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. 2024 Aug 29;34(11):cwae067. doi: 10.1093/glycob/cwae067

Development of new NGLY1 assay systems – toward developing an early screening method for NGLY1 deficiency

Hiroto Hirayama 1,✉, Haruhiko Fujihira 2, Tadashi Suzuki 3,✉
PMCID: PMC11442003  PMID: 39206713

Abstract

Cytosolic peptide: N-glycanase (PNGase/NGLY1 in mammals) is an amidase (EC:3.5.1.52) widely conserved in eukaryotes. It catalyzes the removal of N-glycans on glycoproteins, converting N-glycosylated Asn into Asp residues. This enzyme also plays a role in the quality control system for nascent glycoproteins. Since the identification of a patient with an autosomal recessive genetic disorder caused by NGLY1 gene dysfunction, known as NGLY1 deficiency or NGLY1 congenital disorder of deglycosylation (OMIM: 615273), in 2012, more than 100 cases have been reported worldwide. NGLY1 deficiency is characterized by a wide array of symptoms, such as global mental delay, intellectual disability, abnormal electroencephalography findings, seizure, movement disorder, hypolacrima or alacrima, and liver dysfunction. Unfortunately, no effective therapeutic treatments for this disease have been established. However, administration of adeno-associated virus 9 (AAV9) vector harboring human NGLY1 gene to an NGLY1-deficient rat model (Ngly1−/− rat) by intracerebroventricular injection was found to drastically improve motor function defects. This observation indicated that early therapeutic intervention could alleviate various symptoms originating from central nervous system dysfunction in this disease. Therefore, there is a keen interest in the development of facile diagnostic methods for NGLY1 deficiency. This review summarizes the history of assay development for PNGase/NGLY1 activity, as well as the recent progress in the development of novel plate-based assay systems for NGLY1, and also discusses future perspectives.

Keywords: ELISA, FRET, luciferase assay, NGLY1, Peptide: N-glycanase

Introduction

Cytosolic peptide:N-glycanase (PNGase/NGLY1 in mammals) is an amidase (EC:3.5.1.52) that is widely conserved in eukaryotes. It catalyzes the removal of N-glycans on the consensus sequences (Asn-Xaa-Ser/Thr; Xaa is any amino acid except Pro) of glycoproteins, converting N-glycosylated Asn into Asp residues through deglycosylation (Fig. 1A). Moreover, this enzyme plays a role in the quality control system for nascent glycoproteins (Suzuki et al. 2002a). In particular, most nascent proteins in the endoplasmic reticulum (ER) are N-glycosylated by oligosaccharyltransferase-dependent glycan transfer from donor substrate (i.e. lipid-linked oligosaccharides in the ER membrane) (Harada Y. et al. 2015; Kornfeld and Kornfeld 1985; Lehle et al. 2006). These immature glycoproteins are then assisted by ER-resident molecular chaperones (such as Bip, calnexin/calreticulin, and protein disulfide isomerases) to achieve correct folding, allowing their transport to their appropriate destinations. Conversely, some fractions of nascent proteins that fail to achieve correct folding are recognized by the ER quality control system and are retrotranslocated to the cytosol. The misfolded glycoproteins thus delivered to the cytosol are deglycosylated by PNGase/NGLY1 when being degraded by the ubiquitin–proteasome system (Xu and Ng 2015; Ninagawa et al. 2021; Suzuki et al. 2022). It has been proposed that removing bulky N-glycans from misfolded proteins can facilitate their efficient degradation by the proteasome (Hirayama et al. 2015).

Fig. 1.

Fig. 1

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Enzyme reaction of peptide: N-glycanase. A) The reaction catalyzed by PNGase/NGLY1 can be divided into two steps. Molecules and groups generated in each reaction are shown in bold red text. X represents high mannose, hybrid, and complex-type N-glycans. Green circles and blue squares represent mannose and GlcNAc, respectively. B) Dr. Noriko Takahashi, a pioneering Japanese female scientist, first reported PNGase activity in almond emulsin.

