The cytoplasmic peptide:N-glycanase (NGLY1) - Structure, expression and cellular functions

Abstract

NGLY1/Ngly1 is a cytosolic peptide:N-glycanase, i.e. de-N-glycosylating enzyme acting on N-glycoproteins in mammals, generating free, unconjugated N-glycans and deglycosylated peptides in which the N-glycosylated asparagine residues are converted to aspartates. This enzyme is known to be involved in the quality control system for the newly synthesized glycoproteins in the endoplasmic reticulum (ER). In this system, misfolded (glyco)proteins are retrotranslocated to the cytosol, where the 26S proteasomes play a central role in degrading the proteins: a process referred to as ER-associated degradation or ERAD in short. PNGase-mediated deglycosylation is believed to facilitate the efficient degradation of some misfolded glycoproteins. Human patients harboring mutations of NGLY1 gene (NGLY1-deficiency) have recently been discovered, clearly indicating the functional importance of this enzyme. This review summarizes the current state of our knowledge on NGLY1 and its gene product in mammalian cells.

Keywords: Cytosol; ER associated degradation; Endo-beta-N-acetylglucosaminidase; Mammalian cells; NGLY1; Peptide:N-glycanase.

Figure 1

Reaction scheme for peptide:N-glycanase (Ngly1)

Figure 2. Schematic representation of the domain structure of PNGase (Ngly1) from human, mouse, yeast, and fungi

Gray bar in the scheme is a putative zinc-binding site (Lee et al., 2005). PUB: a domain present in PNGase and UBA- or UBX-containing proteins (p97 binding motif), TG: transglutaminase domain (catalytic domain), PAW: a domain present in PNGase and other worm protein domains (high mannose-type glycan-binding domain). These domains were based on SMART (Simple Molecular Architecture Research Tool) program (http://smart.embl-heidelberg.de/). X indicates the sites for mutations found in NGLY1-deficeincy patients.

Figure 3. Principle of Ngly1 activity assay using a split Venus system (Grotzke et al., 2013)

In this system, the C-terminal Venus fragment is expressed in the cytosol, while the N-terminus fragment with an N-glycosylation site is expressed in the ER to undergo N-glycosylation. The N-terminal fragment is recognized by the ER quality control system and is retrotranslocated into the cytosol. The N- and C-terminal fragment forms a complex, and only when the N-glycan on N-terminal fragment is removed by Ngly1, does the fluorescence increase. One can therefore measure the in vivo activity of Ngly1 through the fluorescence of the Venus protein.

Figure 4. Scheme of N-GlcNAc hypothesis (Huang et al., 2015)

Under normal conditions, Ngly1 preferentially acts on misfolded glycoproteins during their proteasomal degradation, while ENGase acts on free oligosaccharides for non-lysosomal (cytoplasmic) glycan catabolism (Suzuki, 2007; Suzuki and Harada, 2014; Harada et al., 2015a). In the case of the absence of Ngly1, however, ENGase stochastically acts on misfolded glycoproteins. Reaction of ENGase with misfolded glycoproteins results in the formation of N-GlcNAc proteins. The formation of excessive amounts of N-GlcNAc proteins in the cytosol may cause (1) the formation of toxic protein aggregates and/or (2) impairment of proper O-GlcNAc signaling in the cytosol/nucleus.