A new highly toxic protein isolated from the death cap Amanita phalloides is an L-amino acid oxidase Taras Stasyk 1,2 , Maxim Lutsik-Kordovsky 1 , Christer Wernstedt 3 , Volodymyr Antonyuk 1 , Olga Klyuchivska 1 , Serhiy Souchelnytskyi 4 , Ulf Hellman 3 and Rostyslav Stoika 1 1 Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine 2 Biocenter, Innsbruck Medical University, Austria 3 Ludwig Institute for Cancer Research Ltd, Uppsala University, Sweden 4 Karolinska Biomics Center, Karolinska Institutet, Stockholm, Sweden Introduction The death cap (Amanita phalloides) is known to be a deadly poisonous mushroom as a result of the produc- tion of several toxic substances. The first substance was isolated in 1937 by Wieland [1] and was shown to possess an oligopeptide structure. In further studies, Wieland and Faulstich [2] revealed other toxic cyclo- peptides, which were classified into two structural groups (i.e. amanitine and phalloidine), with both Keywords Amanita phalloides; apoptosis; death cap; L-amino acid oxidase; toxic protein Correspondence R. Stoika, Institute of Cell Biology, National Academy of Sciences of Ukraine, Drahomanov Street 14 ⁄ 16, 79005, Lviv, Ukraine Fax: +38 032 261 22 87 Tel: +38 032 261 22 87 E-mail: stoika@cellbiol.lviv.ua Database Nucleotide sequence data have been submitted to the GenBank database under the accession number GU220069 (Received 8 October 2009, revised 2 December 2009, accepted 21 December 2009) doi:10.1111/j.1742-4658.2010.07557.x A new highly cytotoxic protein, toxophallin, was recently isolated from the fruit body of the death cap Amanita phalloides mushroom [Stasyk et al. (2008) Studia Biologica 2, 21–32]. The physico-chemical, chemical and bio- logical characteristics of toxophallin differ distinctly from those of another death cap toxic protein, namely phallolysin. The interaction of toxophallin with target cells is not mediated by a specific cell surface receptor. It induces chromatin condensation, as well as DNA and nucleus fragmenta- tion, which are typical for apoptosis. However, caspase III inhibitor [ben- zyloxycarbonyl-Asp(OMe)-fluoromethylketone] did not stop toxophallin- induced DNA fragmentation. Thus, toxophallin uses a caspase-independent pathway of apoptosis induction. In the present study, we applied a comple- mentary approach based on a combination of proteomics and molecular biology tools for the protein identification of toxophallin. The primary structure of toxophallin was partially studied via direct sequencing of its tryptic peptides, followed by PCR-based cloning of the corresponding cDNA. A subsequent bioinformatic search revealed a structural homology of toxophallin with the L-amino acid oxidase of the Laccaria bicolor mush- room. This demonstrates the usefulness of our approach for the identifica- tion of proteins in organisms with unknown genomes. We also found a broad substrate specificity of toxophallin with respect to oxidizing selected amino acids. Ascorbic acid inhibited the cytotoxic effect of toxophallin, most likely as a result of scavenging hydrogen peroxide, which is the product of oxidase catalysis. Thus, in addition to highly toxic cyclopeptides and toxic lectin phallolysin, the death cap fruit body contains another cytotoxic protein in the form of an enzyme, namely L-amino acid oxidase. Abbreviations CM, carboxymethyl; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. 1260 FEBS Journal 277 (2010) 1260–1269 ª 2010 The Authors Journal compilation ª 2010 FEBS exhibiting different mechanisms of toxic action. Although amanitine inhibits mRNA transcription, phalloidine binds actin and suppresses the functions of the cytoskeleton. These cyclopeptides are frequently used as experimental tools in scientific studies because their intracellular molecular targets and mechanisms of action have been well characterized. In addition to these toxic polypeptides, the death cap also contains antitoxin antamanide, a cyclodecapeptide that blocks the effects of phalloidine [1,2]. Another toxic polypep- tide, phallolysin, possessing hemolytic activity, was also detected in the fruit body of the death cap [3–7]. Its chemical properties and biological activity, as well as its mechanisms of action, have been described previ- ously [2,8]. Many mushroom species have been shown to con- tain polypeptide substances that possess antitumor and immunomodulating activity [9–11]. Lectin-like proteins demonstrating antiproliferative activity towards tumor cells were isolated from the mushrooms Tricholoma mongolicum [12] and Agaricus bisporus [13]. Another protein with antineoplastic activity, volvarin, which belongs to the family of ribosome inactivating proteins type I, was isolated from the edible mush- room Volvariella volvacea [14]. The poisonous mush- room Boletus satanas Lenz contains a toxic lectin bolesatine that inhibits protein synthesis both in vitro and in vivo [15]. Recently, a new cytotoxic protein, toxophallin, was isolated from the fruit body of the death cap A. phalloides [16]. Its physico-chemical, chemical and biological properties differ distinctly from those of the other known toxic proteins of this mushroom. Toxo- phallin was not