Isolation and characterization of new isoforms of trichosanthin from Trichosantheskirilowii

Plant Science 162 (2002) 79 – 85 www.elsevier.com/locate/plantsci

Isolation and characterization of new isoforms of trichosanthin from Trichosanthes kirilowii Pushpa Narayanan, Nai Ki Mak, Phuong Bich Luong, Ricky Ngok Shun Wong * Department of Biology, Hong Kong Baptist Uni6ersity, Kowloon Tong, Kowloon, Hong Kong Received 6 June 2001; received in revised form 10 September 2001; accepted 10 September 2001

Abstract Three ribosome-inactivating proteins (RIPs) were isolated from the previously characterized ‘trichosanthin’ fraction prepared from the root tuber of Trichosanthes kirilowii. They were designated as a-, b-, and g-trichosanthin (TCS). All three trichosanthin isoforms exhibited similar biochemical activities, namely in vitro inhibition of protein synthesis, N-glycosidase activity on 28S rRNA, and conversion of supercoiled DNA to open-circular and linear DNA. SDS-PAGE analysis indicated that their molecular weights were around 26 kDa. Amino acid composition analysis revealed that a-TCS, the major protein, corresponded closely to the previously reported trichosanthin. Protein fingerprinting analysis indicated that g-TCS is structurally distinct from a-, and b-TCS while the latter two are very similar. The identity of g-TCS was confirmed by N-terminal and internal protein sequencing. Cytotoxicity assay indicated that a-TCS is distinctively more toxic towards lymphocytes than b- and g-TCS, while for fibroblast, b-TCS is the least toxic. Their difference in cytotoxicity in spite of their structural similar warrant further investigation into their structure and function relationship. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Trichosanthin; Ribosome-inactivating protein; Isoforms; Trichosanthes kirilowii

1. Introduction Ribosome-inactivating proteins (RIPs) are a group of plant toxins that inactivate ribosomes by virtue of their highly specific rRNA N-glycosidase activities [1–3]. RIPs are widely distributed in the plant kingdom. They have been found in different parts of the plant including seeds, leaves and roots. Different isoforms of RIPs have been isolated from the same or different parts of the same plants [4]. These isoforms with slight differences in protein sequence were not due to post-translational modifications but due to difference in gene sequence [5]. Thus isoforms of RIP that are derived from the same plant are probably encoded as a multigene family. Trichosanthin is a type 1 RIP isolated from the root-tuber of Trichosanthes kirilowii [6]. Several related proteins have been isolated from tissues of the same * Corresponding author. Tel.: + 852-2339-7057; fax: +852-23395995. E-mail address: [email protected] (R.N.S. Wong).

plant. These include the Trichosanthes antiviral protein (TAP29) [7], karasurin from the root-tuber [8], and trichokirin [9], a- and b-kirilowins from the seeds [10]. Also, Southern blot analysis of EcoR1 -digested T. kirilowii DNA showed at least three hybridizing bands of about 7, 4 and 1.4 kbp in size [11]. Thus, it is probable that RIPs in T. kirilowii comprise a multigene family. Furthermore, cDNA cloning experiments have also identified differences in the nucleotide sequence of trichosanthin gene [11]. We report here the isolation and characterization of two new isoforms of trichosanthin.

2. Material and methods Root tubers of T. kirilowii were obtained from Pinghu of Zhejiang and Beijing, China. Chromatographic medium including CM-Sepharose CL-6B and Mono-S (FPLC) column HR 5/5 were purchased from Pharmacia. Rabbit reticulocyte lysates were obtained from Promega.

