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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.

In order to ascertain the role of plant transglutaminases (TGase) in growth and abiotic stress response, the AtPng1 knock out (KO) line of A. thaliana has been analyzed during plant development and under heat and wound stress. Comparing wild type (WT) and KO lines a 58-kDa band was immunodetected by anti-AtPng1p antibody in the cell wall and chloroplasts only in the WT line. A residual TGase activity, not showing correlation with development nor stress response, was still present in the KO line. The KO line was less developed, with a juvenile phenotype characterized by fewer, smaller and less differentiated cells. Chloroplast TGase activity was insensitive to mutation. Data on stressed plants showed that (i) KO plants under heat stress were more juvenile compared to WT, (ii) different responses between WT and KO lines after wounding took place. TGase activity was not completely absent in the KO line, presenting high activity in the plastidial fraction. In general, the mutation affected A. thaliana growth and development, causing less differentiated cytological and anatomical features.

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.

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 elucidation of glycans biological function is essential to understand their role in biological processes, both normal and pathological. Immobilized glycoenzymes are excellent tools for this purpose as they can selectively release glycans from glycoproteins without altering their backbone. They can be easily removed from the reaction mixture avoiding their interference in subsequent experiments. Here, we describe the immobilization of peptide-N-glycosidase F (PNGase F) onto silica magnetic nanoparticles with immobilization yields of 86% and activity yields of 12%. Immobilized PNGase F showed higher thermal stability than its soluble counterpart, and could be reused for at least seven deglycosylation cycles. It was efficient in the deglycosylation of several glycoproteins (ribonuclease B, bovine fetuin, and ovalbumin) and a protein lysate from the parasite Fasciola hepatica under native conditions, with similar performance to that of the soluble enzyme. Successful deglycosylation was evidenced by a decrease in specific lectin recognition of the glycoproteins (40%-80%). Moreover, deglycosylated F. hepatica lysate allowed us to confirm the role of parasite N-glycans in the inhibition of the lipopolysaccharide-induced maturation of dendritic cells. Immobilized PNGase F probed to be a robust biotechnological tool for deglycosylation of glycoproteins and complex biological samples under native conditions.

Serum N-glycan patterns from 50 Crohn's disease (CD) patients and 50 healthy controls were acquired using a carbon matrix, from which eight N-glycans with significant difference were screened out to reveal remarkale performance for CD diagnosis. This research is expected to help future glycan-based disease detection not limited to CD.

The analysis of N-linked glycans using liquid chromatography and mass spectrometry (LC-MS) presents significant challenges, particularly owing to their hydrophilic nature. To address these difficulties, a variety of derivatization methods has been developed to facilitate improved ionization and detection sensitivity. One such method, the Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT)™ strategy for labeling glycans, has previously been utilized in the analysis of N- and O-linked glycans in biological samples. To assess the maximum sensitivity and separability of the INLIGHT ™ preparation and analysis pipeline, several critical steps were investigated. First, recombinant and nonrecombinant sources of PNGase F were compared to assess variations in the released glycans. Second, modifications in the INLIGHT™ derivatization step were evaluated including temperature optimization, solvent composition changes, and reaction condition length and tag concentration. Optimization of the modified method resulted in 20–100 times greater peak areas for the detected N-linked glycans in fetuin and horseradish peroxidase compared to the standard method. Furthermore, the identification of low abundance glycans, such as (Fuc)(1)(Gal)(2)(GlcNAc)(4)(Man)(3)(NeuAc)(1) and (Gal)(3)(GlcNAc)(5)(Man)(3)(NeuAc)(3,) was possible. Finally, the optimal LC setup for the INLIGHT™ derivatized N-linked glycan analyses was found to be a C18 reverse-phase (RP) column with mobile phases typical of RPLC.

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.

NGLY1 deficiency is a rare recessive genetic disease caused by mutations in the NGLY1 gene which codes for N-glycanase 1 (NGLY1). Here, we report the generation of two gene corrected iPSC lines using a patient-derived iPSC line (NCATS-CL6103) that carried a homozygous p.R401X mutation in the NGLY1 gene. These lines contain either one (NCATS-CL6104) or two (NCATS-CL6105) CRISPR/Cas9 corrected alleles of NGLY1. This pair of NGLY1 mutation corrected iPSC lines can be used as a control for the NCATS-CL6103 which serves as a cell-based NGLY1 disease model for the study of the disease pathophysiology and evaluation of therapeutics under development.

