Identification of a claudin-4 residue important for mediating the host cell binding and action of Clostridium perfringens enterotoxin

Abstract

The 24-member claudin protein family plays a key role in maintaining the normal structure and function of epithelial tight junctions. Previous studies with fibroblast transfectants and naturally sensitive Caco-2 cells have also implicated certain claudins (e.g., Claudin-4) as receptors for Clostridium perfringens enterotoxin (CPE). The present study first provided evidence that the second extracellular loop (ECL-2) of claudins is specifically important for mediating the host cell binding and cytotoxicity of native CPE. Rat fibroblast transfectants expressing a Claudin-4 chimera, where the natural ECL-2 was replaced by ECL-2 from Claudin-2, exhibited no CPE-induced cytotoxicity. Conversely, CPE bound to, and killed, CPE-treated transfectants expressing a Claudin-2 chimera with a substituted ECL-2 from Claudin-4. Site-directed mutagenesis was then used to alter an ECL-2 residue that invariably aligns as N in claudins known to bind native CPE but as D or S in claudins that cannot bind CPE. Transfectants expressing a Claudin-4(N149D) mutant lost the ability to bind or respond to CPE, while transfectants expressing a Claudin-1 mutant with the corresponding ECL-2 residue changed from D to N acquired CPE binding and sensitivity. Identifying carriage of this N residue in ECL-2 as being important for native CPE binding helps to explain why only certain claudins can serve as CPE receptors. Finally, preincubating CPE with soluble recombinant Claudin-4, or Claudin-4 fragments containing ECL-2 specifically blocked the cytotoxicity on Caco-2 cells. This result opens the possibility of using receptor claudins as therapeutic decoys to ameliorate CPE-mediated intestinal disease.

FIG. 1.

Receptor inhibition studies using soluble rClaudins or rClaudin fragments. (A) Caco-2 cell culture morphology after treatment with 2.5 μg of CPE/ml that had been preincubated with a 100-fold (per weight) excess of the specified rClaudin or rClaudin fragment for 20 min at 37°C. (B) Cytotoxicity after Caco-2 cell cultures were treated or 60 min at 37°C with the indicated preincubation mix containing 1 μg/ml (light gray bars) or 2.5 μg (dark gray bars) of CPE/ml and 100 μg or 250 μg, respectively, of the specified rClaudin or rClaudin fragment/ml. Cytotoxicity was calculated as percentage dead cells by using the Invitrogen Live/Dead assay. (C) Western immunoblot analysis of Caco-2 cells after treatment with the preincubation mix used in panel B containing 1 μg of CPE/ml. The locations of CH-1 and CH-2 complexes are noted on the left hand side of the figure. (D) Western blot of coimmunoprecipitation reactions after preincubation of CPE with rClaudin-4 or rClaudin-1.

FIG. 1.

Receptor inhibition studies using soluble rClaudins or rClaudin fragments. (A) Caco-2 cell culture morphology after treatment with 2.5 μg of CPE/ml that had been preincubated with a 100-fold (per weight) excess of the specified rClaudin or rClaudin fragment for 20 min at 37°C. (B) Cytotoxicity after Caco-2 cell cultures were treated or 60 min at 37°C with the indicated preincubation mix containing 1 μg/ml (light gray bars) or 2.5 μg (dark gray bars) of CPE/ml and 100 μg or 250 μg, respectively, of the specified rClaudin or rClaudin fragment/ml. Cytotoxicity was calculated as percentage dead cells by using the Invitrogen Live/Dead assay. (C) Western immunoblot analysis of Caco-2 cells after treatment with the preincubation mix used in panel B containing 1 μg of CPE/ml. The locations of CH-1 and CH-2 complexes are noted on the left hand side of the figure. (D) Western blot of coimmunoprecipitation reactions after preincubation of CPE with rClaudin-4 or rClaudin-1.

FIG. 2.

