title: 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.
  
  
  OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 1 Department of Molecular Cell Physiology, Leibniz-Institut für Molekulare Pharmakologie , Berlin, Germany .
authors.forename: Sebastian
authors.initials: S
authors.lastname: Dabrowski
authors.forename: Christian
authors.initials: C
authors.lastname: Staat
authors.forename: Denise
authors.initials: D
authors.lastname: Zwanziger
authors.forename: Reine-Solange
authors.initials: RS
authors.lastname: Sauer
authors.forename: Christian
authors.initials: C
authors.lastname: Bellmann
authors.forename: Ramona
authors.initials: R
authors.lastname: Günther
authors.forename: Eberhard
authors.initials: E
authors.lastname: Krause
authors.forename: Reiner Fritz
authors.initials: RF
authors.lastname: Haseloff
authors.forename: Heike
authors.initials: H
authors.lastname: Rittner
authors.forename: Ingolf Ernst
authors.initials: IE
authors.lastname: Blasig
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D062445:Claudin-1 / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D051381:Rats; D017208:Rats, Wistar; D019108:Tight Junctions / Q000378:metabolism
chemical_list: D062445:Claudin-1