Since the identification of a patient with an autosomal recessive genetic disorder caused by NGLY1 gene dysfunction, known as NGLY1 deficiency or NGLY1 congenital disorder of deglycosylation (NGLY1-CDDG) [OMIM: 615273], in the US in 2012 (Need et al. 2012), more than 100 cases have been reported worldwide, including in Europe, North and South America, Australia, India, China, and Japan (Pandey and Jafar-Nejad 2022; Sonoda et al. 2023). This disorder manifests a broad spectrum of symptoms, including global developmental delay and/or intellectual disability, abnormal electroencephalography (EEG) findings, seizures, movement disorders, hypolacrima or alacrima, and liver dysfunction (Need et al. 2012; Enns et al. 2014; Lam et al. 2017; van Keulen et al. 2019; Ge et al. 2020; Lipari Pinto et al. 2020; Kariminejad et al. 2021; Sonoda et al. 2023). Although some aspects of the molecular pathology of NGLY1-CDDG remain unclear, recent studies have disclosed the versatile functions of NGLY1 in various biological processes, which are well-summarized in recent review articles (Pandey et al. 2022; Suzuki and Fujihira 2024). These functions include not only facilitating the degradation of misfolded proteins but also activating the glycosylated transcription factor, nuclear factor erythroid 2-like 1 (NFE2L1), by editing its N-glycosylated Asn into Asp (Lehrbach and Ruvkun 2016; Tomlin et al. 2017; Lehrbach et al. 2019; Lehrbach 2022; Tachida et al. 2023), thereby aiding in the retrotranslocation of misfolded proteins from the ER to the cytosol (Galeone et al. 2020), maintaining mitochondrial homeostasis (Kong et al. 2018; Yang et al. 2018), innate immunity related to the cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes pathway (Yang et al. 2018), AMPK signaling pathway (Han et al. 2020), BMP signaling (Galeone et al. 2017; Galeone et al. 2020), preventing the accumulation of ubiquitinated N-glycoproteins (Yoshida et al. 2021), and supporting the maturation of neuronal cells during early brain development (Lin et al. 2022; Abbott et al. 2023).

Unfortunately, no effective therapeutic treatments for this disease have been established. Nevertheless, it is presumed that enzyme replacement and/or gene therapies could be effective for patients because (i) almost all NGLY1 mutation alleles derived from patients lack enzyme activity (Hirayama and Suzuki 2022) and (ii) most symptoms appear to be associated with dysfunction of the central nervous system (CNS). Recent studies have demonstrated that administration of an adeno-associated viral vector serotype 9 (AAV9) harboring the human NGLY1 gene to 3 or 5–7 week-old NGLY1-deficient model rats (Ngly1−/− rats) by intracerebroventricular injection drastically improved motor function defects in the rats (Asahina et al. 2021; Fujihira et al. 2022; Zhu et al. 2022). On the basis of these findings, the FDA has granted investigational new drug clearance for the intracerebroventricular administration of AAV9 gene therapy, GS-100, to patients with NGLY1 deficiency (ClinicalTrials.gov ID: NCT06199531). Considering the effective therapeutic time windows for gene therapies, early therapeutic intervention could be critical to alleviate the various symptoms caused by CNS dysfunction in this disease. Therefore, there is a keen interest in the development of facile diagnostic methods for NGLY1 deficiency. Two different approaches are available for developing diagnostic methods for this disease, viz., (i) direct measurement of endogenous NGLY1 activity in patient specimens (e.g. fibroblasts and peripheral blood mononuclear cells (PBMCs)) and (ii) detection of potential NGLY1-specific biomarkers, such as Neu5Ac-Hex-GlcNAc-Asn (Hall et al. 2018) and GlcNAc-Asn (Haijes et al. 2019; Asahina et al. 2021; Mueller et al. 2022). In this regard, it is remarkable that novel facile NGLY1/PNGase assay methods, compatible with the 96-well format, have been reported using extracts from cells or tissues as an enzyme source (Takahashi et al. 2022; Fujihira et al. 2024; Hirayama et al. 2024). This review summarizes the history of assay development for PNGase/NGLY1, as well as newly developed assay systems, and discusses future perspectives.

