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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-β signaling in NGLY1-deficient cells. Given the important role of CCN2 and TGF-β pathway in mediating systemic fibrosis, we propose a potential link of increased CCN2 and TGF-β signaling to microscopic liver fibrosis in NGLY1 patients.

N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing four different cell types derived from 14 NGLY1 deficient patients and 17 controls. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared with parents, residual and likely non-functional NGLY1 protein was detectable in all patient-derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosome biogenesis and mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cysteine ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients.

Protein glycosylation is one of the ubiquitous post-translational modifications in eukaryotic cells, which play important roles in plant growth and adverse response. In this study, we performed the first comprehensive wheat plasma membrane N-glycoproteome analysis under drought stress via glycopeptide HILIC enrichment and LC-MS/MS identification. In total, 414 glycosylated sites corresponding to 407 glycopeptides and 312 unique glycoproteins were identified, of which 173 plasma membrane glycoproteins with 215 N-glycosylation sites were significantly regulated by drought stress. Functional enrichment analysis reveals that the significantly regulated N-glycosylation proteins were particularly related to protein kinase activity involved in the reception and transduction of extracellular signal and plant cell wall remolding. The motifs and sequence structures analysis showed that the significantly regulated N-glycosylation sites were concentrated within [NxT] motif, and 79.5% of them were located on the random coil that is always on the protein surface and flexible regions, which could facilitate protein glycosylated modification and enhance protein structural stability via reducing protein flexibility. PNGase F enzyme digestion and glycosylation site mutation further indicated that N-glycosylated modification could increase protein stability. Therefore, N-glycosylated modification is involved in plant adaptation to drought stress by improving the stability of cell wall remodeling related plasma membrane proteins.

Cytosolic peptide:N-glycanase (NGLY1 in mammals), a highly conserved enzyme in eukaryotes, catalyses the deglycosylation of N-glycans that are attached to glycopeptide/glycoproteins. In 2012, an autosomal recessive disorder related to the NGLY1 gene, which was referred to as NGLY1 deficiency, was reported. Since then, more than 100 patients have been identified. Patients with this disease exhibit various symptoms, including various motor deficits and other neurological problems. Effective therapeutic treatments for this disease, however, have not been established. Most recently, it was demonstrated that the intracerebroventricular administration of an adeno-associated virus 9 vector expressing human NGLY1 during the weaning period allowed some motor functions to be recovered in Ngly1−/− rats. This observation led us to hypothesize that a therapeutic intervention for improving these motor deficits or other neurological symptoms found in the patients might be possible. To achieve this, it is critical to establish robust and facile methods for assaying NGLY1 activity in biological samples, for the early diagnosis and evaluation of the therapeutic efficacy for the treatment of NGLY1 deficiency. In this mini review, we summarize progress made in the development of various assay methods for NGLY1 activity, as well as a recent progress in the identification of NGLY1 deficiency-specific biomarkers.

Recessive mutations in human N-glycanase 1 (NGLY1) cause a multisystem disorder with various phenotypes including global developmental delay. One of the models utilized to understand the biology of NGLY1 and the pathophysiology of NGLY1 deficiency is Drosophila melanogaster, a well-established, genetically tractable organism broadly used to study various biological processes and human diseases. Loss of the Drosophila NGLY1 homolog (Pngl) causes a host of phenotypes including developmental delay and lethality. Phenotypic, transcriptomic and genome-wide association analyses on Drosophila have revealed links between NGLY1 and several critical developmental and cellular pathways/processes. Further, repurposing screens of Food and Drug Administration (FDA)-approved drugs have identified potential candidates to ameliorate some of the Pngl-mutant phenotypes. Here, we will summarize the insights gained into the functions of NGLY1 from Drosophila studies. We hope that the current review article will encourage additional studies in Drosophila and other model systems towards establishing a therapeutic strategy for NGLY1 deficiency patients.

N-glycanase 1 (NGLY1) deficiency is a rare inherited disorder characterized by developmental delay, hypolacrima or alacrima, seizure, intellectual disability, motor deficits, and other neurological symptoms. The underlying mechanisms of the NGLY1 phenotype are poorly understood, and no effective therapy is currently available. Similar to human patients, the rat model of NGLY1 deficiency, Ngly1−/−, shows developmental delay, movement disorder, somatosensory impairment, scoliosis, and learning disability. Here we show that single intracerebroventricular administration of AAV9 expressing human NGLY1 cDNA (AAV9-hNGLY1) to Ngly1−/− rats during the weaning period restored NGLY1 expression in the brain and spinal cord, concomitant with increased enzymatic activity of NGLY1 in the brain. hNGLY1 protein expressed by AAV9 was found predominantly in mature neurons, but not in glial cells, of Ngly1−/− rats. Strikingly, intracerebroventricular administration of AAV9-hNGLY1 normalized the motor phenotypes of Ngly1−/− rats assessed by the rota-rod test and gait analysis. The reversibility of motor deficits in Ngly1−/− rats by central nervous system (CNS)-restricted gene delivery suggests that the CNS is the primary therapeutic target organs for NGLY1 deficiency, and that the Ngly1−/− rat model may be useful for evaluating therapeutic treatments in pre-clinical studies.

The cytosolic peptide:N-glycanase (PNGase; NGLY1 in humans) is a deglycosylating enzyme that is widely conserved in eukaryotes. This enzyme is involved in the degradation of misfolded N-glycoproteins that are destined for proteasomal degradation in the cytosol, a process that is called endoplasmic reticulum-associated degradation. Although the physiological significance of NGLY1 remained unknown until recently, the discovery of NGLY1 deficiency, a human genetic disorder bearing mutations in the NGLY1 gene, has led to explosive research progress regarding the functional characterization of this enzyme. For example, it is now known that NGLY1 can also act as an ‘editing enzyme’ to convert N-glycosylated asparagine residues to aspartate residues, thus introducing negative charges into a core peptide and modulating the function of the target molecule. Diverse biological processes have also been found to be affected by compromised NGLY1 activity. In this special issue, recent research progress on the functional characterization of NGLY1 and its orthologues in worm/fly/rodents, assay methods/biomarkers useful for the development of therapeutics and the comprehensive transcriptome/proteome of NGLY1-KO cells as well as patient-derived cells are discussed.

The presentation of post-translationally modified (PTM) peptides by cell surface HLA molecules has the potential to increase the diversity of targets for surveilling T cells. Although immunopeptidomics studies routinely identify thousands of HLA-bound peptides from cell lines and tissue samples, in-depth analyses of the proportion and nature of peptides bearing one or more PTMs remains challenging. Here we have analyzed HLA-bound peptides from a variety of allotypes and assessed the distribution of mass spectrometry-detected PTMs, finding deamidation of asparagine or glutamine to be highly prevalent. Given that asparagine deamidation may arise either spontaneously or through enzymatic reaction, we assessed allele-specific and global motifs flanking the modified residues. Notably, we found that the N-linked glycosylation motif NX(S/T) was highly abundant across asparagine-deamidated HLA-bound peptides. This finding, demonstrated previously for a handful of deamidated T cell epitopes, implicates a more global role for the retrograde transport of nascently N-glycosylated polypeptides from the ER and their subsequent degradation within the cytosol to form HLA-ligand precursors. Chemical inhibition of Peptide:N-Glycanase (PNGase), the endoglycosidase responsible for the removal of glycans from misfolded and retrotranslocated glycoproteins, greatly reduced presentation of this subset of deamidated HLA-bound peptides. Importantly, there was no impact of PNGase inhibition on peptides not containing a consensus NX(S/T) motif. This indicates that a large proportion of HLA-I bound asparagine deamidated peptides are generated from formerly glycosylated proteins that have undergone deglycosylation via the ER-associated protein degradation (ERAD) pathway. The information herein will help train deamidation prediction models for HLA-peptide repertoires and aid in the design of novel T cell therapeutic targets derived from glycoprotein antigens.

