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Anti‐proinflammatory cytokine therapies against interleukin (IL)‐6, tumor necrosis factor (TNF)‐α, and IL‐1 are major advancements in treating inflammatory diseases, especially rheumatoid arthritis. Such therapies are mainly performed by injection of antibodies against cytokines or cytokine receptors. We initially found that the glycolytic inhibitor 2‐deoxy‐d‐glucose (2‐DG), a simple monosaccharide, attenuated cellular responses to IL‐6 by inhibiting N‐linked glycosylation of the IL‐6 receptor gp130. Aglycoforms of gp130 did not bind to IL‐6 or activate downstream intracellular signals that included Janus kinases. 2‐DG completely inhibited dextran sodium sulfate‐induced colitis, a mouse model for inflammatory bowel disease, and alleviated laminarin‐induced arthritis in the SKG mouse, an experimental model for human rheumatoid arthritis. These diseases have been shown to be partially dependent on IL‐6. We also found that 2‐DG inhibited signals for other proinflammatory cytokines such as TNF‐α, IL‐1β, and interferon ‐γ, and accordingly, prevented death by another inflammatory disease, lipopolysaccharide (LPS) shock. Furthermore, 2‐DG prevented LPS shock, a model for a cytokine storm, and LPS‐induced pulmonary inflammation, a model for acute respiratory distress syndrome of coronavirus disease 2019 (COVID‐19). These results suggest that targeted therapies that inhibit cytokine receptor glycosylation are effective for treatment of various inflammatory diseases. [ABSTRACT FROM AUTHOR]

Ferroptosis is an oxidative form of nonapoptotic cell death whose transcriptional regulation is poorly understood. Cap'n'collar (CNC) transcription factors including nuclear factor erythroid-2-related factor 1 (NFE2L1/NRF1) and NFE2L2 (NRF2) are important regulators of oxidative stress responses. NFE2L1 abundance and function are regulated posttranslationally by N-glycosylation. Functional maturation of NFE2L1 requires deglycosylation by cytosolic peptide:N-glycanase 1 (NGLY1). We find that NGLY1 and NFE2L1 work in a common pathway to enhance ferroptosis resistance, independent of NFE2L2, by promoting expression of the key antiferroptotic enzyme glutathione peroxidase 4 (GPX4). Enhanced ferroptosis sensitivity in NFE2L1-knockout cells can be reverted by expression of an NFE2L1 mutant containing eight asparagine-to-aspartate protein sequence substitutions, which mimic NGLY1-catalyzed protein sequence editing. These results suggest that ferroptosis sensitivity may be regulated by NGLY1-catalyzed NFE2L1 deglycosylation. These results highlight a role for the disease-associated NGLY1/NFE2L1 pathway in ferroptosis regulation. [ABSTRACT FROM AUTHOR]