Early history of PNGase/NGLY1 assay system

Historically, Dr. Noriko Takahashi, a pioneering Japanese female scientist, first reported PNGase activity in almond emulsin (Takahashi 1977) (Fig. 1B). She conducted meticulous biochemical experiments to detect liberated glycans as well as deglycosylated peptides and found that almond PNGase catalyzed deglycosylation and the conversion of glycosylated Asn into Asp, thereby demonstrating that this enzyme is actually an amidase (i.e. cleaving an amide bond), not a glycosidase. As summarized in Table 1, various assays for PNGase activity have been established since the report of PNGase presence in nature. In the early era of PNGase research, it was essential to confirm two different reaction products to unequivocally define PNGase activity, viz., (i) liberated N-glycans from glycopeptide/protein substrates and (ii) peptides possessing Asp residues converted from N-glcosylated Asn, through laborious biochemical experiments. Various methods were applied for identifying the liberated N-glycans from the substrates, such as coloring reaction using the phenol/sulfuric acid method (Plummer et al. 1984; Taga et al. 1984; Chu 1986), quantification of 1-NH2 group in the liberated N-glycans (Takahashi and Nishibe 1981; Taga et al. 1984), composition analysis of released sugars (Plummer and Tarentino 1981; Taga et al. 1984; Tarentino et al. 1985; Chu 1986; Seko et al. 1991; Suzuki et al. 1993; Suzuki et al. 1994; Seko et al. 1999), and quantification of the glycans by labeling the reducing end sugar with NaB3H4 (Takahashi and Nishibe 1981; Hirani et al. 1987; Seko et al. 1991; Seko et al. 1999). More recently, a colorimetric detection method for the reducing ends of released glycans using a tetrazolium compound (WST1) was also developed (Wang et al. 2019). Furthermore, two major assay methods have been used for detecting deglycosylated peptides, viz., (i) detection and identification of the deglycosylated peptides labeled with radioisotopes (3H or 14C) (Takahashi 1977; Plummer and Tarentino 1981; Plummer et al. 1984; Tarentino et al. 1985; Chu 1986; Seko et al. 1991; Suzuki et al. 1993; Kitajima et al. 1995; Suzuki et al. 1997; Suzuki et al. 1998; Seko et al. 1999; Suzuki et al. 2000) or with fluorescence (Taga et al. 1984) at their N-termini and (ii) quantification of amino acid composition in the deglycosylated peptides using an amino acid analyzer (Plummer and Tarentino 1981; Takahashi and Nishibe 1981; Plummer et al. 1984; Taga et al. 1984; Chu 1986; Seko et al. 1991; Suzuki et al. 1993).

Table 1.

Summary of assays for investigating PNGase/NGLY1 activity.

Substrate for the assay Detection method Enzyme source used in the studies References
Glycopeptides from various glycoproteins (i.e. stem bromelain, ovalbumin, ovotransferrin) Paper electrophoresis Almond emulsin (Takahashi 1977; Takahashi and Nishibe 1981)
Glycopeptide from ovalbumin and IgM Amino acid analyzer Almond emulsin (Plummer and Tarentino 1981)
Bromelain glycopeptide, ovomucoid glycopeptide from turkey, dabsyl-labeled fetuin and ovalbumin peptide Reversed-phase HPLC (detected by UV absorbance) Almond emulsin, Elizabethkingia meningoseptica (Plummer et al. 1984; Taga et al. 1984; Tretter et al. 1991)
Fetuin, transferrin, invertase, ribonuclease B (RNaseB), α-1-acid glycoprotein SDS-PAGE and lectin blot E. meningoseptica (Tarentino et al. 1985; Chu 1986; Hirani et al. 1987; Suzuki 2005; Tanabe et al. 2006; Hosomi et al. 2010; Huang et al. 2015)
Ovomucoid glycopeptide from turkey 1H NMR Almond emulsin (Risley and Van Etten 1985)
Fetuin asialoglycopeptide and hyosophorin Paper electrophoresis/paper chromatography Medaka fish, mouse tissue, and mammalian cultured cells (Seko et al. 1991; Suzuki et al. 1993; Suzuki et al. 1994; Kitajima et al. 1995; Seko et al. 1999)
Nontoxic mutant of ricin A subunit (RTA∆)a SDS-PAGE and immunoblot Mammalian cells and yeast (Tanabe et al. 2006; Hosomi et al. 2010; Masahara-Negishi et al. 2012; Huang et al. 2015; Galeone et al. 2017)
ddVenus FACS or microscopies Culture cell lines (Grotzke et al. 2013)
Horseradish peroxidase, RNaseB Colorimetric assay Recombinant PNGase F (expressed in E. coli) (Wang et al. 2019)
5FAM-glycosylated cyclopeptide (5FAM-GCP) HPLC Cell-free system, fibroblast, blood cells, and culture cell lines (Hirayama et al. 2022)
GlcNAc2-modified split intein fused with NanoLuc NanoLuc luciferase Purified ScPng1, culture cell lines (Takahashi et al. 2022)
FRET-based GCP probe (fGCP) FRET Fibroblast, blood cells, culture cell lines, and rat tissues (Hirayama et al. 2024)
BSA glycopeptide-tag substrate (BSA-Gp) ELISA Human fibroblast and culture cells (Fujihira et al. 2024)
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a

It should be noted that the occurrence of “nonglycosylated form” does not necessarily confirm the deglycosylation by NGLY1 (Huang and Suzuki 2020).