N-Glycanase 1 (NGLY1) deficiency is an ultra-rare, complex and devastating neuromuscular disease. Patients display multi-organ symptoms including developmental delays, movement disorders, seizures, constipation and lack of tear production. NGLY1 is a deglycosylating protein involved in the degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER). NGLY1-deficient cells have been reported to exhibit decreased deglycosylation activity and an increased sensitivity to proteasome inhibitors. We show that the loss of NGLY1 causes substantial changes in the RNA and protein landscape of K562 cells and results in downregulation of proteasomal subunits, consistent with its processing of the transcription factor NFE2L1. We employed the CMap database to predict compounds that can modulate NGLY1 activity. Utilizing our robust K562 screening system, we demonstrate that the compound NVP-BEZ235 (Dactosilib) promotes degradation of NGLY1-dependent substrates, concurrent with increased autophagic flux, suggesting that stimulating autophagy may assist in clearing aberrant substrates during NGLY1 deficiency.

N-glycanase 1 (NGLY1) deficiency, an autosomal recessive disease caused by mutations in the NGLY1 gene, is characterized by developmental delay, hypolacrima or alacrima, seizure, intellectual disability, movement disorders and other neurological phenotypes. Because of few animal models that recapitulate these clinical signatures, the mechanisms of the onset of the disease and its progression are poorly understood, and the development of therapies is hindered. In this study, we generated the systemic Ngly1-deficient rodent model, Ngly1−/− rats, which showed developmental delay, movement disorder, somatosensory impairment and scoliosis. These phenotypes in Ngly1−/− rats are consistent with symptoms in human patients. In accordance with the pivotal role played by NGLY1 in endoplasmic reticulum-associated degradation processes, cleaving N-glycans from misfolded glycoproteins in the cytosol before they can be degraded by the proteasome, loss of Ngly1 led to accumulation of cytoplasmic ubiquitinated proteins, a marker of misfolded proteins in the neurons of the central nervous system of Ngly1−/− rats. Histological analysis identified prominent pathological abnormalities, including necrotic lesions, mineralization, intra- and extracellular eosinophilic bodies, astrogliosis, microgliosis and significant loss of mature neurons in the thalamic lateral and the medial parts of the ventral posterior nucleus and ventral lateral nucleus of Ngly1−/− rats. Axonal degradation in the sciatic nerves was also observed, as in human subjects. Ngly1−/− rats, which mimic the symptoms of human patients, will be a useful animal model for preclinical testing of therapeutic options and understanding the detailed mechanisms of NGLY1 deficiency.

High resolution analysis of N-glycans can be performed after their endoglycosidase mediated removal from proteins. N-glycosidase F peptide (PNGase F) is one the most frequently used enzyme for this purpose. Because of the significant demand for PNGase F both in basic and applied research, rapid and inexpensive methods are of great demand for its large-scale production, preferably in immobilizable form to solid supports or surfaces. In this paper, we report on the high-yield production of N-terminal 6His-PNGase F enzyme in a bacterial Escherichia coli SHuffle expression system. The activity profile of the generated enzyme was compared to commercially available PNGase F enzymes, featuring higher activity for the former. The method described here is thus suitable for the cost-effective production of PNGase F in an active, immobilizable form.

Akkermansia muciniphila is key member of the human gut microbiota that impacts many features of host health. A major characteristic of this bacterium is its interaction with host mucin, which is abundant in the gut environment, and its ability to metabolize mucin as a nutrient source. The machinery deployed by A. muciniphila to enable this interaction appears to be extensive and sophisticated, yet it is incompletely defined. The uncharacterized protein AMUC_1438 is encoded by a gene that was previously shown to be upregulated when the bacterium is grown on mucin. This uncharacterized protein has features suggestive of carbohydrate-recognition and peptidase activity, which led us to hypothesize that it has a role in mucin depolymerization. Here, we provide structural and functional support for the assignment of AMUC_1438 as a unique O-glycopeptidase with mucin-degrading capacity. O-glycopeptidase enzymes recognize glycans but hydrolyze the peptide backbone and are common in host-adapted microbes that colonize or invade mucus layers. Structural, kinetic, and mutagenic analyses point to a metzincin metalloprotease catalytic motif but with an active site that specifically recognizes a GalNAc residue α-linked to serine or threonine (i.e., the Tn-antigen). The enzyme catalyzes hydrolysis of the bond immediately N-terminal to the glycosylated residue. Additional modeling analyses suggest the presence of a carbohydrate-binding module that may assist in substrate recognition. We anticipate that these results will be fundamental to a wider understanding of the O-glycopeptidase class of enzymes and how they may contribute to host adaptation.

Protein glycosylation is a diverse form of post-translational modification. Two to three consecutive O-linked N-acetylgalactosamines (Tn-antigens) are recognized by antibodies such as MLS128. MLS128 mAb inhibited cell growth and bound to a 110 kDa glycoprotein (GP) in LS180 and HT29 colon cancer cells. However, purification and identification of the 110 kDa GP was unsuccessful due to its low abundance. The present study used a highly sophisticated and sensitive mass spectrometry method to identify proteins immunoprecipitated with MLS128 and separated by two-dimensional gel electrophoresis. Three desmosome components were identified. Of these, desmocollin and desmoglein shared many similar characteristics, including molecular mass, pI, and potential Tn-antigen sites. Western blotting analyses of LS180 cell lysates revealed a common 110 kDa band recognized by MLS128 and anti-desmocollin, but not by anti-desmoglein. Immunofluorescence microscopy of LS180 cells revealed that desmocollin is membrane-bound, while desmoglein is primarily localized in the cytosol. Confocal microscopy demonstrated colocalization of the desmocollin-specific antibody with the MLS128 antibody on the cell membrane, suggesting that desmocollin may contain Tn-antigens recognized by MLS128. Treatment of LS180 cells with siRNA to knock down desmocollin expression or a desmocollin-specific antibody decreased cell viability, suggesting a critical role for this protein in cell growth and survival. N-glycosidase F digestion of the 110 kDa GP and desmocollin suggested that although both proteins contain N-glycosylation sites, they are not identical. These findings suggest that desmocollin colocalizes with the 110 kDa GP and that growth inhibition induced by the MLS128 antibody may be mediated through a mechanism that involves desmocollin.

NGLY1 deficiency, a rare disease with no effective treatment, is caused by autosomal recessive, loss-of-function mutations in the N-glycanase 1 (NGLY1) gene and is characterized by global developmental delay, hypotonia, alacrima, and seizures. We used a Drosophila model of NGLY1 deficiency to conduct an in vivo, unbiased, small molecule, repurposing screen of FDA-approved drugs to identify therapeutic compounds. Seventeen molecules partially rescued lethality in a patient-specific NGLY1 deficiency model, including multiple serotonin and dopamine modulators. Exclusive dNGLY1 expression in serotonin and dopamine neurons, in an otherwise dNGLY1 deficient fly, was sufficient to partially rescue lethality. Further, genetic modifier and transcriptomic data supports the importance of serotonin signaling in NGLY1 deficiency. Connectivity Map analysis identified glycogen synthase kinase 3 (GSK3) inhibition as a potential therapeutic mechanism for NGLY1 deficiency, which we experimentally validated with TWS119, lithium, and GSK3 knockdown. Strikingly, GSK3 inhibitors and a serotonin modulator rescued size defects in dNGLY1 deficient larvae upon proteasome inhibition, suggesting that these compounds act through NRF1, a transcription factor that is regulated by NGLY1 and regulates proteasome expression. This study reveals the importance of the serotonin pathway in NGLY1 deficiency, and serotonin modulators or GSK3 inhibitors may be effective therapeutics for this rare disease.

