title: Mutated C-terminal fragments of Clostridium perfringens enterotoxin have increased affinity to claudin-4 and reversibly modulate tight junctions in vitro.
abstract: Passage across epithelial cell sheets is the first step in drug absorption. Tight junctions (TJs) are located between adjacent epithelial cells and seal the intercellular space preventing leakage of solutes. Claudin, a tetra-transmembrane protein family, is a pivotal functional and structural component of the TJ barrier. Modulation of the claudin-based TJ seal is a strategy for mucosal drug absorption. We previously found that a claudin-4 binder, a C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE194), was a modulator of the TJ seal and a potent mucosal absorption enhancer. In the present study, we attempted to improve claudin-4 binders by modification of C-CPE194. Substitution of Asn at position 309 and Ser at position 313 with Ala increased the affinity to claudin-4 by 9.9-fold as compared to C-CPE194. Deletion of 10 amino acids in the N-terminal domain of the double-alanine-substituted mutant increased affinity to claudin-4 by 23.9-fold as compared to C-CPE194. These C-CPE194 mutants reversibly modulated the TJ seal in human intestinal epithelial cell sheets. The N-terminal-truncated mutant was the most potent modulator of the TJ seal. These findings indicate that the C-CPE mutant may be a promising lead for the development of a clinical TJ modulator.
authors.affiliation: Laboratory of Bio-Functional Molecular Chemistry, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
authors.forename: Azusa
authors.initials: A
authors.lastname: Takahashi
authors.forename: Masuo
authors.initials: M
authors.lastname: Kondoh
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Uchida
authors.forename: Yohei
authors.initials: Y
authors.lastname: Kakamu
authors.forename: Takao
authors.initials: T
authors.lastname: Hamakubo
authors.forename: Kiyohito
authors.initials: K
authors.lastname: Yagi
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001216:Asparagine / Q000737:chemistry / Q000235:genetics; D018938:Caco-2 Cells; D062506:Claudin-4; D004768:Enterotoxins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D012694:Serine / Q000737:chemistry / Q000235:genetics; D019108:Tight Junctions / Q000378:metabolism*
chemical_list: C568157:CLDN4 protein, human; D062506:Claudin-4; D004768:Enterotoxins; D008565:Membrane Proteins; C035598:enterotoxin, Clostridium; D012694:Serine; D001216:Asparagine