After demonstrating the enzymes as PNGases, two methods were primarily applied to routinely detect PNGase activity. One method involves assays using 14C-labeled glycopeptide prepared from bovine fetuin (14C-labeled asialofetuin) as a substrate. In this system, the substrate was reacted with enzyme sources, separated by paper chromatography or paper electrophoresis, and the reaction product was detected by autoradiography (Hirani et al. 1987; Seko et al. 1991; Suzuki et al. 1993; Suzuki et al. 1994; Kitajima et al. 1995; Suzuki et al. 1997; Suzuki et al. 1998; Seko et al. 1999; Suzuki et al. 2000). One advantage of using fetuin with crude extracts of animal origin as an enzyme source is that its glycan structure (tri (2/4, 2)-antennary complex-type glycans) is resistant to the action of endo-β-N-acetylglucosaminidase (ENGase) (Tachibana et al. 1982), another cytosolic deglycosylating enzyme that acts on N-glycans (Suzuki et al. 2002b). This method enables PNGase researchers to identify PNGase activities in various organisms. The other method uses S-alkylated RNaseB (alk-RNaseB), which has its disulfide bonds reduced and thiol groups alkylated by iodoacetamide, as a substrate (Tarentino et al. 1985; Chu 1986; Hirani et al. 1987; Hirsch et al. 2004; Suzuki 2005). In this assay, after the incubation of alk-RNaseB with the enzyme source, the substrate and product are separated by SDS-PAGE and detected by CBB staining or immunoblotting. Although this assay is semiquantitative, it remains popular for measuring PNGase activity because the product can be easily detected using commonly used techniques (i.e. SDS-PAGE followed by CBB staining or immunoblotting).

To detect intracellular NGLY1 activity in living cells, a reporter fluorescent protein known as deglycosylation-dependent Venus (ddVenus) was also reported (Grotzke et al. 2013). ddVenus consists of an ER-entry signal sequence and a mutant Venus protein, in which the DFT sequence at positions 82–84 is substituted with an N-glycosylation site (NFT). This protein becomes a functional fluorescent protein only when it is initially glycosylated in the ER and subsequently undergoes NGLY1-dependent editing of its N-glycosylated Asn into Asp (N82FT to D82FT). To evaluate the intracellular activity of NGLY1 in patient-derived fibroblasts, these cells were transfected with ddVenus-expressing plasmids. After transfection, the fibroblasts were treated with the proteasome inhibitor MG132 to enhance the fluorescent signal of deglycosylated ddVenus. However, FACS-based population analysis by measuring the fluorescence intensity of ddVenus was required to evaluate NGLY1 activity because of the variable level of fluorescence among cells (Grotzke et al. 2013; He et al. 2015).

Since the first report of the patient with NGLY1 deficiency in 2012, developing quantitative assay methods for measuring endogenous NGLY1 activity in cells/tissues extracts has become a critical need for NGLY1 research. Unfortunately, the previously used methods, such as those involving 14C-labeled asialofetuin peptide or alk-RNaseB, are not suitable for diagnosing this disease. For instance, the preparation of 14C-labeled asialofetuin and the use of radioactive molecules make it challenging to perform this assay in a standard laboratory. Moreover, alk-RNaseB can be susceptible to proteolytic degradation by endogenous peptidases/proteases present in crude enzyme sources (e.g. cell or tissue extracts), thus compromising the precise detection of endogenous PNGase/NGLY1 activity. Therefore, it was imperative to establish quantitative methods applicable for diagnosis. Recently, our research group investigated various chemically synthesized N-glycopeptides and found that 5FAM-labeled glycosylated hepta-cyclopeptides (5FAM-GCP) can be used to measure endogenous NGLY1 activity in crude extracts without the generation of subproducts (i.e. proteolytically degraded deglycosylated peptides by the contaminating protease/peptidase) (Hirayama et al. 2022; Hirayama and Suzuki 2022) (Fig. 2A). This was accomplished by separating the substrate and product by HPLC to confirm that essentially no proteolytic degradation product was detected. Furthermore, endogenous NGLY1 activity in fibroblasts and PBMCs could be quantified using 5FAM-GCP, indicating that the assay using 5FAM-GCP could be a useful tool for evaluating NGLY1 activity in patient-derived cells. Nonetheless, this assay requires product separation and quantitation by HPLC, which is often unavailable in standard clinical laboratories. Therefore, it was further required to develop an HPLC-free NGLY1 assay system.