bound by any target cell surface spe- cific receptors. Furthermore, it induced apoptosis (chromatin condensation, DNA and nucleus fragmen- tation) but this was not blocked by caspase III inhibi- tor [benzyloxycarbonyl-Asp(OMe)-fluoromethylketone] [16]. In the present study, we carried out a more pre- cise structural analysis of toxophallin by directly sequencing its tryptic peptides, followed by PCR-based cloning of cDNA. A bioinformatics approach allowed us to demonstrate the sequence homology of toxophal- lin with the recently identified l-amino acid oxidase of Laccaria bicolor [17]. Results Purification of toxophallin The purification procedure consisted of four main steps: (a) ammonium sulfate precipitation of total protein from the juice of thawed and grinded mush- rooms; (b) elimination of pigmented material from the obtained protein bulk by ion-exchange chroma- tography on a DEAE-cellulose column; (c) affinity chromatography on the immobilized ovomucin to remove cytolytic lectin, phallolysin; and (d) purifica- tion of toxophallin by the repeated ion-exchange chromatography on a carboxymethyl (CM)-cellulose column. Native gel electrophoresis of water-soluble proteins upon elimination of pigmented materials revealed three main protein bands (Fig. 1). The prom- inent band corresponds to phallolysin, the death cap lectin with high cytolytic activity. Phallolysin was effi- ciently removed from the protein extract using affinity chromatography on the immobilized ovomucin. Pro- tein exhibiting cytotoxic activity, and found in the nonlectin fraction, was further purified by two-step ion-exchange chromatography (see Materials and methods). Purified toxophallin migrated as a homogenous protein band by nondenaturing gel electrophoresis (Fig. 1A). A single protein band of 55 kDa was also detected by SDS-PAGE (Fig. 1B). The toxophallin purity was approximately 95% according to native electrophoresis, and approxi- mately 85% according to SDS-PAGE, presumably because of partial protein degradation. Protein characterization Amino acid analysis revealed three cysteines, six methionines and 36 proline residues in the toxophallin molecule, which makes up approximately 7% of the AB Fig. 1. Electrophoretic study of extracted proteins of Amanita phalloides. (A) b-alanin-acetate electrophoretic system, pH 4.5. (1) Crude extract; (2) nonlectin proteins (not retained by the affinity sorbents); and (3) cytotoxic protein purified by the ion-exchange chromatography on CM-cellulose column. (B) SDS-PAGE in 14% gel. (1) Molecular mass protein markers (Sigma) and (2) purified protein (55 kDa) under study. Coomassi R-250 staining. Reproduced with permission [16]. T. Stasyk et al. Cytotoxic L-amino oxidase from A. phalloides FEBS Journal 277 (2010) 1260–1269 ª 2010 The Authors Journal compilation ª 2010 FEBS 1261 amino acid residues present in this 55 kDa protein, which consists of 503 amino acid residues (Table S1). The relatively high content of proline residues suggests a significant rigidity of the polypeptide chain of toxo- phallin. For MS analysis, toxophallin was in-gel digested using trypsin, and the peptide mixture was analyzed by matrix-assisted laser desorption ionization time-of- flight (MALDI-TOF) MS. Sixty-nine tryptic peptides were identified (Table S2) and used for the database search. Because we could not find any homology with other proteins in the databases by peptide mass finger- printing, sequencing of the separated tryptic peptides was carried out. Tryptic peptides of the toxophallin were isolated by microbore reversed phase liquid chro- matography (Fig. S1) and several isolated peptides were subjected to Edman degradation. The amino acid sequences of ten peptides that were sequenced, differ- ing in length by five to 16 amino acid residues, are shown in Fig. 2A. According to the amino acid com- position of toxophallin (Table S1), with 29 K and 14 R identified, 43 tryptic peptides could be expected. This suggests that the other 26 peptides represent modified peptides or miss cuts of the digestion. Unexpectedly, all ten peptides sequenced by Edman degradation were not modified because the masses cal- culated from amino acid composition of the sequenced peptides amounted to the values of the corresponding peptides obtained by the MALDI-TOF MS. Direct sequencing of the toxophallin N-terminus from sam- ples after blotting onto poly(vinylidene difluoride) membrane did not reveal any signal, thereby suggest- ing that the N-terminus of the protein was blocked. In total, sequenced peptides account for 20% of the whole molecule, with 107 amino acids being identified in the ten peptides analyzed. To obtain an mRNA sequence of toxophallin, we performed RT-PCR-based cloning. Ten oligonucleotide primers were designed using the amino acid sequence of the identified peptides. PCR reactions with different combinations of primers were performed with cDNA from mRNA isolated from the whole fruit body. The primer combinations TTC CCA GAG ATC GAG TCA ATG CGT (3¢-to5¢) and TCT GTC GTA CCA ACC AGT TGA (5¢-to3¢), designed on the basis of peptides 15 (FPEIESMR) and nine (STGWYDR), respectively, resulted in a PCR product. This PCR