PNGase F

Related Reading

Introduction

Cellular metabolism involves not only small molecules, but also the biosynthesis and catabolism of macromolecules such as proteins, glycans and lipids. Compared with the biosynthesis of macromolecules, our understanding of their catabolism is limited. For example, N-glycosylation is one of the most common protein modifications that occur in all domains of life (eukaryotes, bacteria, or archaea). Although the biosynthetic pathway of N-glycosylation has been well elucidated in mammals and yeast, even in this "post-genome" era, many issues related to their catabolism still need to be clarified. It is well known that the degradation of N-glycoprotein mainly occurs in lysosomes, but the existence of "non-lysosomal" degradation pathways has also been discovered recently. Cytoplasmic PNGase (peptide: N-glycanase; NGLY1/Ngly1 in human/mice) is a conservative deglycosylating enzyme that participates in the non-lysosomal degradation of N-glycoprotein. PNGase releases intact N-glycans from glycoproteins and is responsible for removing misfolded glycoproteins from the cytoplasm. Current research has found that NGLY1-deficiency is a human genetic disorder, which clearly shows the importance of this enzyme for the normal development of mammals.

Figure 1. The involvement of cytoplasmic PNGase in ER-associated degradation (Suzuki, T. 2015)

The cytoplasmic PNGase - distribution and enzymatic characteristics

PNGase catalyzes the hydrolysis of the amide bond between the innermost N-acetylglucosamine (GlcNAc) and an Asn residue on an N-glycoprotein, generating a de-N-glycosylated protein, and a 1-amino-GlcNAc-containing free oligosaccharide. Then at physiological pH, ammonia is spontaneously released from the reducing end, and a free oligosaccharide with an N,N'-diacetylchitobiose is generated at the reducing end.

Figure 2. Reaction scheme for peptide: N-glycanase (Ngly1) (Suzuki, T.; et al. 2016)

Cytoplasmic PNGase activity in mammals was first reported in several cultured cells. This enzyme is different from other PNGases from plants (glycoamidase/PNGase A) or bacteria (N-glycanase/PNGase F) in several enzymatic properties, for example, it requires a reducing agent for activity, and a neutral pH for optimal activity. In addition, Ngly1 has intrinsic carbohydrate-binding properties. The catalytic core of PNGase contains a GlcNAc2-binding site, and there is a PAW domain that serves as a carbohydrate-binding domain for high mannose-type glycans. On the other hand, the N-terminal PUB domain has been shown to be serve as a p97 binding module. The gene encoding cytoplasmic PNGase was first found in budding yeast, Saccharomyces cerevisiaeand gene orthologues have since been found in wide variety of eukaryotes including mammals. The tissue distribution study of PNGase in mouse cytoplasm showed that Ngly1 mRNA was widely present in all tissues examined, with the highest level in the testis. No such activity was found in the sera, indicating that the enzyme was not secreted. Among the organs tested, the total activity of the liver was the highest, while the thymus showed the highest specific activity.

Figure 3. Schematic representation of the domain structure of PNGase (Ngly1) from human, mouse, yeast, and fungi (Suzuki, T.; et al.2016)

NGLY1-deficiency

In 2012, a human genetic disorder involving mutations in the NGLY1 gene locus was identified through an exome analysis. The clinical features of 12 patients have been reported, including global developmental delay, movement disorder, hypotonia, and the absence of tears. These studies have clearly demonstrated the importance of cytoplasmic PNGase. However, the details of the molecular mechanisms leading to the pathology of NGLY1 deficiency remain to be studied. Among NGLY1-deficiency patients, a nonsense mutation (c.1201A>T (p.R401X)) is the most frequently detected allele. This p.R401X allele has been observed in 2 of the 8598 chromosomes of European ancestry reported in the Exome Variant Server resource. Based on this, some people speculate that compared with patients bearing other alleles, homozygous mutations associated with this allele will show more severe phenotypes. We obviously need further research to explain how this mutation can cause more severe symptoms. All patients reported to date are of Caucasian ethnic origin, which means that the frequency of this genetic disorder may vary between ethnic groups.