ELISA-based highly sensitive assay system for the detection of endogenous NGLY1 activity.

Cytosolic peptide:N-glycanase (NGLY1, PNGase) is an enzyme that cleaves N-glycans from misfolded glycoproteins. In 2012, a human genetic disorder, NGLY1 deficiency, was first reported to be caused by mutations of the NGLY1 gene. Since then, there has been rapid progresses on NGLY1 biology, and gene therapy has been proposed as a promising therapeutic option for NGLY1 deficiency. While a plasma/urine biomarker has also been developed for this disease, detection of NGLY1 activity could be another viable option for early diagnosis of NGLY1 deficiency. Thus far, several in vitro and in cellulo NGLY1 assays have been reported, but those assay systems have several issues that must be addressed in order to develop an assay system compatible for routine clinical examination. Here, we show a facile, highly sensitive in vitro assay system that could be used to detect NGLY1 activity by utilizing its sequence editing function, i.e. conversion of glycosylated Asn into Asp, followed by a detection of newly generated epitope (HA)-tag by anti-HA antibody. Using this ELISA-based assay, we detected endogenous NGLY1 activity in as little as 2 µg of crude extract, which is the equivalent of 5 × 103 cells. Our system also detects NGLY1 activity from cells with compromised NGLY1 activity, such as iPS cells from patient samples. This assay system could be applied in future clinical examinations to achieve an early diagnosis of NGLY1 deficiency.

Intranasal oxytocin suppresses seizure-like behaviors in a mouse model of NGLY1 deficiency.

NGLY1 deficiency is a genetic disease caused by biallelic mutations of the Ngly1 gene. Although epileptic seizure is one of the most severe symptoms in patients with NGLY1 deficiency, preclinical studies have not been conducted due to the lack of animal models for epileptic seizures in NGLY1 deficiency. Here, we observed the behaviors of male and female Ngly1-/- mice by video monitoring and found that these mice exhibit spontaneous seizure-like behaviors. Gene expression analyses and enzyme immunoassay revealed significant decreases in oxytocin, a well-known neuropeptide, in the hypothalamus of Ngly1-/- mice. Seizure-like behaviors in Ngly1-/- mice were transiently suppressed by a single intranasal administration of oxytocin. These findings suggest the therapeutic potential of oxytocin for epileptic seizure in patients with NGLY1 deficiency and contribute to the clarification of the disease mechanism.

Site-Specific Conjugation of Native Antibody: Transglutaminase-Mediated Modification of a Conserved Glutamine While Maintaining the Primary Sequence and Core Fc Glycan via Trimming with an Endoglycosidase.

A versatile chemo-enzymatic tool to site-specifically modify native (nonengineered) antibodies is using transglutaminase (TGase, E.C. 2.3.2.13). With various amines as cosubstrates, this enzyme converts the unsubstituted side chain amide of glutamine (Gln or Q) in peptides and proteins into substituted amides (i.e., conjugates). A pleasant surprise is that only a single conserved glutamine (Gln295) in the Fc region of IgG is modified by microbial TGase (mTGase, EC 2.3.2.13), thereby providing a highly specific and generally applicable conjugation method. However, prior to the transamidation (access to the glutamine residue by mTGase), the steric hindrance from the nearby conserved N-glycan (Asn297 in IgG1) must be reduced. In previous approaches, amidase (PNGase F, EC 3.5.1.52) was used to completely remove the N-glycan. However, PNGase F also converts a net neutral asparagine (Asn297) to a negatively charged aspartic acid (Asp297). This charge alteration may markedly change the structure, function, and immunogenicity of an IgG antibody. In contrast, in our new method presented herein, the N-glycan is trimmed by an endoglycosidase (EndoS2, EC 3.2.1.96), hence retaining both the core N-acetylglucosamine (GlcNAc) moiety and the neutral asparaginyl amide. The trimmed glycan also reduces or abolishes Fc receptor-mediated functions, which results in better imaging agents by decreasing nonspecific binding to other cells (e.g., immune cells). Moreover, the remaining core glycan allows further derivatization such as glycan remodeling and dual conjugation. Practical and robust, our method generates conjugates in near quantitative yields, and both enzymes are commercially available.