Morus nigra L. (black mulberry-BM) is a promising nutraceutical fruit containing biologically active polyphenols like anthocyanins, proanthocyanidins, catechins, and stilbenes, with well-established anti-inflammatory, antidiabetic, anti-obesity, and anticancer biofunctions. However, these health-promoting properties in raw fruit are greatly masked due to the presence of soluble and insoluble carbohydrates in excess amounts restricting daily intake of the required dose to achieve targeted effects. In the current study, different protein sources (defatted whey and chickpea flours) were optimized through different conditions to capture polyphenols from BM juice while diminishing its glucose content. To optimize polyphenol-protein interactions, various pHs (3.7, 4.2, and 4.7), matrix concentrations (20, 50, and 80 g protein/L), and incubation times (5, 20, and 45 min) were tested. In the present work, optimized BM polyphenol enriched whey matrix inhibited pro-inflammatory mediators and promoted Nrf-2 dependent cytoprotective enzyme expressions in lipopolysaccharide (LPS) induced macrophages at low doses. In addition, whey proteins were also subjected to an enzymatic deglycosylation process by using recently identified EndoBI-1 enzyme for the specific cleavage of N-glycan core in all glycan types including high mannoses, hybrids as well as complex glycans found on defatted whey proteins. After this process, the polyphenol sorption capacity of deglycosylated whey proteins was found to be significantly higher (37%) than the capacity of non-treated normal whey protein under optimized conditions. In conclusion, deglycosylation of protein matrices could be a novel strategy for efficient sorption/concentration of polyphenols from fruits and vegetables, however, more detailed studies are needed to understand this effect. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cytosolic peptide: N -glycanase (PNGase; NGLY1), an enzyme responsible for de-glycosylation of N -glycans on glycoproteins, is known to play pivotal roles in a variety of biological processes. In 2012, NGLY1 deficiency, a rare genetic disorder, was reported and since then, more than 100 patients have now been identified worldwide. Patients with this disease exhibit several common symptoms that are caused by the dysfunction of NGLY1. However, correlation between the severity of patient symptoms and the extent of the reduction in NGLY1 activity in these patients remains to be clarified, mainly due to the absence of a facile quantitative assay system for this enzyme, especially in a crude extract as an enzyme source. In this study, a quantitative, non-radioisotope (RI)-based assay method for measuring recombinant NGLY1 activity was established using a BODIPY-labeled asialoglycopeptide (BODIPY-ASGP) derived from hen eggs. With this assay, the activities of 27 recombinant NGLY1 mutants that are associated with the deficiency were examined. It was found that the activities of three (R469X, R458fs and H494fs) out of the 27 recombinant mutant proteins were 30–70% of the activities of wild-type NGLY1. We further developed a method for measuring endogenous NGLY1 activity in crude extracts derived from cultured cells, patients' fibroblasts, iPS cells or peripheral blood mononuclear cells (PBMCs), using a glycosylated cyclopeptide (GCP) that exhibited resistance to the endogenous proteases in the extract. Our methods will not only provide new insights into the molecular mechanism responsible for this disease but also promises to be applicable for its diagnosis. [ABSTRACT FROM AUTHOR]

Abstract Sugar units in natural products are pharmacokinetically important but often redundant and therefore obstructing the study of the structure and function of the aglycon. Therefore, it is recommended to remove the sugars before a theoretical or experimental study of a molecule. Deglycogenases, enzymes that specialized in sugar removal from small molecules, are often used in laboratories to perform this task. However, there is no standardized computational procedure to perform this task in silico. In this work, we present a systematic approach for in silico removal of ring and linear sugars from molecular structures. Particular attention is given to molecules of biological origin and to their structural specificities. This approach is made available in two forms, through a free and open web application and as standalone open-source software.

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.

This study reports a novel BglA9 gene of 1345 bp encoding β-glucosidase from Anoxybacillus ayderensis A9, which was amplified and expressed in E. coli BL21 (DE3): pLysS cells, purified with Ni-NTA column having molecular weight of 52.6 kDa and was used in the bioconversion of polydatin to resveratrol. The kinetic parameters values using p NPG as substrate were K m (0.28 mM), V max (43.8 μmol/min/mg), k cat (38.43 s−1) and k cat / K m (135.5 s−1 mM−1). The BglA9 was active in a broad pH range and had an activity half-life around 24 h at 50 °C. The de-glycosylation efficiency of BglA9 for polydatin was determined by estimating the amount of glucose released after enzymatic reaction by a dinitrosalicylic acid (DNS) assay. The kinetic parameters of BglA9 for polydatin were 5.5 mM, 20.84 μmol/min/mg, 18.28 s−1and 3.27 s−1 mM−1 for K m , V max , k cat , and k cat / K m values, respectively. The K i value for glucose was determined to be 1.7 M. The residues Gln19, His120, Glu355, Glu409, Glu178, Asn222 may play a crucial role in the deglycosylation as revealed by the 3D structure of enzyme docked with polydatin. [ABSTRACT FROM AUTHOR]

