Molecular determinants of the interaction between Clostridium perfringens enterotoxin fragments and claudin-3

Abstract

Clostridium perfringens enterotoxin (CPE) binds to the extracellular loop 2 of a subset of claudins, e.g. claudin-3. Here, the molecular mechanism of the CPE-claudin interaction was analyzed. Using peptide arrays, recombinant CPE-(116-319) bound to loop 2 peptides of mouse claudin-3, -6, -7, -9, and -14 but not of 1, 2, 4, 5, 8, 10-13, 15, 16, 18-20, and 22. Substitution peptide mapping identified the central motif (148)NPL(150)VP, supposed to represent a turn region in the loop 2, as essential for the interaction between CPE and murine claudin-3 peptides. CPE-binding assays with claudin-3 mutant-transfected HEK293 cells or lysates thereof demonstrated the involvement of Asn(148) and Leu(150) of full-length claudin-3 in the binding. CPE-(116-319) and CPE-(194-319) bound to HEK293 cells expressing claudin-3, whereas CPE-(116-319) bound to claudin-5-expressing HEK293 cells, also. This binding was inhibited by substitutions T151A and Q156E in claudin-5. In contrast, removal of the aromatic side chains in the loop 2 of claudin-3 and -5, involved in trans-interaction between claudins, increased the amount of CPE-(116-319) bound. These findings and molecular modeling indicate different molecular mechanisms of claudin-claudin trans-interaction and claudin-CPE interaction. Confocal microscopy showed that CPE-(116-319) and CPE-(194-319) bind to claudin-3 at the plasma membrane, outside cell-cell contacts. Together, these findings demonstrate that CPE binds to the hydrophobic turn and flanking polar residues in the loop 2 of claudin-3 outside tight junctions. The data can be used for the specific design of CPE-based modulators of tight junctions, to improve drug delivery, and as chemotherapeutics for tumors overexpressing claudins.

FIGURE 1.

A, CPE-(116–319) binds much more strongly to the ECL2 peptide of Cld3 than does CPE-(290–319). ECL2 peptide (amino acids 140–159) of mouse Cld3 was immobilized and superinfused with different concentrations of GST-CPE-(116–319) and GST-CPE-(290–319), respectively. The binding of GST was negligible (<1 fmol for <20 μm GST). The binding was analyzed by surface plasmon resonance spectroscopy. Mean ± S.E., n > 3 except for 18 μm GST-CPE-(290–319), n = 1. Incubation of Caco-2 cells with CPE-(116–319) reduces junctional resistance (B) and transepithelial electrical resistance, TEER (C), and increases the permeation coefficient (P_c) of fluorescein (D). For B, cells were incubated for 12 h with 10 μg/ml GST or GST-CPE-(116–319) and the junctional resistance relative to the value before GST or GST-CPE-(116–319) incubation was determined by electric cell-substrate impedance sensing. Mean ± S.E. (error bars); n ≥ 9; *, p < 0.001. For C and D, cells were incubated with 25 μg/ml GST or GST-CPE-(116–319) for 16 h and TEER determined relative to the value before incubation. Mean ± S.E. n = 6, *, p < 0.001.

FIGURE 2.

Single amino acid substitution analysis of the ECL2 of Cld3 (Cld3-ECL2) identified the motif ¹⁴⁸NPLVP¹⁵² as essential for the association of GST-CPE-(116–319). The C-terminal flank (Cld3-(154–157)) also contains amino acids involved in the interaction. 15-mer peptides of Cld3-ECL2 were immobilized on a membrane. In the left column, all spots contain the wild type sequence (wt); the letter and number give the position of the amino acid replaced in the same line of the second column. The membrane was incubated with 10 μg/ml GST-CPE-(116–319). Bound CPE was visualized via mouse anti-GST and HRP-conjugated anti-mouse antibodies. Binding was considered to be decreased or increased if the mean chemiluminescence was increased or decreased by a factor of 4, respectively, compared with the wt (n = 2). AA, amino acid; X, every amino acid except Cys and those mentioned as less or stronger. Dotted circles, wt sequence; dashed boxes, substitutions resulting in decreased binding. A representative membrane is shown.

FIGURE 3.

Binding of full-length Cld3 to CPE is affected by amino acid substitutions in the ECL2 of Cld3. HEK cells were transfected with Cld3_wt or a Cld3_mutant. Lysates thereof were used for pull-down assays with GST-CPE-(116–319) or GST immobilized on glutathione-Sepharose. Bound and unbound fractions were analyzed by SDS-PAGE and Western blot. A, representative blots are shown. Cld3_wt bound specifically to GST-CPE-(116–319) but not to GST. The substitutions N148D and L150A reduced the binding strongly, Y147A increased the amount of bound Cld3. B, the intensity of the immunoreactive bands was quantified. For each mutant the ratio of intensity in the bound fraction to that in the unbound fraction relative to the ratio for Cld3_wt (internal standard in each experiment) was determined (bound Cld3/unbound Cld3). Mean ± S.E. (error bars); n > 4; *, p < 0.05; ***, p < 0.001 to Cld3_wt.

FIGURE 4.

Binding of GST-CPE to Cld3-transfected HEK cells. Three days after transfection, cells were incubated with 1 μg/ml GST-CPE-(116–319) (left, middle) or GST-CPE-(194–319) (right) for 1 h at 37 °C, washed with PBS, and the amount of bound GST-CPE and claudin in the lysate analyzed by SDS-PAGE and Western blot using anti-GST (top) and anti-Cld3 (bottom) antibodies. Left, GST-CPE-(116–319) bound to Cld3_wt-transfected (Cld3) but not to non-transfected (n.t.) cells. Middle, GST-CPE-(116–319) bound to Cld3-N148D and Cld3-L150A to a similar extent as to Cld3_wt. In contrast, binding to Cld3-N148D/L150A was strongly reduced. Right, similar results were obtained for GST-CPE-(194–319). Representative blots are shown.

