Human CLDN3 Stable Cell Line - HEK293T

Clostridium perfringens enterotoxin (CPE) is a common foodborne pathogen-derived factor and a pore-forming toxin; it induces Ca2+ influx into intestinal epithelial cells, leading to cellular damage. Upon binding to specific claudin proteins (such as claudin-4), CPE forms oligomeric pores in the cell membrane. Although the mechanism of CPE-claudin interaction is well-understood, the structure and assembly mode of the pore complex remain unclear. Here, researchers employed AlphaFold2 complex prediction, structural alignment, and molecular dynamics simulations to construct structural models of the CPE/claudin-4 complex in both prepore and pore states. The CPE pore adopts a hexameric form, differing from the typical heptameric "stem-and-cap" architecture found in aerolysin-like β-barrel pore-forming toxins (β-PFTs). The inner wall of the pore is lined with three hexameric glutamate rings-a feature distinguishing it from other β-PFTs and conferring specific cation selectivity upon CPE. Furthermore, the study reveals that the pore center is anchored by a dodecameric claudin ring, formed through a cis variant of the interface interactions found in claudin tight-junction strands. Mutating key residues at this interface inhibits CPE-induced cellular damage in vitro. Based on these findings, the researchers propose that the claudin ring acts as an anchoring structure involved in a twisting mechanism that drives the extension of CPE β-hairpins and their subsequent membrane insertion.

Here, the viability of both HEK293-CLDN3WT and HEK293-CLDN3-S68E cell lines decreased with increasing CPE concentrations (Figure 1A). However, the CLDN3-S68E mutation attenuated CPE-mediated cell damage to some extent, resulting in an increased EC50 value for cell viability (Figure 1B). Furthermore, saturation binding assays revealed that the CLDN3-S68E mutation slightly reduced the affinity of cCPE for CLDN3-expressing HEK293 cells (Figure 1C). The dissociation constant (Kd) for this mutant was 38 ± 3 nM, approximately double that of the wild-type (WT) (14 ± 5 ​​nM) (Figure 1D). In summary, in vitro data indicate that when the S68E mutation inhibits linear cis-interactions of claudin, both CPE-induced toxicity and cCPE affinity are diminished; this supports the view that interactions associated with these linear cis-interactions contribute to the formation of claudin-anchored CPE-pore complexes.

Figure 1. Cell viability and cCPE binding assays with HEK293 cells expressing either mouse CLDN3 wild type (mCLDN3-WT) or mCLDN3-S68E. (Nagarajan S K, et al., 2025)