Development of a fluorescence and quencher-based FRET assay for detection of endogenous peptide:N-glycanase/NGLY1 activity.

Cytosolic peptide:N-glycanase (PNGase/NGLY1 in mammals) catalyzes deglycosylation of N-glycans on glycoproteins. A genetic disorder caused by mutations in the NGLY1 gene leads to NGLY1 deficiency with symptoms including motor deficits and neurological problems. Effective therapies have not been established, though, a recent study used the administration of an adeno-associated viral vector expressing human NGLY1 to dramatically rescue motor functions in young Ngly1-/- rats. Thus, early therapeutic intervention may improve symptoms arising from central nervous system dysfunction, and assay methods for measuring NGLY1 activity in biological samples are critical for early diagnostics. In this study, we established an assay system for plate-based detection of endogenous NGLY1 activity using a FRET-based probe. Using this method, we revealed significant changes in NGLY1 activity in rat brains during aging. This novel assay offers reliable disease diagnostics and provides valuable insights into the regulation of PNGase/NGLY1 activity in diverse organisms under different stress conditions.

Progressive myoclonic epilepsy as an expanding phenotype of NGLY1-associated congenital deglycosylation disorder: A case report and review of the literature.

INTRODUCTION: NGLY1-associated congenital disorder of deglycosylation (CDDG1: OMIM #615273) is a rare autosomal recessive disorder caused by a functional impairment of endoplasmic reticulum in degradation of glycoproteins. Neurocognitive dysfunctions have been documented in patients with CDDG1; however, deteriorating phenotypes of affected individuals remain elusive. CASE PRESENTATION: A Japanese boy with delayed psychomotor development showed ataxic movements from age 5 years and myoclonic seizures from age 12 years. Appetite loss, motor and cognitive decline became evident at age 12 years. Electrophysiological studies identified paroxysmal discharges on myoclonic seizure and a giant somatosensory evoked potential. Perampanel was effective for controlling myoclonic seizures. Exome sequencing revealed that the patient carried compound heterozygous variants in NGLY1, NM_018297.4: c.857G > A and c.-17_12del, which were inherited from mother and father, respectively. A literature review confirmed that myoclonic seizures were observed in 28.5% of patients with epilepsy. No other patients had progressive myoclonic epilepsy or cognitive decline in association with loss-of-function variations in NGLY1. CONCLUSION: Our data provides evidence that a group of patients with CDDG1 manifest slowly progressive myoclonic epilepsy and cognitive decline during the long-term clinical course.

A commentary on 'Patient-derived gene and protein expression signatures of NGLY1 deficiency'.

The cytosolic peptide:N-glycanase (PNGase; NGLY1 in human and PNG1 in budding yeast) is a deglycosylating enzyme widely conserved in eukaryotes. Initially, functional importance of this enzyme remained unknown as the png1Δ mutant in yeast did not exhibit any significant phenotypes. However, the discovery of NGLY1 deficiency, a rare genetic disorder with biallelic mutations in NGLY1 gene, prompted an intensification of research that has resulted in uncovering the significance of NGLY1 as well as the proteins under its influence that are involved in numerous cellular processes. A recent report by Rauscher et al. (Patient-derived gene and protein expression signatures of NGLY1 deficiency. J. Biochem. 2022; 171: 187-199) presented a comprehensive summary of transcriptome/proteome analyses of various cell types derived from NGLY1-deficient patients. The authors also provide a web application called 'NGLY1 browser', which will allow researchers to have access to a wealth of information on gene and protein expression signature for patients with NGLY1 deficiency.

A Natural Compound Containing a Disaccharide Structure of Glucose and Rhamnose Identified as Potential N-Glycanase 1 (NGLY1) Inhibitors.

