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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.

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Claudins expressed in MDCK I cells. (A) Immunoblotting. The total lysate of E. coli (E. coli lysate) expressing MBP fusion protein with the cytoplasmic domains of claudin-1 (1) and GST fusion proteins with the cytoplasmic domains of claudin-2 (2), -3 (3), and -4 (4), and the total lysate of MDCK I cells (MDCK I lysate) were separated by SDS-PAGE, followed by immunoblotting with anti–claudin-1 pAb (anti-cln-1 pAb), anti–claudin-2 pAb (anti-cln-2 pAb), anti–claudin-3 pAb (anti-cln-3 pAb), and anti–claudin-4 pAb (anti-cln-4 pAb). Only claudin-1 and -4 were detected in MDCK I cells. (B) Immunofluorescence microscopy. Confluent cultures of MDCK I cells were stained with anti–claudin-1 pAb (claudin-1), anti–claudin-2 pAb (claudin-2), anti–claudin-3 pAb (claudin-3), or anti–claudin-4 pAb (claudin-4). Both claudin-1 and -4 were concentrated at cell–cell borders in large amounts, where only a trace amount of claudin-3 was also detected. Claudin-2 signal was undetectable. Bar, 20 μm.
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Figure 1: Claudins expressed in MDCK I cells. (A) Immunoblotting. The total lysate of E. coli (E. coli lysate) expressing MBP fusion protein with the cytoplasmic domains of claudin-1 (1) and GST fusion proteins with the cytoplasmic domains of claudin-2 (2), -3 (3), and -4 (4), and the total lysate of MDCK I cells (MDCK I lysate) were separated by SDS-PAGE, followed by immunoblotting with anti–claudin-1 pAb (anti-cln-1 pAb), anti–claudin-2 pAb (anti-cln-2 pAb), anti–claudin-3 pAb (anti-cln-3 pAb), and anti–claudin-4 pAb (anti-cln-4 pAb). Only claudin-1 and -4 were detected in MDCK I cells. (B) Immunofluorescence microscopy. Confluent cultures of MDCK I cells were stained with anti–claudin-1 pAb (claudin-1), anti–claudin-2 pAb (claudin-2), anti–claudin-3 pAb (claudin-3), or anti–claudin-4 pAb (claudin-4). Both claudin-1 and -4 were concentrated at cell–cell borders in large amounts, where only a trace amount of claudin-3 was also detected. Claudin-2 signal was undetectable. Bar, 20 μm.

Mentions: As shown in Fig. 1 A, in the total cell lysate of MDCK I cells, claudin-1 and -4 were reproducibly detected by immunoblotting as bands with expected molecular masses. When confluent cultures of MDCK I cells were immunofluorescently stained with antibodies for claudin-1 to -4, intense signals for claudin-1 and -4 and very weak signals for claudin-3 were detected at cell–cell borders, but claudin-2 was undetectable (Fig. 1 B). Confocal microscopy confirmed that claudin-1 and -4 were concentrated at the most apical part of lateral membranes of MDCK I cells (data not shown).


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)

Claudins expressed in MDCK I cells. (A) Immunoblotting. The total lysate of E. coli (E. coli lysate) expressing MBP fusion protein with the cytoplasmic domains of claudin-1 (1) and GST fusion proteins with the cytoplasmic domains of claudin-2 (2), -3 (3), and -4 (4), and the total lysate of MDCK I cells (MDCK I lysate) were separated by SDS-PAGE, followed by immunoblotting with anti–claudin-1 pAb (anti-cln-1 pAb), anti–claudin-2 pAb (anti-cln-2 pAb), anti–claudin-3 pAb (anti-cln-3 pAb), and anti–claudin-4 pAb (anti-cln-4 pAb). Only claudin-1 and -4 were detected in MDCK I cells. (B) Immunofluorescence microscopy. Confluent cultures of MDCK I cells were stained with anti–claudin-1 pAb (claudin-1), anti–claudin-2 pAb (claudin-2), anti–claudin-3 pAb (claudin-3), or anti–claudin-4 pAb (claudin-4). Both claudin-1 and -4 were concentrated at cell–cell borders in large amounts, where only a trace amount of claudin-3 was also detected. Claudin-2 signal was undetectable. Bar, 20 μm.
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Related In: Results  -  Collection

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Figure 1: Claudins expressed in MDCK I cells. (A) Immunoblotting. The total lysate of E. coli (E. coli lysate) expressing MBP fusion protein with the cytoplasmic domains of claudin-1 (1) and GST fusion proteins with the cytoplasmic domains of claudin-2 (2), -3 (3), and -4 (4), and the total lysate of MDCK I cells (MDCK I lysate) were separated by SDS-PAGE, followed by immunoblotting with anti–claudin-1 pAb (anti-cln-1 pAb), anti–claudin-2 pAb (anti-cln-2 pAb), anti–claudin-3 pAb (anti-cln-3 pAb), and anti–claudin-4 pAb (anti-cln-4 pAb). Only claudin-1 and -4 were detected in MDCK I cells. (B) Immunofluorescence microscopy. Confluent cultures of MDCK I cells were stained with anti–claudin-1 pAb (claudin-1), anti–claudin-2 pAb (claudin-2), anti–claudin-3 pAb (claudin-3), or anti–claudin-4 pAb (claudin-4). Both claudin-1 and -4 were concentrated at cell–cell borders in large amounts, where only a trace amount of claudin-3 was also detected. Claudin-2 signal was undetectable. Bar, 20 μm.
Mentions: As shown in Fig. 1 A, in the total cell lysate of MDCK I cells, claudin-1 and -4 were reproducibly detected by immunoblotting as bands with expected molecular masses. When confluent cultures of MDCK I cells were immunofluorescently stained with antibodies for claudin-1 to -4, intense signals for claudin-1 and -4 and very weak signals for claudin-3 were detected at cell–cell borders, but claudin-2 was undetectable (Fig. 1 B). Confocal microscopy confirmed that claudin-1 and -4 were concentrated at the most apical part of lateral membranes of MDCK I cells (data not shown).

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.

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