 4751 a novel salicylic acid-independent pathway. Plant Mol. Biol. 44, 219–229. 9. Nielsen, K. & Boston, R.S. (2001) Ribosome-inactivating pro- teins: a plant perspective. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 785–816. 10. Wool, I.G., Gluck, A. & Endo, Y. (1992) Ribotoxin recognition of ribosomal RNA and a proposal for the mechanism of transloca- tion. Trends Biochem. Sci. 17, 266–269. 11. Wang, H.X. & Ng, T.B. (2001) Isolation of pleuturegin, a novel ribosome-inactivating protein from fresh sclerotia of the edible mushroom Pleurotus tuber-regium. Biochem. Biophys. Res. Com- mun. 288, 718–721. 12. Lam, S.K. & Ng, T.B. (2001) Hypsin, a novel thermostable ribosome-inactivating protein with antifungal and antiprolifera- tive activities from fruiting bodies of the edible mushroom Hypsizigus marmoreus. Biochem. Biophys. Res. Commun. 285, 1071–1075. 13. Bolognesi, A., Polito, L., Lubelli, C., Barbierr, L., Parente, A. & Stirpe, F. (2002) Ribosome-inactivating and adenine polynucleo- tide glycosylase activity in Mirabilis jalapa L. tissues. J. Biol. Chem. 277, 13709–13716. 14. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York. 15. Spedding, G. (1990) Ribosomes and Protein Synthesis: A Practical Approach. IRL Press, New York. 16. Zamboni, M., Brigotti, M., Rambelli, F., Montanaro, L. & Sperti, S. (1989) High-pressure-liquid-chromatographic and fluorimetric methods for the determination of adenine released from ribosomes by ricin and gelonin. Biochem. J. 259, 639–643. 17. Endo, Y., Gluck, A. & Wool, I.G. (1991) Ribosomal RNA identity elements for ricin A-chain recognition and catalysis. J. Mol. Biol. 221, 193–207. 18. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. 19. Ansorge, W. (1985) Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate. J. Bio- chem. Biophys. Methods 11, 13–20. 20. Neuhoff, V., Stamm. R. & Eibl, H. (1985) Clear background and highly sensitive protein staining with coomassie blue dyes inpoly- acrylamide gels: a systematic analysis. Eletrophoresis 6, 427–448. 21. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 142, 336–339. 22. Orita, M.N., Ishikawa, F., Shimayama, T., Taira, K., Endo, Y. & Nishikawa, S. (1993) High-resolution NMR study of a synthetic oligoribonucleotide with a tetranucleotide GAGA loop that is a substrate for the cytotoxic protein, ricin. Nucleic Acids Res. 21, 5670–5678. 23. Endo, Y. & Gluck, A. (1990) A new assay to measure RNA N-glycosidase activity. Nucleic Acids Symp. Series 22, 21–22. 24. Percival, E. & McDowell, H. (1987) Plant Carbohydrates. Springer-Verlag Press, New York. 25. Jordan, P. & Vilter, H. (1991) Extraction of proteins from material rich in anionic mucilages: partition and fractionation of vanadate- dependent bromoperoxidases from the brown algae Laminaria digitata and L. saccharina in aqueous polymer two-phase systems. Biochem. Biophy. Acta 1073, 98–106. 26. Brigotti, M., Barbieri, L., Valbonesi, P., Stirpe, F., Montanaro, L. & Sperti, S. (1998) A rapid and sensitive method to measure the enzymatic activity of ribosome-inactivating proteins. Nucleic Acids Res. 18, 4306–4307. 27. Correll, C.C., Munishkin, A., Chan, Y.L., Ren, Z., Wool, I.G. & Steitz, T.A. (1998) Crystal structure of the ribosomal RNA domain essential for binding elongation factors. Proc. Natl Acad. Sci. USA 95, 13436–13441. 28. Barbieri, L., Ferreras, J.M., Barraco, A., Ricci. P. & Stirpe. F. (1992) Some ribosome-inactivating proteins depurinate ribosomal RNA at multiple sites. Biochem. J. 286, 1–4. 29. Di Maro, A., Valbonesi, P., Bolognesi, A., Stirpe, F., De Luca, P., Gigliano, G., Gaudio, L., Delli Bovi, P., Ferranti, P., Malomi, A. & Parente, A. (1999) Isolation and characterization of four type-1 ribosome-inactivating proteins, with polynucleotide: adenosine glycosidase activity, from leaves of Phytolacca Dioica L. Planta 208, 125–131. 30. Barbieri, L., Battelli, M.G. & Stirpe, F. (1993) Ribosome- inactivating proteins from plants. Biochim. Biophys. Acta 1154, 237–282. 31. Gasperi-Campani, A., Barbieri, L., Battelli, M.G. & Stirpe, F. (1985) On the distribution of ribosome-inactivating proteins amongst plants. J. Nat. Prod. 48, 446–454. 4752 R s. Liu et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . Isolation and enzymatic characterization of lamjapin, the first ribosome-inactivating protein from cryptogamic algal plant ( Laminaria japonica A) Ren-shui. 52 column retained the most of the RIP activity and also separated the lamjapin from the other impurities. The proteins obtained from the CM-cellulose 52