Document Detail


Domain mapping of a claudin-4 modulator, the C-terminal region of C-terminal fragment of Clostridium perfringens enterotoxin, by site-directed mutagenesis.
MedLine Citation:
PMID:  18342294     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
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; Yoshiteru Watanabe; Masuo Kondoh
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-01-05
Journal Detail:
Title:  Biochemical pharmacology     Volume:  75     ISSN:  1873-2968     ISO Abbreviation:  Biochem. Pharmacol.     Publication Date:  2008 Apr 
Date Detail:
Created Date:  2008-03-31     Completed Date:  2008-04-22     Revised Date:  2009-05-21    
Medline Journal Info:
Nlm Unique ID:  0101032     Medline TA:  Biochem Pharmacol     Country:  England    
Other Details:
Languages:  eng     Pagination:  1639-48     Citation Subset:  IM    
Affiliation:
Department of Pharmaceutics and Biopharmaceutics, Showa Pharmaceutical University, Tokyo 194-8543, Japan.
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MeSH Terms
Descriptor/Qualifier:
Amino Acids / chemistry,  genetics,  metabolism
Animals
Caco-2 Cells
Cell Line
Enterotoxins / chemistry*,  genetics,  pharmacology
Humans
Jejunum / metabolism
Male
Membrane Proteins / metabolism*
Mice
Mutagenesis, Site-Directed
Protein Structure, Tertiary
Rats
Rats, Wistar
Tight Junctions / metabolism
Chemical
Reg. No./Substance:
0/Amino Acids; 0/Enterotoxins; 0/Membrane Proteins; 0/claudin 4; 0/enterotoxin, Clostridium

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