Mechanism of Clostridium perfringens enterotoxin interaction with claudin-3/-4 protein suggests structural modifications of the toxin to target specific claudins.

Claudins (Cld) are essential constituents of tight junctions. Domain I of Clostridium perfringens enterotoxin (cCPE) binds to the second extracellular loop (ECL2) of a subset of claudins, e.g. Cld3/4 and influences tight junction formation. We aimed to identify interacting interfaces and to alter claudin specificity of cCPE. Mutagenesis, binding assays, and molecular modeling were performed. Mutation-guided ECL2 docking of Cld3/4 onto the crystal structure of cCPE revealed a common orientation of the proposed ECL2 helix-turn-helix motif in the binding cavity of cCPE: residues Leu(150)/Leu(151) of Cld3/4 bind similarly to a hydrophobic pit formed by Tyr(306), Tyr(310), and Tyr(312) of cCPE, and Pro(152)/Ala(153) of Cld3/4 is proposed to bind to a second pit close to Leu(223), Leu(254), and Leu(315). However, sequence variation in ECL2 of these claudins is likely responsible for slightly different conformation in the turn region, which is in line with different cCPE interaction modes of Cld3 and Cld4. Substitutions of other so far not characterized cCPE residues lining the pocket revealed two spatially separated groups of residues (Leu(223), Asp(225), and Arg(227) and Leu(254), lle(258), and Asp(284)), which are involved in binding to Cld3 and Cld4, albeit differently. Involvement of Asn(148) of Cld3 in cCPE binding was confirmed, whereas no evidence for involvement of Lys(156) or Arg(157) was found. We show structure-based alteration of cCPE generating claudin binders, which interact subtype-specific preferentially either with Cld3 or with Cld4. The obtained mutants and mechanistic insights will advance the design of cCPE-based modulators to target specific claudin subtypes related either to paracellular barriers that impede drug delivery or to tumors.

Overexpression of claudin-5 but not claudin-3 induces formation of trans-interaction-dependent multilamellar bodies.

Tight junctions (TJs) regulate paracellular barriers and claudins (Cld) form the backbone of TJ strands. To elucidate the molecular mechanism of claudin polymer formation, TJs were reconstituted by claudin transfection of TJ-free HEK293 cells. Therewith, typical TJ stands can be found at cell-cell contacts. In addition, overexpression of Cld5-YFP induces formation of huge intracellular multilamellar bodies. In contrast, Cld3 does not induce similar structures. Inhibition of trans-interaction of Cld5 by Y148A substitution diminished formation of multilamellar bodies. These results demonstrate claudin subtype-specific oligomerization. Cld3 and Cld5 localize to the plasma membrane differentially. Phosphorylation at T207 of Cld5 was suggested to participate in regulation of Cld5 internalization. However, prevention of potential phosphorylation by T207A substitution did not increase Cld5 amount in the plasma membrane of transfected cells. Taken together, if carefully evaluated, transfection of claudin constructs in nonpolar cells is a powerful strategy to improve understanding of subcellular targeting and assembly of TJ proteins.

Determinants contributing to claudin ion channel formation.

Pore-forming properties of claudins (Cld) are likely defined by residues of their first extracellular loop (ECL1). Detailed mechanisms are unclear. MDCK cells overexpressing FLAG-Cld-1 wild-type and mutants were characterized by transepithelial resistance (TER) and ion permeability measurements. Replacing ECL1 residues of sealing Cld-1 by corresponding Cld-2 residues we aimed to identify new determinants responsible for sealing and/or pore formation. We found that E48K and S53E substitutions in human Cld-1 strongly reduced TER and increased permeability for Na(+) and Cl(-) . In contrast, K65D, D68S, and other single substitutions showed no significant change of TER and permeability for Na(+) and Cl(-) . Double substitution S53E/K65D did not change TER and ion permeability, whereas S53E/D68S decreased TER, albeit weaker than S53E. Ratio of permeabilities for Na(+) and Cl(-) revealed no clear charge specificity of the pore induced by S53E or S53E/D68S in Cld-1, suggesting that primarily S53 and potentially D68 in Cld-1 are involved in sealing of the paracellular cleft and that charge-unselective pores may be induced by substituting S53E.

Segmental expression of claudin proteins correlates with tight junction barrier properties in rat intestine.

In tubular epithelia, barrier function varies in a segment-specific way. The aim of this study was to correlate the presence of tight junction proteins and paracellular barrier properties along rat intestine. Tissue segments of duodenum, jejunum, ileum, and colon were stripped of submucosal cell layers and mounted in Ussing chambers for impedance spectroscopy to measure epithelial resistance (R (epi)). In parallel, expression of tight junction proteins was analysed by Western blots and immune fluorescence confocal microscopy. Colon showed highest R (epi), followed by duodenum, jejunum, and ileum. In small intestine, common transepithelial resistance (R (trans) or TER) overestimated true R (epi) by approximately 60%. In colon, strongest expression of "tightening" claudins 1, 3, 4, 5, and 8 was detected. In accordance with R (epi) the most proximal of the small intestinal segments, duodenum exhibited highest expression of "tightening" claudins and lowest expression of claudins mediating permeability, namely claudin-2, -7, and -12, compared to jejunum and ileum. These results correspond to the specific role of the duodenum as the first segment facing the acidic gastric content.

On the interaction of Clostridium perfringens enterotoxin with claudins.

Clostridium perfringens causes one of the most common foodborne illnesses, which is largely mediated by the Clostridium perfringens enterotoxin (CPE). The toxin consists of two functional domains. The N-terminal region mediates the cytotoxic effect through pore formation in the plasma membrane of the mammalian host cell. The C-terminal region (cCPE) binds to the second extracellular loop of a subset of claudins. Claudin-3 and claudin-4 have been shown to be receptors for CPE with very high affinity. The toxin binds with weak affinity to claudin-1 and -2 but contribution of these weak binding claudins to CPE-mediated disease is questionable. cCPE is not cytotoxic, however, it is a potent modulator of tight junctions. This review describes recent progress in the molecular characterization of the cCPE-claudin interaction using mutagenesis, in vitro binding assays and permeation studies. The results promote the development of recombinant cCPE-proteins and CPE-based peptidomimetics to modulate tight junctions for improved drug delivery or to treat tumors overexpressing claudins.