Fig. 2.

Fig. 2

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Development of 5FAM-GCP assay to FRET-based GCP assay (Hirayama et al. 2022; Hirayama et al. 2024). A) Schematic of assaying NGLY1 activity using 5FAM-GCP. 5FAM-GCP and the reaction were separated and quantified by HPLC. B) Detection of NGLY1 activity using a FRET-based probe. NGLY1 activity was detected by real-time measurement of fluorescence using a plate reader. Green circle and blue square represent mannose and GlcNAc, respectively.

Development of novel microplate-based quantitative assay systems for measuring endogenous NGLY1 activity in cells and tissues

Recent studies have reported three novel, microplate-compatible NGLY1 assays (i.e. FRET-based GCP assay, PTS-based luciferase assay, and ELISA-based BSA-Gp assay) (Table 2) (Takahashi et al. 2022; Fujihira et al. 2024; Hirayama et al. 2024). The details of these three assays will be summarized in the following sections.

Table 2.

Comparison table for the three different plate-based methods for measuring endogenous NGLY1 activity.

FRET-based assay PTS-based split Luc assay ELISA-based assay
Probe used in each assay fGCP NLs-3GN2-IntN and IntCmut3-NLlg BSA-Gp
Availability of the probe Commercially available (GlyTech. Inc) Not for sale Commercially available (GlyTech. Inc)
Number of cells required for one reaction 1–5 × 106 cells 8 × 103 cell/well (HeLa)
1.5 × 104 cells/well (3T3-L1)		 5 × 103 cells
Reaction time (h) 0.5–12 0.5–8 1.0–16
Applied for live imaging Not applicable Possible Not applicable
Detection Fluorescence Luciferase activity (luminescence) Anti-HA, and anti-IgG conjugated with HRP
Background signal Weak Weak Modest
Resistance against ENGase Excellent Excellent Not determined
Resistance against proteases Excellent Potentially problematic Potentially problematic
Reference (Hirayama et al. 2024) (Takahashi et al. 2022) (Fujihira et al. 2024)
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(Pros and cons of each assay are denoted by bold letters with underlining and italic letters with underlining, respectively.)

FRET-based GCP assay for measuring NGLY1 activity

Encouraged by various FRET-based fluorescence-quenching systems, such as a FRET probe for detecting ENGase activity (MANT-M3GN2-DNP) (Ishii et al. 2023) and a FRET-based assay for protease activity (Bickett et al. 1993; Tanskul et al. 2003), a novel FRET-based assay for measuring endogenous NGLY1 activity has been developed by further improving the 5FAM-GCP assay (Hirayama et al. 2024). In the previous 5FAM-GCP assay, not only the NGLY1-dependent product (deglycosylated GCP) but also a byproduct, N-GlcNAc-GCP, generated by endogenous ENGase, was produced when the assay was performed using crude cell extracts (Fig. 2A). The generation of N-GlcNAc-GCP will cause a problem in the development of FRET-based probes because the ENGase-dependent cleavage of N-glycans might also produce FRET-based fluorescence, thereby causing high nonspecific signals. To overcome this problem, the researchers exploited the fact that ENGase generally has narrower substrate specificities than NGLY1/PNGase (Tachibana et al. 1982; Maley et al. 1989; Suzuki et al. 1994; Tarentino and Plummer Jr 1994; Suzuki et al. 1995; Murakami et al. 2013; Hirayama et al. 2024). In the recent study, the researchers constructed a FRET-based GCP probe (fGCP) that exhibited strong resistance to the action of ENGase (Hirayama et al. 2024). fGCP consists of a glyco-cyclopeptide, including a lysine residue derivatized with a quencher, dabcyl, and an agalacto-biantenna glycan labeled with a fluorophore, AMCA, via bisected GlcNAz (Fig. 2B). Although a relatively larger amount of cells was required for this assay (1–5 × 106 cells), it enabled microplate-based, real-time measurement of endogenous NGLY1 activity in various enzyme sources, including cell lines, rodent tissues, and PBMCs (Hirayama et al. 2024). The researchers also demonstrated that the fGCP assay could quantitatively measure NGLY1 activity in NGLY1-deficient, patient-derived fibroblasts, in which NGLY1 activity was severely compromised (Hirayama et al. 2024). These findings indicate that fGCP is a powerful tool for characterizing endogenous NGLY1 activities in patient-derived cells.