product was cloned and sequenced, as described in the A B Fig. 2. Partial sequence of toxophallin. (A) Amino acid sequence of ten identified tryptic peptides of toxophallin (Edman degradation analysis; Fig. 2). (B) Partial nucleotide sequence and deduced amino acid sequence of the toxophallin. The amino acid translation is under the second nucleotide of the corresponding codon. The masses of underlined peptides correspond to the masses of tryptic peptides (Table S2) identified by MALDI-TOF MS. Cytotoxic L-amino oxidase from A. phalloides T. Stasyk et al. 1262 FEBS Journal 277 (2010) 1260–1269 ª 2010 The Authors Journal compilation ª 2010 FEBS Materials and methods, and 429 nucleotides were iden- tified (Fig. 2B). This sequence, in combination with primers, corresponds to a polypeptide consisting of 158 amino acid residues, and comprises approximately one-third of the molecule (approximately 503 amino acid residues; Table S1). In the internal part of the identified cDNA, sequences corresponding to two other peptides sequenced by Edman degradation were found: peptides 22 and 57 (Fig. 2). Moreover, the obtained partial sequence of toxophallin was also con- firmed by the MS data when comparing the in silico tryptic digest of the translated amino acid sequence with the list of masses of tryptic peptides obtained by the MALDI-TOF analysis. In total, nine peptides from the list of toxophallin tryptic peptides (numbers 9, 15, 19, 22, 27, 57, 58, 63, 65; Fig. 2 and Table S2), includ- ing four peptides sequenced by Edman degradation, matched the corresponding tryptic peptides of the sequenced toxophallin mRNA fragment (see under- lined peptides in Fig. 2), thereby unambiguously con- firming our RT-PCR-based cloning strategy in combination with MS and Edman sequencing. A database search for similar protein sequences was carried out using the blast algorithm. We found sequence homology of toxophallin with the amine oxidase of L. bicolor (Fig. S2). The partial amino acid sequence deduced from the cloned mRNA fragment was found to be related to two predicted proteins from L. bicolor S238N-H82 according to the recently published genome of this mushroom [17] (accession numbers EDR00058.1 and EDR12198.1) with 49% and 45% identities (i.e. the extent to which two sequences are invariant) and 60% and 57% positives (i.e. changes at a specific position of an amino acid sequence that preserves the physico-chemical proper- ties of the original residue), respectively. Moreover, we could align the remaining six sequenced peptides to the C-terminal part of the L. bicolor protein EDR12198.1 (Fig. S2). A high degree of similarity between the partial sequence of toxophallin and the amine oxidases sequences available in the database strongly suggests a putative amine oxidase activity of toxophallin. Biological activity of toxophallin The cytotoxic activity of the purified toxophallin was monitored by measuring its effect towards human leu- kemia CEM-T4 and murine leukemia L1210 cells (Fig. 3). Toxophallin possesses a distinct cytotoxic effect (as detected by the trypan blue exclusion assay) that was much stronger in the case of CEM-T4 cells compared to L1210 cells. The IC 50 of purified toxophallin was 0.5 lgÆmL )1 . The IC 50 values with respect to the action of toxophallin in the cell viability test as estimated by the trypan blue exclusion assay (0.5 lgÆmL )1 ), as well as by cell proliferation as deter- mined by [ 3 H]-thymidine incorporation (0.25–0.45 lgÆmL )1 ) [16], were of similar concentration depen- dence, indicating that the activity of toxophallin is cytotoxic, rather than antiproliferative. In a previous study, we have shown that toxophallin promotes cell death via apoptosis, which was demonstrated by a DNA fragmentation assay performed in different mammalian cell lines (murine leukemia L1210, mink lung epithelial CCL-64, human lung carcinoma A549 and human breast carcinoma MCF-7 cells) [16]. The proapoptotic action of toxophallin, as revealed in a DNA-laddering bio-assay, was demonstrated by the results of a cytomorphological study, using 4¢,6¢- diamidino-2-phenylindole staining and terminal 0.0 0.5 1.0 1.5 2.0 2.5 0 20 40 60 80 100 A B L1210 CEMT4 % of trypan-positive (dead) cells L-amino-acid oxidase (µg·mL –1 ) 0.0 0.5 1.0 1.5 2.0 2.5 0 20 40 60 80 100 L1210 CEMT4 Cell number (% of control) L-amino-acid oxidase (µg·mL –1 ) Fig. 3. Dose-dependent effect of toxophallin (L-amino acid oxidase) towards target (human CEM-T4 and murine L1210) cells. Approximately 300 000 cells of the L1210 line per well in 1 mL, and 200 000 cells of the CEM-T4 line per well in 1 mL were present at the beginning of the experiment. After 24 h, the tested substances were added at different concentrations. The number of viable cells was counted in the hemocytometric chamber. The ratio of dead cells was defined subsequent to staining with trypan blue (0.1%, w ⁄ v) and observation under a light microscope. T. Stasyk et al. Cytotoxic L-amino oxidase from A. phalloides FEBS Journal 277 (2010) 1260–1269 ª 2010 The Authors Journal compilation ª 2010 FEBS 1263 deoxynucleotidyl-transferase-mediated dUDP nick-end labeling (i.e. assay for apoptosis