Receptor inhibition studies using soluble recombinant claudins or chimeric claudins. (A) Caco-2 cell culture morphology after treatment with 2.5 μg of CPE/ml that had been preincubated with a 100-fold excess (per weight) of the specified recombinant claudin or chimeric claudin for 20 min at 37°C. (B) Cytotoxicity after Caco-2 cell cultures were treated with the indicated preincubation mix containing 1 μg (light gray bars) or 2.5 μg (dark gray bars) of CPE/ml and 100 or 250 μg, respectively, of the specified recombinant claudin or chimeric claudin/ml. Cytotoxicity was calculated as the percentage of dead cells by using the Invitrogen Live/Dead assay. (C) Western immunoblot analysis of Caco-2 cells after treatment with preincubation mix used in panel A containing the 1 μg of CPE/ml. The locations of CH-1 and CH-2 complexes are noted on the left-hand side of the figure.

FIG. 3.

CPE binding ability and sensitivity of Rat1-R12 fibroblast transfectants expressing chimeric claudins. (A) Confluent Rat1-R12 fibroblast transfectants expressing the indicated claudin or chimeric claudin were treated with or without 2.5 μg of CPE/ml for 30 min at 37°C to visualize morphological damage. (B) Fibroblast transfectants expressing the indicated claudin or chimeric claudin were treated with 1 μg of CPE/ml (light gray bar) or 2.5 μg of CPE/ml (dark gray bar) for 60 min at 37°C. CPE-induced cell cytotoxicity was expressed as a percentage of total dead cells. (C) Parental or transfectant fibroblasts expressing the indicated claudin or chimeric claudin were (+) or were not (−) treated as a monolayer with 1 μg of CPE/ml for 60 min at 37°C. CPE immunoreactivity was then detected by Western blot analysis with rabbit polyclonal anti-CPE serum. The location of CH-1 complex is noted on the left-hand side of the figure.

FIG. 4.

Comparative alignment of the C-terminal half of CPE-sensitive and CPE-resistant claudins based upon previous results using transfectants (9, 36). The claudin residue in the middle of the ECL-2 region suggested to be important for CPE binding is shown in black.

FIG. 5.

Receptor inhibition studies using soluble, affinity-purified, rClaudin ECL-2 site-directed mutants. (A) Caco-2 cell culture morphology after treatment with 2.5 μg of CPE/ml that had been preincubated with a 100-fold excess (per weight) of the specified rClaudin ECL-2 mutant for 20 min at 37°C. (B) Cytotoxicity after Caco-2 cell cultures were treated with the indicated preincubation mix containing 1 μg (light gray bars) or 2.5 μg (dark gray bars) of native CPE/ml and 100 or 250 μg of the specified rClaudin ECL-2 mutant/ml, respectively. Cytotoxicity was calculated by using the Invitrogen Live/Dead assay. (C) Western immunoblot analysis of Caco-2 cells after treatment for 60 min at 37°C with the indicated preincubation mix containing 1 μg of CPE/ml. The locations of the CH-1 and CH-2 complexes are noted on the left side of the figure.

FIG. 6.

Characterization of CPE effects on fibroblast transfectants expressing claudin point mutants with amino acid substitutions at ECL-2 residue 149. (A) Transfectants expressing the indicated native claudin or claudin ECL-2 point mutant were treated with CPE as described for Fig. 3 and then analyzed for morphological damage. (B) Cytotoxic effects of CPE on rat fibroblast transfectants expressing native claudins or claudin ECL-2 point mutants. Rat1-R12 fibroblast transfectants were treated with 1 μg (light gray bars) or 2.5 μg (dark gray bars) of CPE/ml for 60 min at 37°C. The percentage of dead cells was calculated by using the Invitrogen Live/Dead assay. (C) CPE Western immunoblot analysis of CPE large complex formation by transfectants expressing native claudins or claudin ECL-2 point mutants. Fibroblast transfectants were either treated (+) or not treated (−) with 1 μg of CPE/ml for 60 min at 37°C. A large complex was then detected by Western blot analysis with rabbit polyclonal anti-CPE serum. The location of the CH-1 complex is noted on the left of the figure.