Construction of a detection method for endo-β- N -acetylglucosaminidase activity using engineered split inteins and glycopeptides

Abstract Cytosolic peptide:N-glycanase (Ngly1 in mammals) and endo-β-N-acetylglucosaminidase (ENGase) are deglycosylating enzymes. Detection methods for the Ngly1 and ENGase activities in the cytosol are necessary for understanding the deglycosylation of the N-glycoproteins during endoplasmic reticulum-associated degradation. We previously reported the development of a detection method for Ngly1 using an engineered split intein that self-catalyzes protein trans-splicing (PTS) and NanoLuc luciferase. Here, we report a detection method for the ENGase activity using a PTS-based approach. By employing bioluminescence resonance energy transfer (BRET), we successfully detected the endogenous levels of Ngly1 and ENGase. The PTS-based BRET assay can be used to characterize the enzyme functions in the cell cytosol.