Creation and biochemical analysis of a broad-specific claudin binder.
Claudins (CL) are a family of tetra-transmembrane proteins that are the structural and functional components of tight junctions (TJ). CLs are promising targets for drug development because of their role in mucosal drug absorption and cancer. However, CL-targeted drug development has been delayed because CLs have low antigenicity and preparing CL proteins is difficult. We developed a CL binder by using the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) and a baculoviral display system. After screening CL binders from a C-CPE mutant-displaying library by using CL-displaying budded baculovirus (BV) we isolated a C-CPE mutant called m19, which bound to CL1, CL2, CL4 and CL5. A 3-dimensional analysis showed that m19 has a structural backbone similar to C-CPE. The charge density of the CL-binding domains of m19 and C-CPE differed, suggesting that electrostatic interactions may occur between m19 and CLs. Treatment of epithelial cells with m19 decreased the paracellular but not transcellular integrity, and m19 enhanced jejunal absorption. Thus, we successfully created a CL binder with broad specificity. These findings will contribute to future preparation of CL binders for CL-targeted drug development.
Laboratory of Bio-Functional Molecular Chemistry, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan.
Azusa
A
Takahashi
Yumiko
Y
Saito
Masuo
M
Kondoh
Kyohei
K
Matsushita
Susanne M
SM
Krug
Hidehiko
H
Suzuki
Hirofumi
H
Tsujino
Xiangru
X
Li
Hiroshi
H
Aoyama
Koji
K
Matsuhisa
Tadayuki
T
Uno
Michael
M
Fromm
Takao
T
Hamakubo
Kiyohito
K
Yagi
D000818:Animals; D016367:Baculoviridae / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D018938:Caco-2 Cells; D002460:Cell Line; D057167:Claudins / Q000378:metabolism*; D003016:Clostridium perfringens / Q000737:chemistry* / Q000378:metabolism; D016503:Drug Delivery Systems / Q000379:methods; D004768:Enterotoxins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007313:Insecta / Q000166:cytology; D051379:Mice; D008958:Models, Molecular; D019151:Peptide Library; D011485:Protein Binding; D019108:Tight Junctions / Q000378:metabolism
D057167:Claudins; D004768:Enterotoxins; D019151:Peptide Library; C035598:enterotoxin, Clostridium