Redox-sensitive structure and function of the first extracellular loop of the cell-cell contact protein claudin-1: lessons from molecular structure to animals

Sebastian Dabrowski, Christian Staat, Denise Zwanziger, Reine-Solange Sauer, Christian Bellmann, Ramona Günther, Eberhard Krause, Reiner Fritz Haseloff, Heike Rittner, and Ingolf Ernst Blasig

Antioxidants & Redox Signaling
2014 vol: 22 issue: 1 doi: 10.1089/ars.2013.5706

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 β-sheet binding surface flanked by an α-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 β-sheet, stabilized by a shielded and redox-sensitive disulfide bond.

CONCLUSION:
Since the β-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.

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Topics: Fusion proteins, Peptides, Monolith – MicroScale Thermophoresis, MST, Proteins, Publications