Figure 4.

Junction opener 1 (JO-1). (A) Schematic structure of JO-1 The Ad serotype 3 fiber knob domain and one fiber shaft motif was fused through a flexible linker to a homodimerizing K-coil domain (182). The protein is self-dimerizing and can be purified by His-Ni-NTA affinity chromatography. (B) Mode of action. JO-1 binds with picomolar avidity to DSG2. In epithelial cancer cells, DSG2 is overexpressed and exposed on the cell surface with preferential localization to desmosomes. JO-1 binding to DSG2 triggers cleavage of DSG2 dimers between neighboring cells and the transient activation of EMT pathways. This triggers junction opening and relocalization of target receptors that are often trapped in epithelial junctions. Junction opening allows for access of drugs (for example mAbs) to their target receptors. (C) Transmission electron microscopy of junctional areas of T84 cells. Cells were either treated with PBS (upper panel) or JO-1 (lower panel) for 1 h on ice, washed, and then incubated for 15 min at 37°C. At this time, the electron-dense dye ruthenium red (Ru) (1) was added together with the fixative. If tight junctions (above the desmosomes) are closed, the dye only stains the apical membrane (black line). If tight junctions are open, the dye penetrates between the cells and stains the baso-lateral membrane. JO-1 also mediates the partial dissociation of desmosomes (D). The scale bar is 0.5 μm.