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2.1. Isolation of trichosanthin isoforms

2.4. Amino acid analysis and partial protein sequencing

Root tubers of T. kirilowii (200 g) were chopped into small pieces and soaked in 600 ml of phosphate buffered saline. After stirring at 4 °C overnight, the root tubers were homogenized in a Waring blender and the homogenate was filtered through four layers of cheese cloth. The filtrate was centrifuged at 10,000× g for 30 min. The supernatant was dialyzed against deionized water for 2 days and lyophilized. Approximately 5 g of crude powder could be recovered. The crude powder was dissolved in 50 mM phosphate buffer and fractionated by ion-exchange chromatography on CM-Sepharose CL-6B column (1.5×29 cm). Around 450 mg of crude powder was applied onto the column which was first equilibrated with 50 mM phosphate buffer, pH 6.3. After the unabsorbed material had been eluted, a linear gradient of 0– 0.3 N NaCl in 50 mM phosphate buffer (pH 6.3) was applied to elute the trichosanthin (TCS) fraction. The TCS fraction, eluted as a major peak at around 80 mM of NaCl, was further purified by MPLC on Mono-S HR 5/5 column. In a typical run, around 3– 4 mg of protein from the trichosanthin fraction was dissolved in 20 mM phosphate buffer (pH 7.5, buffer A) and applied to the Mono-S HR 5/5 column which had been equilibrated with the same buffer. Elution was performed with a linear gradient of buffer B (0.2 M NaCl in buffer A) from 0 to 50% in 50 min. However, successful separation of the TCS isoforms was obtained using a linear gradient of 0–10% buffer B (200 mM NaCl in buffer A) for 15 min followed by 10–30% buffer B for 10 min. The flow rate was maintained at 1 ml/min and the protein level was monitored at 280 nm. The corresponding isoforms were collected, dialyzed and freeze-dried.

Amino acid composition and protein sequence analysis were performed at the Molecular Biology Resource Facility at the University of Oklahoma Health Sciences Center in Oklahoma City, OK, USA. Protein hydrolysis was performed in vacuum sealed tubes with 6 N HCl at 110 °C for 20–24 h. Hydrolyzed samples were vacuum dried, dissolved in 0.01 N HCl, and filtered through 0.45 mm nylon filter before analysis. Amino acids were separated by cation exchange chromatography using a two-buffer system. Amino acids were detected by on-line post-column reaction with ninhydrin. This procedure was performed on a fully automated Beckman system Gold HPLC amino acid analyzer. For protein sequence determination, protein samples after separation by SDS-PAGE were transblotted onto PVDF membrane. The transblotted protein was sequenced with an Applied Biosystems 479A gas-phase sequencer with an on-line PTH-amino acid identification using an Applied Biosystem 120 PTH analyzer.

2.2. SDS-polyacrylamide gel electrophoresis SDS-polyacrylamide gel electrophoresis (SDSPAGE) was used to monitor the isolation as well as the estimation of the apparent molecular weights of the TCS isoforms [12]. The Bio-Rad low range protein standards were used as markers.

2.3. Protein fingerprinting by chemical methods Cleavage at the methionine residues using cyanogen bromide (CNBr) followed by SDS-PAGE was used for protein fingerprinting of the TCS isoforms [13]. Purified TCS isoforms (2 mg) were incubated in 70% formic acid with equal weight of CNBr crystals in the dark for 24 h at room temperature. After incubation, the mixture was diluted 15 times with water and freeze-dried. The resulting peptides were analyzed by SDS-PAGE. The peptide pattern was visualized by staining with Coomassie blue.

2.5. N-glycosidase acti6ity assay The N-glycosidase activity of the TCS isoforms was determined by a modified method according to Fong et al [14]. Briefly, TCS isoforms (10 mg) were incubated at 37 °C for 30 min with rabbit reticulocyte lysate in buffer H (90 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 50 mM NaCl). The reaction was terminated by adding SDS to a final concentration of 0.5%. Total RNA was extracted with phenol/chloroform followed by ethanol precipitation. The depurinated site on the rRNA was cleaved by incubation with aniline at 60 °C for 3 min. The treated RNA sample was electrophoresed at a constant voltage of 100 V for 1.5 h in 2.5% polyacrylamide/0.5% agarose composite gel containing 50% formamide. The rRNA was stained with ethidium bromide and observed under UV illumination.

2.6. Inhibition of protein synthesis The in vitro translation system developed by Pelham and Jackson was used to determine the protein synthesis inhibitory activity of the TCS isoforms [15]. Various concentrations of the TCS isoforms were incubated with rabbit reticulocyte lysate (Promega) at 30 °C for 1 h in the presence of L-[4,5-3H] leucine. The reaction was terminated by transferring 5 ml of the reaction mixture to 95 ml of 1 N NaOH/2% H2O2. Proteins were precipitated by the addition of 900 ml of 25% TCA, 2% casamino acid mixture followed by incubation on ice for 30 min. L-[4,5-3H]-leucine labelled protein precipitates were collected by filtration onto glass fiber discs and processed for liquid scintillation counting.

