| Trichosanthin (TCS) is a ribosome-inactivating protein isolated from the root tuber of Trichosanthes kirillowii Maximowicz of the Cucurbitaceae family. Its multiple pharmacological properties have aroused intensive research in basic structure/function studies, in vitro/in vivo activities and clinical application. Crystal structures of TCS show that the molecule constitutes of a large N-terminal domain of 202 amino acids and a small C-terminal domain of 45 amino acids that contains no active site residues. To generate a minimum TCS retaining potent activities with reduced antigenicity, the C-terminal amino, acids were systemically deleted and the mutant proteins expressed in E. coli. We found that the last four residues at the C-terminal of TCS were not important for in vitro ribosome-inactivating activity, secondary structure and stability of the protein. Deletion of the last five or six amino acids reduced the stability of TCS, while deletion of the last seven residues significantly decreased both the ribosome-inactivating activity and stability. Further deletion caused aberrant folding of the mutants. We conclude that TCS and other related RIPs are highly evolved proteins that only very little part of their C-terminal regions can be deleted without impairment in structure or function.;Mammalian proteins that interact with TCS were identified by the yeast two-hybrid system and an in vitro affinity trap. By the yeast two-hybrid system, human ribosomal phosphoproteins P0 and P1, and a putative human cell cycle protein MAD2B (mitotic arrest deficiency protein 2B) were identified to interact with TCS in the yeast cell. The three proteins were cloned and expressed in E. coli. The partially purified proteins interacted with TCS in an in vitro binding assays. These experiments show that P0, P1 and MAD2B interact with TCS both in vivo and in vitro. For P0, the domain where interaction with TCS takes place was implicated to be at the amino acids 220 to 273. On the other hand, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), inhibitor 2 of protein phosphatase, 2A (I2PP2A), heat shock protein 86 (hsp86), heat shock cognate protein 70 (hsc70) and transformation sensitive protein (TSP or IEF SSP 3521) were found to have possible interaction with TCS by the affinity trap using cytosol of human carcinoma cell JAR. The equilibrium dissociation constant of TCS with commercial porcine muscle GAPDH was found to be 0.32 +/- 0.08 muM. Further studies are required to confirm the interaction between these candidate proteins and TCS and the implications of these interactions. |