N-glycanase 1 (NGLY1) is an essential enzyme involved in the deglycosylation of misfolded glycoproteins through the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway, which could hydrolyze N-glycan from N-glycoprotein or N-glycopeptide in the cytosol. Recent studies indicated that NGLY1 inhibition is a potential novel drug target for antiviral therapy. In this study, structure-based virtual analysis was applied to screen candidate NGLY1 inhibitors from 2960 natural compounds. Three natural compounds, Poliumoside, Soyasaponin Bb, and Saikosaponin B2 showed significantly inhibitory activity of NGLY1, isolated from traditional heat-clearing and detoxifying Chinese herbs. Furthermore, the core structural motif of the three NGLY1 inhibitors was a disaccharide structure with glucose and rhamnose, which might exert its action by binding to important active sites of NGLY1, such as Lys238 and Trp244. In traditional Chinese medicine, many compounds containing this disaccharide structure probably targeted NGLY1. This study unveiled the leading compound of NGLY1 inhibitors with its core structure, which could guide future drug development.

NGLY1 mutations cause protein aggregation in human neurons.

Biallelic mutations in the gene that encodes the enzyme N-glycanase 1 (NGLY1) cause a rare disease with multi-symptomatic features including developmental delay, intellectual disability, neuropathy, and seizures. NGLY1's activity in human neural cells is currently not well understood. To understand how NGLY1 gene loss leads to the specific phenotypes of NGLY1 deficiency, we employed direct conversion of NGLY1 patient-derived induced pluripotent stem cells (iPSCs) to functional cortical neurons. Transcriptomic, proteomic, and functional studies of iPSC-derived neurons lacking NGLY1 function revealed several major cellular processes that were altered, including protein aggregate-clearing functionality, mitochondrial homeostasis, and synaptic dysfunctions. These phenotypes were rescued by introduction of a functional NGLY1 gene and were observed in iPSC-derived mature neurons but not astrocytes. Finally, laser capture microscopy followed by mass spectrometry provided detailed characterization of the composition of protein aggregates specific to NGLY1-deficient neurons. Future studies will harness this knowledge for therapeutic development.

Amino acid editing of NFE2L1 by PNGase causes abnormal mobility on SDS-PAGE.

NFE2L1 (also known as NRF1) is a member of the nuclear erythroid 2-like family of transcription factors and is critical for counteracting various types of cellular stress such as oxidative, proteotoxic or metabolic stress. This unique transcription factor is also known to undergo changes, including post-translational modifications, limited proteolysis or translocation into the nucleus, before it exerts full transcriptional activity. As a result, there are various molecular forms with distinct sizes for this protein, while the precise nature of each form remains elusive. In this study, the N-glycosylated status of NFE2L1 in cells was examined. The findings revealed that when NFE2L1 was deglycosylated by PNGase F, the size-shift on SDS-PAGE was minimal. This was in contrast to deglycosylation by Endo H, which resulted in a clear size-shift, even though N-linked GlcNAc residues remained on the protein. It was found that this unusual behavior of PNGase-deglycosylated NFE2L1 was dependent on the conversion of the glycosylated-Asn to Asp, resulting in the introduction of more negative charges into the core peptide of NFE2L1. We also demonstrate that NGLY1-mediated deglycosylation and DDI2-mediated proteolytic processing of NFE2L1 are not strictly ordered reactions. Our study will allow us to better understand the precise structures as well as biochemical properties of the various forms of NFE2L1.

Development of a PNGase Rc Column for Online Deglycosylation of Complex Glycoproteins during HDX-MS.

Protein glycosylation is one of the most common PTMs and many cell surface receptors, extracellular proteins, and biopharmaceuticals are glycosylated. However, HDX-MS analysis of such important glycoproteins has so far been limited by difficulties in determining the HDX of the protein segments that contain glycans. We have developed a column containing immobilized PNGase Rc (from Rudaea cellulosilytica) that can readily be implemented into a conventional HDX-MS setup to allow improved analysis of glycoproteins. We show that HDX-MS with the PNGase Rc column enables efficient online removal of N-linked glycans and the determination of the HDX of glycosylated regions in several complex glycoproteins. Additionally, we use the PNGase Rc column to perform a comprehensive HDX-MS mapping of the binding epitope of a mAb to c-Met, a complex glycoprotein drug target. Importantly, the column retains high activity in the presence of common quench-buffer additives like TCEP and urea and performed consistent across 114 days of extensive use. Overall, our work shows that HDX-MS with the integrated PNGase Rc column can enable fast and efficient online deglycosylation at harsh quench conditions to provide comprehensive analysis of complex glycoproteins.

