A strategy for cell-based multiplex diagnostics of Myasthenia gravis and autoimmune encephalitis by modifying the subcellular localization of cell membrane autoantigens

Many autoimmune diseases are characterized by autoantibodies directed against cell membrane proteins. We were intrigued to develop a strategy for targeting individual cell membrane proteins to various subcellular compartments as a prerequisite for their simultaneous immunofluorescence detection. We first employed GFP and RFP reporters that were equipped with defined intracellular localization signals. Expressing these protein reporters in HEp-2 cells we found by using fluorescence microscopy that protein localization in cytoplasm or at mitochondria can be clearly discriminated from localization at Golgi, ER or lysosomes. We then tested for muscle-specific kinase, a relevant cell membrane autoantigen in Myasthenia gravis, and NMDA receptor which is relevant for autoimmune encephalitis, whether these autoantigens can be localized to the same intracellular compartments. To this end, we successfully targeted muscle-specific kinase to Golgi apparatus, mitochondria and cytoplasm. We found that its Golgi localization can be clearly distinguished from its natural cell membrane localization. The same we found for Golgi-localized NMDA receptor 1. Interestingly, cell membrane proteins kept at the Golgi system accumulated in higher amounts than their wild-type counterparts. The obtained results are the basis for the further development of multiplex assays for the immunofluorescence diagnostics of Myasthenia gravis and autoimmune encephalitis.