Platycosides are the functional saponins present in balloon flowers that exert diverse biological effects, and which can be further improved by their deglycosylation. Deapiose-xylosylated platycodin D, which is absent in balloon flowers, can be generated only by cytolase PCL5 by acting on platycoside E. To improve cytolase PCL5-catalyzed production of deapiose-xylosylated platycodin D from platycoside E, we explored the use of high hydrostatic pressure (HHP). At an HHP of 150 MPa, the optimal temperature of cytolase PCL5 activity for converting platycoside E into deapiose-xylosylated platycodin D shifted from 50 to 55 °C, and increased the activity and stability of the enzyme by 5- and 4.9-fold, respectively. Under HHP, the enzyme completely converted 1 mM platycoside E into deapiose-xylosylated platycodin D within 4 h, with a 3.75-fold higher productivity than that under atmospheric pressure. Our results suggest that the application of HHP is a potential method for the economical production of platycosides and enzyme-catalyzed biotransformation of functional saponins. [ABSTRACT FROM AUTHOR]

Background: Xenogeneic pericardium has been used largely for various applications in cardiovascular surgery. Nevertheless, xenogeneic pericardial patches fail mainly due to their antigenic components. The xenoantigens identified as playing a major role in recipient immune response are the Galα1‐3Gal (α‐Gal) epitope, the non‐human sialic acid N‐glycolylneuraminic acid (Neu5Gc), and the porcine SDa antigen, associated with both proteins and lipids. The reduction in glycans from porcine pericardium might hinder or reduce the immunogenicity of xenogeneic scaffolds. Methods: Decellularized porcine pericardia were further treated at different time points and dilutions with digestive enzymatic supplements and enzymatic mixtures applied for food industry, for the removal of potentially immunogenic carbohydrates. Carbohydrates removal was investigated using up to 8 different lectin stains for the identification of N‐ and O‐glycosylations, as well as glycolipids. Histoarchitectural changes in the ECM were assessed using Elastica van Gieson stain, whereas changes in mechanical properties were investigated via uniaxial tensile test and burst pressure test. Results: Tissues after enzymatic treatments showed a dramatic decrease in lectin stainings in comparison to tissues which were only decellularized. Histological assessment revealed cell‐nuclei removal after decellularization. Some of the enzymatic treatments induced elastic lamellae disruption. Tissue strength decreased after enzymatic treatment; however, treated tissues showed values of burst pressure higher than physiological transvalvular pressures. Conclusions: The application of these enzymatic treatments for tissue deglycosylation is totally novel, low cost, and appears to be very efficient for glycan removal. The immunogenic potential of treated tissues will be further investigated in subsequent studies, in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Milk glycoproteins are involved in different functions and contribute to different cellular processes, including adhesion and signaling, and shape the development of the infant microbiome. Methods have been developed to study the complexities of milk protein glycosylation and understand the role of N-glycans in protein functionality. Endo-β-N-acetylglucosaminidase (EndoBI-1) isolated from Bifidobacterium longum subsp. infantis ATCC 15697 is a recently isolated heat-stable enzyme that cleaves the N-N'-diacetyl chitobiose moiety found in the N-glycan core. The effects of different processing conditions (pH, temperature, reaction time, and enzyme/protein ratio) were evaluated for their ability to change EndoBI-1 activity on bovine colostrum whey glycoproteins using advanced mass spectrometry. This study shows that EndoBI-1 is able to cleave a high diversity of N-glycan structures. Nano-LC-Chip-Q-TOF MS data also revealed that different reaction conditions resulted in different N-glycan compositions released, thus modifying the relative abundance of N-glycan types. In general, more sialylated N-glycans were released at lower temperatures and pH values. These results demonstrated that EndoBI-1 is able to release a wide variety of N-glycans, whose compositions can be selectively manipulated using different processing conditions.