detection) [16]. The time dependence of the effect of toxophallin as studied using the trypan blue exclusion test and 4¢,6¢- diamidino-2-phenylindole staining demonstrated that toxophallin-induced cell death became noticeable after 5 h [16]. Taking into account that toxophallin of A. phalloides displays structural homology with amine oxidase isolated from L. bicolor, we have examined the amine oxidase activity of toxophallin, as reported previously [18]. The results obtained in that study are presented in Table 1 (see also Table S3). Toxophallin did not use benzylamine, ethanolamine, diethylamine, meta- and para-phenylendiamine, ortho-, meta- and para- aminophenols, or putrescin as a substrate for the enzymatic reaction, which testifies to the absence of its mono- and diamine oxidase activity. The highest oxi- dase activity was observed towards dl-methionine and l-methionine, l-phenylalanine, dl-norleucine, l-isoleu- cine, l-arginine, l-tyrosine, and dl-leucine; oxidase activity was relatively low towards dl-lysine and l-lysine, dl-asparagine, dl-valine, l-histidine, dl -threo- nine, dl-thryptophane, and l-glutamic acid; and there was a lack of oxidase activity towards l-cysteine, l-glycine, l-proline, l-oxyproline, dl-serine, and dl-aspartic acid. These results indicate that the novel toxic protein, purified from A. phalloides mushroom is an l-amino acid oxidase. Ascorbic acid (10 lgÆmL )1 ) inhibited the cytotoxic effect (measured by the trypan blue exclusion test) caused by toxophallin (l-amino acid oxidase) (Fig. 4). The mechanisms of such inhibition could be based on inactivating the H 2 O 2 that appears as a result of the amine oxidase reaction and is toxic for cells. Both ascorbic acid and reduced glutathione also inhibited amine oxidase reaction in vitro (Fig. 5). Discussion When studying toxic proteins isolated from the fruit bodies of the death cap A. phalloides, we detected a novel cytotoxic protein that differed from all previ- ously described toxic proteins from that mushroom species. It differs distinctly from phallolysin, which was isolated and characterized by Faulstich et al. [3,4] and Seeger et al. [5–7]. Both proteins differ substan- tially in their biological activity. Phallolysin is highly toxic in animals, reaching a lethal dose at 40 lgÆkg )1 in rabbits [3]. Its hemolytic activity towards rabbit erythrocytes in vitro was 5 lgÆmL )1 [16]. Toxophallin was found to exhibit high toxicity towards various mammalian cells; for example, in cells of A549 and T47D lines, IC 50 = 0.25 lgÆmL )1 and, in cells of CCL-64 and MCF 7 lines, IC 50 = 0.45 lgÆmL )1 [16]. Toxophallin preparations did not possess hemolytic Table 1. Toxophallin is L-amino acid oxidase. Different amino acids were studied as toxophallin substrates using an amine oxidase enzymatic activity assay, as described in the Materials and methods. No. Substrate Relative activity 1 DL-tyrosine 1.0 2 L-tyrosine 1.9 3 DL-lysine 0.3 4 L-lysine 0.6 5 DL-asparagine 0.4 6 L-asparagine 0.8 7 DL-phenylalanine 1.3 8 L-phenylalanine 2.6 9 DL-methionine 2.7 10 L-methionine 3.6 012345 0 20 40 60 80 100 A B Cell number (% of control) L-amino-acid oxidase (µg·mL –1 ) Control + 10 µg·mL –1 ascorbic acid 012345 0 10 20 30 40 50 Control + 10 µg·mL –1 ascorbic acid % of trypan-positive (dead) cells L-amino-acid oxidase (µg·mL –1 ) Fig. 4. Ascorbic acid inhibits cytotoxic effect of toxophallin (L-amino acid oxidase) towards the murine L1210 cell line. The experiment conditions are as in Fig. 3. Ascorbic acid (a concentration of 10 lgÆmL )1 was selected as being the most effective with respect to inhibiting the cytotoxicity of toxophallin) was added to cell culture simultaneously with toxophallin used at different concentrations. A statistical significant difference (P < 0.05) was observed at a concentration of toxophallin of 5 lgÆmL )1 . Cytotoxic L-amino oxidase from A. phalloides T. Stasyk et al. 1264 FEBS Journal 277 (2010) 1260–1269 ª 2010 The Authors Journal compilation ª 2010 FEBS activity, whereas the hemolytic activity of phallolysin reached 24 000 unitsÆmg )1 [4]. In the present study, we used a complementary approach (i.e. a combination of proteomic and molec- ular biology tools) to identify biologically active pro- teins in organisms with unknown genomes. The sequence of toxophallin was partially studied by a combination of MS and direct Edman sequencing of tryptic peptides with a PCR-based cloning of the cDNA. The high level of overlap between the sequenced peptides and the cDNA indicated strong support for the partial protein sequence obtained, and allowed us to find a homology of toxophallin with the l-amino acid oxidase of L. bicolor according to the recently published genome of this mushroom [17]. The mRNA of toxophallin (2.1 kb) was detected only in the stem and, to a lesser extent, in the cap of A. phalloides fruit bodies by Northern blot analysis using a RT-PCR fragment of the cloned toxophallin cDNA as a probe for the hybridization reaction (data not shown). Recently, the l-amino acid oxidase gene of L. bicolor has been shown to be expressed at protein level [19]. In this study, it was suggested that amine oxidases are enzymes of cellular amino