Protein trans-splicing-based luciferase assay for measuring NGLY1 activity

In a separate study, (Takahashi et al. 2022) demonstrated a novel protein trans-splicing (PTS)-based luciferase assay for measuring NGLY1 activity (Takahashi et al. 2022). They observed that intein is responsible for self-catalytic PTS, wherein an internal protein segment (intein) can excise itself and connect the remaining proteins, the exteins, with a peptide bond (Skretas and Wood 2005; Sakamoto et al. 2013; Takahashi and Saito 2016; Gramespacher et al. 2017). The authors previously demonstrated that PTS is also mediated by a pair of split inteins, IntN and IntCmut3, derived from Nostoc punctiforme (Npu) DnaE (Kawase et al. 2021) (Fig. 3A). They also showed that the interaction of a pair of fusion proteins, the C-terminal region of NanoLuc fused with IntN (NLs-IntN) and IntCmut3 fused with a NanoLuc fragment, 11S (IntCm3-NLlg) (Fig. 3B), promoted PTS and generated active NanoLuc (Fig. 3C) (Takahashi et al. 2022).

Fig. 3.

Fig. 3

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Measurement of NGLY1 activity by PTS-based luciferase assay (Takahashi et al. 2022). A) Schematic of PTS using engineered split inteins. Asp5 in IntN and Arg67 in IntCmut3 enhance their interaction. B) Amino acid sequences and the structure of NLS-IntN and IntCmut3-NLlg. 11S represents codon-optimized N-terminus fragment of NanoLuc 1-156 (Dixon et al. 2016). Sequences shaded by red and blue represent the C-terminal short peptide of NanoLuc, which can interact with 11S. C) PTS-based luciferase assay for detecting PNGase/NGLY1 activity. Blue square represents GlcNAc.

To develop their split intein system for a read-out of NGLY1 activity, the authors prepared NLs-3GN2-IntN, in which a GlcNAc2 moiety is incorporated into NLs-IntN. In this system, active NanoLuc is generated in a PTS-dependent manner only when Asn67-GlcNAc2, which is the minimum glycan structure serving as a PNGase/NGLY1 substrate, is converted into Asp67 by the action of PNGase/NGLY1. Using this setup, they successfully detected PNGase/NGLY1 activity with luciferase-derived luminescence as a read-out, by incubating enzyme sources (e.g. purified yeast Png1 and cytosolic fractions from cultured cell lines) with chemically synthesized NLs-3GN2-IntN and recombinantly expressed IntCm3-NLlg proteins (Takahashi et al. 2022) (Fig. 3C). The authors further developed this system for monitoring NGLY1 activity in living cells. In this system, they prepared NLs-3GN2-R9, which incorporates a stretch of nine arginine residues into GN2-IntN to facilitate its endocytotic transport into cells of interest. By treating cells that exogenously express IntCm3-NLlg with the NLs-3GN2-R9 peptide and a substrate for luciferase, furimazine, the authors were able to detect intracellular NGLY1 activity in cultured cell lines, HeLa and 3T3-L1 cells (Takahashi et al. 2022). These data indicate the usefulness of their assay system for detecting endogenous NGLY1 activity both in vitro and in cellulo.