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2.7. DNase acti6ity

3. Results

The DNase activity of the TCS isoforms was determined using super-coiled plasmid DNA (pBluescript II KS+) as substrate [16]. The plasmid DNA (2 mg) was incubated at 37 °C for 2.5 h with different amounts of protein in a final volume of 20 ml of One-Phor-All buffer (Pharmacia) containing 10 mM Tris– acetate (pH 7.5), 10 mM magnesium acetate and 50 mM potassium acetate. The reaction was terminated by adding 3.5 ml of 6× sample dye (50% glycerol, 1 mM EDTA, 0.25% bromophenol blue and 0.25% xylene cyanol). The samples were electrophoresed (1.2% agarose gel) at a constant voltage of 100 V for 90 min. DNA bands were visualized and photographed under UV after staining with ethidium bromide. The DNA cleavage activities were quantitated by digitizing the photographic image using a scanner. The digital image was then analyzed using a computer software— QuantiScan from Biosoft. The DNase cleavage activity was expressed as the percentage of conversion of supercoiled DNA to open-circular and linear form DNA.

3.1. Isolation and physical characterization

2.8. Cytotoxicity assay Trypan blue exclusion and neutral red uptake assay were used to measure the cytotoxicity of TCS isoforms on normal spleen cells and L929 fibroblast, respectively. Spleen cells from C57Bl/6 mice were re-suspended in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics PSN (50 U/ml penicillin G, 50 mg/ml streptomycin, and 100 mg/ml neomycin). Dead cells and red blood cells were removed by centrifugation on a Ficoll-isopaque gradient. Cytotoxicity of TCS isoforms was determined by incubating spleen cells (1×105/well) with various concentrations of protein samples in a final volume of 0.2 ml RPMI medium in 96-well flat-bottom tissue culture plates. The mixtures were incubated at 37 °C in a humidified 5% CO2 incubator for 24 h. Dead cells were stained with 0.1% trypan blue, and a total of 500 cells were counted for each sample. Cytotoxicity of TCS isoforms on L929 fibroblasts was determined by incubating L929 cells (4×104 cells/well) with various concentrations of TCS isoforms in a final volume of 0.2 ml RPMI medium in 96-well flat-bottom tissue culture plates. After 24 h of incubation, 0.1 ml of culture medium in each well was replaced with 0.1 ml neutral red (0.036%, w/v). The mixtures were incubated at 37 °C for 20 min. The cell monolayer was then washed with phosphate buffered saline. Acidified ethanol (0.2 ml) was added into each well to release dye from viable cells. Optical density of the released dye was measured at 540 nm.

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Results from previous studies showed that trichosanthin prepared from the root tubers of T. kirilowii appeared as a single band with an apparent molecular weight of 26 kDa in SDS-PAGE [17]. When the trichosanthin preparation was further purified using MPLC on Mono-S column, three distinct protein peaks were observed (Fig. 1). The three protein peaks were designated as a-, b-, and g-TCS according to their reverse order of elution from the Mono-S column. The size and homogeneity of the three isolated proteins were checked by SDS-PAGE (Fig. 2). A single band with an apparent molecular weight of 26 kDa was observed for the three protein samples, suggesting that these three proteins had similar molecular mass.

3.2. Protein fingerprinting The primary structure of the three isolated TCS isoforms was analyzed using protein fingerprinting method. After cleavage by CNBr, the patterns of peptide fragments from the three isoforms were obtained (Fig. 3). The pattern of peptide fragments generated from g-TCS was quite distinct from the digests pre-

Fig. 1. Elution profile of trichosanthin isoforms from the Mono-S column. Trichosanthin fraction obtained from CM-Sepharose CL-6B column was fractionated using Mono-S HR 5/5 column. The three isoforms were eluted with a linear gradient of buffer B (0.2 M NaCl in 20 mM phosphate buffer, pH 7.5) from 0 to 50% in 50 min. Flow rate: 1 ml/min.