Plant acidic peptide: N-glycanase (aPNGase) release N-glycans from glycopeptides during the degradation process of glycoproteins in developing or growing plants. We have previously developed a new method to detect the aPNGase activity in crude extracts, which is prerequisite for the construction of aPNGase knockout or overexpression lines. However, this method has the disadvantage of requiring de-sialylation treatment and a lectin chromatography. In this study, therefore, we improved the simple and accurate method for detecting aPNGase activity using anion-exchange HPLC requiring neither the desialylation treatment nor the lectin affinity chromatography.

Cytosolic peptide:N-glycanase (cPNGase), which occurs ubiquitously in eukaryotic cells, is involved in the de-N-glycosylation of misfolded glycoproteins in the protein quality control system. In this study, we aimed to provide direct evidence of plant cPNGase activity against a denatured glycoprotein using a crude extract prepared from a mutant line of Arabidopsis thaliana lacking 2 acidic PNGase genes.

Mutations in human N-glycanase 1 (NGLY1) cause the first known congenital disorder of deglycosylation (CDDG). Patients with this rare disease, which is also known as NGLY1 deficiency, exhibit global developmental delay and other phenotypes including neuropathy, movement disorder, and constipation. NGLY1 is known to regulate proteasomal and mitophagy gene expression through activation of a transcription factor called "nuclear factor erythroid 2-like 1" (NFE2L1). Loss of NGLY1 has also been shown to impair energy metabolism, but the molecular basis for this phenotype and its in vivo consequences are not well understood. Using a combination of genetic studies, imaging, and biochemical assays, here we report that loss of NGLY1 in the visceral muscle of the Drosophila larval intestine results in a severe reduction in the level of AMP-activated protein kinase α (AMPKα), leading to energy metabolism defects, impaired gut peristalsis, failure to empty the gut, and animal lethality. Ngly1–/– mouse embryonic fibroblasts and NGLY1 deficiency patient fibroblasts also show reduced AMPKα levels. Moreover, pharmacological activation of AMPK signaling significantly suppressed the energy metabolism defects in these cells. Importantly, the reduced AMPKα level and impaired energy metabolism observed in NGLY1 deficiency models are not caused by the loss of NFE2L1 activity. Taken together, these observations identify reduced AMPK signaling as a conserved mediator of energy metabolism defects in NGLY1 deficiency and suggest AMPK signaling as a therapeutic target in this disease., Author summary Thousands of proteins in most organisms harbor a type of sugar modification called N-glycan. Addition of N-glycans to proteins has long been known to affect protein quality control and function. However, recent studies have shown that removing N-glycans from proteins also plays key roles in cellular biology and animal development. Importantly, children with mutations in the enzyme responsible for N-glycan removal (NGLY1) exhibit developmental delay, seizures, movement disorder, and other abnormalities. NGLY1 is responsible for adjusting the activity of proteasome, a cellular machinery involved in protein degradation. NGLY1 has also been linked to the function and homeostasis of mitochondria, the major energy production engine in animal cells. However, the link between these processes and patient symptoms is not clear, and no therapies exist for this disease. We now report that loss of NGLY1 results in reduced levels of a cellular energy sensor called AMPKα in fruit flies, mouse cells and patient cells. Restoring AMPKα level or its pharmacological activation improves the energy homeostasis defects in all three systems and significantly increases the survival of NGLY1-mutant fruit flies independently of proteasome. Our data suggest that enhancement of AMPKα activity can serve as a potential therapeutic approach in NGLY1 deficiency patients.

Here, we present for the first time, a site-specific N-glycosylation analysis of proteins from a Brazilian Zika virus (ZIKV) strain. The virus was propagated with high yield in an embryo-derived stem cell line (EB66, Valneva SE), and concentrated by g-force step-gradient centrifugation. Subsequently, the sample was proteolytically digested with different enzymes, measured via a LC–MS/MS-based workflow, and analyzed in a semi-automated way using the in-house developed glyXtoolMS software. The viral non-structural protein 1 (NS1) was glycosylated exclusively with high-mannose structures on both potential N-glycosylation sites. In case of the viral envelope (E) protein, no specific N-glycans could be identified with this method. Nevertheless, N-glycosylation could be proved by enzymatic de-N-glycosylation with PNGase F, resulting in a strong MS-signal of the former glycopeptide with deamidated asparagine at the potential N-glycosylation site N444. This confirmed that this site of the ZIKV E protein is highly N-glycosylated but with very high micro-heterogeneity. Our study clearly demonstrates the progress made towards site-specific N-glycosylation analysis of viral proteins, i.e. for Brazilian ZIKV. It allows to better characterize viral isolates, and to monitor glycosylation of major antigens. The method established can be applied for detailed studies regarding the impact of protein glycosylation on antigenicity and human pathogenicity of many viruses including influenza virus, HIV and corona virus.

Background Hashimoto's thyroiditis (HT) is an autoimmune disease characterized by chronic inflammation of thyroid gland. Although HT is the most common cause of hypothyroidism, the pathogenesis of this disease is not fully understood. Glycosylation of serum proteins was examined in HT only to a limited extent. The study was designed to determine the glycosylation pattern of IgG-depleted sera from HT patients. Methods Serum N-glycans released by N-glycosidase F (PNGase F) digestion were analyzed by normal-phase high-performance liquid chromatography (NP-HPLC). N-glycan structures in each collected HPLC fraction were determined by liquid chromatography-mass spectrometry (LC-MS) and exoglycosidase digestion. Fucosylation and sialylation was also analyzed by lectin blotting. Results The results showed an increase of monosialylated tri-antennary structure (A3G3S1) and disialylated diantennary N-glycan with antennary fucose (FA2G2S2). Subsequently, we analyzed the serum N-glycan profile by lectin blotting using lectins specific for fucose and sialic acid. We found a significant decrease of Lens culinaris agglutinin (LCA) staining in HT samples, which resulted from the reduction of α1,6-linked core fucose in HT serum. We also observed an increase of Maackia amurensis II lectin (MAL-II) reaction in HT due to the elevated level of α2,3-sialylation in HT sera. Conclusions The detected alterations of serum protein sialylation might be caused by chronic inflammation in HT. The obtained results complete our previous IgG N-glycosylation analysis in autoimmune thyroid patients and show that the altered N-glycosylation of serum proteins is characteristic for autoimmunity process in HT. General Significance Thyroid autoimmunity is accompanied by changes of serum protein sialylation.

