Trichosathin Interacts With Ribosomal Protein L10a And Plasma Membrane Proteins

Trichosanthin is a type I ribosome-inactivating protein (RIP) with manypharmacological activities. The trichosanthin-coupled Sepharose affinity purificationrevealed a protein, which was identified by mass spectrometry as the ribosomalprotein L10a. The interaction between trichosanthin and recombinant L10a wasfurther confirmed by in vitro binding assay. Kinetic analysis by surface plasmonresonance technology revealed that L10a had a high affinity to trichosanthin with aKD of 7.78nM. The study with mutated forms of trichosanthin demonstrated that thisspecific association correlates with the ribosome-inactivating activity of trichosanthin.This finding might provide insight into the mechanisms in which trichosanthininactivates ribosome and that underlies its pharmacological effect.Trichosanthin is effective in inducing abortion (94% successfully) with mildside effects. Study of trichosanthin in vitro cytotoxicity demonstrated trichosanthinselectively injured choriocarcinoma cells . Observation of Ca2+ changes by confocallaser scanning microscopy revealed a high fluo-3 fluorescence of Jar cells treated withtrichosanthin. The distribution of trichosanthin-binding proteins on Jar cells wasdetermined by immunoflurescence and flow cytometry. Radioligand binding assayshowed that two saturable classes of binding sites for trichosanthin on Jar cells. Todetermine the biochemical nature of these binding proteins, Jar cells were cell surfacebiotinylated and the trichosanthin-binding proteins were isolated by pull-down withtrichosanthin-Sepharose beads. Two specific bands with molecular weight of about50kDa and 60kDa were identified in a SDS-polyacrylamide gel, which furthercharacterization is expected to provide clues regarding the selective cytotoxicitymechanism of trichosanthin on cells.