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P. Narayanan et al. / Plant Science 162 (2002) 79–85 Table 1 Analysis of total amino acid composition Amino acids

Fig. 2. SDS Polyacrylamide gel electrophoresis of TCS isoforms. Lane 1, molecular weight markers: phosphorylase b (97 KDa), bovine serum albumin (66.2 KDa), ovalbumin (45 KDa), carbonic anhydrase (31 KDa), soybean trypsin inhibitor (21.5 KDa), and a-lactalbumin (14 KDa). Lane 2, g-TCS. Lane 3, b-TCS. Lane 4, a-TCS.

pared from a- and b-TCS. Similar observations were obtained when the proteins were digested with enzymes, such as Staphylococcal V8 protease, Glu-C endoprotease, Lys-C endoprotease and alkaline protease (data not shown). These results indicated that g-TCS is structurally distinct from a- and b-TCS while the latter two are very similar.

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan Total number of amino acids

Amino acid residues/molecule g-TCS

b-TCS

a-TCS

Trichosanthin

26.9 14.2 21.7 24.0 8.0 22.1 26.9 0.0 17.7 1.9 14.9 24.3 10.8 11.9 8.7 2.6 13.4 0.0 250

29.8 15.2 25.6 20.9 6.9 14.5 28.2 0.0 14.2 3.7 18.1 26.7 12.3 9.7 9.8 1.3 13.1 0.0 251

28.8 14.9 24.1 20.7 8.0 12.8 29.6 0.0 14.2 4.0 18.2 27.0 13.2 9.8 9.9 1.0 13.9 0.0 250

29.0 16.0 25.0 20.0 8.0 11.0 28.0 0.0 15.0 4.0 19.0 25.0 13.0 9.0 10.0 1.0 13.0 1.0 247

3.3. Partial amino acid sequence of TCS The relationship of a-, b-, and g-TCS was further examined by analyzing their amino acid composition and protein sequences. The amino acid composition of a-TCS is almost identical to the previously reported ‘trichosanthin’, while the composition for g-TCS is clearly distinct from the other two (Table 1). There is no cysteine residues in a-, b-, g-TCS, and ‘trichosanthin’. g-TCS has a lower content of serine, methionine,

and isoleucine, while the amounts of glycine and histidine are almost two-folds of the a-TCS, b-TCS and ‘trichosanthin’. Results from both partial N-terminal and internal sequencing (residues 140–159) showed that g-TCS was distinct from ‘trichosanthin’ (Table 2). On the other hand, the partial sequence of b-trichosanthin (residues 101– 120) obtained from its CNBr fragment was found to be identical to trichosanthin. This result further confirmed the relatedness between b-TCS and ‘trichosanthin’.

3.4. Enzymatic acti6ities of the three isoforms

Fig. 3. Electrophoresis of peptide fragments generated from cyanogens bromide cleavage of TCS and its isoforms. Lane 1, trichosanthin fraction. Lane 2, molecular weight marker. Lane 3, aTCS. Lane 4, b-TCS. Lane 5, g-TCS.

All three TCS isoforms exhibited the specific N-glycosidase activity as indicated by the release of a diagnostic RNA fragment of about 500 bases from the 28S rRNA (Fig. 4). In the cell-free protein synthesis assay, all three isoforms also inhibited the incorporation of 3 H-leucine into trichloroacetic acid precipitable proteins (Fig. 5). a-TCS, being most active, exhibited 78% inhibition of incorporation at a concentration of 4 ng/ml. The other two isoforms b- and g-TCS exhibited similar level of protein synthesis inhibitory activity. Recently, RIPs were found to possess DNase activity. RIP has been shown to convert supercoiled plasmid DNA into closed-circular and linear form DNA. The concentration of a-, b-, and g-TCS required to convert 50% of supercoiled plasmid DNA into open circular and linear DNA is 6, 6 and 17.4 mg/ml, respectively (Fig. 6).