Genomics methodologies have significantly improved elucidation of Mendelian disorders. The combination with high-throughput functional-omics technologies potentiates the identification and confirmation of causative genetic variants, especially in singleton families of recessive inheritance. In a cohort of 99 individuals with abnormal Golgi glycosylation, 47 of which being unsolved, glycomics profiling was performed of total plasma glycoproteins. Combination with whole-exome sequencing in 31 cases revealed a known genetic defect in 15 individuals. To identify additional genetic factors, hierarchical clustering of the plasma glycomics data was done, which indicated a subgroup of four patients that shared a unique glycomics signature of hybrid type N-glycans. In two siblings, compound heterozygous mutations were found in SLC10A7, a gene of unknown function in human. These included a missense mutation that disrupted transmembrane domain 4 and a mutation in a splice acceptor site resulting in skipping of exon 9. The two other individuals showed a complete loss of SLC10A7 mRNA. The patients' phenotype consisted of amelogenesis imperfecta, skeletal dysplasia, and decreased bone mineral density compatible with osteoporosis. The patients' phenotype was mirrored in SLC10A7 deficient zebrafish. Furthermore, alizarin red staining of calcium deposits in zebrafish morphants showed a strong reduction in bone mineralization. Cell biology studies in fibroblasts of affected individuals showed intracellular mislocalization of glycoproteins and a defect in post-Golgi transport of glycoproteins to the cell membrane. In contrast to yeast, human SLC10A7 localized to the Golgi. Our combined data indicate an important role for SLC10A7 in bone mineralization and transport of glycoproteins to the extracellular matrix. ispartof: HUMAN MOLECULAR GENETICS vol:27 issue:17 pages:3029-3045 ispartof: location:England status: published

N-glycanase deficiency (NGLY1 deficiency, NGLY1-CDDG), the first autosomal recessive congenital disorder of N-linked deglycosylation (CDDG), is caused by pathogenic variants in NGLY1. The majority of affected individuals have been identified using exome or genome sequencing. To date, no reliable, clinically available biomarkers have been identified. Urine oligosaccharide analysis was included as part of a routine evaluation for possible biomarkers in patients with confirmed NGLY1-CDDG. During the qualitative review of oligosaccharide profiles by an experienced laboratory director an abnormal analyte with a proposed structure of Neu5Ac1Hex1GlcNAc1-Asn was identified in NGLY1-CDDG patient urine samples. The same species has been observed in profiles from individuals affected with aspartylglucosaminuria, although the complete spectra are not identical. Additional studies using tandem mass spectrometry confirmed the analyte's structure. In addition to the known NGLY1-CDDG patients identified by this analysis, a single case was identified in a population referred for clinical testing who subsequently had a diagnosis of NGLY1-CDDG confirmed by molecular testing. Urine oligosaccharide screening by MALDI-TOF MS can identify individuals with NGLY1-CDDG. In addition, this potential biomarker might also be used to monitor the effectiveness of therapeutic options as they become available.

Autosomal recessive loss-of-function mutations in N-glycanase 1 (NGLY1) cause NGLY1 deficiency, the only known human disease of deglycosylation. Patients present with developmental delay, movement disorder, seizures, liver dysfunction and alacrima. NGLY1 is a conserved cytoplasmic component of the Endoplasmic Reticulum Associated Degradation (ERAD) pathway. ERAD clears misfolded proteins from the ER lumen. However, it is unclear how loss of NGLY1 function impacts ERAD and other cellular processes and results in the constellation of problems associated with NGLY1 deficiency. To understand how loss of NGLY1 contributes to disease, we developed a Drosophila model of NGLY1 deficiency. Loss of NGLY1 function resulted in developmental delay and lethality. We used RNAseq to determine which processes are misregulated in the absence of NGLY1. Transcriptome analysis showed no evidence of ER stress upon NGLY1 knockdown. However, loss of NGLY1 resulted in a strong signature of NRF1 dysfunction among downregulated genes, as evidenced by an enrichment of genes encoding proteasome components and proteins involved in oxidation–reduction. A number of transcriptome changes also suggested potential therapeutic interventions, including dysregulation of GlcNAc synthesis and upregulation of the heat shock response. We show that increasing the function of both pathways rescues lethality. Together, transcriptome analysis in a Drosophila model of NGLY1 deficiency provides insight into potential therapeutic approaches.

The biologic activity of IgG molecules is modulated by its crystallizable fragment N-glycosylation, and thus, the analysis of IgG glycosylation is critical. A standard approach to analyze glycosylation of IgGs involves the release of the N-glycans by the enzyme peptide N-glycosidase F, which cleaves the linkage between the asparagine residue and innermost N-acetylglucosamine (GlcNAc) of all N-glycans except those containing a 3-linked fucose attached to the reducing terminal GlcNAc residue. The importance of obtaining complete glycan release for accurate quantitation led us to investigate the kinetics of this de-glycosylation reaction for IgG glycopeptides and to determine the effect of glycan structure and amino acid sequence on the rate of glycan release from glycopeptides of IgGs. This study revealed that the slight differences in amino acid sequences did not lead to a statistically different deglycosylation rate. However, statistically significant differences in the deglycosylation rate constants were observed between glycopeptides differing only in glycan structure (i.e., nonfucosylated, fucosylated, bisecting-GlcNAc, sialylated, etc.). For example, a single sialic acid residue was found to decrease the rate by a factor of 3. Similar reductions in rate were associated with the presence of a bisecting-GlcNAc. We predict the differences in release kinetics can lead to significant quantitative variations of the glycosylation study of IgGs.

Background N-Glycosylation, one of the most prominent post-translational modifications of proteins, is found in all domains of life, i.e. eukaryotes, bacteria and archaea, and has been shown to play a crucial role in modulating the physicochemical/physiological properties of carrier proteins. Deglycosylating enzymes that act on N-glycans are widely used in analyzing the structures/functions of N-glycans. Fungi are known to produce various deglycosylating enzymes, some of which are fungi-specific. While such enzymes likely are biologically relevant in fungal biology, their properties as well as their functions have not been explored in detail. Scope of review In this review, we summarize the current knowledge of fungal deglycosylating enzymes and discuss their biological significance. Major conclusions As of this writing, five types of deglycosylating enzymes that act on N-glycans are known to occur in fungi; (1) the cytosolic peptide: N-glycanase (PNGase), (2) the acidic PNGase, (3) the glycoside hydrolase family (GH) 85 endo-β-N-acetylglucosaminidase (ENGase), (4) the GH18 cytosolic ENGase, and (5) the GH18 secreted ENGase. Interestingly, genome surveys indicate that the loss of cytosolic PNGase activity in certain fungi coincide with the occurrence of GH18 cytosolic ENGase, implying that the GH18 ENGase serves to replace the deglycosylation function of the cytosolic PNGase in those filamentous ascomycete fungi. General significance Our review concludes that fungi promise to be valuable organisms for developing an understanding of the biological functions of PNGases/ENGases.

N-glycosylation of proteins is important for protein folding and function. We have recently reported that FAM5C/BRINP3 contributes to the tumor necrosis factor-α-induced expression of leukocyte adhesion molecules in vascular endothelial cells (ECs). However, regulatory mechanism of the FAM5C biosynthesis is poorly understood. Co-immunoprecipitation assay revealed the interaction of FAM5C with UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1), a glycoprotein folding-sensor enzyme. FAM5C ectopically expressed in HEK293 cells was localized to the endoplasmic reticulum and co-localized with endogenously expressed UGGT1. Molecular size of FAM5C was reduced by treatment with N-glycosidase F and in FAM5C-expressing cells cultured in the presence of the N-glycosylation inhibitor tunicamycin. FAM5C was secreted by the cells and the secretion of FAM5C was blocked by tunicamycin. Among six potential N-glycosylation sites, the potential site at Asn168 was not N-glycosylated, and Asn337, Asn456, Asn562, Asn609, and Asn641 mutants were poorly secreted by the cells. These results demonstrated that FAM5C is an N-glycosylated protein and N-glycosylation is necessary for the secretion of FAM5C.

Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze endoglycosidase released and fluorophore-labeled N-glycans from formalin-fixed paraffin-embedded (FFPE) mouse tissue samples of lung, brain, heart, spleen, liver, kidney and intestine. The FFPE samples were first deparaffinized followed by solubilization and glycoprotein retrieval. PNGase F mediated release of the N-linked oligosaccharides was followed by labeling with aminopyrene trisulfonate. After CE-LIF glycoprofiling of the FFPE mouse tissues, the N-glycan pool of the lung specimen was subject to further investigation by exoglycosidase array based carbohydrate sequencing. Structural assignment of the oligosaccharides was accomplished by the help of the GUcal software and the associated database, based on the mobility shifts after treatments with the corresponding exoglycosidase reaction mixtures. Sixteen major N-linked carbohydrate structures were sequenced from the mouse lung FFPE tissue glycome and identified, as high mannose (3) neutral biantennary (3) sialylated monoantennary (1) and sialylated bianennary (9) oligosaccharides. Two of these latter ones also possessed alpha(1-3) linked galactose residues.

N-Glycosylated substrates that undergo ER-associated degradation (ERAD) are often deglycosylated by the cytosolic peptide:N-glycanase (Ngly1) during their proteasomal degradation in the cytosol. Consequently, the presence of non- or deglycosylated forms of such proteins after treatment with proteasome inhibitors is widely used as evidence for cytosolic deglycosylation by Ngly1. However, in this study, the accumulation of nonglycosylated RTA∆m, a model ERAD substrate, was still observed in mouse cells lacking cytosolic de-N-glycosylating enzymes, when treated with proteasome inhibitors. It was found that RTA∆m is normally partially N-glycosylated, while the nonglycosylated form is rapidly degraded by proteasomes in the cytosol. Our results suggest that the occurrence of 'nonglycosylated' ERAD substrates upon treatment with proteasome inhibitors is not necessarily a clue for cytosolic deglycosylation.

We introduce an efficient sample preparation workflow to facilitate deep N-glycomics analysis of the human serum by capillary electrophoresis with laser induced fluorescence (CE-LIF) detection and to accommodate the higher sample concentration requirement of electrospray ionization mass spectrometry connected to capillary electrophoresis (CE-ESI-MS). A novel, temperature gradient denaturing protocol was applied on amine functionalized magnetic bead partitioned glycoproteins to circumvent the otherwise prevalent precipitation issue. During this process, the free sugar content of the serum was significantly decreased as well, accommodating enhanced PNGase F mediated release of the N-linked carbohydrates. The liberated oligosaccharides were tagged with aminopyrene-trisulfonate, utilizing a modified evaporative labeling protocol. Processing the samples with this new workflow enabled deep CE-LIF analysis of the human serum N-glycome and provided the appropriate amount of material for CE-ESI-MS analysis in negative ionization mode.

Background NGLY1-CDDG is a congenital disorder of deglycosylation caused by a defective peptide:N-glycanase (PNG). To date, all but one of the reported patients have been diagnosed through whole-exome or whole-genome sequencing, as no biochemical marker was available to identify this disease in patients. Recently, a potential urinary biomarker was reported, but the data presented suggest that this marker may be excreted intermittently. Methods In this study, we performed untargeted direct-infusion high-resolution mass spectrometry metabolomics in seven dried blood spots (DBS) from four recently diagnosed NGLY1-CDDG patients, to test for small-molecule biomarkers, in order to identify a potential diagnostic marker. Results were compared to 125 DBS of healthy controls and to 238 DBS of patients with other diseases. Results We identified aspartylglycosamine as the only significantly increased compound with a median Z-score of 4.8 (range: 3.8–8.5) in DBS of NGLY1-CDDG patients, compared to a median Z-score of −0.1 (range: −2.1–4.0) in DBS of healthy controls and patients with other diseases. Discussion The increase of aspartylglycosamine can be explained by lack of function of PNG. PNG catalyzes the cleavage of the proximal N-acetylglucosamine residue of an N-glycan from the asparagine residue of a protein, a step in the degradation of misfolded glycoproteins. PNG deficiency results in a single N-acetylglucosamine residue left attached to the asparagine residue which results in free aspartylglycosamine when the glycoprotein is degraded. Thus, we here identified aspartylglycosamine as the first potential small-molecule biomarker in DBS for NGLY1-CDDG, making a biochemical diagnosis for NGLY1-CDDG potentially feasible.

The proteasome mediates selective protein degradation and is dynamically regulated in response to proteotoxic challenges. SKN-1A/Nrf1, an ER-associated transcription factor that undegoes N-linked glycosylation, serves as a sensor of proteasome dysfunction and triggers compensatory upregulation of proteasome subunit genes. Here, we show that the PNG-1/NGLY1 peptide:N-glycanase edits the sequence of SKN-1A protein by converting particular N-glycosylated asparagine residues to aspartic acid. Genetically introducing aspartates at these N-glycosylation sites bypasses the requirement for PNG-1/NGLY1, showing that protein sequence editing rather than deglycosylation is key to SKN-1A function. This pathway is required to maintain sufficient proteasome expression and activity, and SKN-1A hyperactivation confers resistance to the proteotoxicity of human amyloid beta peptide. Deglycosylation-dependent protein sequence editing explains how ER-associated and cytosolic isoforms of SKN-1 perform distinct cytoprotective functions corresponding to those of mammalian Nrf1 and Nrf2. Thus, we uncover an unexpected mechanism by which N-linked glycosylation regulates protein function and proteostasis.

The cytoplasmic peptide:N-glycanase (Ngly1) is a de-N-glycosylating enzyme that cleaves N-glycans from misfolded glycoproteins and is involved in endoplasmic reticulum-associated degradation. The recent discovery of NGLY1-deficiency, which causes severe systemic symptoms, drew attention to the physiological function of Ngly1 in mammals. While several studies have been carried out to reveal the physiological necessity of Ngly1, the semi-lethal nature of Ngly1-deficient animals made it difficult to analyze its function in adults. In this study, we focus on the physiological function of Ngly1 in liver (hepatocyte)-specific Ngly1-deficient mice generated using the cre-loxP system. We found that hepatocyte-specific Ngly1-deficient mice showed abnormal hepatocyte nuclear size/morphology with aging but did not show other notable defects in unstressed conditions. This nuclear phenotype did not appear to be related to the function of the only gene currently reported to rescue Ngly1-deficient murine lethality so far, endo-β-N-acetylglucosaminidase. We also found that under a high fructose diet induced stress, the hepatocyte-specific Ngly1-deletion resulted in liver transaminases elevation and increased lipid droplet accumulation. We showed that the processing and localization of the transcription factor, nuclear factor erythroid 2-like 1 (Nfe2l1), was impaired in the Ngly1-deficient hepatocytes. Therefore, Nfe2l1, at least partially, contributes to the phenotypes observed in hepatocyte-specific Ngly1-deficient mice. Our results indicate that Ngly1 plays important roles in the adult liver impacting nuclear morphology and lipid metabolism. Hepatocyte-specific Ngly1-deficient mice could thus serve as a valuable animal model for assessing in vivo efficacy of drugs and/or treatment for NGLY1-deficiency.

ALG3-CDG is one of the very rare types of congenital disorder of glycosylation (CDG) caused by variants in the ER-mannosyltransferase ALG3. Here, we summarize the clinical, biochemical, and genetic data of four new ALG3-CDG patients, who were identified by a type I pattern of serum transferrin and the accumulation of Man5 GlcNAc2 -PP-dolichol in LLO analysis. Additional clinical symptoms observed in our patients comprise sensorineural hearing loss, right-descending aorta, obstructive cardiomyopathy, macroglossia, and muscular hypertonia. We add four new biochemically confirmed variants to the list of ALG3-CDG inducing variants: c.350G>C (p.R117P), c.1263G>A (p.W421*), c.1037A>G (p.N346S), and the intron variant c.296+4A>G. Furthermore, in Patient 1 an additional open-reading frame of 141 bp (AAGRP) in the coding region of ALG3 was identified. Additionally, we show that control cells synthesize, to a minor degree, a hybrid protein composed of the polypeptide AAGRP and ALG3 (AAGRP-ALG3), while in Patient 1 expression of this hybrid protein is significantly increased due to the homozygous variant c.160_196del (g.165C>T). By reviewing the literature and combining our findings with previously published data, we further expand the knowledge of this rare glycosylation defect.

