The mechanism of action of trichosanthin on eukaryotic ribosomes—RNA N-glycosidase activity of the cytotoxin

Abstract

Trichosanthin is a ribosome-inactivating protein from root tubers of Trichosanthes kirilowii Maxim. In this paper, the mechanism of action of trichosanthin on eukaryotic ribosomes was studied. A fragment of about 450 nucleotides was released from 28S ribosomal RNA after treatment of rat liver ribosome with trichosanthin and its isolated ribosomal RNAs were treated with aniline. Analysis of nucleotide sequence of 5' terminus of this fragment revealed that the aniline-sensitive site of the phosphodiester bond was between positions A4324 and G4325 in the 28S rRNA. Adenine was recovered by ion-exchange column chromatography from the 50% ethanol soluble fraction of the reaction mixture in which rat liver ribosomes were treated with trichosanthin. Thin-layer chromatographic analysis indicated that 1 mol of adenine was released from 1 mol of ribosomes. When the ribosomes were incubated with trichosanthin in the presence of inorganic [32P]phosphate, little incorporation of radioactivity into 28S rRNA was observed, indicating that the release of adenine was not mediated by phosphorolysis. These results demonstrate that trichosanthin inactivates the ribosomes by cleaving the N-C glycosidic bond of adenylic acid at 4324 of 28S rRNA in a hydrolytic fashion.

Mapping the antigenic determinants and reducing the immunogenicity of trichosanthin by site-directed mutagenesis

“…By site-directed mutagenesis, three TCS mutants namely TCS YFF81-83ACS , TCS KR173-174CG and TCS YFF-KR were constructed successfully, and expressed at high level in soluble form in E. coli BL21 (DE3) and purified easily to homogeneity by using Ni-NTA purification system. TCS is classified as a type I RIP that inhibits protein synthesis [1][2][3]. It is generally believed that the anti-HIV activity of TCS is related to its RI activity.…”

“…It is processed from a larger precursor protein, containing a 23 amino acid amino (N)-terminal sequence (pre sequence) and a 19 amino acid carboxy (C)-terminal extension (pro sequence). TCS is classified as a type I ribosomeinactivating protein (RIP) that cleaves specifically the N-glycosidic bond of A-4324 of 28S rRNA and inhibits protein synthesis [1][2][3]. TCS also has DNase-like activity and cleaves the supercoiled double-stranded DNA to produce nicked circular and linear DNA [4].…”

Reactive oxygen species involved in trichosanthin-induced apoptosis of human choriocarcinoma cells

“…TCS can inactivate the ribosome by removing adenine-4324 in 28 S rRNA, by N-glycosidase activity [30]. The increasing evidence to suggest that TCS has a broad spectrum of biological and pharmacological activities, including abortifacient, anti-tumour and anti-HIV activities [10][11][12][13][14][15][16][17][18], the proposed mechanisms of which include anti-tumour activity of the N-terminal sequences of TCS and anti-HIV action by the inhibition of HIV-1 integrase [13,14].…”

Trichosanthin Induces Leakage and Membrane Fusion of Liposome

“…It can depurinate adenine 4324 of 28S rRNA through its N-glycosidase activity, which renders the ribosome incapable of binding EF-2 and GTP so that protein synthesis is arrested (3). TCS consists of a single peptide (247 amino acids, 27 kd) homologous to the A chain of type II RIPs (4).…”