Endo-β-N-acetylglucosaminidase forms N-GlcNAc protein aggregates during ER-associated degradation in Ngly1-defective cells

Significance In the endoplasmic reticulum (ER), N-glycans on glycoproteins play important roles in dictating the folding status of proteins by a sophisticated N-glycan–dependent protein quality control machinery. In this study we identified the dysregulation of ER-associated degradation (ERAD) in cells that were defective in the cytosolic deglycosylating enzyme, Ngly1. ERAD dysregulation was caused by an unexpected deglycosylating activity of endo-β- N -acetylglucosaminidase, another cytosolic deglycosylation enzyme, and this action resulted in the intracellular formation of protein aggregates. Our results clearly point to the critical role of N-glycans even in cytosolic events of the ERAD process by controlling the conformation/solubility of proteins. This study may also provide a potential mechanism for explaining the pathology of a human genetic disorder caused by mutations in the NGLY1 gene. The cytoplasmic peptide:N-glycanase (PNGase; Ngly1 in mice) is a deglycosylating enzyme involved in the endoplasmic reticulum (ER)-associated degradation (ERAD) process. The precise role of Ngly1 in the ERAD process, however, remains unclear in mammals. The findings reported herein, using mouse embryonic fibroblast (MEF) cells, that the ablation of Ngly1 causes dysregulation of the ERAD process. Interestingly, not only delayed degradation but also the deglycosylation of a misfolded glycoprotein was observed in Ngly1 ⁻/⁻ MEF cells. The unconventional deglycosylation reaction was found to be catalyzed by the cytosolic endo-β- N -acetylglucosaminidase (ENGase), generating aggregation-prone N- GlcNAc proteins. The ERAD dysregulation in cells lacking Ngly1 was restored by the additional knockout of ENGase gene. Thus, our study underscores the functional importance of Ngly1 in the ERAD process and provides a potential mechanism underlying the phenotypic consequences of a newly emerging genetic disorder caused by mutation of the human NGLY1 gene.