Peptide:N-glycanase (NGLY1) is an enzyme responsible for cleaving oligosaccharide moieties from misfolded glycoproteins to enable their proper degradation. Deletion and truncation mutations in this gene are responsible for an inherited disorder of the endoplasmic reticulum-associated degradation pathway. However, the literature is unclear whether the disorder is a result of mutations leading to loss-of-function, loss of substrate specificity, loss of protein stability or a combination of these factors. In this communication, without burdening ourselves with the mechanistic underpinning of disease causation because of mutations on the NGLY1 protein, we demonstrate the successful application of virtual ligand screening (VLS) combined with experimental high-throughput validation to the discovery of novel small-molecules that show binding to the transglutaminase domain of NGLY1. Attempts at recombinant expression and purification of six different constructs led to successful expression of five, with three constructs purified to homogeneity. Most mutant variants failed to purify possibly because of misfolding and the resultant exposure of surface hydrophobicity that led to protein aggregation. For the purified constructs, our threading/structure-based VLS algorithm, FINDSITEcomb, was employed to predict ligands that may bind to the protein. Then, the predictions were assessed by high-throughput differential scanning fluorimetry. This led to the identification of nine different ligands that bind to the protein of interest and provide clues to the nature of pharmacophore that facilitates binding. This is the first study that has identified novel ligands that bind to the NGLY1 protein as a possible starting point in the discovery of ligands with potential therapeutic applications in the treatment of the disorder caused by NGLY1 mutants.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a proteolytic pathway for handling misfolded or improperly assembled proteins that are synthesized in the ER. Cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme that cleaves N-glycans that are attached to ERAD substrates. While the critical roles of N-glycans in monitoring the folding status of carrier proteins in the ER lumen are relatively well understood, the physiological role of PNGase-mediated deglycosylation in the cytosol remained poorly understood. We report herein the identification of endogenous substrates for the cytoplasmic PNGase in Saccharomyces cerevisiae. Using an isotope-coded glycosylation site-specific tagging (IGOT) method-based LC/MS analysis, 11 glycoproteins were specifically detected in the cytosol of PNGase-deletion cells (png1Δ). Among these molecules, at least five glycoproteins were clearly identified as ERAD substrates in vivo. Moreover, four out of the five proteins were found to be either deglycosylated by PNGase in vivo or the overall degradation was delayed in a png1Δ mutant. Our results clearly indicate that the IGOT method promises to be a powerful tool for the identification of endogenous substrates for the cytoplasmic PNGase.

Conbercept is an Fc fusion protein with very complicated carbohydrate profiles which must be carefully monitored through manufacturing process. Here, we introduce an optimized fluorescence derivatization high-performance liquid chromatographic method for glycan mapping in conbercept. Compared with conventional glycan analysis method, this method has much better resolution and higher reproducibility making it excellent for product quality control.

During the development of recombinant monoclonal antibody (rMAb) drugs, glycosylation receives particular focus because changes in the attached glycans can have a significant impact on the antibody effector functions. The vast heterogeneity of structures that exist across glycosylation sites hinders the in-depth analysis of glycan changes specific to an individual protein within a complex mixture. In this study, we established a sensitive and specific method for monitoring site-specific glycosylation in rMAbs using multiple reaction monitoring (MRM) on an ultra high performance liquid chromatography – triple quadrupole MS (UHPLC-QqQ-MS). Our results showed that irrespective of the IgG subclass expressed in the drugs, the N-glycopeptide profiles are nearly the same but differ in abundances. In all rMAb drugs, a single subclass of IgG comprised over 97% of the total IgG content and showed over 97% N-glycan site occupancy. This study demonstrates the utility of an MRM-based method to rapidly characterize over 130 distinct glycopeptides and determine the extent of site occupancy within minutes. Such multi-level structural characterization is important for the successful development of therapeutic antibodies.

Various studies in the past have revealed that molluscs can produce a wide range of rather complex N-glycan structures, which vary from those occurring in other invertebrate animals; particularly methylated glycans have been found in gastropods, and there are some reports of anionic glycans in bivalves. Due to the high variability in terms of previously described structures and methodologies, it is a major challenge to establish glycomic workflows that yield the maximum amount of detailed structural information from relatively low quantities of sample. In this study, we apply differential release with peptide:N-glycosidases F and A followed by solid-phase extraction on graphitized carbon and reversed-phase materials to examine the glycome of Volvarina rubella (C. B. Adams, 1845), a margin snail of the clade Neogastropoda. The resulting four pools of N-glycans were fractionated on a fused core RP-HPLC column and subject to MALDI-TOF MS and MS/MS in conjunction with chemical and enzymatic treatments. In addition, selected N-glycan fractions, as well as O-glycans released by β-elimination, were analyzed by porous graphitized carbon-LC-MS and MS(n). This comprehensive approach enabled us to determine a number of novel modifications of protein-linked glycans, including N-methyl-2-aminoethylphosphonate on mannose and N-acetylhexosamine residues, core β1,3-linked mannose, zwitterionic moieties on core Galβ1,4Fuc motifs, additional mannose residues on oligomannosidic glycans, and bisubstituted antennal fucose; furthermore, typical invertebrate N-glycans with sulfate and core fucose residues are present in this gastropod.

© 2020 Elsevier Ltd. All rights reserved., Methods that allow for chemical site-selective dual protein modification are scarce. Here, we provide proof-of-concept for the orthogonality and compatibility of a method for regioselective lysine modification with strategies for protein modification at cysteine and genetically encoded ketone-tagged amino acids. This sequential, orthogonal approach was applied to albumin and a therapeutic antibody to create functional dual site-selectively labelled proteins., We thank funding from Xunta de Galicia (Galician Plan of research, innovation and growth 2011–2015, ED481B 2014/086-0 and ED481B 2018/007 to MJM), FAPESP (17/13168-8 to BB), FCT Portugal (FCT Investigator to GJLB, IF/00624/2015 and PhD studentship to AG, SFRH/BD/115932/2016), the Spanish Ministry of Science and Innovation (MCI) co-financed with FEDER funds (RTI2018-099592-B-C22 to G. J.O) and the BBSRC (iCASE studentship to LB). GJLB is a Royal Society University Research Fellow (URF\R\180019).

Gene knock outs (KOs) are efficiently engineered through CRISPR-Cas9-induced frameshift mutations. While the efficiency of DNA editing is readily verified by DNA sequencing, a systematic understanding of the efficiency of protein elimination has been lacking. Here we devised an experimental strategy combining RNA sequencing and triple-stage mass spectrometry to characterize 193 genetically verified deletions targeting 136 distinct genes generated by CRISPR-induced frameshifts in HAP1 cells. We observed residual protein expression for about one third of the quantified targets, at variable levels from low to original, and identified two causal mechanisms, translation reinitiation leading to N-terminally truncated target proteins or skipping of the edited exon leading to protein isoforms with internal sequence deletions. Detailed analysis of three truncated targets, BRD4, DNMT1 and NGLY1, revealed partial preservation of protein function. Our results imply that systematic characterization of residual protein expression or function in CRISPR-Cas9-generated KO lines is necessary for phenotype interpretation.