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.

Reliable biomarkers for oral cancer (OC) remain scarce, and routine tests for the detection of precancerous lesions are not routine in the clinical setting. This study addresses a current unmet need for more sensitive and quantitative tools for the management of OC. Whole saliva was used to identify and characterize the nature of glycans present in saliva and determine their potential as OC biomarkers. Proteins obtained from whole saliva were subjected to PNGase F enzymatic digestion. The resulting N-glycans were analyzed with weak anion exchange chromatography, exoglycosidase digestions coupled to ultra-high performance liquid chromatography and/or mass spectrometry. To determine N-glycan changes, 23 individuals with or without cancerous oral lesions were analyzed using Hydrophilic interaction ultra performance liquid chromatography (HILIC–UPLC), and peak-based area relative quantitation was performed. An abundant and complex salivary N-glycomic profile was identified. The main structures present in saliva were neutral oligosaccharides consisting of high mannose, hybrid and complex structures, followed by smaller fractions of mono and di-sialylated structures. To determine if differential N-glycosylation patterns distinguish between OC and control groups, Mann–Whitney testing and principle component analysis (PCA) were used. Eleven peaks were shown to be statistically significant (P ≤ 0.05), while PCA analysis showed segregation of the two groups based on their glycan profile. N-glycosylation changes are active in the oral carcinogenic process and may serve as biomarkers for early detection to reduce morbidity and mortality. Identifying which N-glycans contribute most in the carcinogenic process may lead to their use in the detection, prognosis and treatment of OC.

Entamoeba histolytica harbors an extensive intracellular distribution of ubiquitin-proteasome systems important for numerous cellular processes. However, glycosylation studies of ubiquitin-proteasome components have not yet been elucidated. Here we report the partial characterization of N-linked glycosylation profile in E. histolytica ubiquitin by Fluorophore-Assisted Carbohydrate Electrophoresis (FACE), Nanoelectrospray Ionization-Tandem Mass Spectrometry (NSI-MS), Matrix-Assisted Laser-Desorption time-of-flight Mass Spectrometry (MALDI-TOF MS) and Gas Chromatography-Mass Spectrometry (GC-MS) analysis. To our knowledge, the data presented in this report represents the first structural glycomics analysis of E. histolytica ubiquitin, while most of the reports are performed on whole parasitic glycan profiles. The glycan profile of E. histolytica ubiquitin has high mannose N-glycan structures. The N-linked glycan profile showed fragments from Hex3HexNAc2 to Hex9HexNAc2. Based in our findings and ubiquitin function, we hypothesize that the same ubiquitin Asn-Asp-Ser sequon carries heterogenic glycosylations, at different metabolic pathway stages according to ubiquitin functional requirements. Finally, we propose a set of possible high mannose N-glycan structures that will help to elucidate the ubiquitin biochemical composition and may well represent good targets for anti-amoebic drugs.

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.

NGLY1 deficiency is a rare disorder caused by mutations in the NGLY1 gene which codes for the highly conserved N-glycanase1 (NGLY1). This enzyme functions in cytosolic deglycosylation of N- linked glycoproteins. An induced pluripotent stem cell (iPSC) line was generated from the dermal fibroblasts of a 2-year-old patient carrying compound heterozygous mutations, p.R390P and p.L318P in the NGLY1 gene. This cell-based iPSC disease model provides a resource to study disease pathophysiology and to develop a cell-based disease model for drug development for NGLY1 patients.

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.

Talaromyces marneffei is a dimorphic fungus that has emerged as an opportunistic pathogen particularly in individuals with HIV/AIDS. Since its dimorphism has been associated with its virulence, the transition from mold to yeast-like cells might be important for fungal pathogenesis, including its survival inside of phagocytic host cells. We investigated the expression of yeast antigen of T.marneffei using a yeast-specific monoclonal antibody (MAb) 4D1 during phase transition. We found that MAb 4D1 recognizes and binds to antigenic epitopes on the surface of yeast cells. Antibody to antigenic determinant binding was associated with time of exposure, mold to yeast conversion, and mammalian temperature. We also demonstrated that MAb 4D1 binds to and recognizes conidia to yeast cells’ transition inside of a human monocyte-like THP-1 cells line. Our studies are important because we demonstrated that MAb 4D1 can be used as a tool to study T.marneffei virulence, furthering the understanding of the therapeutic potential of passive immunity in this fungal pathogenesis.

