Takahashi Azusa, Kondoh Masuo, Masuyama Akane, Fujii Makiko, Mizuguchi Hiroyuki, Horiguchi Yasuhiko, Watanabe Yoshiteru
Department of Pharmaceutics and Biopharmaceutics, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan.
J Control Release. 2005 Nov 2;108(1):56-62. doi: 10.1016/j.jconrel.2005.07.008. Epub 2005 Aug 8.
Claudin family proteins, which contain 4 transmembrane domains, play a pivotal role in the barrier function of tight junctions (TJs) in epithelial sheets. We previously found that a modulator of claudin-4, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), is a potent enhancer of jejunal drug absorption in rats. But the effects of C-CPE on the barrier function of TJs have never been fully understood. In the present study, we investigated the effects of C-CPE on the barrier function of TJs in Caco-2 monolayer and characterized the functional domain of C-CPE that is responsible for interaction with claudin-4. To evaluate the effects of C-CPE on the barrier function of TJs, we measured transepithelial electric resistance (TER) in Caco-2 monolayer cells seeded onto polycarbonate filters. Treatment of Caco-2 cells with C-CPE resulted in a decrease in TER. But, deletion of the 30 C-terminal amino acids of C-CPE, which is the putative binding domain for claudin, attenuated the decrease in TER values. Moreover, ablation of the 16 C-terminal amino acids of C-CPE also resulted in attenuation of the decrease in TER values. The C-terminal-deleted C-CPEs did not interact with claudin-4 or the extracellular domain 2 of claudin-4, which is the C-CPE binding site. These results suggest that the 16 C-terminal amino acids of C-CPE are responsible for the interaction of C-CPE and claudin-4 following the disruption of TJ barrier function.
闭合蛋白家族蛋白含有4个跨膜结构域，在上皮组织紧密连接（TJ）的屏障功能中起关键作用。我们之前发现，产气荚膜梭菌肠毒素的C端片段（C-CPE）作为闭合蛋白-4的调节剂，是大鼠空肠药物吸收的有效增强剂。但C-CPE对紧密连接屏障功能的影响尚未完全明确。在本研究中，我们研究了C-CPE对Caco-2单层细胞紧密连接屏障功能的影响，并确定了C-CPE与闭合蛋白-4相互作用的功能结构域。为了评估C-CPE对紧密连接屏障功能的影响，我们测量了接种在聚碳酸酯滤膜上的Caco-2单层细胞的跨上皮电阻（TER）。用C-CPE处理Caco-2细胞导致TER降低。但是，删除C-CPE的30个C端氨基酸（这是推测的与闭合蛋白的结合结构域）可减弱TER值的降低。此外，删除C-CPE的16个C端氨基酸也导致TER值降低的减弱。C端缺失的C-CPE不与闭合蛋白-4或闭合蛋白-4的胞外结构域2（即C-CPE结合位点）相互作用。这些结果表明，C-CPE的16个C端氨基酸在紧密连接屏障功能破坏后负责C-CPE与闭合蛋白-4的相互作用。
