Tricellulin (Tric) is a tight junction protein at tricellular contacts; however its exact structure, function and regulation are unclear. Tric contributes to the paracellular tightening by intercellular trans-interactions between its extracellular loops 2 (ECL2). Consequently, trictide, a peptide derived from the Tric ECL2 was designed as a potential drug enhancer to specifically overcome tissue barriers. This work is aimed to investigate the functional and molecular modulations of tricellular contacts caused by trictide and to understand essential elements and protein interactions of tricellular tight junctions (tTJ) for barrier formation. Trictide increased the passage of ions, small and larger molecules up to 10 kDa in a concentration dependent manner over duration from 16 h to 47 h after application to human colon epithelial cells. Under normal conditions, lipolysis-stimulated lipoprotein receptor (LSR) and Tric localized at tTJs while occludin (Occl), claudin-5 (Cldn5) and claudin-1 (Cldn1) localized at bicellular TJs (bTJ). After trictide treatment, Tric and LSR moved from tTJs to bTJs, and the bTJ proteins Cldn1 and Occl were internalized. Trictide also decreased the transcellular resistance of brain endothelial cells after 14 h and caused enrichment of Cldn5 around the tri-cellular area. Trictide down-regulated Occl, Tric, Cldn1, Cldn5 in epithelial cells and LSR, Occl, Cldn1 in mouse brain endothelial cells. Trictide-initiated opening of the tricellular sealing tube revealed a Tric-free area at the tricellular region as demonstrated by super resolution microscopy. In different cells, cis-interactions of Tric–Tric (tTJs), Tric–Cldn1, Tric–marvelD3, and Occl–Occl (bTJs) were strongly reduced by trictide treatment. In normal brain capillaries of different species, Tric was localized both at bTJs and tTJs while LSR was found exclusively at tTJs. In vivo, trictide did not increase the uptake of small molecules by mouse kidney and liver but tended to enhance brain uptake. Circular dichroism spectroscopy and molecular modelling suggested that trictide consists of 50% β-sheet structure resulting in an elongated conformation. Contrarily, scrambled trictide has a globular shape. Molecular docking models of trictide support the assumption that outward-directed aromatic amino acids are involved in a binding to tricellulin. In conclusion, trictide is a novel and promising tool for overcoming cellular barriers at bTJs and tTJs with the potential to improve permeation of small molecules. Moreover, after targeting the cellular junctions, a connection has been disclosed between the disturbances of mutual interactions and the resulting redistribution of TJ proteins and functional alterations of the barrier.
