Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine.
FEBS J. 2010 Mar;277(5):1260-9. doi: 10.1111/j.1742-4658.2010.07557.x. Epub 2010 Feb 1.
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.
一种新的高细胞毒性蛋白——鹅膏蕈毒环肽，最近从致命鹅膏菌（Amanita phalloides）的子实体中分离出来[Stasyk 等人（2008）Studia Biologica 2, 21-32]。鹅膏蕈毒环肽的物理化学、化学和生物学特性与另一种致命鹅膏菌的毒性蛋白——鹅膏蕈溶血素（phallolysin）明显不同。鹅膏蕈毒环肽与靶细胞的相互作用不是通过特定的细胞表面受体介导的。它诱导染色质浓缩以及 DNA 和核片段化，这是典型的细胞凋亡特征。然而，半胱天冬酶 III 抑制剂[苄氧羰基-Asp(OMe)-氟甲基酮]不能阻止鹅膏蕈毒环肽诱导的 DNA 片段化。因此，鹅膏蕈毒环肽利用一种不依赖半胱天冬酶的细胞凋亡诱导途径。在本研究中，我们应用了一种基于蛋白质组学和分子生物学工具相结合的互补方法来鉴定鹅膏蕈毒环肽。通过鹅膏蕈毒环肽的胰蛋白酶肽的直接测序部分研究了其一级结构，然后通过基于 PCR 的 cDNA 克隆进行。随后的生物信息学搜索显示，鹅膏蕈毒环肽与双孢蘑菇（Laccaria bicolor）的 L-氨基酸氧化酶具有结构同源性。这证明了我们的方法对于鉴定未知基因组生物中的蛋白质是有用的。我们还发现鹅膏蕈毒环肽对氧化选定氨基酸具有广泛的底物特异性。抗坏血酸抑制了鹅膏蕈毒环肽的细胞毒性作用，这很可能是由于其作为氧化酶催化产物的过氧化氢的清除。因此，除了高细胞毒性环肽和毒性凝集素鹅膏蕈溶血素外，致命鹅膏菌的子实体还含有另一种细胞毒性蛋白形式的酶，即 L-氨基酸氧化酶。