acid catabo- lism, comprising potential candidates for a mechanism that catalyses nitrogen mineralization from amino acids at the ecosystem level. The distribution of l-amino acid oxidase in the stem of the A. phalloides mushroom fruit body fits very well with this hypothe- sis. It should be noted that we also found a protein possessing toxophallin-like activity in the Amanita virosa fruit body (V. Antonyuk et al., in press). A cross-linking receptor study did not reveal specific receptor molecules for this protein on the surface of target cells [16]. The cytotoxic effects were found to develop relatively slowly because the first signs of cell damage were observed only after 5 h of treatment. Target cells underwent apoptosis subsequent to toxophallin treatment and cell death did not depend on the activation of the caspase cascade [16]. The most pronounced destructive changes, namely condensation of nuclear chromatin and DNA fragmentation, were observed in the cell nucleus. Similar processes were characteristic for cell damage caused by the ionizing radiation, and these were mediated by generation of reactive oxygen species [20]. Thus, it is suggested that toxophallin induces cell damage indirectly via the gen- eration of free radicals and oxidant agents that can trigger cell impairment and apoptosis by a caspase- independent pathway. l-amino acid oxidase enzymatic activity of the toxophallin is well suited for such action. Via the H 2 O 2 generated by the enzyme activity, amine oxidases may act as a defense or attack mecha- nism. l-amino acid oxidase has been described as one of the most common components of snake venom [21–23], as recently reviewed [24]. Although partially purified toxophallin was accessible previously [25], its specific enzymatic activity remained unknown at that time. Recently, a protective action of the radical scavenger N-acetylcysteine upon treatment of A. phalloides poi- soning was demonstrated [26]. It is possible that the products of the l-amino acid oxidase (toxophallin) enzymatic reaction could also be inactivated by this agent. Various biological systems, accompanied by an increased production of reactive oxygen species, are effective as potential anticancer remedies. Cytotoxicity was observed as a result of the action of BSA oxidase in the presence of spermine, and this was attributed to H 2 O 2 and aldehyde production [27]. Increasing the incubation temperature from 37 to 42 °C enhanced cytotoxicity in tumor cells exposed to spermine metab- olites. Moreover, it was found that multidrug-resistant human melanoma cells were more sensitive than their wild-type counterparts to H 2 O 2 and aldehydes [28]. The metabolites formed by BSA oxidase targeting spermine were more toxic than exogenous H 2 O 2 and acrolein, even though their concentration was lower during the initial phase of incubation. The increase of natural polyamines in malignant and actively prolifer- 0 0.1 0.2 0.3 0.4 0.5 13579 E525 Time (min) Control glutathione-SH 0.5 mg·mL –1 Ascorbic acid 0.5 mg·mL –1 Ascorbic acid, glutathione-SH, 5 mg·mL –1 Fig. 5. Ascorbic acid and reduced glutathione inhibit L-amine oxidase activity of the toxophallin in vitro. The reaction mixture consisted of 0.15 mL of 0.1% aqueous solution of toxophallin and 2.5 mL of 0.3 m M o-dianizidine solution to which 0.2 mL of 0.1% horseradish peroxidase (RZ = 0.4–0.6) was added. The reaction was started by adding 0.2 mL of 0.2% solution of L-methionine, after which the absorbance at 525 nm was measured in the spectrophotometer cuv- ette at different time intervals. When ascorbic acid or glutathione-SH were used, they were added at a final concentration of 0.5 and 5mgÆmL )1 before adding L-methionine. T. Stasyk et al. Cytotoxic L-amino oxidase from A. phalloides FEBS Journal 277 (2010) 1260–1269 ª 2010 The Authors Journal compilation ª 2010 FEBS 1265 ating cells has led the use of polyamine depletion as a strategy for inhibiting cell growth [29]. Thus, in the anticancer therapeutic strategy, there is increasing interest in spermine oxidase, which specifically oxidases spermine. Because putrescine was not active as a sub- strate in the enzymatic reaction of toxophallin purified from A. phalloides, this testifies to the absence of mono- and diamine oxidase activity in that protein. However, toxophallin demonstrated itself to be an l-amino acid oxidase, which suggests that it should not have a deficiency of substrates for its catalytic activity. For definite conclusions on the anticancer potential of toxophallin, additional investigations are required. It should be noted that deadly poisonous mush- rooms, such as the death cap, contain various cyto- toxic polypeptide compounds that possess different mechanisms of toxic action. Thus, treatment for poi- soning caused by these mushrooms should be complex and include antidotes against all toxic compounds. Because toxophallin is an l-amino acid oxidase, H 2 O 2 scavengers may be protective during its action in the organism. In conclusion, in the present study, a novel cytotoxic protein was isolated from the death cap and character- ized. The physico-chemical, chemical and biological characteristics of this