Background

Cytosolic peptide: N-glycanase (PNGase; NGLY1 in mammals) is an amidase that catalyzes the removal of N-glycans from the consensus sequences (Asn-Xaa-Ser/Thr, where Xaa represents any amino acid except proline) of glycoproteins, converting N-glycosylated Asn into Asp residues through deglycosylation [1]. Cytosolic PNGase also plays a role in the quality control system for newly synthesized glycoproteins [2].

To explore the biochemical properties of this enzyme, ¹⁴C-labeled glycopeptides, such as pentapeptides that carry asialoglycans derived from fetuin, have been commonly used as substrates for measuring PNGase activity [3]. Enzyme activity was measured based on the radioactivity of the reaction products, which were separated by paper chromatography or paper electrophoresis. However, the preparation of radioisotope-labeled glycopeptides and the use of radioactive molecules make it difficult to perform this assay in standard laboratory settings, especially under the tight regulations for the use of radioactive isotopes in Japan. Although non-radioisotope-based assays have been developed (e.g., assays using S-alkylated RNase [4] or fluorescent-labeled glycopeptides [5]), they are incompatible with crude enzyme sources due to the degradation of reaction products by contaminating endogenous proteases. Therefore, there exists a need to develop an alternative, easy-to-handle assay method.

An autosomal recessive disorder linked to NGLY1, known as NGLY1 deficiency or congenital disorder of deglycosylation (NGLY-CDDG) [OMIM: 615273], was first reported in 2012 [6]. Since then, more than 100 patients have been identified worldwide, including in Europe, America, Australia, India, China, and Japan [7,8]. The disease exhibits a broad spectrum of symptoms, including global developmental delay and/or intellectual disability, abnormal EEG, seizures, movement disorders, hypolacrima or alacrima, and liver dysfunction [8–13]. Unfortunately, there are no effective treatments; however, recent studies have demonstrated that administering an adeno-associated viral vector serotype 9 carrying the human NGLY1 gene to Ngly1-deficient model rats aged 3 or 5 to 7 weeks through intracerebroventricular injection significantly improved their motor function defects [14–16]. Considering the importance of the therapeutic time window for gene therapies, early intervention may be crucial to alleviate the various symptoms caused by the dysfunction of the central nervous system in this disease. Therefore, there exists an urgent need for methods to enable the early diagnosis of NGLY1 deficiency by measuring endogenous NGLY1 activity in specimens from potential disease candidates.

A method for measuring endogenous NGLY1 activity using 5-carboxyfluorescein-labeled glycosylated cyclo-heptapeptide (5FAM-GCP) has been established previously [3,17,18]. This approach enables detecting endogenous PNGase/NGLY1 activities from various enzyme sources without the proteolytic degradation of reaction products during incubation with crude enzyme preparations. However, it requires HPLC for the separation and detection of products, which is often unavailable in standard clinical laboratories. Hence, it is crucial to develop a facile, sensitive probe for enzyme assay similar to MM3D, a fluorescence and quencher-based FRET probe designed for detecting ENGase activity [19]. We recently developed a novel FRET-based GCP probe (fGCP) consisting of a glycan modified with a fluorophore-labeled bisected-GlcNAc [aminomethylcoumarin acetate-labeled GlcNAc (AMCA-GlcNAc)] and a cyclo-heptapeptide modified with a quencher, 4-((4-(dimethylamino)phenyl)azo)benzoic acid (Dabcyl) (Figure 1) [20]. This method allows the detection of endogenous NGLY1 activity in various enzyme sources via fluorescence on multiarray plates. Our novel assay method could provide a reliable diagnostic tool and valuable insights into the regulation of PNGase/NGLY1 activities in various organisms.

Figure 1. Structure of 5-carboxyfluorescein- and dabcyl-labeled glycosylated cyclo-heptapeptide (fGCP) and deglycosylation reaction catalyzed by PNGase [20]. The bisected-GlcNAc and lysine residues on the glycosylated cyclo-heptapeptide were labeled with a fluorophore, aminomethylcoumarin acetate (AMCA), and a quencher, 4-((4-(dimethylamino)phenyl)azo)benzoic acid (Dabcyl), respectively. The right bracket illustrates a schematic of fGCP and deglycosylation reaction catalyzed by PNGase/NGLY1.