amanitine and phalloidine, with both Keywords Amanita phalloides; apoptosis; death cap; L -amino acid oxidase; toxic protein Correspondence R.. Ascorbic acid inhibited the cytotoxic effe
Trang 1Amanita phalloides is an L-amino acid oxidase
Taras Stasyk1,2, Maxim Lutsik-Kordovsky1, Christer Wernstedt3, Volodymyr Antonyuk1,
Olga Klyuchivska1, Serhiy Souchelnytskyi4, Ulf Hellman3and Rostyslav Stoika1
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 theL-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, namelyL-amino acid oxidase
Abbreviations
CM, carboxymethyl; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight.
Trang 2exhibiting 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
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
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].
Trang 3amino 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¢- to 5¢) and TCT GTC GTA CCA ACC AGT TGA (5¢- to 3¢), 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.
Trang 4Materials 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 leuleu-kemia 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 IC50 of purified
toxophallin was 0.5 lgÆmL)1 The IC50 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 [3H]-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 20 40 60 80 100
A
B
L1210 CEMT4
L -amino-acid oxidase (µg·mL –1 )
0 20 40 60 80 100
L1210 CEMT4
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.
Trang 5deoxynucleotidyl-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 H2O2 that appears as a result of the
amine oxidase reaction and is toxic for cells Both
ascorbic acid and reduced glutathione also inhibited
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, IC50= 0.25 lgÆmL)1 and, in cells of CCL-64 and MCF 7 lines, IC50= 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.
0 20 40 60 80 100
A
B
L -amino-acid oxidase (µg·mL –1 )
Control + 10 µg·mL –1 ascorbic acid
0 10 20 30 40
+ 10 µg·mL –1 ascorbic acid
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)1was 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.
Trang 6activity, 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 virosafruit 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 H2O2generated 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
H2O2 and aldehyde production [27] Increasing the incubation temperature from 37 to 42C 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 H2O2 and aldehydes [28] The metabolites formed by BSA oxidase targeting spermine were more toxic than exogenous H2O2 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
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
5 mgÆmL)1before adding L -methionine.
Trang 7ating 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, H2O2
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
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
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
described previously [33] The peptide mixture was analyzed
by MALDI-TOF MS, using a Bruker Biflex III instrument
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)
(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
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
Trang 8mRNA 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,
(Boehringer Mannheim, Mannheim, Germany) and RNAse
inhibitor in the reverse transcriptase buffer (BRL) The
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¢- to 5¢ and 5¢- to 3¢ primers were employed The
cycling program started with 0.5 min of denaturation at
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
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
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
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
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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
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