protein differ distinctly from those of all previously described toxic substances of A. phalloides, such as toxic cyclopeptides or phallolysin. The isolated cytotoxic protein was shown to be an enzyme, namely l-amino acid oxidase. Materials and methods Isolation and purification of cytotoxic proteins from the death cap Fruit bodies of A. phalloides mushrooms were collected in the forests of Lviv region (Ukraine), and stored at )20 °C until use (not longer than 1 month). The mush- room fruit bodies were pressed, subjected to centrifugation at 4000 g for 15 min, and the supernatant was collected. Ammonium sulfate was added to 90% saturation of the supernatant, and precipitated proteins were collected by filtration. For elimination of dark colored pigment mate- rial, the precipitate was dissolved in a small volume of distilled water, dialyzed against buffer solution (50 mm potassium phosphate buffer, pH 7.0 supplemented with 100 mm sodium chloride) and passed through a DEAE- cellulose column (Serva, Heidelberg, Germany), equili- brated with the same buffer. The fraction of unabsorbed protein was collected and precipitated with ammonium sulfate at 90% saturation. For elimination of cytolytic lectin, phallolysin, crude pro- tein fraction was passed through a column filled with affin- ity sorbent, ovomucin immobilized on agarose [30], equilibrated with NaCl ⁄ Pi. The unbound ‘nonlectin’ protein fraction was collected, dialyzed against 30 mm sodium acetate buffer (pH 5.3) and applied onto a CM-cellulose column (CM-32; Whatman Biochem. Ltd., Maidstone, UK), which was equilibrated with 30 mm sodium acetate buffer (pH 5.3). The column was eluted stepwise with 100 mm sodium acetate buffer and, subsequently, with the same buffer supplemented with 75 mm sodium chloride. Protein possessing cytotoxic action was eluted with 75 mm sodium chloride. This protein peak was collected, concen- trated and subjected to re-chromatography on a CM-cellu- lose column in 100 mm sodium acetate buffer (pH 5.3) and 75 mm sodium chloride. The main protein peak corre- sponding to pure cytotoxic protein was collected, dialyzed against distilled water and lyophilized. Two electrophoretic systems were applied for the evaluation of purity of isolated toxophallin: (a) disc-electro- phoresis in 7.5% PAGE using the Reisfeld system in b- alanine-acetate buffer (pH 4.5) and protein staining with Amido Black 10B [31] and (b) SDS-PAGE in 14% slab gel in a Laemmli buffer system [32] with protein visualization by Coomassie Brilliant Blue R-250. Markers of protein molecular mass (GE Healthcare, Uppsala, Sweden) were in the range 14.4–94 kDa. In-gel digestion, MS analysis and Edman sequencing Purified toxopallin sample was subjected to SDS-PAGE, and protein bands were visualized by Coomassie Brilliant Blue R-250 staining. The 55 kDa protein band was excised from the gel and in-gel digested with modified trypsin of sequence grade (Promega, Madison, WI, USA), as described previously [33]. The peptide mixture was analyzed by MALDI-TOF MS, using a Bruker Biflex III instrument (Bruker Daltonics, Bremen, Germany) equipped with delayed extraction and reflector. The sample was prepared by the dried droplet technique, using alpha-cyano-4- hydroxycinnamic acid as matrix. The instrument was externally calibrated with angiotensin II (MH+1046.54) and adrenocorticotropic hormone fragment 18–39 (MH+2465.20). The peptide mass fingerprint analysis was performed using profound (http://prowl.rockefeller.edu/) and mascot (http://www.matrixscience.com/). For Edman degradation, peptides were isolated by microbore reversed phase liquid chromatography on a 1 · 150 mm Kromasil C18 column using a SMART System (GE Healthcare). Selected fractions were subjected to amino acid sequence analysis using a Procise 494 instrument (PE-Biosystems, Foster City, CA, USA), in accordance with the manufac- turer’s instructions. Cytotoxic L-amino oxidase from A. phalloides T. Stasyk et al. 1266 FEBS Journal 277 (2010) 1260–1269 ª 2010 The Authors Journal compilation ª 2010 FEBS mRNA purification and cDNA synthesis Total RNA was extracted using TRIzol Reagent (Life Technologies, Grand Island, NY, USA), according to the manufacturer’s instructions. cDNA from mRNA of the fruit body was synthesized by a reverse transcriptase reac- tion with Moloney murine leukemia virus reverse transcrip- tase and random hexamer primers. Twenty microliters of reaction mixture contained 1 lg of total RNA, 200 U of Moloney murine leukemia virus reverse transcriptase (BRL, Gaithersburg, MD, USA), 10 mm dithiotreitol (BRL), 10 mm of each dNTP (Pharmacia Biotechnology AB, Stockholm, Sweden), 100 pM of random hexamers (Boehringer Mannheim, Mannheim, Germany) and RNAse inhibitor in the reverse transcriptase buffer (BRL). The solutions were incubated at 37 °C for 90 min, and then heated to 98 °C for 10 min, and cooled rapidly to 4 °C. Cloning and sequencing of cDNA The primers for PCR-based cloning were designed on the basis of the peptide sequences identified by Edman sequenc- ing. The selection of alternative codons was random. Five pairs of complementary primers were used and 18 combina- tions of 3¢-to5¢ and 5¢-to3¢ primers