A high-throughput screening platform for enzymes active on mucin-type O-glycoproteins.

Mucin-type O-glycosylation is a post-translational modification present at the interface between cells where it has important roles in cellular communication. However, deciphering the function of O-glycoproteins and O-glycans can be challenging, especially as few enzymes are available for their assembly or selective degradation. Here, to address this deficiency, we developed a genetically encoded screening methodology for the discovery and engineering of the diverse classes of enzymes that act on O-glycoproteins. The method uses Escherichia coli that have been engineered to produce an O-glycosylated fluorescence resonance energy transfer probe that can be used to screen for O-glycopeptidase activity. Subsequent cleavage of the substrate by O-glycopeptidases provides a read-out of the glycosylation state of the probe, allowing the method to also be used to assay glycosidases and glycosyltransferases. We further show the potential of this methodology in the first ultrahigh-throughput-directed evolution of an O-glycopeptidase.

Analyzing the topology of N-linked glycans by PNGase F accessibility assay.

While N-glycans are synthesized in the lumens, some of them reach the cytosolic side of membranes through retro-translocation independent of endoplasmic-reticulum-associated degradation. Here, we present a protocol to measure the topology of N-glycans in a transmembrane protein, based on the principle that cytosolic but not luminal N-glycans are trimmed by PNGase F in the absence of detergent. We describe the procedures for this protocol consisting of microsome preparation from cells, PNGase F accessibility assay, and western blot analysis. For complete details on the use and execution of this protocol, please refer to Wang et al.1.

Site-specific, covalent immobilization of PNGase F on magnetic particles mediated by microbial transglutaminase.

In the workflow of global N-glycosylation analysis, endoglycosidase-mediated removal of glycans from glycoproteins is an essential and rate-limiting step. Peptide-N-glycosidase F (PNGase F) is the most appropriate and efficient endoglycosidase for the removal of N-glycans from glycoproteins prior to analysis. Due to the high demand for PNGase F in both basic and industrial research, convenient and efficient methods are urgently needed to generate PNGase F, preferably in the immobilized form to solid phases. However, there is no integrated approach to implement both efficient expression, and site-specific immobilization of PNGase F. Herein, efficient production of PNGase F with a glutamine tag in Escherichia coli and site-specific covalent immobilization of PNGase F with this special tag via microbial transglutaminase (MTG) is described. PNGase F was fused with a glutamine tag to facilitate the co-expression of proteins in the supernatant. The glutamine tag was covalently and site-specifically transformed to primary amine-containing magnetic particles, mediated by MTG, to immobilize PNGase F. Immobilized PNGase F could deglycosylate substrates with identical enzymatic performance to that of the soluble counterpart, and exhibit good reusability and thermal stability. Moreover, the immobilized PNGase F could also be applied to clinical samples, including serum and saliva.

Release of bifidogenic N-glycans from native bovine colostrum proteins by an endo-ß-N-acetylglucosaminidase.

Milk glycoproteins play various biological roles including antibacterial, antiviral activities, modulating immune responses in living organisms. Released N-glycans from milk glycoproteins act as growth substrates for infant-associated bifidobacteria, which are key members of the breastfed infant's gut. To date, the mechanisms, and contributions of glycans to the biological activities of glycoproteins remain to be elucidated. Only by testing both the released glycans and the deglycosylated protein in their native (i.e., non-denatured) form, can the individual contribution to the biological activity of glycoproteins be elucidated. However, for conventional enzymatic and chemical deglycosylation strategies to work efficiently, glycoprotein denaturation is required, which alters the protein native shape, hindering further investigations of its biological roles. An endo-ß-N-acetylglucosaminidase (EndoBI-1) from Bifidobacterium longum subsp. infantis ATCC 15697 (B. infantis) was characterized as having the ability to release N-glycans from bovine milk glycoproteins efficiently, without the denaturation. In this study, the activity of EndoBI-1 was compared to a commercial enzyme to release N-glycans, the peptide-N-glycosidase F (PNGase F), using dairy glycoproteins as the substrate. The kinetic evaluation showed that EndoBI-1 displayed higher activity on native glycoproteins than PNGase F, with 0.036 mg/mL×min and 0.012 mg/mL×min glycan release, respectively. EndoBI-1 released a broader array of glycan structures compared to PNGase F from native glycoproteins. Thirty-two and fifteen distinct compositions were released from the native glycoproteins by EndoBI-1 and PNGase F, respectively, as characterized by advanced mass spectrometry. EndoBI-1 can be considered a promising enzyme for the release of N-glycans and their protein backbone in the native form, which will enable effective glycan release and will facilitate subsequent investigations to reveal their contribution to glycoproteins' biological roles.