SugarQb (http://www.imba.oeaw.ac.at/sugarqb) is a freely available collection of computational tools for the automated identification of intact glycopeptides from high‐resolution HCD MS/MS datasets in the Proteome Discoverer environment. We report the migration of SugarQb to the latest and free version of Proteome Discoverer 2.1, and apply it to the analysis of PNGase F‐resistant N‐glycopeptides from mouse embryonic stem cells. The analysis of intact glycopeptides highlights unexpected technical limitations to PNGase F‐dependent glycoproteomic workflows at the proteome level, and warrants a critical reinterpretation of seminal datasets in the context of N‐glycosylation‐site prediction.

Acidic peptide:N-glycanase (aPNGase) plays a pivotal role in plant glycoprotein turnover. For the construction of aPNGase-knockout or -overexpressing plants, a new method to detect the activity in crude plant extracts is required because endogenous peptidases present in the extract hamper enzyme assays using fluorescence-labeled N-glycopeptides as a substrate. In this study, we developed a new method for measuring aPNGase activity in crude extracts from plant materials.

Asparagine-linked glycosylation is a common post-translational modification of proteins catalyzed by oligosaccharyltransferase that is important in regulating many aspects of protein function. Analysis of protein glycosylation, including glycoproteomic measurement of the site-specific extent of glycosylation, remains challenging. Here, we developed methods combining enzymatic deglycosylation and protease digestion with SWATH-MS to enable automated measurement of site-specific occupancy at many glycosylation sites. Deglycosylation with peptide-endoglycosidase H, leaving a remnant N-acetylglucosamine on asparagines previously carrying high-mannose glycans, followed by trypsin digestion allowed robust automated measurement of occupancy at many sites. Combining deglycosylation with the more general peptide-N-glycosidase F enzyme with AspN protease digest allowed robust automated differentiation of nonglycosylated and deglycosylated forms of a given glycosylation site. Ratiometric analysis of deglycosylated peptides and the total intensities of all peptides from the corresponding proteins allowed relative quantification of site-specific glycosylation occupancy between yeast strains with various isoforms of oligosaccharyltransferase. This approach also allowed robust measurement of glycosylation sites in human salivary glycoproteins. This method for automated relative quantification of site-specific glycosylation occupancy will be a useful tool for research with model systems and clinical samples.

N-Glycanase 1, encoded by NGLY1, catalyzes the deglycosylation of misfolded N-linked glycoproteins retrotranslocated into the cytosol. We identified nine cases with mutations in NGLY1. The patients show developmental delay, seizures, peripheral neuropathy, abnormal liver function and alacrima (absence of tears). The mutations in NGLY1 resulted in the absence of N-glycanase 1 protein in patient-derived fibroblasts. Applying a recently established cellular deglycosylation-dependent Venus fluorescence assay, we found that patient fibroblasts had dramatically reduced fluorescence, indicating a pronounced reduction in N-glycanase enzymatic activity. Using this assay, we could find no evidence of other related activities. Our findings reveal that NGLY1 mutations destroy both N-glycanase 1 protein and enzymatic activity.

Epidermal growth factor (EGF) domain-specific O-linked N-acetylglucosamine (EOGT) is an endoplasmic reticulum (ER)-resident O-linked N-acetylglucosamine (O-GlcNAc) transferase that acts on EGF domain-containing proteins such as Notch receptors. Recently, mutations in EOGT have been reported in patients with Adams-Oliver syndrome (AOS). Here, we have characterized enzymatic properties of mouse EOGT and EOGT mutants associated with AOS. Simultaneous expression of EOGT with Notch1 EGF repeats in human embryonic kidney 293T (HEK293T) cells led to immunoreactivity with the CTD110.6 antibody in the ER. Consistent with the GlcNAc modification in the ER, the enzymatic properties of EOGT are distinct from those of Golgi-resident GlcNAc transferases; the pH optimum of EOGT ranges from 7.0 to 7.5, and the Km value for UDP N-acetylglucosamine (UDP-GlcNAc) is 25 μm. Despite the relatively low Km value for UDP-GlcNAc, EOGT-catalyzed GlcNAcylation depends on the hexosamine pathway, as revealed by the increased O-GlcNAcylation of Notch1 EGF repeats upon supplementation with hexosamine, suggesting differential regulation of the luminal UDP-GlcNAc concentration in the ER and Golgi. As compared with wild-type EOGT, O-GlcNAcylation in the ER is nearly abolished in HEK293T cells exogenously expressing EOGT variants associated with AOS. Introduction of the W207S mutation resulted in degradation of the protein via the ubiquitin-proteasome pathway, although the stability and ER localization of EOGT(R377Q) were not affected. Importantly, the interaction between UDP-GlcNAc and EOGT(R377Q) was impaired without adversely affecting the acceptor substrate interaction. These results suggest that impaired glycosyltransferase activity in mutant EOGT proteins and the consequent defective O-GlcNAcylation in the ER constitute the molecular basis for AOS.

Anti-Tn antigen MLS128 monoclonal antibody was produced two decades ago by immunizing mice with "cancerous antigens" derived from LS180 colon cancer cells. Previous studies demonstrated that MLS128 bound to 110 kDa glycoprotein (GP) in colon cancer cells, thereby inhibiting cell growth. Extensive attempts have been made towards understanding the inhibitory action of MLS128 on colon cancer cell growth and solving the primary structure of 110 kDa GP. Since limited proteolysis of 110 kDa GP was observed in microdomain fractions that had been kept frozen for several years, susceptibility of 110 kDa GP to trypsin and other proteases as well as N-glycosidase F has been investigated. Furthermore, 110 kDa GP expression was examined in colon cancer cells independently cultured in Akiyama laboratory. In summary, 110 kDa GP contains N-glycans. It does not contain inter-disulfide bonds but appears to have intra-disulfides. It must contain multiple cleavage sites for trypsin and thermolysin since these proteases digested 110 kDa GP to MLS128-undetectable small fragments. It seems to contain cleavage sites for cathepsin D which could cause limited digestion. LS180 cells derived from Akiyama laboratory produced a limited proteolysis product-like 75 kDa GP. This study provides a structural basis for developing cancer diagnostics and therapeutics.

Mitochondrial respiratory chain (RC) diseases and congenital disorders of glycosylation (CDG) share extensive clinical overlap but are considered to have distinct cellular pathophysiology. Here, we demonstrate that an essential physiologic connection exists between cellular N-linked deglycosylation capacity and mitochondrial function. Following identification of altered muscle and liver mitochondrial amount and function in two children with a CDG subtype caused by NGLY1 deficiency, we evaluated mitochondrial physiology in NGLY1 disease human fibroblasts, and in NGLY1-knockout mouse embryonic fibroblasts and C. elegans. Across these distinct evolutionary models of cytosolic NGLY1 deficiency, a consistent disruption of mitochondrial physiology was present involving modestly reduced mitochondrial content with more pronounced impairment of mitochondrial membrane potential, increased mitochondrial matrix oxidant burden, and reduced cellular respiratory capacity. Lentiviral rescue restored NGLY1 expression and mitochondrial physiology in human and mouse fibroblasts, confirming that NGLY1 directly influences mitochondrial function. Overall, cellular deglycosylation capacity is shown to be a significant factor in mitochondrial RC disease pathogenesis across divergent evolutionary species.