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Table 2 Sequence comparison of N-terminal and CNBr fragments of TCS N-Terminal sequences Residue 1 g-TCS D Trichosanthin D

S/R R

L L

L S

G G

A A

10 T T

S S

A S

T S

Y Y

K G

Q V

F F

I I

Q S

20 N N

Internal sequence of CNBr fragments Residue 140 g-TCS F H Y N Trichosanthin F Y Y N

A A

G –

T N

S S

V A

P A

150 K S

A A

F L

I M

V V

I L

I I

Q Q

T S

159 V T

Residue b-TCS Trichosanthin

L L

P P

Y Y

S S

G G

110 N N

Y Y

E E

R R

L L

G G

T T

A A

A A

G G

120 K K

101 R R

V V

K K

N S

V V

F F

T T

The residues underlined are those different from trichosanthin.

3.5. Cytotoxicity assays The cytotoxic effect of a-, b-, and g-TCS on splenocytes and fibroblasts was examined. The cytotoxicity of a-TCS on splenocytes was higher than b- and g-TCS. The LC50 of a-, b-, and g-TCS on splenocytes was 0.31 mg/ml, \ 2.5 mg/ml, and 2.5 mg/ml, respectively (Fig. 7). At the highest concentration (2.5 mg/ml) that we have examined, the percentage of cytotoxicity of a-, b-, and g-TCS on splenocytes was 64.4, 39.1 and 23.1%, respectively. Thus g- and b-trichosanthin are less toxic than a-trichosanthin. Compared with splenocytes, a different degree of cytotoxicity was observed for the three isoforms. The ID50 for g- and a-TCS was observed at a concentration of 1.2 and 0.8 mg/ml, respectively. However, b-TCS displayed only 40% cytotoxicity at concentration as high as 2.5 mg/ml. Judging from these results, g- and a-trichosanthin are more toxic to fibroblasts than bTCS.

showed that a-TCS shares immunochemical identity with ‘trichosanthin’ (data not shown). These observations suggested that a-TCS is probably identical to the previously reported trichosanthin. The isoform b-TCS is structurally similar to a-TCS. Not only the amino acid composition of b-TCS was similar to trichosanthin, the internal amino acid sequence of fragment (residue 101–120) obtained from CNBr digestion was also identical to trichosanthin. However, the cytotoxicity of b-TCS on splenocytes and also the protein synthesis inhibitory activity were much less toxic than a-TCS.

4. Discussion Earlier studies on trichosanthin (TCS) isolated from the root tuber of T. kirilowii suggested that TCS is a type 1 RIP [18]. In this study, we established a fractionation procedure for the isolation of the various isoforms of TCS from the previously reported ‘trichosanthin’ fraction. Three TCS isoforms were identified, and the molecular weight of these three proteins was similar. All three isoforms possess N-glycosidase activities but they exhibit different degree of cytotoxicity. a-TCS, the major protein, exhibits many characteristics similar to the ‘trichosanthin’. Apart from the Nglycosidase activity, DNase activity, and the inhibition of protein synthesis, the total amino acid composition of a-TCS is almost identical to the ‘trichosanthin’. Results from double immunodiffusion assay also

Fig. 4. Characterization of the N-glycosidase activity of the TCS isoforms. Rabbit reticulocyte lysate (50 ml) was incubated with 10 mg of each TCS isoform at 37 °C for 30 min. The ribosomal RNA was extracted and separated by composite gel electrophoresis (2.5% acrylamide– 0.5% agarose) after treatment with aniline. The RNA bands were visualized by ethidium bromide staining. Lane 1, RNA marker; lane 2, negative control (without TCS); lane 3, g-TCS; lane 4, b-TCS; and lane 5, a-TCS. The new RNA fragment produced by the TCS isoform and aniline cleavage was indicated by an arrow.

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Fig. 5. Inhibition of protein synthesis by the TCS isoforms in an in vitro translation system. Different concentrations of TCS isoforms were applied to a rabbit reticulocyte lysate system. Inhibitors of protein synthesis were determined by their effect on the incorporation of 3H-leucine into TCS precipitable material. Results shown represent the mean of two independent experiments. Each data represents the mean of triplicate determinations.

g-TCS, as judged from the elution profile, is the least abundant of the three isoforms. According to the results from protein fingerprinting, amino acid composition and partial sequence analysis, g-TCS is structurally more distinct from a- and b-TCS. Partial sequence analysis of a CNBr fragment of g-TCS corresponding to the residue numbers 140– 159 of trichosanthin indicated a 50% sequence homology. Cytotoxicity assay indicated that g-TCS displayed less cytotoxicity towards splenocytes than a-TCS. Although both b- and g-TCS display similar level of cytotoxicity to splenocytes, the toxicity of b-TCS to fibroblast is lower than g-TCS.