EndoBI-1 is a recently isolated endo-β-N-acetylglucosaminidase, which cleaves the N-N'-diacetyl chitobiose moiety found in the N-glycan core of high mannose, hybrid and complex N-glycans. These N-glycans have selective prebiotic activity for a key infant gut microbe, Bifidobacterium longum subsp. infantis. The broad specificity of EndoBI-1 suggests the enzyme may be useful for many applications, particularly for deglycosylating milk glycoproteins in dairy processing. To facilitate its commercial use, we determined kinetic parameters for EndoBI-1 on the model substrates ribonuclease B and bovine lactoferrin, as well as on concentrated bovine colostrum whey. Km values ranging from 0.25 to 0.49, 0.43 to 1.00 and 0.90 to 3.18 mg/mL and Vmax values ranging from 3.5×10(-3) to 5.09×10(-3), 4.5×10(-3) to 7.75×10(-3) and 1.9×10(-2)to 5.2×10(-2) mg/mL×min were determined for ribonuclease B, lactoferrin and whey, respectively. In general, EndoBI-1 showed the highest apparent affinity for ribonuclease B, while the maximum reaction rate was the highest for concentrated whey. EndoBI-1-released N-glycans were quantified by a phenol-sulphuric total carbohydrate assay and the resultant N-glycan structures monitored by nano-LC-Chip-Q-TOF MS. The kinetic parameters and structural characterization of glycans released suggest EndoBI-1 can facilitate large-scale release of complex, bioactive glycans from a variety of glycoprotein substrates. Moreover, these results suggest that whey, often considered as a waste product, can be used effectively as a source of prebiotic N-glycans.

Background: Despite clinical success with anti-spike vaccines, the effectiveness of neutralizing antibodies and vaccines has been compromised by rapidly spreading SARS-CoV-2 variants. Viruses can hijack the glycosylation machinery of host cells to shield themselves from the host's immune response and attenuate antibody efficiency. However, it remains unclear if targeting glycosylation on viral spike protein can impair infectivity of SARS-CoV-2 and its variants. Methods: We adopted flow cytometry, ELISA, and BioLayer interferometry approaches to assess binding of glycosylated or deglycosylated spike with ACE2. Viral entry was determined by luciferase, immunoblotting, and immunofluorescence assays. Genome-wide association study (GWAS) revealed a significant relationship between STT3A and COVID-19 severity. NF-κB/STT3A-regulated N-glycosylation was investigated by gene knockdown, chromatin immunoprecipitation, and promoter assay. We developed an antibody-drug conjugate (ADC) that couples non-neutralization anti-spike antibody with NGI-1 (4G10-ADC) to specifically target SARS-CoV-2-infected cells. Findings: The receptor binding domain and three distinct SARS-CoV-2 surface N-glycosylation sites among 57,311 spike proteins retrieved from the NCBI-Virus-database are highly evolutionarily conserved (99.67%) and are involved in ACE2 interaction. STT3A is a key glycosyltransferase catalyzing spike glycosylation and is positively correlated with COVID-19 severity. We found that inhibiting STT3A using N-linked glycosylation inhibitor-1 (NGI-1) impaired SARS-CoV-2 infectivity and that of its variants [Alpha (B.1.1.7) and Beta (B.1.351)]. Most importantly, 4G10-ADC enters SARS-CoV-2-infected cells and NGI-1 is subsequently released to deglycosylate spike protein, thereby reinforcing the neutralizing abilities of antibodies, vaccines, or convalescent sera and reducing SARS-CoV-2 variant infectivity. Interpretation: Our results indicate that targeting evolutionarily-conserved STT3A-mediated glycosylation via an ADC can exert profound impacts on SARS-CoV-2 variant infectivity. Thus, we have identified a novel deglycosylation method suitable for eradicating SARS-CoV-2 variant infection in vitro. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section