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.

N-Glycanase 1 (NGLY1) is a cytoplasmic deglycosylating enzyme. Loss-of-function mutations in the NGLY1 gene cause NGLY1 deficiency, which is characterized by developmental delay, seizures, and a lack of sweat and tears. To model the phenotypic variability observed among patients, we crossed a Drosophila model of NGLY1 deficiency onto a panel of genetically diverse strains. The resulting progeny showed a phenotypic spectrum from 0 to 100% lethality. Association analysis on the lethality phenotype, as well as an evolutionary rate covariation analysis, generated lists of modifying genes, providing insight into NGLY1 function and disease. The top association hit was Ncc69 (human NKCC1/2), a conserved ion transporter. Analyses in NGLY1-/- mouse cells demonstrated that NKCC1 has an altered average molecular weight and reduced function. The misregulation of this ion transporter may explain the observed defects in secretory epithelium function 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.

The glycosylation of proteins is typically considered as a stabilizing modification, including resistance to proteolysis. A class of peptidases, referred to as glycopeptidases or O-glycopeptidases, circumvent the protective effect of glycans against proteolysis by accommodating the glycans in their active sites as specific features of substrate recognition. IMPa from Pseudomonas aeruginosa is such an O-glycopeptidase that cleaves the peptide bond immediately preceding a site of O-glycosylation, and through this glycoprotein-degrading function contributes to the host-pathogen interaction. IMPa, however, is a relatively large multidomain protein and how its additional domains may contribute to its function remains unknown. Here, through the determination of a crystal structure of IMPa in complex with an O-glycopeptide, we reveal that the N-terminal domain of IMPa, which is classified in Pfam as IMPa_N_2, is a proline recognition domain that also shows the properties of recognizing an O-linked glycan on the serine/threonine residue following the proline. The proline is bound in the center of a bowl formed by four functionally conserved aromatic amino acid side chains while the glycan wraps around one of the tyrosine residues in the bowl to make classic aromatic ring-carbohydrate CH-π interactions. This structural evidence provides unprecedented insight into how the ancillary domains in glycoprotein-specific peptidases can noncatalytically recognize specific glycosylated motifs that are common in mucin and mucin-like molecules.

In cells, asparagine/N-linked glycans are added to glycoproteins cotranslationally, in an attachment process that supports proper folding of the nascent polypeptide. We found that following pruning of N-glycan by the amidase PNGase F, the principal influenza vaccine antigen and major viral spike protein hemagglutinin (HA) spontaneously reattached N-glycan to its de-N-glycosylated positions when the amidase was removed from solution. This reaction, which we term N-glycanation, was confirmed by site-specific analysis of HA glycoforms by mass spectrometry prior to PNGase F exposure, during exposure to PNGase F, and after amidase removal. Iterative rounds of de-N-glycosylation followed by N-glycanation could be repeated at least three times and were observed for other viral glycoproteins/vaccine antigens, including the envelope glycoprotein (Env) from HIV. Covalent N-glycan reattachment was nonenzymatic as it occurred in the presence of metal ions that inhibit PNGase F activity. Rather, N-glycanation relied on a noncovalent assembly between protein and glycan, formed in the presence of the amidase, where linearization of the glycoprotein prevented this retention and subsequent N-glycanation. This reaction suggests that under certain experimental conditions, some glycoproteins can organize self-glycan addition, highlighting a remarkable self-assembly principle that may prove useful for re-engineering therapeutic glycoproteins such as influenza HA or HIV Env, where glycan sequence and structure can markedly affect bioactivity and vaccine efficacy.

