Change in pH-dependent membrane insertion characteristics of trichosanthin caused by deletion of its last seven C-terminal amino acid residues.
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.
Zhang
Fan
F
Department of Biological Sciences and Biotechnology, State Key Laboratory of Biomembranes, Tsinghua University, Beijing, 100084, P. R. China.
Lu
Ying-Jie
YJ
Shaw
Pang Chui
PC
Sui
Sen-Fang
SF
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D002942:Circular Dichroism; D003600:Cytosol / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D012964:Sodium / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D015978:Trichosanthin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; D015978:Trichosanthin; D012964:Sodium