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

Abstract

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.PNGase (Ngly1) | ENGase | protein aggregates | glycoprotein | ERAD

Non-lysosomal Degradation of Singly Phosphorylated Oligosaccharides Initiated by the Action of a Cytosolic Endo-β-N-acetylglucosaminidase

“…These results clearly show that the POSs were mainly monophosphorylated, consistent with the predictions made by previous biochemical studies (10,11,14). Man␣1,2Man␣1,3(Man␣1,6)Man␤1, 4GlcNAc␤1,4GlcNAc-P-Because ENGase catalyzes the hydrolysis of ␤1,4-linkage of the N,NЈ-diacetylchitobiose core of protein-bound (24) and unbound (19,21) forms of high mannosetype glycans in the cytosol of mammalian cells, we speculated that POSs, which are exclusively localized in the cytosol (10, 13, 14), may also serve as physiological substrates for ENGase. To test this hypothesis, the amounts and glycan structures of the POSs were compared between wild-type and Engase Ϫ/Ϫ MEFs.…”

Lethality of mice bearing a knockout of the Ngly1-gene is partially rescued by the additional deletion of the Engase gene

“…As shown in Fig 1D, the loss of Ngly1 in MEF cells from Ngly1 −/− mice was confirmed. In the case of ENGase, we confirmed the loss of the ENGase activity through an activity assay in a previous study [27] as well as by conducting a detailed structural analysis of the free oligosaccharides (fOSs) in the cytoplasm of MEF cells [28]. The Ngly1 heterozygous ( Ngly1 −/+ ) mice were fertile and did not show any obviously recognizable phenotypes.…”

“…To this end, we used mice (C57BL/6 background mice) that had been generated in a previous study, in which the Ngly1 and Engase genes had been knocked out [27]. The knockout constructs of Ngly1 and Engase are shown in Fig 1B and 1C, respectively.…”

“…According to our previous cell-based study using an ERAD model substrate [27], we hypothesized that ENGase could have the ability to function as a deglycosylating enzyme and that its deletion could rescue the defects caused by the lack of Ngly1 . In this study, we attempted to verify the effect of the additional Engase deletion of Ngly1 −/− in mice at an individual level.…”

Repurposing of Proton Pump Inhibitors as first identified small molecule inhibitors of endo -β- N -acetylglucosaminidase (ENGase) for the treatment of NGLY1 deficiency, a rare genetic disease

“…Interestingly, this ERAD dysregulation could be reversed in cells by an additional ENGASE gene knockout. 4 Furthermore, the most recent mouse model showed that the ENGASE gene deletion could partially rescue the lethality of Ngly1-deficient mice and mitigate the phenotypes. 5 Therefore, ENGase is proposed as a promising drug target for treating human NGLY1 deficiency (Scheme 1).…”