Molecular determinants of the interaction between Clostridium perfringens enterotoxin fragments and claudin-3.
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.
Leibniz Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse, 10, 13125 Berlin, Germany.
Lars
L
Winkler
Claudia
C
Gehring
Ariane
A
Wenzel
Sebastian L
SL
Müller
Christian
C
Piehl
Gerd
G
Krause
Ingolf E
IE
Blasig
Jörg
J
Piontek
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D018938:Caco-2 Cells; D002460:Cell Line; D062465:Claudin-3; D003016:Clostridium perfringens / Q000378:metabolism*; D004768:Enterotoxins / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D019108:Tight Junctions / Q000378:metabolism
C568122:CLDN3 protein, human; D062465:Claudin-3; C568123:Cldn3 protein, mouse; D004768:Enterotoxins; D008565:Membrane Proteins; D010455:Peptides