Redox-Sensitive Structure and Function of the First Extracellular Loop of the Cell–Cell Contact Protein Claudin-1: Lessons from Molecular Structure to Animals

Abstract

The paracellular cleft within epithelia/endothelia is sealed by tight junction (TJ) proteins. Their extracellular loops (ECLs) are assumed to control paracellular permeability and are targets of pathogenes. We demonstrated that claudin-1 is crucial for paracellular tightening. Its ECL1 is essential for the sealing and contains two cysteines conserved throughout all claudins. Aims: We prove the hypothesis that this cysteine motif forms a redox-sensitive intramolecular disulfide bridge and, hence, the claudin-1-ECL1 constitutes a functional structure which is associated to ECLs of this and other TJ proteins. Results: The structure and function of claudin-1-ECL1 was elucidated by investigating sequences of this ECL as synthetic peptides, C1C2, and as recombinant proteins, and exhibited a b-sheet binding surface flanked by an a-helix. These sequences bound to different claudins, their ECL1, and peptides with nanomolar binding constants. C-terminally truncated C1C2 (-4aaC) opened cellular barriers and the perineurium. Recombinant ECL1 formed oligomers, and bound to claudin-1 expressing cells. Oligomerization and claudin association were abolished by reducing agents, indicating intraloop disulfide bridging and redox sensitivity. Innovation: The structural and functional model based on our in vitro and in vivo investigations suggested that claudin-1-ECL1 constitutes a functional and ECL-binding bsheet, stabilized by a shielded and redox-sensitive disulfide bond. Conclusion: Since the b-sheet represents a consensus sequence of claudins and further junctional proteins, a general structural feature is implied. Therefore, our model is of general relevance for the TJ assembly in normal and pathological conditions. C1C2-4aaC is a new drug enhancer that is used to improve pharmacological treatment through tissue barriers.

Redox Regulation of Cell Contacts by Tricellulin and Occludin: Redox-Sensitive Cysteine Sites in Tricellulin Regulate Both Tri- and Bicellular Junctions in Tissue Barriers as Shown in Hypoxia and Ischemia

“…The unique properties of Tric are supported by the short peptide double chain of Tric-ECL2, including two specific prolines, but lacking b-sheet and a-helix elements. Consequently, the Tric-ECL2 structure is neither compatible with the Occl-ECL2 (2) nor with the ECL1 of Clds, both of which share the latter structural features (8). This further explains why heterophilic associations are impossible for Tric in the 3-cell contact.…”

“…MDCK-II, Madin-Darby canine kidney cell line; TJ, tight junction; Tric, tricellulin; TM, transmembrane domain. 8 CORDING ET AL.…”

Claudin peptidomimetics modulate tissue barriers for enhanced drug delivery

“…C5C2/shifted is truncated (4 aa, C‐terminal) and prolonged (5 aa, N‐terminal). C2C2 was derived from the ECL1 of murine claudin‐2 (aa 53–81) and used as a control . Native Cys were replaced by Ser.…”

“…The claudin‐1 peptidomimetic C1C2 dose‐dependently and reversibly opens the perineurial barrier of peripheral nerves in rats, allowing small drugs that typically do not permeate the perineurium to do so in a defined time frame . Analysis of the mode of action revealed that C1C2 causes redistribution of claudins from junctions to the cytosol, disturbs TJ‐strand morphology, and reduces claudin‐1 expression …”

Identity crisis in the PMP-22/EMP/MP20/Claudin superfamily (Pfam00822)

“…The two conserved cysteines in the signature motif of the claudins have been found to form an intramolecular disulfide bond and mediate paracellular tightening. 11 In EMP-1 the intramolecular disulfide bond cannot be formed, as only one of these cysteines is conserved (Fig. 1, left).…”