The cytoplasmic peptide:N-glycanase (Ngly1 in mammals) is a de-N-glycosylating enzyme that is highly conserved among eukaryotes. It was recently reported that subjects harboring mutations in the NGLY1 gene exhibited severe systemic symptoms (NGLY1-deficiency). While the enzyme obviously has a critical role in mammals, its precise function remains unclear. In this study, we analyzed Ngly1-deficient mice and found that they are embryonic lethal in C57BL/6 background. Surprisingly, the additional deletion of the gene encoding endo-β-N-acetylglucosaminidase (Engase), which is another de-N-glycosylating enzyme but leaves a single GlcNAc at glycosylated Asn residues, resulted in the partial rescue of the lethality of the Ngly1-deficient mice. Additionally, we also found that a change in the genetic background of C57BL/6 mice, produced by crossing the mice with an outbred mouse strain (ICR) could partially rescue the embryonic lethality of Ngly1-deficient mice. Viable Ngly1-deficient mice in a C57BL/6 and ICR mixed background, however, showed a very severe phenotype reminiscent of the symptoms of NGLY1-deficiency subjects. Again, many of those defects were strongly suppressed by the additional deletion of Engase in the C57BL/6 and ICR mixed background. The defects observed in Ngly1/Engase-deficient mice (C57BL/6 background) and Ngly1-deficient mice (C57BL/6 and ICR mixed background) closely resembled some of the symptoms of patients with an NGLY1-deficiency. These observations strongly suggest that the Ngly1- or Ngly1/Engase-deficient mice could serve as a valuable animal model for studies related to the pathogenesis of the NGLY1-deficiency, and that cytoplasmic ENGase represents one of the potential therapeutic targets for this genetic disorder., Author summary Ngly1 is a cytoplasmic de-N-glycosylating enzyme that is ubiquitously found in eukaryotes. This enzyme is involved in a process referred to as endoplasmic reticulum-associated degradation (ERAD), one of the quality control mechanisms for newly synthesized proteins. A genetic disorder, NGLY1-deficiency, caused by mutations in the NGLY1 gene has recently been discovered. However, the precise mechanism for the pathogenesis of this devastating disease continues to remain unclear. We report herein that Ngly1-deficient mice are embryonically lethal in a C57BL/6 background. Surprisingly, the lethality was suppressed by crossing the mice with an outbred mouse strain (ICR), suggesting that the phenotypic consequence of Ngly1 is greatly influenced by their genetic background. In both cases, the additional deletion of Engase in Ngly1-deficient mice could strongly mitigate the phenotypes. Interestingly, the remaining defects in Ngly1-deficient or Ngly1/Engase-deficient mice were reminiscent of the symptoms of subjects with an NGLY1-deficiency. Our results clearly point to the importance of Ngly1 in mammals and show that the inhibition of ENGase represents an effective therapy for treating an NGLY1-deficiency. Most importantly, the mice described herein could serve as valuable viable model mice for studies related to the pathophysiology of an NGLY1-deficiency.

Protein misfolding cyclic amplification (PMCA) is an in vitro simulation of prion replication, which relies on the use of normal brain homogenate derived from host species as substrate for the specific amplification of abnormal prion protein, PrP(Sc). Studies showed that recombinant cellular PrP, PrP(C), expressed in Escherichia coli lacks N-glycosylation and an glycophosphatidyl inositol anchor (GPI) and therefore may not be the most suitable substrate in seeded PMCA reactions to recapitulate prion conversion in vitro. In this study, we expressed 2 PRNP genotypes of sheep, V136L141R154Q171 and A136F141R154Q171, and one genotype of white-tailed deer (Q95G96, X132,Y216) using the baculovirus expression system and evaluated their suitability as substrates in seeded-PMCA. It has been reported that host-encoded mammalian RNA molecules and divalent cations play a role in the pathogenesis of prion diseases, and RNA molecules have also been shown to improve the sensitivity of PMCA assays. Therefore, we also assessed the effect of co-factors, such as prion-specific mRNA molecules and a divalent cation, manganese, on protein conversion. Here, we report that baculovirus-expressed recombinant PrP(C) shows a glycoform and GPI-anchor profile similar to mammalian brain-derived PrP(C) and supports amplification of PrP(Sc) and PrP(CWD) derived from prion-affected animals in a single round of seeded PMCA in the absence of exogenous co-factors. Addition of species-specific in vitro transcribed PrP mRNA molecules stimulated the conversion efficiency resulting in increased PrP(Sc) or PrP(CWD) production. Addition of 2 to 20 μM of manganese chloride (MnCl2) to unseeded PMCA resulted in conversion of recombinant PrP(C) to protease-resistant PrP. Collectively, we demonstrate, for the first time, that baculovirus expressed sheep and deer PrP can serve as a substrate in protein misfolding cyclic amplification for sheep and deer prions in the absence of additional exogenous co-factors.

Glycosylation is one of the most common and important protein modifications in biological systems. Many glycoproteins naturally occur at low abundances, which makes comprehensive analysis extremely difficult. Additionally, glycans are highly heterogeneous, which further complicates analysis in complex samples. Lectin enrichment has been commonly used, but each lectin is inherently specific to one or several carbohydrates, and thus no single or collection of lectin(s) can bind to all glycans. Here we have employed a boronic acid-based chemical method to universally enrich glycopeptides. The reaction between boronic acids and sugars has been extensively investigated, and it is well known that the interaction between boronic acid and diols is one of the strongest reversible covalent bond interactions in an aqueous environment. This strong covalent interaction provides a great opportunity to catch glycopeptides and glycoproteins by boronic acid, whereas the reversible property allows their release without side effects. More importantly, the boronic acid-diol recognition is universal, which provides great capability and potential for comprehensively mapping glycosylation sites in complex biological samples. By combining boronic acid enrichment with PNGase F treatment in heavy-oxygen water and MS, we have identified 816 N-glycosylation sites in 332 yeast proteins, among which 675 sites were well-localized with greater than 99% confidence. The results demonstrated that the boronic acid-based chemical method can effectively enrich glycopeptides for comprehensive analysis of protein glycosylation. A general trend seen within the large data set was that there were fewer glycosylation sites toward the C termini of proteins. Of the 332 glycoproteins identified in yeast, 194 were membrane proteins. Many proteins get glycosylated in the high-mannose N-glycan biosynthetic and GPI anchor biosynthetic pathways. Compared with lectin enrichment, the current method is more cost-efficient, generic, and effective. This method can be extensively applied to different complex samples for the comprehensive analysis of protein glycosylation.

Peptide: N-glycosidase F (PNGase F) is an asparagine amidase produced by Flavobacterium meningosepticum that serves as a useful tool in the research on protein N-glycosylation. However, native PNGase F purified from F. meningosepticum and recombinant PNGase F expressed in Escherichia coli are obtained only at low levels, with the culture yield being no more than 15mg/L. Here, we report the efficient production of large amounts of recombinant PNGase F. First, a codon-optimized sequence encoding F. meningosepticum PNGase F was cloned into the pPICZaA vector, which was used to transform Pichia pastoris GS115. Clones were screened directly by dot blotting with an anti-6His-tag antibody, and then protein expression was induced in glass tubes to conduct validation assays. The clone expressing the highest level of PNGase F was selected for fermentation at a 5-L scale, and then the recombinant enzyme produced was purified in a single step using affinity chromatography, which yielded 800mg of the protein per liter of culture. The partly glycosylated recombinant PNGase F exhibited an identical specific activity as commercially available PNGase F when using RNase B or other N-glycoproteins as substrates. Thus, the method developed in this study can facilitate the large-scale production and use of PNGase F in the rapidly developing research field of N-glycomics.