BAT3, a Faithful Partner of RTA: Identification of the Interactive Sites on Ricin A Chain and Human HLA-B Associated Transcript-3

• Main Authors: Pei-Chin Tsai, 蔡佩衿
• Other Authors: Jung-Yaw Lin
• Format: Others
• Language: en_US
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/83901598782095143815

碩士 === 國立臺灣大學 === 生化學研究所 === 89 === Ricin, a member of type II ribosome-inactivating protein (RIP), is composed of an A and a B chains. The A chain of ricin is an N-glycosidase, which plays the main role in cytotoxicity, while B chain is a lectin and directs the whole protein into cells. We used ricin A-chain (RTA) as bait to screen the target proteins from a human Jurkat cDNA library by yeast two-hybrid system. One of the clones interacts with RTA, and it was identified as a fragment of human HLA-B-associated transcript 3 (BAT3), from amino acid residues 614-1044. BAT3 is a functional-unknown protein with high proline content. Present investigation is to study the function of RTA via interacting with BAT3 in mammalian cells, and to examine the interactive sites of RTA and BAT3. In the experiment, RTA was found to interact specifically with BAT3 in vivo, and the interaction was about 2.5 fold higher than that of p53 to SV40 large T antigen. The 3D structure of RTA has already been solved, and it is composed of three structural and functional different domains, namely RTA I, RTA II, and RTA III. In this study, the three domains of RTA were constructed into a GAL4 DNA binding domain (DNA-BD) cloning vector, pAS2-1, in order to examine which domain interacts with BAT3. By using PCGENE software to simulate the secondary structure of BAT3 through computer analysis, two amphipathic α-helixes were identified which might be involved in its interaction with RTA. The two candidate amphipathicα-helixes were mutated by site directed mutagenesis to affect its amphipathic characteristics, and several systematic deletion clones were also constructed to study its specific binding sites with RTA. Present investigation showed that RTA III can interact with BAT3. However, it is only 2% of the original intensity, which indicated that other domains of RTA also involved in the interaction. This investigation also suggests that RTA C-terminus seemed to be important for the interaction. The fragment, amino acid residues 740-1000 of BAT3, was shown to be sufficient for the interaction with RTA. Within this region, one or both of F846 and N847 in the simulatedα-helixes B are important sites for the interaction. Because when they are mutated to F846R and N847I, BAT3 lost the interaction ability. From this experiment, we not only identified the interactive sites on RTA and BAT3, but also speculated that the mechanism of ricin triggered cell death and protein synthesis inhibition are mediated by different portions of RTA.