Analysis of glycans in glycoproteins is often performed by liquid chromatography (LC) separation coupled with fluorescence detection and/or mass spectrometric detection. Enzymatically or chemically released glycans from glycoproteins are usually labeled by reductive amination with a fluorophore reagent. Although labeling techniques based on reductive amination have been well-established as sample preparation methods for fluorometric HPLC-based glycan analysis, they often include time-consuming and tedious purification steps. Here, we reported an alternative fluorescent labeling method based on the synthesis of hydrazone and its reduction using 9-fluorenylmethyl carbazate (Fmoc-hydrazine) as a fluorophore reagent. Using isomaltopentaose and N-glycans from human IgG, we optimized the Fmoc-labeling conditions and purification procedure of Fmoc-labeled N-glycans and applied the optimized method for the analysis of N-glycans released from four glycoproteins (bovine RNase B, human fibrinogen, human α1-acid glycoprotein, and bovine fetuin). The complete workflow for preparation of fluorescent-labeled N-glycans takes a total of 3.5 h and is simple to implement. The method presented here lowers the overall cost of a fluorescently labeled N-glycan and will be practically useful for the screening of disease-related glycans or routine analysis at an early stage of development of biopharmaceuticals.

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.

Background Although NGLY1 is known as a pivotal enzyme that catalyses the deglycosylation of denatured glycoproteins, information regarding the responses of human cancer and normal cells to NGLY1 suppression is limited. Methods We examined how NGLY1 expression affects viability, tumour growth, and responses to therapeutic agents in melanoma cells and an animal model. Molecular mechanisms contributing to NGLY1 suppression-induced anticancer responses were revealed by systems biology and chemical biology studies. Using computational and medicinal chemistry-assisted approaches, we established novel NGLY1-inhibitory small molecules. Results Compared with normal cells, NGLY1 was upregulated in melanoma cell lines and patient tumours. NGLY1 knockdown caused melanoma cell death and tumour growth retardation. Targeting NGLY1 induced pleiotropic responses, predominantly stress signalling-associated apoptosis and cytokine surges, which synergise with the anti-melanoma activity of chemotherapy and targeted therapy agents. Pharmacological and molecular biology tools that inactivate NGLY1 elicited highly similar responses in melanoma cells. Unlike normal cells, melanoma cells presented distinct responses and high vulnerability to NGLY1 suppression. Conclusion Our work demonstrated the significance of NGLY1 in melanoma cells, provided mechanistic insights into how NGLY1 inactivation leads to eradication of melanoma with limited impact on normal cells, and suggested that targeting NGLY1 represents a novel anti-melanoma strategy.

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

Yang et al. show that NGLY1, a deglycosylation enzyme, regulates mitochondrial homeostasis and mitophagy through transcription factor NRF1. In the absence of NGLY1, cellular clearance of damaged mitochondria by mitophagy is impaired, resulting in chronic activation of innate immune nucleic acid–sensing pathways., Mutations in the NGLY1 (N-glycanase 1) gene, encoding an evolutionarily conserved deglycosylation enzyme, are associated with a rare congenital disorder leading to global developmental delay and neurological abnormalities. The molecular mechanism of the NGLY1 disease and its function in tissue and immune homeostasis remain unknown. Here, we find that NGLY1-deficient human and mouse cells chronically activate cytosolic nucleic acid–sensing pathways, leading to elevated interferon gene signature. We also find that cellular clearance of damaged mitochondria by mitophagy is impaired in the absence of NGLY1, resulting in severely fragmented mitochondria and activation of cGAS–STING as well as MDA5–MAVS pathways. Furthermore, we show that NGLY1 regulates mitochondrial homeostasis through transcriptional factor NRF1. Remarkably, pharmacological activation of a homologous but nonglycosylated transcriptional factor NRF2 restores mitochondrial homeostasis and suppresses immune gene activation in NGLY1-deficient cells. Together, our findings reveal novel functions of the NGLY1–NRF1 pathway in mitochondrial homeostasis and inflammation and uncover an unexpected therapeutic strategy using pharmacological activators of NRF2 for treating mitochondrial and immune dysregulation.

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.