As the main saponin component of Platycodon grandiflorum A.DC, Platycodin D has been reported to have an anti-obesity effect. Due to poor oral absorption, the intestinal microflora usually transforms saponins into potential bioactive substances. In this study, we profiled the metabolic changes of platycodin D by incubating it with intestinal microflora extracted from mice feces subjected to either a standard control diet or a high-fat diet. A UPLC-LTQ-Orbitrap-MS method was used for rapid analysis of the metabolic profile of platycodin D. A total of 10 compounds were identified 9 of which were assessed to be metabolized by intestinal microflora. Dehydroxylation and deglycosylation were the major metabolic process of platycodin D. The metabolic profile of platycodin D biotransformed by intestinal microflora was elucidated based on the metabolite information. Platycodin D and its metabolites had anti-inflammatory effects in LPS-stimulated RAW 264.7 cells. Only platycodin D could alleviate lipid accumulation in FFA-treated HepG2 cells. • The biotransformation of platycodin D by intestinal microflora of mice fed a high-fat diet was firstinvestigated. • 10 metabolites were tentatively characterized. • The gut microbiota fed control diet metabolize PD faster than fed high-fat diet. • The anti-inflammatory and anti-lipid accumulation effects of platycodin D and metabolites were compared. • It was first found that platycodigenin has anti-inflammatory effect in vitro. [ABSTRACT FROM AUTHOR]

[Display omitted] • The genome of Deinococcus aerius contains a gene cluster with three genes responsible for deglycosylation of C -glycosyl compounds. • The enzyme system is specific for flavone compounds with a glucosyl moiety attached at the C6 position of the aromatic aglycone group. • The enzyme (Da CGD) that catalyze cleavage of the C–C bond features a new oligomeric state not previously observed for CGDs produced by soil or gut bacteria. • Lysine 261 in DaCGD is suitably positioned to activate a catalytic metal-coordinated hydroxide ion, which we propose may play a role in the catalytic mechanism. Plant C -glycosylated aromatic polyketides are important for plant and animal health. These are specialized metabolites that perform functions both within the plant, and in interaction with soil or intestinal microbes. Despite the importance of these plant compounds, there is still limited knowledge of how they are metabolized. The Gram-positive aerobic soil bacterium Deinococcus aerius strain TR0125 and other Deinococcus species thrive in a wide range of harsh environments. In this work, we identified a C -glycoside deglycosylation gene cluster in the genome of D. aerius. The cluster includes three genes coding for a GMC-type oxidoreductase (Da CGO1) that oxidizes the glucosyl C3 position in aromatic C -glucosyl compounds, which in turn provides the substrate for the C -glycoside deglycosidase (Da CGD; composed of α+β subunits) that cleaves the glucosyl-aglycone C–C bond. Our results from size-exclusion chromatography, single particle cryo-electron microscopy and X-ray crystallography show that Da CGD is an α 2 β 2 heterotetramer, which represents a novel oligomeric state among bacterial CGDs. Importantly, the high-resolution X-ray structure of Da CGD provides valuable insights into the activation of the catalytic hydroxide ion by Lys261. Da CGO1 is specific for the 6- C -glucosyl flavones isovitexin, isoorientin and the 2- C -glucosyl xanthonoid mangiferin, and the subsequent C–C-bond cleavage by Da CGD generated apigenin, luteolin and norathyriol, respectively. Of the substrates tested, isovitexin was the preferred substrate (Da CGO1, K m 0.047 mM, k cat 51 min−1; Da CGO1/ Da CGD, K m 0.083 mM, k cat 0.42 min−1). [ABSTRACT FROM AUTHOR]

Abstract Background Flavone C-glycosides are difficult to be deglycosylated using traditional chemical methods due to their solid carbon–carbon bond between sugar moieties and aglycones; however, some bacteria may easily cleave this bond because they generate various specific enzymes. Results A bacterial strain, named W12-1, capable of deglycosylating orientin, vitexin, and isovitexin to their aglycones, was isolated from human intestinal bacteria in this study and identified as Enterococcus faecalis based on morphological examination, physiological and biochemical identification, and 16S rDNA sequencing. The strain was shown to preferentially deglycosylate the flavone C-glycosides on condition that the culture medium was short of carbon nutrition sources such as glucose and starch, and its deglycosylation efficiency was negatively correlated with the content of the latter two substances. Conclusion This study provided a new bacterial resource for the cleavage of C-glycosidic bond of flavone C-glycosides and reported the carbon nutrition sources reduction induced deglycosylation for the first time.

