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 regulatory relevant. Under normal conditions, tricellulin tightens tricellular junctions against macromolecules. Traces of tricellulin occur in bicellular junctions. Aim: 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 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. Innovation: Tricellulin forms TJs via trans- and cis-association in 3-cell contacts 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: TAMPs' 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 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