An efficient deglycosylation process is a key requirement for the identification and characterization of glycosylation during the production and purification of therapeutic antibodies. PNGase F is widely used for the deglycosylation of N-linked glycans. The commonly-used in-solution deglycosylation method is relatively time-consuming and requires several hours up to overnight for complete removal of all N-linked glycans. In order to develop a simple and efficient method for the rapid release of N-linked glycans from glycoproteins, we fabricated trypsin- and PNGase F-impregnated polyacrylamide gels in a commercial 200 μL volume pipette tip. Our enzyme reactor is based on simple photochemical copolymerization of monomers using the following procedure: (1) a pipette tip was filled with a gel solution comprising acrylamide, N,N’-methylene-bis-acrylamide containing PNGase F or trypsin with 2,2-azobis(2-methyl-N-(2-hydroxyethyl) propionamide) as a photocatalytic initiator; and (2) in situ polymerization of gel solution approximately 30 mm from the tip was performed by irradiation with a 365 nm blue LED beam from a distance 10 mm. The fixed enzymes maintained their activities in the polyacrylamide gel and the reaction was completed by 40 iterations of suction and discharge with a pipette (hereafter referred to as manual pipetting times) for 8 min with each enzyme digestion. Capillary electrophoresis (CE) of released glycans labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS) demonstrated quantitative recovery of glycans from selected glycoproteins.

NGLY1 deficiency is the first and only autosomal recessive congenital disorder of N-linked deglycosylation (NGLY1-CDDG). To date, no patients with NGLY1 deficiency has been reported from mainland China or East Asia in English literature. Here, we present six patients with a diagnosis of NGLY1-CDDG on the basis of clinical phenotype, genetic testing, and functional studies. We retrospectively analyzed clinical phenotypes and NGLY1 genotypes of six cases from four families. Informed consent was obtained for diagnosis and treatment. In-silico tools and in vitro enzyme activity assays were used to determine pathogenicity of NGLY1 varaints. All patients had typical features of NGLY1-CDDG, including global developmental delay, microcephaly, hypotonia, hypertransaminasemia, alacrimia, and feeding difficulty. Dysmorphic features found in our patients include flat nasal bridge, loose and hollow cheeks, short stature, malnutrition, and ptosis. Pachylosis could be a novel cutaneous feature that may be explained by lack of sweat. We found three novel variants, including one missense (c.982C > G/p.Arg328Gly), one splice site (c.1003+3A > G), and one frame-shift (c.1637-1652delCATCTTTTGCTTATAT/p.Ser546PhefsTer) variant. All mutations were predicted to be disease causing with in-silico prediction tools, and affected at least one feature of gene splicing. Protein modeling showed missense variants may affect covalent bonding within the protein structure, or interrupt active/binding amino-acid residues. In vitro studies indicated that proteins carrying missense variants (p.Arg328Gly and p.Tyr342Cys) lost the enzyme activity. We expanded clinical phenotype and genetic mutation spectrum of NGLY1-CDDG by reporting six cases, three novel variants, and novel clinical features from mainland China.

Extracellular vesicles (EVs) are important mediators of cell-cell communication in a broad variety of physiological contexts. However, there is ambiguity around the fundamental mechanisms by which these effects are transduced, particularly in relation to their uptake by recipient cells. Multiple modes of cellular entry have been suggested and we have further explored the role of glycans as potential determinants of uptake, using EVs from the murine hepatic cell lines AML12 and MLP29 as independent yet comparable models. Lectin microarray technology was employed to define the surface glycosylation patterns of EVs. Glycosidases PNGase F and neuraminidase which cleave N-glycans and terminal sialic acids, respectively, were used to analyze the relevance of these modifications to EV surface glycans on the uptake of fluorescently labelled EVs by a panel of cells representing a variety of tissues. Flow cytometry revealed an increase in affinity for EVs modified by both glycosidase treatments. High-content screening exhibited a broader range of responses with different cell types preferring different vesicle glycosylation states. We also found differences in vesicle charge after treatment with glycosidases. We conclude that glycans are key players in the tuning of EV uptake, through charge-based effects, direct glycan recognition or both, supporting glycoengineering as a toolkit for therapy development.

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.

NGLY1 deficiency is a rare genetic disease caused by mutations in the NGLY1 gene that encodes N-glycanase 1. The disease phenotype in patient cells is unclear. A human induced pluripotent stem cell (iPSC) line was generated from skin dermal fibroblasts of a patient with NGLY1 deficiency that has compound heterozygous mutations of a p.Q208X variant (c.622C > T) in exon 4 and a p.G310G variant (c.930C > T) in exon 6 of the NGLY1 gene. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for drug development to treat NGLY1 deficiency.

