Domain mapping of a claudin-4 modulator, the C-terminal region of C-terminal fragment of Clostridium perfringens enterotoxin, by site-directed mutagenesis

Biochem Pharmacol. 2008 Apr 15;75(8):1639-48. doi: 10.1016/j.bcp.2007.12.016. Epub 2008 Jan 5.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / chemistry
  • Amino Acids / genetics
  • Amino Acids / metabolism
  • Animals
  • Caco-2 Cells
  • Cell Line
  • Claudin-4
  • Enterotoxins / chemistry*
  • Enterotoxins / genetics
  • Enterotoxins / pharmacology
  • Humans
  • Jejunum / metabolism
  • Male
  • Membrane Proteins / metabolism*
  • Mice
  • Mutagenesis, Site-Directed
  • Protein Structure, Tertiary
  • Rats
  • Rats, Wistar
  • Tight Junctions / metabolism


  • Amino Acids
  • CLDN4 protein, human
  • Claudin-4
  • Cldn4 protein, mouse
  • Enterotoxins
  • Membrane Proteins
  • enterotoxin, Clostridium