were employed. The cycling program started with 0.5 min of denaturation at 95 °C, which was followed by 30 cycles consisting of 0.5 min of annealing at 38, 45 or 50 °C, 1.5 min of exten- sion at 72 °C, and 0.5 min of denaturation at 95 °C. The amplified DNA fragments were cloned in pCR-Script vector (Stratagene, La Jolla, CA, USA), according to manufac- turer’s instructions. Double-stranded DNA fragments were sequenced in both directions with Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) using an ABI Prism 310 Genetic Analyzer. Sequences of PCR products translated into amino acid sequences were also analyzed using gpmaw software (Light- house Data, Odense, Denmark) after in silico tryptic diges- tion of the corresponding peptides. Cells: culturing and testing Human lung carcinoma epithelial A549 cells, mink lung epithelial CCL-64 cells, human breast adenocarcinoma MCF-7 and T47D cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Murine leukemia L1210 cells and human leukemia T-cells CEM-T4 line were obtained from the collection at R. E. Kavetsky Institute of Experimental Pathology, Oncology and Radio- biology (National Academy of Sciences of Ukraine, Kyiv). Cells were cultured in DMEM (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% fetal bovine serum and 100 unitsÆmL )1 of gentamycin (Sigma-Aldrich). For mea- surement of the cytotoxic effects of toxophallin, cells were seeded in 24-well plastic dishes in DMEM in the presence of 10% fetal bovine serum. After 24 h, tested substances were added at different concentrations. The number of via- ble cells was counted in the hemocytometric chamber at various time intervals. The ratio of dead cells was defined subsequent to staining with trypan blue (0.1%, w ⁄ v) and observation under a light microscope. Assay of enzymatic activity of amine oxidase The enzymatic activity of toxophallin was measured as described by Haywood and Large [18], with minor modifi- cations. The mixture of 0.2 mL of 0.1% horseradish peroxi- dase (RZ = 0.4–0.6) and 0.15 mL of 0.1% aqueous solution of toxophallin was added to 2.5 mL of 0.3 mm o-dianizidine solution, placed in the spectrophotometer cuvette, and measured at a wavelength of 525 nm. The reaction was started by adding various amine containing compounds. The absorbance of the reaction mixture was measured at different time intervals. The reaction was initi- ated by adding 0.2 mL of 0.1–1% solution of various amino compounds, and the absorbance was determined at various time intervals. Enzymatic activity towards dl-tyro- sine was considered as 1.0, and the corresponding activities towards other amino compounds were calculated relative to this value. When ascorbic acid or glutathione-SH was used for inhibition of the enzymatic reaction, they were added at a final concentration of 0.5 and 5 mgÆmL )1 . Statistical analysis All experiments were repeated at least three times with minimum three parallels. The standard deviation was calculated, and the statistical significance of difference was evaluated using Student’s t-test (P < 0.05). Acknowledgements The present study was supported by a grant awarded by the Royal Swedish Academy of Sciences. T.S. was also partially supported by a grant from the West- Ukrainian BioMedical Research Center. The technical assistance of Mrs Galina Shafranska during the cell culturing is highly appreciated. References 1 Wieland T (1983) The toxic peptides from Amanita mushrooms. Int J Pept Protein Res 23, 257–276. 2 Wieland T & Faulstich H (1978) Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms. CRC Crit Rev Biochem 5, 185–260. T. Stasyk et al. Cytotoxic L-amino oxidase from A. phalloides FEBS Journal 277 (2010) 1260–1269 ª 2010 The Authors Journal compilation ª 2010 FEBS 1267 3 Faulstich H & Weckauf-Bloching M (1974) Isolation and toxicity of two cytolytic glycoproteins from Amanita phalloides mushrooms. Hoppe Seylers Z Physiol Chem 355, 1489–1494. 4 Faulstich H, Buhring H-J & Seitz J (1983) Physical properties and function of phallolysin. Biochemistry 22, 4574–4580. 5 Seeger R, Scharrer H & Haupt M (1973) Phallolysin, ein hochmolekulares Toxin aus Amanita phalloides. Experientia 29, 829–830. 6 Seeger R (1975) Demonstration and isolation of phallolysin, a haemolytic toxin from Amanita phalloides. Naunyn-Schmiedebergs Arch Pharmacol 287, 277–287. 7 Seeger R & Wachter B (1981) Rubescenslysin and phallolysin release marker molecules from phospholipid cholesterol liposomes. Biochim Biophys Acta 645, 59. 8 Herrmann M, Lorenz H-M, Voll R, Grunke M, Woith W & Kalden JR (1994) A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic Acid Res 22, 5506–5507. 9 Borchers AT, Stern JS, Hackman RM, Keen CL & Gershwin ME (1999) Mushrooms, tumors, and immu- nity. Proc Soc Exp Biol Med 221, 281–293. 10 Hobbs Ch (2003) Medicinal Mushrooms: an Exploration of Tradition, Healing, and Culture. Botanica Press, Summertown, Tennessee. pp. 64–67. 11 Mizuno T, Saito H, Nishitoba T & Kawagishi H (1995) Antitumor-active substances from mushrooms. Food Rev Int 11, 23–61. 12 Wang HX, Ng TB, Liu WK, Ooi VE & Chang ST (1995) Isolation and characterization of two distinct lectins with antiproliferative activity from the cultured mycelium of the edible mushroom Tricholoma mongolicum. Int J Pept Protein Res 46, 508–513. 