A plant expression platform with eukaryotic post-translational modification (PTM) machinery has many advantages compared to other protein expression systems. This promising technology is useful for the production of a variety of recombinant proteins including, therapeutic proteins, vaccine antigens, native additives, and industrial enzymes. However, plants lack some of the important PTMs, including furin processing, which limits this system for the production of certain mammalian complex proteins of therapeutic value. Furin is a ubiquitous proprotein convertase that is involved in the processing (activation) of a wide variety of precursor proteins, including blood coagulation factors, cell surface receptors, hormones and growth factors, viral envelope glycoproteins, etc. and plays a critical regulatory role in a wide variety of cellular events. In this study, we engineered the human furin gene for expression in plants and demonstrated the production of a functional active recombinant truncated human furin in N. benthamiana plant. We demonstrate that plant produced human furin is highly active both in vivo and in vitro and specifically cleaved the tested target proteins, Factor IX (FIX) and Protective Antigen (PA83). We also demonstrate that both, enzymatic deglycosylation and proteolytic processing of target proteins can be achieved in vivo by co-expression of deglycosylating and furin cleavage enzymes in a single cell to produce deglycosylated and furin processed target proteins. It is highly expected that this strategy will have many potential applications in pharmaceutical industry and can be used to produce safe and affordable therapeutic proteins, antibodies, and vaccines using a plant expression system.

Etanercept is a recombinant Fc fusion protein widely used to treat rheumatic diseases. This protein is highly glycosylated and contains numerous O- and N-glycosylation sites. Since glycosylation is recognized as an important critical quality attribute (CQA) that can affect immunogenicity, solubility, and stability of Fc fusion proteins, it should be thoroughly characterized. In this work, hydrophilic interaction chromatography (HILIC) was combined with high-resolution mass spectrometry (HRMS) by using a quadrupole time-of flight mass spectrometer to assess glycosylation of etanercept at the middle-up level of analysis (fragments of ca. 25−30 kDa). In addition, a combination of different enzymatic digestion procedures (i.e., glycosidase, sialidase, and protease) was systematically employed to facilitate spectra deconvolution. With the developed procedure, the main post-translational modifications (PTMs) of etanercept were assessed, and a global overview of the subunit-specific distribution of the glycosylation pattern was obtained at a middle-up level of analysis.

N-glycosylation profiling of glycoprotein biotherapeutics is an essential step in each phase of product development in the biopharmaceutical industry. For example, during clone selection, hundreds of clones should be analyzed quickly from limited amounts of samples. On the other hand, identification of disease related glycosylation alterations can serve as early indicators (glycobiomarkers) for various pathological conditions in the biomedical field. Therefore, there is a growing demand for rapid and easy to automate sample preparation methods for N-glycosylation analysis. In this paper, we report on the design and implementation of immobilized recombinant glutathione-S-transferase (GST) tagged PNGase F enzyme microcolumns for rapid and efficient removal of N-linked carbohydrates from glycoproteins. Digestion speed and efficiency were compared to conventional in-solution based protocols. The use of PNGase F functionalized microcolumns resulted in efficient N-glycan removal in 10min from all major N-linked glycoprotein types of: (i) neutral (IgG), (ii) highly sialylated (fetuin), and (iii) high mannose (ribonuclease B) carbohydrate containing glycoprotein standards. The approach can be readily applied to automated sample preparation systems, such as liquid handling robots.

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.

Plasma membrane proteome analysis is of significance for screening candidate biomarkers and drug targets. However, due to their low abundance and lack of specific groups that can enable their capture, the plasma membrane proteins (PMPs) are under-represented. On the basis of the fact that PMPs are embedded in or anchored to the phospholipid bilayer of the plasma membrane and the glycan moieties of proteins and lipids located on the plasma membrane are exposed outside of the cell surface, we proposed a strategy to capture PMPs, termed as glycan moieties-directed PMPs enrichment (GMDPE). With the glycan moieties exposed outside of the cells as bait to ensure the selectivity and the phospholipid bilayer as raft to provide the sensitivity, we applied this strategy into the plasma membrane proteome analysis of HeLa cells, and in total, 772 PMPs were identified, increased by 4.5 times compared to those identified by the reported cell surface biotinylation method. Notably, among them, 86 CD antigens and 16 ion channel proteins were confidently identified. All these results demonstrated that our proposed approach has great potential in the large scale plasma membrane proteome profiling.

