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.
Highlights
tight junctions (TJ) consist of transmembrane proteins, mainly the tetraspan proteins of the claudin family, as well as occludin and tricellulin [8]
These findings demonstrate that Clostridium perfringens enterotoxin (CPE) binds to the hydrophobic turn and flanking polar residues in the loop 2 of claudin-3 outside tight junctions
CPE causes one of the most common food-borne diseases [13]. It consists of two functional domains, an N-terminal region that mediates the cytotoxic effect and the C-terminal region (CPE-(184 –319)), which binds to extracellular loop 2 (ECL2) of Cld3 but not of Cld1 nor to the ECL1 of Cld3 [12]
Summary
Brigham and Women’s Hospital, Harvard Medical School, 77 Louis Pasteur Ave., Boston, MA 02115. Claudins (Cld) are the major functional constituent of TJ [10]. It was proposed that tissue-specific drug delivery via the paracellular route would be possible by modulation of the barrier-function of claudins in a subtype-specific manner [7]. A subset of claudins, e.g. Cld and -4 but not -1 and -2, have been shown to be receptors for Clostridium perfringens enterotoxin (CPE) with high association constants of about 108 MϪ1 [12]. CPE is a promising tool to modulate claudins, the key constituents of TJ, and thereby to enhance paracellular drug delivery. The design of CPE-based TJ modulators could permit efficient claudin subtype-specific modulation, which would be tissue-specific modulation of TJ. We identify the residues within the ECL2 of Cld that are involved in interaction with CPE
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