Peptide-N-glycosidase F or A treatment and procainamide-labeling for identification and quantification of N-glycans in two types of mammalian glycoproteins using UPLC and LC-MS/MS.

BACKGROUND: N-glycans in glycoproteins can affect physicochemical properties of proteins; however, some reported N-glycan structures are inconsistent depending on the type of glycoprotein or the preparation methods. OBJECTIVE: To obtain consistent results for qualitative and quantitative analyses of N-glycans, N-glycans obtained by different preparation methods were compared for two types of mammalian glycoproteins. METHODS: N-glycans are released by peptide-N-glycosidase F (PF) or A (PA) from two model mammalian glycoproteins, bovine fetuin (with three glycosylation sites) and human IgG (with a single glycosylation site), and labeled with a fluorescent tag [2-aminobenzamide (AB) or procainamide (ProA)]. The structure and quantity of each N-glycan were determined using UPLC and LC-MS/MS. RESULTS: The 21 N-glycans in fetuin and another 21 N-glycans in IgG by either PF-ProA or PA-ProA were identified using LC-MS/MS. The N-glycans in fetuin (8-13 N-glycans were previously reported) and in IgG (19 N-glycans were previously reported), which could not be identified by using the widely used PF-AB, were all identified by using PF-ProA or PA-ProA. The quantities (%) of the N-glycans (>0.1 %) relative to the total amount of N-glycans (100 %) obtained by AB- and ProA-labeling using LC-MS/MS had a similar tendency. However, the absolute quantities (pmol) of the N-glycans estimated using UPLC and LC-MS/MS were more efficiently determined with ProA-labeling than with AB-labeling. Thus, PF-ProA or PA-ProA allows for more effective identification and quantification of N-glycans than PF-AB in glycoprotein, particularly bovine fetuin. This study is the first comparative analysis for the identification and relative and absolute quantification of N-glycans in glycoproteins with PF-ProA and PA-ProA using UPLC and LC-MS/MS.

NGLY1 deficiency: estimated incidence, clinical features, and genotypic spectrum from the NGLY1 Registry.

PURPOSE: NGLY1 Deficiency is an ultra-rare, multisystemic disease caused by biallelic pathogenic NGLY1 variants. The aims of this study were to (1) characterize the variants and clinical features of the largest cohort of NGLY1 Deficiency patients reported to date, and (2) estimate the incidence of this disorder. METHODS: The Grace Science Foundation collected genotypic data from 74 NGLY1 Deficiency patients, of which 37 also provided phenotypic data. We analyzed NGLY1 variants and clinical features and estimated NGLY1 disease incidence in the United States (U.S.). RESULTS: Analysis of patient genotypes, including 10 previously unreported NGLY1 variants, showed strong statistical enrichment for missense variants in the transglutaminase-like domain of NGLY1 (p < 1.96E-11). Caregivers reported global developmental delay, movement disorder, and alacrima in over 85% of patients. Some phenotypic differences were noted between males and females. Regression was reported for all patients over 14 years old by their caregivers. The calculated U.S. incidence of NGLY1 Deficiency was ~ 12 individuals born per year. CONCLUSION: The estimated U.S. incidence of NGLY1 indicates the disease may be more common than the number of patients reported in the literature suggests. Given the low frequency of most variants and proportion of compound heterozygotes, genotype/phenotype correlations were not distinguishable.

The Effect of Sample Glucose Content on PNGase F-Mediated N-Glycan Release Analyzed by Capillary Electrophoresis.