NGLY1/Ngly1 is a cytosolic peptide:N-glycanase, i.e. de-N-glycosylating enzyme acting on N-glycoproteins in mammals, generating free, unconjugated N-glycans and deglycosylated peptides in which the N-glycosylated asparagine residues are converted to aspartates. This enzyme is known to be involved in the quality control system for the newly synthesized glycoproteins in the endoplasmic reticulum (ER). In this system, misfolded (glyco)proteins are retrotranslocated to the cytosol, where the 26S proteasomes play a central role in degrading the proteins: a process referred to as ER-associated degradation or ERAD in short. PNGase-mediated deglycosylation is believed to facilitate the efficient degradation of some misfolded glycoproteins. Human patients harboring mutations of NGLY1 gene (NGLY1-deficiency) have recently been discovered, clearly indicating the functional importance of this enzyme. This review summarizes the current state of our knowledge on NGLY1 and its gene product in mammalian cells.

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.

Reactivation of T cell immunity by PD-1/PD-L1 immune checkpoint blockade has been shown to be a promising cancer therapeutic strategy. However, PD-L1 immunohistochemical readout is inconsistent with patient response, which presents a clinical challenge to stratify patients. Because PD-L1 is heavily glycosylated, we developed a method to resolve this by removing the glycan moieties from cell surface antigens via enzymatic digestion, a process termed sample deglycosylation. Notably, deglycosylation significantly improves anti-PD-L1 antibody binding affinity and signal intensity, resulting in more accurate PD-L1 quantification and prediction of clinical outcome. This proposed method of PD-L1 antigen retrieval may provide a practical and timely approach to reduce false-negative patient stratification for guiding anti-PD-1/PD-L1 therapy.

Eel follicle-stimulating hormone (eelFSH) is composed of a common α-subunit and a hormone specific β-subunit, both of which contain two N-linked carbohydrate residues. We characterized the biologically active single chains by fusing the α-subunit to the carboxyl terminal region of the eelFSH β-subunit. Expression vectors were constructed and the biological activity of the recombinant hormones (rec-hormones) was characterized using Chinese hamster ovary (CHO) K1 cells expressing the eelFSH receptor gene. Mutagenesis of the individual and double glycosylated sites was performed to determine the functions of the oligosaccharide chains on signal transduction. The absence of the Asn22 (eelFSHβΔ22/α) and Asn5.22 (eelFSHβΔ5.22/α) N-linked oligosaccharide chain in the eelFSH β-subunit completely reduced the secretion level in the medium and cell lysate of CHO-K1 cells. The expression levels of eelFSHβ/α wild-type in CHO suspension (CHO-S) cells was approximately 4-fold higher in CHO-k1 cells. The molecular weight of rec-eelFSHβ/α wild-type by western blotting analysis was found to be 34 kDa. Mutants (β/αΔ56, β/αΔ79, and βΔ5/α) lacking single oligosaccharide sites showed molecular weights that were reduced by approximately 10%. The digestion of N-linked oligosaccharides using PNGaseF treatment showed that the molecular weights of all mutants were reduced to 27-kDa. The oligosaccharide chains in rec-eelFSHβ/α wild-type were modified to a molecular weight of approximately 7–10 kDa in CHO-K1 and CHO-S cells. Oligosaccharide site deletions at positions Asn56 and Asn79 on the α-subunit and Asn5 on the β-subunit were found to play an essential role in cAMP signal transduction through the eelFSH receptor. The EC50 values of Asn56 and Asn5 resulted in a significant decrease in potency to 64% and 53% of the wild type, respectively. Specifically, the removal of the carbohydrates at Asn79 of the α-subunit (β/αΔ79) was drastically reduced to 53.8% of the wild-type levels in maximum response. These results have allowed for the identification of the site-specific roles of carbohydrate residues in eel FSH. Our data suggest that N-linked oligosaccharide chains play a pivotal role in biological activity through the eelFSH receptor as suggested in similar studies of other mammalian FSH hormones.