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.
Footnotes
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This study is partly supported by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (Japan).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.105.085399.
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ABBREVIATIONS: TJ, tight junction; CPE, Clostridium perfringens enterotoxin; C-CPE, C-terminal fragment of Clostridium perfringens enterotoxin; CPE-R, Clostridium perfringens enterotoxin receptor; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; FD-4, fluorescein isothiocyanate-dextran with a molecular mass of 4000 Da; AUC0–4, area under the plasma concentration-time curve from 0 to 4 h; Ab, antibody; GST, glutathione S-transferase; EC2hCld-4, extracellular domain 2 of human claudin-4; aa, amino acid; TER, transepithelial electric resistance; AUC, the area under the plasma concentration-time curve; Ni-NTA, nickel-nitrilotriacetic acid.
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↵1 These authors equally contributed to this work.
- Received February 24, 2005.
- Accepted April 14, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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