Fig. 6. Conversion of supercoiled DNA to open circular and linear forms by the TCS isoforms.

Fig. 7. The differential cytotoxicity of TCS isoforms towards mouse splenocytes. A total of 500 cells were counted for each data point.

These results demonstrated the presence of multiple isoforms with different biological activity in the root tubers of T. kirilowii. It is generally believed that RIPs exert their cytotoxic effect through the N-glycosidase activity on the 28S rRNA. This ribosome-inactivating property is considered as a form of defense mechanism in the plant cells. We have previously demonstrated that trichosanthin can be induced to accumulate by exposing the sterile plant to soil microorganisms [19]. Furthermore, induction of RIP upon environmental stress was also reported [20]. It has been suggested that RIP may have a function in the normal turnover of ribosomes during normal development or under stress conditions. However, one is still intrigued by the abundance and the presence of multiple forms even in the same tissue. Recently, the view of DNase activity of RIPs, such as ricin-A chain and PAP, has been challenged by the observation that the DNase activity is due to the presence of contaminating nucleases [21]. The activity was not observed in recombinant RIPs. Our results showed that the three isoforms displayed various levels of DNase activity, a- and b-TCS exhibit similar activity while g-TCS is the least active. Recombinant TCS isoform as well as its site-directed mutants also displayed different levels of DNase activities (manuscript in preparation). Taken together, our results indicated that the observed DNase activity of the isolated TCS is unlikely due to nuclease contamination. The existence of RIP isoforms in the same plant or even within the same organ is a common phenomenon [1]. This can be exemplified by the purification of seven major RIPs (saporins) from the leaves, roots and seeds of Saponaria officinalis [4]. Although the exact function of individual isoforms would be difficult to define, they seem to possess different protein inhibitory activity as well as cytotoxicity [5]. Differential cytotoxicity of the

P. Narayanan et al. / Plant Science 162 (2002) 79–85

isoforms towards different cells was confirmed by the present study, which may be related to their targets for each isoforms. If this hypothesis is right, one should be able to demonstrate differential expression of isoforms in response to different stimuli.

Acknowledgements This study was supported by an earmarked grant (RGC/96-97/28) from the Research Grants Committee, Hong Kong SAR Government. P. Narayanan was supported by a post-graduate studentship, Hong Kong Baptist University.

References [1] L. Barbieri, M.G. Battelli, F. Stirpe, Ribosome-inactivating proteins from plants, Biochim. Biophys. Acta 1154 (1993) 237 – 282. [2] W.J. Peumans, Q. Hao, E.J. Van Damme, Ribosome-inactivating proteins more than RNA N-glycosidase?, FASEB J. 15 (9) (2001) 1493 – 1506. [3] W.P. Fong, R.N. Wong, T.T. Go, H.W. Yeung, Minireview: enzymatic properties of ribosome-inactivating proteins (RIPs) and related toxins, Life Sci. 49 (1991) 1859 –1869. [4] J.M. Ferreras, L. Barbieri, T. Girbes, M.G. Battelli, M.A. Rojo, F.J. Arias, M.A. Rocher, F. Soriano, E. Mendez, F. Stirpe, Distribution and properties of major ribosome-inactivating proteins (28 S rRNA N-glycosidases) of the plant Saponaria officinalis L. (Caryophyllaceae), Biochim. Biophys. Acta 1216 (1993) 31 – 42. [5] M.S. Fabbrini, E. Rappocciolo, D. Carpani, M. Solinas, B. Valsasina, U. Breme, U. Cavallaro, A. Nykjaer, E. Rovida, G. Legname, M.R. Soria, Characterization of a saporin isoform with lower ribosome-inhibiting activity, Biochem. J. 322 (Pt 3) (1997) 719 – 727. [6] P.C. Shaw, W.L. Chan, H.W. Yeung, T.B. Ng, Minireview: trichosanthin— a protein with multiple pharmacological properties, Life Sci. 55 (1994) 253 –262. [7] S. Lee-Huang, P.L. Huang, H.F. Kung, B.Q. Li, P.L. Huang, P. Huang, H.I. Huang, H.C. Chen, TAP 29: an anti-human immunodeficiency virus protein from Trichosanthes kirilowii that is nontoxic to intact cells, Proc. Natl. Acad. Sci. USA 88 (1991) 6570 – 6574.

