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Published on: May 13, 2020
Development of a fluorescence and quencher-based FRET assay for detection of endogenous peptide:N-glycanase/NGLY1
Hiroto Hirayama1, Yuriko Tachida1, Reiko Fujinawa1
1Glycometabolic Biochemistry Laboratory, RIKEN Cluster for Pioneering Research (CPR), Riken, Wako Saitama, Japan; Takeda-CiRA Joint Program (T-CiRA), Fujisawa, Kanagawa, Japan.
Abstract:
Cytosolic peptide:N-glycanase (PNGase/NGLY1 in mammals) catalyzes deglycosylation of N-glycans on glycoproteins. A genetic disorder caused by mutations in the NGLY1 gene leads to NGLY1 deficiency with symptoms including motor deficits and neurological problems. Effective therapies have not been established, though, a recent study used the administration of an adeno-associated viral vector expressing human NGLY1 to dramatically rescue motor functions in young Ngly1-/- rats. Thus, early therapeutic intervention may improve symptoms arising from central nervous system dysfunction, and assay methods for measuring NGLY1 activity in biological samples are critical for early diagnostics. In this study, we established an assay system for plate-based detection of endogenous NGLY1 activity using a FRET-based probe. Using this method, we revealed significant changes in NGLY1 activity in rat brains during aging. This novel assay offers reliable disease diagnostics and provides valuable insights into the regulation of PNGase/NGLY1 activity in diverse organisms under different stress conditions.
