Domain mapping of a claudin-4 modulator, the C-terminal region of C-terminal fragment of Clostridium perfringens enterotoxin, by site-directed mutagenesis
- 作者：Azusa Takahashi ; Eriko Komiya ; Hideki Kakutani ; Takeshi Yoshida ; Makiko Fujii ; Yasuhiko Horiguchi ; Hiroyuki Mizuguchi ; Yasuo Tsutsumi ; Shin-ichi Tsunoda ; Naoya Koizumi ; Katsuhiro Isoda ; Kiyohito Yagi
- 关键词：Clostridium perfringens enterotoxin ; Tight junction ; Claudin ; Site-directed mutagenesis
- 刊名：Biochemical Pharmacology
- 出版年：2008
- 期刊代码：2_00062952
- 类别：pha
- 出版时间：15 April 2008
- 卷：75
- 期：8
- 页码：1639-1648
- 文件大小：715 K
摘要
A C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) is a modulator of claudin-4. We previously found that upon deletion of the C-terminal 16 amino acids, C-CPE lost its ability to modulate claudin-4. Tyrosine residues in the 16 amino acids were involved in the modulation of claudin-4. In the present study, we performed functional domain mapping of the 16-amino acid region of C-CPE by replacing individual amino acids with alanine. To evaluate the ability of the alanine-substituted mutants to interact with claudin-4, we carried out a competition analysis using claudin-4-targeting protein synthesis inhibitory factor. We found that Tyr306Ala, Tyr310Ala, Tyr312Ala, and Leu315Ala mutants had reduced binding to claudin-4 compared to C-CPE. Next, we investigated effects of each alanine-substituted mutant on the TJ-barrier function in Caco-2 monolayer cells. The TJ-disrupting activity of C-CPE was reduced by the Tyr306Ala and Leu315Ala substitutions. Enhancement of rat jejunal absorption was also decreased by each of these mutations. The double mutant Tyr306Ala/Leu315Ala lost the ability to interact with claudin-4, modulate TJ-barrier function, and enhance jejunal absorption. These data indicate that Tyr306 and Leu315 are key residues in the modulation of claudin-4 by C-CPE. This information should be useful for the development of a novel claudin modulator based on C-CPE.