Harada Motoki, Kondoh Masuo, Ebihara Chiaki, Takahashi Azusa, Komiya Eriko, Fujii Makiko, Mizuguchi Hiroyuki, Tsunoda Shin-Ichi, Horiguchi Yasuhiko, Yagi Kiyohito, Watanabe Yoshiteru
Department of Pharmaceutics and Biopharmaceutics, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan.
Biochem Pharmacol. 2007 Jan 15;73(2):206-14. doi: 10.1016/j.bcp.2006.10.002. Epub 2006 Oct 6.
The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) modulates the barrier function of claudin-4 via its C-terminal 16 amino acids. In the current study, we investigated the roles of tyrosine residues (Y306, Y310 and Y312) in this region in the modulation of TJs by C-CPE. Single mutations of Y306, Y310 and Y312 to alanine resulted in partial reduction of claudin-4 binding. We also prepared double mutants of C-CPE to further evaluate the roles of these tyrosine residues. Replacement of Y310 and Y312 with alanine (Y310A/Y312A) partly reduced the ability of C-CPE to bind to claudin-4. Double mutants Y306A/Y310A and Y306A/Y312A, however, lost the ability to bind to claudin-4 and to modulate the TJ barrier. We also found that a triple mutant (Y306A/Y310A/Y312A) lost the ability to bind claudin-4, modulate the TJ barrier, and enhance jejunal absorption in rats. These results indicate that tyrosines 306, 310, and 312 are critical for the interaction of C-CPE with claudin-4 and for the modulation of TJ barrier function by C-CPE. This study provides information that should help in the development of claudin modulators based on C-CPE.
产气荚膜梭菌肠毒素的C端片段（C-CPE）通过其C端的16个氨基酸调节claudin-4的屏障功能。在本研究中，我们调查了该区域酪氨酸残基（Y306、Y310和Y312）在C-CPE调节紧密连接（TJ）中的作用。Y306、Y310和Y312突变为丙氨酸的单突变导致claudin-4结合部分减少。我们还制备了C-CPE的双突变体以进一步评估这些酪氨酸残基的作用。用丙氨酸取代Y310和Y312（Y310A/Y312A）部分降低了C-CPE与claudin-4结合的能力。然而，双突变体Y306A/Y310A和Y306A/Y312A失去了与claudin-4结合以及调节TJ屏障的能力。我们还发现三突变体（Y306A/Y310A/Y312A）失去了结合claudin-4、调节TJ屏障以及增强大鼠空肠吸收的能力。这些结果表明酪氨酸306、310和312对于C-CPE与claudin-4的相互作用以及C-CPE对TJ屏障功能的调节至关重要。本研究提供的信息应有助于基于C-CPE开发claudin调节剂。
