Limits...
Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier.

Sonoda N, Furuse M, Sasaki H, Yonemura S, Katahira J, Horiguchi Y, Tsukita S - J. Cell Biol. (1999)

Bottom Line: We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4.At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased.These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.

Show MeSH

Related in: MedlinePlus

Specific interaction of Clostridium perfringens enterotoxin (CPE) with claudin-3 and -4. (A) L transfectants expressing claudin-1 (C1L), claudin-2 (C2L), claudin-3 (C3L), or claudin-4 (C4L) were cultured in the absence (CPE(−)) or presence of 500 ng/ml full-length CPE (CPE(+)) or 2.5 μg/ml COOH-terminal half fragment of CPE (C-CPE(+)). Phase-contrast microscopic images taken at 1 h after incubation revealed that CPE, but not C-CPE, showed cytotoxicity only to C3L and C4L cells. Bar, 20 μm. (B) L transfectants (1 × 105 cells) expressing claudin-1, -2, -3, or -4 were incubated with various concentration of 125I-CPE, and binding was determined as reported previously (Katahira et al. 1997a). The binding of 125I-CPE to claudin-1 or -2 was negligible. Each symbol represents mean value (n = 6). (C) Scatchard plot of the specific binding of 125I-CPE to L transfectants expressing claudin-3 (closed triangles; Ka = 8.4 × 107 M−1) or claudin-4 (open circles; Ka = 1.1 × 108 M−1).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2164970&req=5

Figure 2: Specific interaction of Clostridium perfringens enterotoxin (CPE) with claudin-3 and -4. (A) L transfectants expressing claudin-1 (C1L), claudin-2 (C2L), claudin-3 (C3L), or claudin-4 (C4L) were cultured in the absence (CPE(−)) or presence of 500 ng/ml full-length CPE (CPE(+)) or 2.5 μg/ml COOH-terminal half fragment of CPE (C-CPE(+)). Phase-contrast microscopic images taken at 1 h after incubation revealed that CPE, but not C-CPE, showed cytotoxicity only to C3L and C4L cells. Bar, 20 μm. (B) L transfectants (1 × 105 cells) expressing claudin-1, -2, -3, or -4 were incubated with various concentration of 125I-CPE, and binding was determined as reported previously (Katahira et al. 1997a). The binding of 125I-CPE to claudin-1 or -2 was negligible. Each symbol represents mean value (n = 6). (C) Scatchard plot of the specific binding of 125I-CPE to L transfectants expressing claudin-3 (closed triangles; Ka = 8.4 × 107 M−1) or claudin-4 (open circles; Ka = 1.1 × 108 M−1).

Mentions: We next examined the sensitivity of claudin-1 to -4 to full-length CPE using L transfectants expressing respective claudins (C1L, C2L, C3L, and C4L cells). When purified CPE at 500 ng/ml was added into the culture medium followed by 1-h incubation, C3L and C4L cells formed characteristic bleb balloons and underwent cell death as expected from the results of previous studies (Katahira et al. 1997a,Katahira et al. 1997b), whereas either C1L or C2L cells did not exhibit any morphological changes up to 24-h incubation (Fig. 2 A), suggesting differences in the affinity to CPE between claudin-1/2 and claudin-3/4.


Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier.

Sonoda N, Furuse M, Sasaki H, Yonemura S, Katahira J, Horiguchi Y, Tsukita S - J. Cell Biol. (1999)

Specific interaction of Clostridium perfringens enterotoxin (CPE) with claudin-3 and -4. (A) L transfectants expressing claudin-1 (C1L), claudin-2 (C2L), claudin-3 (C3L), or claudin-4 (C4L) were cultured in the absence (CPE(−)) or presence of 500 ng/ml full-length CPE (CPE(+)) or 2.5 μg/ml COOH-terminal half fragment of CPE (C-CPE(+)). Phase-contrast microscopic images taken at 1 h after incubation revealed that CPE, but not C-CPE, showed cytotoxicity only to C3L and C4L cells. Bar, 20 μm. (B) L transfectants (1 × 105 cells) expressing claudin-1, -2, -3, or -4 were incubated with various concentration of 125I-CPE, and binding was determined as reported previously (Katahira et al. 1997a). The binding of 125I-CPE to claudin-1 or -2 was negligible. Each symbol represents mean value (n = 6). (C) Scatchard plot of the specific binding of 125I-CPE to L transfectants expressing claudin-3 (closed triangles; Ka = 8.4 × 107 M−1) or claudin-4 (open circles; Ka = 1.1 × 108 M−1).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2164970&req=5

Figure 2: Specific interaction of Clostridium perfringens enterotoxin (CPE) with claudin-3 and -4. (A) L transfectants expressing claudin-1 (C1L), claudin-2 (C2L), claudin-3 (C3L), or claudin-4 (C4L) were cultured in the absence (CPE(−)) or presence of 500 ng/ml full-length CPE (CPE(+)) or 2.5 μg/ml COOH-terminal half fragment of CPE (C-CPE(+)). Phase-contrast microscopic images taken at 1 h after incubation revealed that CPE, but not C-CPE, showed cytotoxicity only to C3L and C4L cells. Bar, 20 μm. (B) L transfectants (1 × 105 cells) expressing claudin-1, -2, -3, or -4 were incubated with various concentration of 125I-CPE, and binding was determined as reported previously (Katahira et al. 1997a). The binding of 125I-CPE to claudin-1 or -2 was negligible. Each symbol represents mean value (n = 6). (C) Scatchard plot of the specific binding of 125I-CPE to L transfectants expressing claudin-3 (closed triangles; Ka = 8.4 × 107 M−1) or claudin-4 (open circles; Ka = 1.1 × 108 M−1).
Mentions: We next examined the sensitivity of claudin-1 to -4 to full-length CPE using L transfectants expressing respective claudins (C1L, C2L, C3L, and C4L cells). When purified CPE at 500 ng/ml was added into the culture medium followed by 1-h incubation, C3L and C4L cells formed characteristic bleb balloons and underwent cell death as expected from the results of previous studies (Katahira et al. 1997a,Katahira et al. 1997b), whereas either C1L or C2L cells did not exhibit any morphological changes up to 24-h incubation (Fig. 2 A), suggesting differences in the affinity to CPE between claudin-1/2 and claudin-3/4.

Bottom Line: We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4.At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased.These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.

Show MeSH
Related in: MedlinePlus