Ovomucoid (OVM), known as the major allergen in egg white, has gained increasing concerns in industrialized countries. Here, we found the deglycosylation and Maillard reaction with galactooligosaccharide (GOS) and fructooligosaccharide (FOS) can induce conformational transformation of OVM from other structures (β‐turn, strang, and random coils) to α‐helix. We also introduced an approach to reduce the allergenicity of Gallus domesticus OVM by Maillard reaction with GOS and FOS. However, the OVM glycated by mannosan (MOS) and deglycosylated OVM exhibited higher allergenicity than native OVM. Therefore, GOS and FOS, especially GOS, could be applied in the reduction of the potential allergenicity of OVM through glycation. Furthermore, these findings may provide new insights into the development of hypoallergenic egg products. Practical Application: In this study, the allergenicity and conformation of OVM treated with deglycosylation and glycation (GOS, FOS, and MOS) were investigated. The results would provide a better understanding of the effects of deglycosylation and Maillard reaction with different reducing sugars on the molecular characteristics of OVM and further provide new insights into the development of hypoallergenic egg products. [ABSTRACT FROM AUTHOR]

In the process of glycosyltransferase-catalyzed transglycosylation, the synthesis of uridine diphosphate sugars is generally thermodynamically disfavored. This study found that GTBP1 from Bacillus pumilus BF1 displayed the potential to reversely synthesize uridine diphosphate glucose (UDPG, UDP-glucose), which catalyzed the deglycosylation of ponasteroside A and the transglycosylation of phenolic compounds in one-pot reaction. Sequence alignment and phylogenetic tree analysis of GTBP1 and other GTs with reversible glycosylation ability were also implemented. Using solvent engineering strategy, the reaction time in water-organic biphasic was shortened and the conversion was improved. The final conversion of ponasterone A was reached 93.4%, and the final yield of ferulic acid glycoside was 92.7%. Using the fed-batch technology with the coupled reaction, the cumulative product of ponasterone A was about 1.97 g/L, and the level of produced ferulic acid glycoside was about 1.52 g/L. The substrate specificity of the GTBP1 was also confirmed. It implies the diversity of GTBP1's ability to construct UDP cycles. [ABSTRACT FROM AUTHOR]

The binding of mouse IgG3 to Fcγ receptors (FcγR) and the existence of a mouse IgG3-specific receptor have been discussed for 40 years. Recently, integrin beta-1 (ITGB1) was proposed to be a part of an IgG3 receptor involved in the phagocytosis of IgG3-coated pathogens. We investigated the interaction of mouse IgG3 with macrophage-like J774A.1 and P388D1 cells. The existence of an IgG3-specific receptor was verified using flow cytometry and a rosetting assay, in which erythrocytes clustered around the macrophage-like cells coated with an erythrocyte-specific IgG3. Our findings confirmed that receptors binding antigen-free IgG3 are present on J774A.1 and P388D1 cells. We demonstrated for the first time that the removal of N-glycans from IgG3 completely abolished its binding to the cells. Moreover, we discovered that the cells treated with Accutase did not bind IgG3, indicating that IgG3-specific receptors are substrates of this enzyme. The results of antibody-mediated blocking of putative IgG3 receptors suggested that apart from previously proposed ITGB1, FcγRII, FcγRIII, also additional, still unknown, receptor is involved in IgG3 binding. These findings indicate that there is a complex network of glycan-dependent interactions between mouse IgG3 and the surface of effector immune cells. [ABSTRACT FROM AUTHOR]

