title: Domain mapping of a claudin-4 modulator, the C-terminal region of C-terminal fragment of Clostridium perfringens enterotoxin, by site-directed mutagenesis.
abstract: 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.
authors.affiliation: Department of Pharmaceutics and Biopharmaceutics, Showa Pharmaceutical University, Tokyo 194-8543, Japan.
authors.forename: Azusa
authors.initials: A
authors.lastname: Takahashi
authors.forename: Eriko
authors.initials: E
authors.lastname: Komiya
authors.forename: Hideki
authors.initials: H
authors.lastname: Kakutani
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yoshida
authors.forename: Makiko
authors.initials: M
authors.lastname: Fujii
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Horiguchi
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Mizuguchi
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Tsutsumi
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Tsunoda
authors.forename: Naoya
authors.initials: N
authors.lastname: Koizumi
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Isoda
authors.forename: Kiyohito
authors.initials: K
authors.lastname: Yagi
authors.forename: Yoshiteru
authors.initials: Y
authors.lastname: Watanabe
authors.forename: Masuo
authors.initials: M
authors.lastname: Kondoh
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D018938:Caco-2 Cells; D002460:Cell Line; D062506:Claudin-4; D004768:Enterotoxins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D007583:Jejunum / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D019108:Tight Junctions / Q000378:metabolism
chemical_list: D000596:Amino Acids; C568157:CLDN4 protein, human; D062506:Claudin-4; C568158:Cldn4 protein, mouse; D004768:Enterotoxins; D008565:Membrane Proteins; C035598:enterotoxin, Clostridium