ELISA-based BSA-Gp assay for measuring NGLY1 activity

A recent study reported a novel ELISA-based NGLY1 assay through the detection of NGLY1-dependent amino acid editing (i.e. conversion of N-glycosylated Asn into Asp) (Fujihira et al. 2024). In that study, a glycopeptide-tag, in which the Asp residue at the 4th position in the hemagglutinin tag (HA-tag; YPYDVPDYA) was substituted with N-glycosylated Asn (YPYN (glycans)VPDYA), was chemically synthesized (Fig. 4A). The glycopeptides were conjugated, through the thiol group at the N-terminus cysteine, with a carrier protein, maleimide-activated BSA, via a maleimide-thiol reaction to synthesize the BSA glycopeptide-tag substrate (BSA-Gp) (Fig. 4A). After confirming the detection of the HA-epitope in BSA-Gp treated with PNGase/NGLY1 by immunoblotting, the authors demonstrated that BSA-Gp coated onto a 96-well plate can specifically detect PNGase/NGLY1 activity through the ELISA-based system only when the HA epitope is exposed through PNGase/NGLY1-specific editing of N-glycosylated Asn into Asp (Fig. 4B) (Fujihira et al. 2024). Remarkably, this assay system can detect endogenous NGLY1 activity in a highly sensitive manner, only requiring cell extracts from as little as 5 × 103 cells (equivalent to 2 μg total protein). Moreover, the system is sensitive enough to detect weak endogenous NGLY1 activity in samples from patients with NGLY1 deficiency (~5% of the activity observed in healthy patients) (Fujihira et al. 2024). This observation strongly indicates that the ELISA-based assay system could be easily applied to clinical examinations for screening NGLY1 activity, as ELISAs are routinely applicable for clinical laboratory tests.

Fig. 4.

Fig. 4

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ELISA-based NGLY1 assay system (Fujihira et al. 2024). A) Schematic of the synthesis of BSA-Gp by maleimide-thiol reaction. N- and C-termini of glycopeptide-tag were modified with acetyl and amide-group, respectively, to confer the peptide some resistance against proteases. B) Schematic of ELISA-based NGLY1 assay system. The conversion of amino acid where the N-glycosylated Asn to asp turns into HA epitope. HRP represents horseradish peroxidase.

Concluding remarks

This review summarizes the early history as well as recent advancements of the PNGase/NGLY1 assay system, particularly focusing on microplate-based assay systems for NGLY1 activity. The features and pros/cons of newly developed microplate-based assay systems are summarized in Table 2. Further refinement of the probes for real-time monitoring of intracellular NGLY1 activity in both cultured cell lines and tissues would provide powerful tools for gaining insights into the function and regulation of PNGase/NGLY1 activity across diverse organisms. Although each assay can quantitatively detect endogenous NGLY1 activity, it will be crucial to optimize the chemical structure of each probe for further improving sensitivity, a prerequisite for its diagnostic utility. In particular, further refinement of these assay systems to detect endogenous NGLY1 activity in a minute amount of blood-derived cells holds promise for establishing a highly effective diagnostic method for NGLY1 deficiency.

Acknowledgments

We wish to thank Professor Tsuyoshi Takahashi (Gunma University) and Dr. Akinobu Honda (RIKEN) for critical reading of the manuscript. We are also grateful to Dr. Hirokazu Yagi (Nagoya City University) for providing a portrait photograph of Dr. Noriko Takahashi. Our research was supported by Takeda-CiRA Joint Program (T-CiRA: NGLY1 deficiency project) (to T.S.), RIKEN Pioneering Research Project (“Glyco-lipidologue Initiative”) (to T.S.), Japan Agency for Medical Research and Development Core Research for Evolutional Science and Technology (AMED-CREST), Grant Number JP24gm1410003 (to T.S. and H.F.), and KAKENHI Grant Number JP22K06155 (to H.H.), JP21K06092 (to H.F.) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT)/Japan Society for the Promotion of Science (JSPS). We thank Enago (https://www.enago.jp) for the English language review.

Contributor Information

Hiroto Hirayama, Glycometabolic Biochemistry Laboratory, RIKEN Cluster for Pioneering Research (CPR), Riken, 2-1 Hirosawa, Wako Saitama 351-0198, Japan.

Haruhiko Fujihira, Glycometabolic Biochemistry Laboratory, RIKEN Cluster for Pioneering Research (CPR), Riken, 2-1 Hirosawa, Wako Saitama 351-0198, Japan.

Tadashi Suzuki, Glycometabolic Biochemistry Laboratory, RIKEN Cluster for Pioneering Research (CPR), Riken, 2-1 Hirosawa, Wako Saitama 351-0198, Japan.

Author contributions

Hiroto Hirayama (Visualization [equal], Writing—original draft-Equal, Writing—review & editing [equal]), Haruhiko Fujihira (Visualization [equal], Writing—original draft [equal], Writing—review & editing [equal]), and Tadashi Suzuki (Writing—review & editing [equal]).

 

Conflict of interest statement. None declared.

References

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