Discovery of new protein partners of anterior gradient 2 (AGR2) from cell-cell adhesion pathway using genetically-encoded photo-crosslinker DiZPK

Anterior gradient 2 (AGR2) has recently been reported as a tumor biomarker in various cancers including breast, prostate, and lung cancers. Thus, there have been great efforts to unveil the roles of AGR2 as an extracellular signaling molecule and intracellular endoplasmic reticulum (ER) chaperone. However, it remains challenging to capture the whole protein interactome of AGR2 because both weak and transient interacting partners tend to be washed off during purification. In order to capture weak and transient interacting protein partners, a genetically-encoded photo-crosslinker, DiZPK (3-(3-methyl-3H-diazirine-3-yl)-propaminocarbonyl-N^ε-l-lysine), was introduced at the essential domains of AGR2. Photo-crosslinking reaction of the recombinant mutant AGR2^Q85DiZPK was subsequently initiated by UV light (365 nm) in MCF-7 breast cancer cell lysate. After purification and further analysis by LC-MS/MS, we identified a number of AGR2 partner proteins that are involved in the cell–cell adhesion pathway. Namely, they are desmosomes-related proteins including γ-catenin, desmocollin-1 (DSC-1), desmoglein-1 (DSG-1), desmoplakin (DSP), and plakophillin-1 (PKP-1). Further protein–protein interaction studies, including immunofluorescence and molecular dynamic simulation analysis, especially using γ-catenin as an example, corroborated with the observation that AGR2 directly interacts with γ-catenin. We believe the identification of these new protein partners would help to unveil how AGR2 renders its roles in cell adhesion, migration, and ultimately metastasis of cancers, and these proteins are worthy of future in-depth study for the elucidation of AGR2-mediated cellular functions.