Claudin targeting as an effective tool for directed barrier modulation of the viable epidermis

Abstract

Tight junction (TJ) formation is vital for epidermal barrier function. We aimed to specifically manipulate TJ barriers in the reconstructed human epidermis (RHE) by claudin‐1 and ‐4 knockdown (KD) and by claudin‐binding fusion proteins of glutathione S‐transferase and modified C‐terminal fragments of Clostridium perfringens enterotoxin (GST‐cCPE). Impedance spectroscopy and tracer permeability imaging were employed for functional barrier assessment and investigation of claudin contribution. KD of claudin‐1, but not claudin‐4, impaired the paracellular barrier in vitro. Similarly, claudin‐binding GST‐cCPE variants weakened the paracellular but not the stratum corneum barrier. Combining both TJ targeting methods, we found that claudin‐1 targeting by GST‐cCPE after claudin‐4 KD led to a marked decrease in paracellular barrier properties. Conversely, after claudin‐1 KD, GST‐cCPE did not further impair the barrier. Comparison of GST‐cCPE variants with different claudin‐1/claudin‐4 affinities, NHS‐fluorescein tracer detection, and immunostaining of RHE paraffin sections showed that GST‐cCPE variants bind to extrajunctional claudin‐1 and ‐4, which are differentially distributed along the stratum basale–stratum granulosum axis. GST‐cCPE binding blocks these claudins, thereby specifically opening the paracellular barrier of RHE. The data indicate a critical role for claudin‐1 in regulating paracellular permeability for ions and small molecules in the viable epidermis. Claudin targeting is presented as a proof‐of‐concept for precise barrier modulation.

cCPE Fusion Proteins as Molecular Probes to Detect Claudins and Tight Junction Dysregulation in Gastrointestinal Cell Lines, Tissue Explants and Patient-Derived Organoids

“…Previously, we used GST-cCPE fusion proteins as claudin binders and claudin-specific TJ modulators. cCPE-wt and different mutants thereof were used to target different claudin subtypes [ 33 , 37 , 39 , 40 , 44 , 45 ]. To enable direct detection of cCPE fusion proteins, for instance for live-cell imaging, YFP-cCPE fusion proteins were generated by subcloning, expression in E. coli and his-tag-based purification (see Section 2 and Figure 1 A).…”

“…The high-affinity interaction between cCPE and a subset of members of the claudin protein family—the CPE receptor claudins (CLDN3, −4, −6 to −9, −14, −19)—is well established [ 33 , 35 , 36 , 37 , 38 , 39 , 46 , 66 ]. Mutations in cCPE have been used to expand, narrow or shift the claudin subtype preference [ 37 , 39 , 40 , 41 , 42 , 44 , 45 ]. In general, all of these cCPE variants bind to more than one claudin subtype.…”

“…However, if this effect is unwanted, as in this study, it can be prevented by reducing the concentration and incubation time of cCPE. It was shown that long-term incubation (>4 h) with high concentrations of cCPE promotes tight junction disassembly and barrier opening, whereas short-term incubation (<1 h) and low concentrations do not impair epithelial barriers [ 37 , 39 , 40 , 44 , 45 , 47 , 48 , 49 , 50 ].…”

“…To foster the implementation of cCPE as a claudin-targeting tool for diverse applications, claudin-binding properties of cCPE have been altered by structure-based mutagenesis [ 37 , 39 , 40 , 41 , 42 , 43 ]. The variant cCPE-S305P/S307R/S313H (cCPE-SSS) binds with high affinity to a broad spectrum of claudins (CLDN1 −9, −14, −19) [ 39 , 42 , 44 , 45 ], including relevant claudins that are expressed in the gastrointestinal tract and frequently deregulated during disease. This renders cCPE-SSS a promising and highly sensitive probe to detect disease-specific claudin mislocalization and overexpression.…”

Expression and Localization Profiles of Tight Junction Proteins in Immune Cells Depend on Their Activation Status

“…As the potential expression of claudins on immune cells might be too low for detection in Western blots, we used the ability of the claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) to bind to certain claudins when present on the cell surface. For this, we used a modified variant of cCPE, S305P/S307R/S313H (SSS) [23], which is known to bind to a variety of claudins including Cldn1 to -9, Cldn14, and Cldn19 [23][24][25][26]. As negative controls, unstained cells and, to exclude unspecific binding, the inactive Y306A/L315A (YALA) [23] variant of the aforementioned cCPE were used.…”