title: A new highly toxic protein isolated from the death cap Amanita phalloides is an L-amino acid oxidase.
abstract: 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 biological 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 fragmentation, which are typical for apoptosis. However, caspase III inhibitor [benzyloxycarbonyl-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 complementary 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 mushroom. This demonstrates the usefulness of our approach for the identification 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.
authors.affiliation: Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine.
authors.forename: Taras
authors.initials: T
authors.lastname: Stasyk
authors.forename: Maxim
authors.initials: M
authors.lastname: Lutsik-Kordovsky
authors.forename: Christer
authors.initials: C
authors.lastname: Wernstedt
authors.forename: Volodymyr
authors.initials: V
authors.lastname: Antonyuk
authors.forename: Olga
authors.initials: O
authors.lastname: Klyuchivska
authors.forename: Serhiy
authors.initials: S
authors.lastname: Souchelnytskyi
authors.forename: Ulf
authors.initials: U
authors.lastname: Hellman
authors.forename: Rostyslav
authors.initials: R
authors.lastname: Stoika
mesh_terms: D000545:Amanita / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002477:Cells / Q000187:drug effects; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics* / Q000633:toxicity*; D006801:Humans; D050830:L-Amino Acid Oxidase / Q000737:chemistry* / Q000302:isolation & purification; D007938:Leukemia / Q000188:drug therapy; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009183:Mycotoxins / Q000235:genetics* / Q000633:toxicity*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000970:Antineoplastic Agents; D005656:Fungal Proteins; D009183:Mycotoxins; C549162:toxophallin, Amanita phalloides; D050830:L-Amino Acid Oxidase