Role of tyrosine residues in modulation of claudin-4 by the C-terminal fragment of Clostridium perfringens enterotoxin.
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.
Harada
Motoki
M
Department of Pharmaceutics and Biopharmaceutics, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan.
Kondoh
Masuo
M
Ebihara
Chiaki
C
Takahashi
Azusa
A
Komiya
Eriko
E
Fujii
Makiko
M
Mizuguchi
Hiroyuki
H
Tsunoda
Shin-Ichi
S
Horiguchi
Yasuhiko
Y
Yagi
Kiyohito
K
Watanabe
Yoshiteru
Y
D001483:Base Sequence; D018938:Caco-2 Cells; D062506:Claudin-4; D003016:Clostridium perfringens / Q000737:chemistry*; D017931:DNA Primers; D004768:Enterotoxins / Q000737:chemistry / Q000633:toxicity*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D014443:Tyrosine / Q000378:metabolism*
C568157:CLDN4 protein, human; D062506:Claudin-4; D017931:DNA Primers; D004768:Enterotoxins; D008565:Membrane Proteins; C035598:enterotoxin, Clostridium; D014443:Tyrosine