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.
Tight junctions (TJs) seal paracellular clefts in epithelia/endothelia and form tissue barriers for proper organ function. TJ-associated marvel proteins (TAMPs; tricellulin, occludin, marvelD3) are thought to be relevant to regulation. Under normal conditions, tricellulin tightens tricellular junctions against macromolecules. Traces of tricellulin occur in bicellular junctions.


OBJECTIVE
As pathological disturbances have not been analyzed, the structure and function of human tricellulin, including potentially redox-sensitive Cys sites, were investigated under reducing/oxidizing conditions at 3- and 2-cell contacts.


RESULTS
Ischemia, hypoxia, and reductants redistributed tricellulin from 3- to 2-cell contacts. The extracellular loop 2 (ECL2; conserved Cys321, Cys335) trans-oligomerized between three opposing cells. Substitutions of these residues caused bicellular localization. Cys362 in transmembrane domain 4 contributed to bicellular heterophilic cis-interactions along the cell membrane with claudin-1 and marvelD3, while Cys395 in the cytosolic C-terminal tail promoted homophilic tricellullar cis-interactions. The Cys sites included in homo-/heterophilic bi-/tricellular cis-/trans-interactions contributed to cell barrier tightness for small/large molecules.


METHODS
Tricellulin forms TJs via trans- and cis-association in 3-cell contacts, as demonstrated electron and quantified fluorescence microscopically; it tightens 3- and 2-cell contacts. Tricellulin's ECL2 specifically seals 3-cell contacts redox dependently; a structural model is proposed.


CONCLUSIONS
TAMP ECL2 and claudins' ECL1 share functionally and structurally similar features involved in homo-/heterophilic tightening of cell-cell contacts. Tricellulin is a specific redox sensor and sealing element at 3-cell contacts and may compensate as a redox mediator for occludin loss at 2-cell contacts in vivo and in vitro. Molecular interaction mechanisms were proposed that contribute to tricellulin's function. In conclusion, tricellulin is a junctional redox regulator for ischemia-related alterations.
1 Leibniz-Institut für Molekulare Pharmakologie , Berlin, Germany .
Jimmi
J
Cording
1 Leibniz-Institut für Molekulare Pharmakologie , Berlin, Germany .
Ramona
R
Günther
2 Department of Nephrology, Max Delbrueck Center for Molecular Medicine , Charite Berlin, Berlin, Germany .
Emilia
E
Vigolo
1 Leibniz-Institut für Molekulare Pharmakologie , Berlin, Germany .
Christian
C
Tscheik
1 Leibniz-Institut für Molekulare Pharmakologie , Berlin, Germany .
Lars
L
Winkler
1 Leibniz-Institut für Molekulare Pharmakologie , Berlin, Germany .
Isabella
I
Schlattner
1 Leibniz-Institut für Molekulare Pharmakologie , Berlin, Germany .
Dorothea
D
Lorenz
1 Leibniz-Institut für Molekulare Pharmakologie , Berlin, Germany .
Reiner F
RF
Haseloff
2 Department of Nephrology, Max Delbrueck Center for Molecular Medicine , Charite Berlin, Berlin, Germany .
Kai M
KM
Schmidt-Ott
3 Department of General Pathology, Institute of Pathology and Neuropathology, Medical School, University of Tübingen , Tübingen, Germany .
Hartwig
H
Wolburg
1 Leibniz-Institut für Molekulare Pharmakologie , Berlin, Germany .
Ingolf E
IE
Blasig
D000818:Animals; D001665:Binding Sites; D015687:Cell Hypoxia; D002463:Cell Membrane Permeability; D003545:Cysteine / Q000378:metabolism*; D004285:Dogs; D004847:Epithelial Cells / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D007511:Ischemia / Q000378:metabolism* / Q000473:pathology; D007668:Kidney / Q000098:blood supply* / Q000378:metabolism / Q000473:pathology; D062794:MARVEL Domain Containing 2 Protein / Q000737:chemistry / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells; D008297:Male; D008810:Mice, Inbred C57BL; D062793:Occludin / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D019108:Tight Junctions / Q000378:metabolism*
D062794:MARVEL Domain Containing 2 Protein; C508309:Marveld2 protein, mouse; D062793:Occludin; C568986:Ocln protein, mouse; D003545:Cysteine