NGLY1 deficiency is an ultra-rare autosomal recessive disorder caused by loss of function variants in the NGLY1 gene, representing the first known congenital disorder of deglycosylation. The disorder is characterized by a range of core features, including global developmental delay and/or intellectual disability, hyperkinetic movement disorder, transient elevation of liver transaminases, hypo- or alacrima, and peripheral neuropathy. Additional symptoms such as microcephaly, hypotonia, EEG abnormalities, brain imaging abnormalities, GI disturbances, and a history of intrauterine growth restriction have been reported in some cases. NGLY1 gene mutations lead to reduced or absent N-glycanase 1 activity, resulting in the accumulation of abnormal glycoproteins within the endoplasmic reticulum and impairing protein quality control mechanisms. Diagnosis is primarily achieved through genetic testing, while the identification of the biomarker GlcNAc-Asn may facilitate diagnosis rates. Treatment options are currently limited to supportive care, although gene therapy is being developed. To advance understanding and intervention in NGLY1 deficiency, participation in patient registries, natural history studies, and interventional research is crucial. Genetic counseling is strongly recommended for carriers and individuals at risk, with consideration given to DNA preservation for future evaluations and targeted treatments. [ABSTRACT FROM AUTHOR]

BackgroundThe hygiene hypothesis suggests a link between parasitic infections and immune disorders, such as allergic diseases. We previously showed that infection with Toxoplasma gondii or systemic application of T. gondii tachyzoites lysate antigen (TLA) in a prophylactic, but not therapeutic protocol, prevented allergic airway inflammation in mice. Here we tested the effect of prophylactic and therapeutic application of TLA via the mucosal route.MethodsMice were intranasally treated with TLA either i) prior to sensitization, ii) during sensitization and challenge, or iii) after sensitization with ovalbumin (OVA). Recruitment of inflammatory cells to the lung, cytokine levels in restimulated lung and spleen cell cultures as well as levels of OVA-specific antibodies in serum were measured. In parallel, the effect of native TLA, heat-inactivated (hiTLA) or deglycosylated TLA (dgTLA) on sensitized splenocytes was evaluated ex vivo.ResultsWhen applied together with OVA i) during systemic sensitization and local challenge or ii) exclusively during local challenge, TLA reduced infiltration of eosinophils into the lung, OVA-specific type 2 cytokines in restimulated lung cell cultures, and partially, type 2 cytokines in restimulated spleen cell cultures in comparison to allergic controls. No beneficial effect was observed when TLA was applied prior to the start of sensitization. Analysis of epitope sugars on TLA indicated a high abundance of mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. Deglycosylation of TLA, but not heat-inactivation, abolished the potential of TLA to reduce type 2 responses ex vivo, suggesting a significant role of carbohydrates in immunomodulation.ConclusionWe showed that mucosal application of TLA reduced the development of experimental allergy in mice. The beneficial effects depended on the timing of the application in relation to the time point of sensitization. Not only co-application, but also therapy in sensitized/allergic animals with native TLA reduced local allergic responses. Furthermore, we show that TLA is highly glycosylated and glycoconjugates seem to play a role in anti-allergic effects. In summary, given the powerful modulatory effect that TLA exhibits, understanding its exact mechanisms of action may lead to the development of novel immunomodulators in clinical application.

Norathyriol is an aglycone of a xanthonoid C -glycoside mangiferin that possesses different bioactive properties useful for humans compared to mangiferin. Mangiferin is more readily available in nature than norathyriol; thus, efficient mangiferin conversion into norathyriol is desirable. There are a few reports regarding mangiferin C -deglycosylation because of the C–C bond resistance toward acid, alkaline, and enzyme hydrolysis. In this study, we isolated a mangiferin-deglycosylating bacterium strain KM7-1 from the mouse intestine. 16S rDNA sequencing indicated that KM7-1 belongs to the Bacillus genus. Compared to the taxonomically similar bacteria, the growth characteristic of facultative anaerobic and thermophilic resembled, yet only Bacillus sp. KM7-1 was able to convert mangiferin into norathyriol. Resting cells of Bacillus sp. KM7-1 obtained from aerobic cultivation at 50 °C showed high norathyriol formation from 1 m  m of mangiferin. Norathyriol formation can be conducted either under aerobic or anaerobic conditions, and the reaction depended on time and bacterial amount. [ABSTRACT FROM AUTHOR]