13 Parslew R, Jones K, Rhodes JM & Sharpe GR (1999) The antiproliferative effect of lectin from edible mush- room Agaricus bisporus on human keratinocytes: preli- minary studies of its use in psoriasis. Brit J Dermatol 140, 56–60. 14 Yao QZ, Yu MM, Ooi LS, Ng TB, Chang ST, Sun SS & Ooi VE (1998) Isolation and characterization of a type 1 ribosome-inactivating protein from fruiting bodies of the edible mushroom (Volvariella volvacea). J Agr Food Chem 46, 788–792. 15 Kretz O, Creppy EE, Boulanger Y & Dirheimer G (1989) Purification and some properties of bolesatine, a protein inhibiting in vitro protein synthesis, from the mushroom Boletus satanas Lenz (Boletaceae). Arch Toxicol Suppl 13, 422–427. 16 Stasyk T, Lootsik M, Hellman U, Wernstedt C, Souchelnytskyi S & Stoika R (2008) A new toxic pro- tein from death cap Amanita phalloides: isolation and study of cytotoxic activity. Studia Biologica 2, 21–32. 17 Martin F, Aerts A, Ahre ´ n D, Brun A, Danchin EG, Coutinho PM, Henrissat B, Tuskan G & Grigoriev IV (2008) The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis. Nature 452, 88–92. 18 Haywood G & Large P (1981) Microbial oxidation of amines. Biochem J 199, 187–201. 19 Nuutinen JT & Timonen S (2008) Identification of nitrogen mineralization enzymes, L-amino acid oxidases, from the ectomycorrhizal fungi Hebeloma spp. and Laccaria bicolor. Mycol Res 112, 1453–1464. 20 Mikkelsen RB & Wardman P (2003) Biological chemis- try of reactive oxygen and nitrogen and radiation- induced signal transduction mechanisms. Oncogene 22, 5734–5754. 21 Zhong SR, Jin Y, Wu JB, Jia YH, Xu GL, Wang GC, Xiong YL & Lu QM (2009) Purification and character- ization of a new L-amino acid oxidase from Daboia russellii siamensis venom. Toxicon 54, 763–771. 22 Zhang L & Wu WT (2008) Isolation and characteriza- tion of ACTX-6: a cytotoxic l-amino acid oxidase from Agkistrodon acutus snake venom. Nat Prod Res 22, 554–563. 23 Jin Y, Lee WH, Zeng L & Zhang Y (2007) Molecular characterization of l-amino acid oxidase from king cobra venom. Toxicon 50, 479–489. 24 Doley R & Kini RM (2009) Protein complexes in snake venom. Cell Mol Life Sci 66, 2851–2871. 25 Lutsik-Kordovsky MD, Stasyk TV & Stoika RS (2001) Analysis of cytotoxicity of lectin and non-lectin proteins from Amanita mushrooms. Exp Oncol 23, 43–45. 26 Ferna ´ ndez de Larrea C, Lozano M, Castro P & Nicola ´ s JM (2008) Prothrombin index prolongation due to N-acetylcysteine during the treatment of Amanita phalloides poisoning. Med Clin (Barc) 131, 717–718. 27 Agostinelli E, Tempera G, Molinari A, Salvi M, Battaglia V, Toninello A & Arancia G (2007) The physiological role of biogenic amines redox reactions in mitochondria. New perspectives in cancer therapy. Amino Acids 33, 175–187. 28 Agostinelli E, Condello M, Molinari A, Tempera G & Arancia G (2009) Cytotoxicity of spermine oxidation products to multidrug resistant melanoma M14 ADR2 cells: sensitization by the MDL 72527 lysosomotropic compound. Int J Oncol 35, 485–498. 29 Amendola R, Cervelli M, Fratini E, Polticelli F, Sallustio DE & Mariottini P (2009) Spermine metabo- lism and anticancer therapy. Curr Cancer Drug Targets 9, 118–130. 30 Antonyuk VA (1989) Method for obtaining affinity sorbent for purification of lectins. Patent of USSR. Number 1554961 from December 8, 1989. 31 Maurer HR (1971) Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophore- sis, 2nd edn. Walter de Gruyter Ltd, New York, NY. 32 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680–685. Cytotoxic L-amino oxidase from A. phalloides T. Stasyk et al. 1268 FEBS Journal 277 (2010) 1260–1269 ª 2010 The Authors Journal compilation ª 2010 FEBS 33 Hellman U (2000) Sample preparation by SDS ⁄ PAGE and in-gel digestion. EXS 88, 43–54. Supporting information The following supplementary material is available: Fig. S1. Isolation of tryptic peptides of toxophallin by microbore reversed phase liquid chromatography. Fig. S2. Toxophallin is homological to amine oxidase from Laccaria bicolor S238N-H82 (accession number EDR12198, XP_001876462). Table S1. Amino acid composition of toxophallin. Table S2. Tryptic peptides of toxophallin. Table S3. Substances tested as toxophallin substrates in amine oxidase enzymatic activity assay, as described in the Materials and methods. This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. T. Stasyk et al. Cytotoxic L-amino oxidase from A. phalloides FEBS Journal 277 (2010) 1260–1269 ª 2010 The Authors Journal compilation ª 2010 FEBS 1269 . A new highly toxic protein isolated from the death cap Amanita phalloides is an L-amino acid oxidase Taras Stasyk 1,2 , Maxim Lutsik-Kordovsky 1 , Christer Wernstedt 3 , Volodymyr Antonyuk 1 , Olga. intracellular molecular targets and mechanisms of action have been well characterized. In addition to these toxic polypeptides, the death cap also contains antitoxin antamanide, a cyclodecapeptide. was suggested that amine oxidases are enzymes of cellular amino acid catabo- lism, comprising potential candidates for a mechanism that catalyses nitrogen mineralization from amino acids at the