Peptide:N-glycanase (PNGase; also called glycoamidase or N-glycanase) is a deglycosylating enzyme which acts upon asparagine-linked (N-linked) glycans on glycoproteins/glycopeptides. PNGases isolated from plants or bacteria have been used as powerful “tool” reagents for structural and functional studies of N-linked glycans on glycoproteins. Curiously enough, the physiological significance of this enzyme itself has long been undocumented until recently.

It has been known that the cytoplasmic PNGase activity distributes ubiquitously in eukaryotic cells. Past studies have revealed that the deglycosylating enzyme is implicated in proteasomal degradation of misfolded glycoproteins synthesized in the ER. This review will summarize the current knowledge of the structural/functional features of this enzyme. For complete review, see references 1, 2.

In eukaryotes, many proteins synthesized in the cytoplasm are translocated across, or integrated into, the membrane of the endoplasmic reticulum (ER), a central organelle for delivery of these soluble or membrane proteins to their respective destination. As this organelle is also responsible for monitoring proper folding/assembly of proteins, those with abnormal structure arising from mutations or metabolic damage can be eliminated. These proofreading and elimination system (“quality control system”) ensures cells to avoid the intracellular accumulation of nonfunctional proteins. In this system, proteins that fail to fold productively are retained in the ER by various lumenal chaperones, assisting their folding and appropriate subunit assembly. However, if some proteins maintain undesired structure despite the effort by chaperone system, they are destined for destruction by a mechanism known as an “ER-associated degradation” (ERAD)³. ERAD involves three successive steps: (I) sensing of aberrant proteins in the ER, (II) the retrotranslocation of substrates into the cytosol, and (III) degradation of them by ubiquitin-proteasome system ⁴. It has also been suggested that there is an evolutionally conserved system specifically dedicated for N-linked glycoproteins, named glycoprotein ERAD (GERAD) ⁵. Understanding the detail of ERAD/GERAD system is significant from not only for our understanding of this basic cellular mechanism but also for the medical application regarding the treatment of various human inherited or acquired disorders caused by the abnormality of this surveillance system.

The cytoplasmic PNGase activity was first suggested to be involved in the ERAD machinery by Wiertz et al. ⁶. Since then the ubiquitous enzyme conserved from yeast to human has been shown to be utilized in GERAD process ¹. The deglycosylation reaction must achieve an efficient degradation of N-glycosylated proteins, since the bulky modification of amino acid side chain such as N-glycosylation limits the substrate access into the proteolytic chamber, i.e. inside the cylinder structure of the 20S proteasome. However, at least in budding yeast, the degradation of misfolded glycoproteins, though delayed, can be completed without the presence of this enzyme ⁷. This result clearly demonstrates the existence of PNGase-independent degradation for misfolded glycoproteins. Nevertheless, considering the current molecular model of the 20S proteasome, it seems unlikely that the efficient two-way traffic of N-glycosylated protein in and out of the central cavity of the 20S proteasome can occur with the bulky N-linked glycan attached to the protein/peptide throughout the process ⁸. Moreover, gene analysis as well as biochemical studies have suggested that there is no redundancy in this cytosolic enzyme ^{7, 8}, not as in the case with ubiquitin-ligases. It remains unclear how the proteasome achieves the degradation of misfolded glycoproteins without the help of PNGase-catalyzed deglycosylation.

Fig. 1 Schematic representation of enzyme reaction by transglutaminase (upper) and peptide:N-glycanase (lower)
While the transglutaminase catalyzes the formation of amide bond between side chains of Glu and Lys residues, PNGase catalyzes the hydrolysis of glycosylamide bond, forming a free oligosaccharide retaining di-N-acetylchitobioase structure at its reducing terminus as well as deglycosylated protein/peptide in which glycosylated Asn residue is converted to Asp.

Fig. 2 Hypothetical model for degradation of a misfolded glycoprotein; cytosolic events
A glycoprotein that fails to acquire the correct structure is extracted from the ER using the novel protein channel, called dislocon. In the cytosol, it is recognized by a glycoprotein-specific ubiquitin ligase, SCF^Fbx2 complex, and ubiquitinated ²³. PNGase then removes the N-linked glycan, and the polyubiquitin chain was removed by Rpn11 protein, a 19S proteasome subunit ^{25, 26}, prior to the proteasomal degradation of protein.