Change in pH-dependent Membrane Insertion Characteristics of Trichosanthin Caused by Deletion of Its Last Seven C-terminal Amino Acid Residues

Fan Zhang¹, Ying-Jie Lu¹, Pang Chui Shaw², and Sen-fang Sui^1*

¹Department of Biological Sciences and Biotechnology, State Key Laboratory of Biomembranes, Tsinghua University, Beijing 100084, P. R. China; fax: (86)-10-62784768; E-mail: suisf@mail.tsinghua.edu.cn

²Department of Biochemistry, Chinese University of Hong Kong, Hong Kong, China

^* To whom correspondence should be addressed.

Received March 7, 2002; Revision received June 27, 2002

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Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) that can selectively kill some types of cells at low concentration (0.1-1 nM). The pH-dependent membrane insertion ability of TCS makes it possible that the internalized toxin avoids degradation in lysosomes and further undergoes transportation into the cytosol by some still unidentified mechanism. Here, we show that deletion of C-terminal residues affects interactions of modified TCS (C7-TCS) with lipids and reduces its pH-dependent membrane insertion ability. Fluorescence measurements indicate that at low pH C7-TCS undergoes profound conformational changes that causes exposure of a hydrophobic region and leads to oligomerization of the C7-TCS molecules. The results suggest that the membrane insertion of TCS at low pH might be important for translocation of TCS into the cytosol, which is important for exertion of the RIP activity of TCS. Deletion of the last seven C-terminal residues of TCS would reduce both its RIP activity in vitro and cytotoxicity in vivo, with the degree of decrease being more significant for the cytotoxicity in vivo.

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KEY WORDS: conformation change, lipid-protein interaction, membrane surface pressure, ribosome-inactivating protein, trichosanthin

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