title: Role of N-terminal amino acids in the absorption-enhancing effects of the c-terminal fragment of Clostridium perfringens enterotoxin.
abstract: We recently found that a polypeptide, the C-terminal of Clostridium perfringens enterotoxin (C-CPE), was a novel type of drug absorption enhancer. The C-terminal of C-CPE is thought to play a role in the binding of C-CPE to its receptor, claudin-4; however, the function of the N-terminal of C-CPE is unclear. In the present study, we evaluated the role of the N-terminal domain of C-CPE in jejunal absorption and claudin-4 binding. The treatment of rat jejunum with C-CPE resulted in enhanced absorption of dextran, with a molecular weight of 4000 Da. However, treatment with C-CPE220, which lacks the 36 N-terminal amino acids of C-CPE, did not enhance jejunal absorption. C-CPE had affinity for claudin-4 in rat jejunum lysates and Caco-2 lysates, but C-CPE220 did not. Interaction of C-CPE with the recombinant extracellular domain 2 of human claudin-4 (EC2hCld-4), which is the putative binding site for C-CPE, was observed, but C-CPE220 had no affinity for EC2hCld-4. To investigate the effect of C-CPE220 on the barrier function of tight junctions, we measured transepithelial electric resistance (TER) in C-CPE- or C-CPE220-treated Caco-2 monolayer cells. Although C-CPE decreased TER in Caco-2 monolayer cells, C-CPE220 did not disrupt the barrier function of tight junctions. Together, these results indicate that the 36 N-terminal amino acids of C-CPE may be necessary for the enhanced absorption mediated by C-CPE and play a partial role in binding to claudin-4.
authors.affiliation: Department of Pharmaceutics and Biopharmaceutics, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan.
authors.forename: Akane
authors.initials: A
authors.lastname: Masuyama
authors.forename: Masuo
authors.initials: M
authors.lastname: Kondoh
authors.forename: Hirotoshi
authors.initials: H
authors.lastname: Seguchi
authors.forename: Azusa
authors.initials: A
authors.lastname: Takahashi
authors.forename: Motoki
authors.initials: M
authors.lastname: Harada
authors.forename: Makiko
authors.initials: M
authors.lastname: Fujii
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Mizuguchi
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Horiguchi
authors.forename: Yoshiteru
authors.initials: Y
authors.lastname: Watanabe
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000502:physiology*; D000818:Animals; D018938:Caco-2 Cells; D002478:Cells, Cultured; D062506:Claudin-4; D004553:Electric Conductivity; D004768:Enterotoxins / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007408:Intestinal Absorption / Q000187:drug effects*; D007583:Jejunum / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D013268:Stimulation, Chemical; D019108:Tight Junctions / Q000187:drug effects
chemical_list: D000596:Amino Acids; C568157:CLDN4 protein, human; D062506:Claudin-4; D004768:Enterotoxins; D008565:Membrane Proteins; D010446:Peptide Fragments; C035598:enterotoxin, Clostridium; D005982:Glutathione Transferase