FIGURE 5.

Binding of GST-CPE-(116–319) and GST-CPE-(194–319) to Cld3 on the surface of transfected HEK cells. Cells were transfected with Cld3_wt-CFP (A and B), Cld3_wt (C and E), or Cld3-N148D/L150A (D and E), 3 days later, the cells were incubated with 10 μg/ml GST-CPE-(116–319)_, GST-CPE-(194–319), or GST for 20 min at 37 °C, washed with PBS, and fixed. GST (red) was stained using mouse anti-GST antibodies, nuclei (blue) with 4′,6-diamidino-2-phenylindole, and Cld3 (green) detected via CFP-fluorescence (A and B) or anti-Cld3 antibodies (C-E). Confocal images are shown. In contrast to GST (A, red), GST-CPE-(116–319) (B, red) and GST-CPE-(194–319) (C, red) bound to cells expressing Cld3 (green) but not to Cld3 negative/4′,6-diamidino-2-phenylindole positive cells (violet arrows). GST-CPE-(116–319) and GST-CPE-(194–319) bound to Cld3 on the cell surface (B and C, arrow) but not to Cld3 in the tight junction area at cell-cell contacts (B and C, arrowhead). For the detection of Cld3, high (C, left and middle) or low (C, right) detector gain was used to visualize colocalization of GST-CPE-(194–319) and Cld3 (arrow) or enrichment of Cld3 at contacts between two Cld3-expressing cells (arrowhead), respectively. Cld3-N148D/L150A (D) showed enrichment at contacts between two Cld3-expressing cells similar to Cld3_wt. Binding of GST-CPE-(194–319) to Cld3-N148D/L150A-transfected cells was barely detected. E, 14 h incubation with 10 μg/ml GST-CPE-(194–319) led to internalization (arrows) of Cld3 (green) and GST-CPE-(194–319) (red) for Cld3_wt, but not Cld3-N148D/L150A expressing cells. Z stacks, xy (quadrat), xz (top), and yz images (side) are shown and the position within the stack indicated by lines; 4′,6-diamidino-2-phenylindole is shown for the xy image only. Equal detector gains were used for A and B; C, right, and D; E, left; and E, right. Bar, 4 μm.

FIGURE 6.

Binding of GST-CPE-(116–319) to Cld5-transfected HEK cells. Cells were incubated with 1 μg/ml GST-CPE-(116–319) (A) or GST-CPE-(194–319) (B) at 37 °C 3 days after transfection, washed with PBS, and the amount of bound GST-CPE and claudin in the lysate analyzed by SDS-PAGE and Western blot using anti-GST and anti-GFP (A) or anti-Cld3 and anti-Cld5 antibodies (B). Representative blots are shown. A, GST-CPE-(116–319) bound to Cld3-YFP- and Cld5-YFP-transfected cells. B, GST-CPE-(194–319) bound to Cld3- but not to Cld5-transfected cells. C, binding of GST-CPE-(116–319) to claudin-5 mutants. Transfected cells were incubated with 10 μg/ml GST-CPE-(116–319) for 1 h at 37 °C. Representative Western blots show that neither GST (n.t. + GST) nor GST-CPE-(116–319) (n.t. + GST-CPE_116–319) bound to non-transfected cells. GST-CPE-(116–319) (Cld5_wt) but not GST (Cld5wt+GST) bound to Cld5_wt-YFP transfected cells. D, quantification of the binding. The ratio of GST-CPE-(116–319) intensity to Cld5-YFP intensity relative to the ratio for Cld5_wt-YFP (internal standard) was determined for each mutant (bound CPE). Substitutions T151A and Q156E decreased whereas F147A, Y148A, Y158A, and E159Q increased the amount of GST-CPE-(116–319) bound. Striped columns, substitutions that block trans-interaction between claudins. Mean ± S.E. (error bars), n > 4. ***, p < 0.001 compared with Cld5_wt.

FIGURE 7.

Schemes of molecular interaction between CPE and the ECL2 of claudin-3. A, topology of claudins with ECL2 marked in a box. B, alignment of ECL2 of Cld3 and -5, with similarity of residues calculated with matrix blossom62 (dot = weak; colon = strong similarity; bar = identity). Secondary structure (SecStr) according to the model, helices (H), turn (t). C, homologous helix-turn-helix model for ECL2 of Cld3 based on the fragment of PDB code 2BDV in front view. N-terminal helix, orange; C-terminal helix, cyan. The hydrophobic residues ¹⁴⁹PLVP (green) constituting the turn region of the loop are found to be important for CPE binding. The residues Asn¹⁴⁸ and Lys¹⁵⁶ (light blue) stabilize the turn-fold of the ECL2 by hydrogen bridges (hydrogens in cyan). Residues Phe¹⁴⁶, Tyr¹⁴⁷, and Arg¹⁵⁷ (yellow) correspond to aromatic residues Phe¹⁴⁷, Tyr¹⁴⁸, and Tyr¹⁵⁸ in Cld5, which were found to be important for trans-interaction. Heteroatoms are red (oxygen) and dark blue (nitrogen). D, according to the results, CPE cannot bind to trans-interacting claudins (left), probably due to steric hindrance, but needs a free ECL2 for binding (right). E, supposed spatial regions of interaction between binding sensitive residues (Tyr³⁰⁶, Tyr³¹⁰, Tyr³¹², and Leu³¹⁵ from literature (36)) visualized at the CPE-(194–319) x-ray structure (PDB code 2QUO) and the residues (¹⁴⁸NPLVP, Gln¹⁵⁵) identified in this study for Cld3.