85

[8] S. Toyokawa, T. Takeda, Y. Ogihara, Isolation and characterization of a new abortifacient protein, karasurin, from root tubers of Trichosanthes kirilowii Max. var. japonicum Kitam, Chem. Pharm. Bull. (Tokyo) 39 (1991) 716 – 719. [9] P. Casellas, D. Dussossoy, A.I. Falasca, L. Barbieri, J.C. Guillemot, P. Ferrara, A. Bolognesi, P. Cenini, F. Stirpe, Trichokirin, a ribosome-inactivating protein from the seeds of Trichosanthes kirilowii Maximowicz. Purification, partial characterization and use for preparation of immunotoxins, Eur. J. Biochem. 176 (1988) 581 – 588. [10] T.X. Dong, T.B. Ng, H.W. Yeung, R.N. Wong, Isolation and characterization of a novel ribosome-inactivating protein, betakirilowin, from the seeds of Trichosanthes kirilowii, Biochem. Biophys. Res. Commun. 199 (1994) 387 – 393. [11] T.P. Chow, R.A. Feldman, M. Lovett, M. Piatak, Isolation and DNA sequence of a gene encoding alpha-trichosanthin, a type I ribosome-inactivating protein, J. Biol. Chem. 265 (1990) 8670 – 8674. [12] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680 –685. [13] P. Matsudaira, Limited N-terminal sequence analysis, Methods Enzymol. 182 (1990) 602 – 613. [14] W.P. Fong, Y.T. Poon, T.M. Wong, J.W. Mock, T.B. Ng, R.N. Wong, Q.Z. Yao, H.W. Yeung, A highly efficient procedure for purifying the ribosome-inactivating proteins alpha- and beta-momorcharins from Momordica charantia seeds, N-terminal sequence comparison and establishment of their N-glycosidase activity, Life Sci. 59 (1996) 901 – 909. [15] H.R. Pelham, R.J. Jackson, An efficient mRNA-dependent translation system from reticulocyte lysates, Eur. J. Biochem. 67 (1976) 247 – 256. [16] M.X. Li, H.W. Yeung, L.P. Pan, S.I. Chan, Trichosanthin, a potent HIV-1 inhibitor, can cleave supercoiled DNA in vitro, Nucleic Acids Res. 19 (1991) 6309 – 6312. [17] R.N. Wong, T.B. Ng, S.H. Chan, T.X. Dong, H.W. Yeung, Characterization of Mirabilis antiviral protein —a ribosome inactivating protein from Mirabilis jalapa L., Biochem. Int. 28 (1992) 585 – 593. [18] J.S. Zhang, W.Y. Liu, The mechanism of action of trichosanthin on eukaryotic ribosomes — RNA N-glycosidase activity of the cytotoxin, Nucleic Acids Res. 20 (1992) 1271 – 1275. [19] R.N. Wong, N.K. Mak, W.T. Choi, P.T. Law, Increased accumulation of trichosanthin in Trichosanthes kirilowii induced by microorganisms, J. Exp. Bot. 36 (1995) 669 – 676. [20] J.F. Rippmann, C.B. Michalowski, D.E. Nelson, H.J. Bohnert, Induction of a ribosome-inactivating protein upon environmental stress, Plant Mol. Biol. 35 (1997) 701 – 709. [21] P.J. Day, J.M. Lord, L.M. Roberts, The deoxyribonuclease activity attributed to ribosome-inactivating proteins is due to contamination, Eur. J. Biochem. 258 (1998) 540 – 545.