Protein therapeutics have recently gained high importance in general health care along with applied clinical research. Therefore, it is important to understand the structure-function relationship of these new generation drugs. Asparagine-bound carbohydrates represent an important critical quality attribute of therapeutic glycoproteins, reportedly impacting the efficacy, immunogenicity, clearance rate, stability, solubility, pharmacokinetics and mode of action of the product. In most instances, these linked N-glycans are analyzed in their unconjugated form after endoglycosidase-mediated release, e.g., PNGase F-mediated liberation. In this paper, first, N-glycan release kinetics were evaluated using our previously reported in-house produced 6His-PNGase F enzyme. The resulting deglycosylation products were quantified by sodium dodecyl sulfate capillary gel electrophoresis to determine the optimal digestion time. Next, the effect of sample glucose content was investigated as a potential endoglycosidase activity modifier. A comparative Michaelis-Menten kinetics study was performed between the 6His-PNGase F and a frequently employed commercial PNGase F product with and without the presence of glucose in the digestion reaction mixture. It was found that 1 mg/mL glucose in the sample activated the 6His-PNGase F enzyme, while did not affect the release efficiency of the commercial PNGase F. Capillary isoelectric focusing revealed subtle charge heterogeneity differences between the two endoglycosidases, manifested by the lack of extra acidic charge variants in the cIEF trace of the 6His-PNGase F enzyme, which might have possibly influenced the glucose-mediated enzyme activity differences.

Luminescence detection of peptide:N-glycanase activity using engineered split inteins.

A split intein-based method has been developed to detect peptide:N-glycanase (PNGase) activity in live cells. PNGase cleaves the linkage between N,N'-diacetylchitobiose and the Asn side-chain of N-intein peptides and the products react rapidly with C-intein by protein trans-splicing to generate an active luciferase.

Comparative proteomics reveals elevated CCN2 in NGLY1-deficient cells.

N-glycanase 1(NGLY1) catalyzes the removal of N-linked glycans from newly synthesized or misfolded protein. NGLY1 deficiency is a recently diagnosed rare genetic disorder. The affected individuals present a broad spectrum of clinical features. Recent studies explored several possible molecular mechanisms of NGLY1 deficiency including defects in proteostasis, mitochondrial homeostasis, innate immunity, and water/ion transport. We demonstrate abnormal accumulation of endoplasmic reticulum-associated degradation (ERAD) substrates in NGLY1-deficient cells. Global quantitative proteomics discovered elevated levels of endogenous proteins in NGLY1-defective human and mouse cells. Further biological validation assays confirmed the altered abundance of several key candidates that were subjected to isobarically labeled proteomic analysis. CCN2 was selected for further analysis due to its significant increase in different cell models of NGLY1 deficiency. Functional assays show elevated CCN2 and over-stimulated TGF-ß signaling in NGLY1-deficient cells. Given the important role of CCN2 and TGF-ß pathway in mediating systemic fibrosis, we propose a potential link of increased CCN2 and TGF-ß signaling to microscopic liver fibrosis in NGLY1 patients.

Development of a novel, label-free N-glycan method using charged aerosol detection.

Monoclonal antibodies (mAbs) are complex glycoproteins that are developed for treatment of various therapeutic indications such as cancer and autoimmune diseases. MAbs are glycosylated at conserved asparagine residues (N-X-S/T) of the Fc region at amino acid position 297 of the heavy chain. Glycans are important in governing the functions of efficacy and serum half-life of protein therapeutics and are part of the critical quality attribute panel for release testing. Traditionally, N-linked glycans are released from glycoproteins after denaturation and enzymatic digestion with PNGase F, followed by fluorescent labeling of the liberated glycans. The labeled glycans are then separated using hydrophilic liquid chromatography (HILIC) with fluorescence detection to generate chromatographic profile. Despite decades of use, this strenuous process remains unchanged, utilizing toxic reagents and extended sample preparation time. As an intervention, this report showcases a novel, label-free approach to detect and quantify N-glycans without using fluorescent labeling. Separation of glycans using mixed-mode PGC column along with detection of non-derivatized glycans using charged aerosol detector, the overall turnaround time can be greatly reduced with significant cost savings. The label-free method provides similar quantitative results as the conventional fluorescent labeled method, confirming the validity of the method for product release.