Background: The hygiene hypothesis suggests a link between parasitic infections and immune disorders, such as allergic diseases. We previously showed that infection with Toxoplasma gondii or systemic application of T. gondii tachyzoites lysate antigen (TLA) in a prophylactic, but not therapeutic protocol, prevented allergic airway inflammation in mice. Here we tested the effect of prophylactic and therapeutic application of TLA via the mucosal route. Methods: Mice were intranasally treated with TLA either i) prior to sensitization, ii) during sensitization and challenge, or iii) after sensitization with ovalbumin (OVA). Recruitment of inflammatory cells to the lung, cytokine levels in restimulated lung and spleen cell cultures as well as levels of OVA-specific antibodies in serum were measured. In parallel, the effect of native TLA, heat-inactivated (hiTLA) or deglycosylated TLA (dgTLA) on sensitized splenocytes was evaluated ex vivo. Results: When applied together with OVA i) during systemic sensitization and local challenge or ii) exclusively during local challenge, TLA reduced infiltration of eosinophils into the lung, OVA-specific type 2 cytokines in restimulated lung cell cultures, and partially, type 2 cytokines in restimulated spleen cell cultures in comparison to allergic controls. No beneficial effect was observed when TLA was applied prior to the start of sensitization. Analysis of epitope sugars on TLA indicated a high abundance of mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. Deglycosylation of TLA, but not heat-inactivation, abolished the potential of TLA to reduce type 2 responses ex vivo , suggesting a significant role of carbohydrates in immunomodulation. Conclusion: We showed that mucosal application of TLA reduced the development of experimental allergy in mice. The beneficial effects depended on the timing of the application in relation to the time point of sensitization. Not only co-application, but also therapy in sensitized/allergic animals with native TLA reduced local allergic responses. Furthermore, we show that TLA is highly glycosylated and glycoconjugates seem to play a role in anti-allergic effects. In summary, given the powerful modulatory effect that TLA exhibits, understanding its exact mechanisms of action may lead to the development of novel immunomodulators in clinical application. [ABSTRACT FROM AUTHOR]

Major cashew allergen, Ana o 1, was purified in its native form from cashew seeds and subjected to enzymatic deglycosylation using PNGase F to assess the potential role of N‐glycans in immunoreactivity. Western and dot blotting with pooled human plasma containing anticashew IgE revealed that deglycosylation increased IgE‐binding of Ana o 1. Removal of N‐glycans may have exposed previously masked Ana o 1 epitopes. Purified glycosylated and deglycosylated Ana o 1 were also subjected to in vitro pepsin digestion at pH 3.0 for 2 hr. Both glycosylated and deglycosylated Ana o 1 remained stable and reactive with IgE antibodies following digestion. Practical Application: Understanding the role of glycosylation in Ana o 1 immunoreactivity may provide insight into the potential development of hypoallergenic cashews/cashew products for sensitive individuals in the future. [ABSTRACT FROM AUTHOR]

The ability of retaining glycoside hydrolases (GHs) to transglycosylate is inherent to the double‐displacement mechanism. Studying reaction intermediates, such as the glycosyl‐enzyme intermediate (GEI) and the Michaelis complex, could provide valuable information to better understand the molecular factors governing the catalytic mechanism. Here, the GEI structure of RBcel1, an endo‐1,4‐β‐glucanase of the GH5 family endowed with transglycosylase activity, is reported. It is the first structure of a GH5 enzyme covalently bound to a natural oligosaccharide with the two catalytic glutamate residues present. The structure of the variant RBcel1_E135A in complex with cellotriose is also reported, allowing a description of the entire binding cleft of RBcel1. Taken together, the structures deliver different snapshots of the double‐displacement mechanism. The structural analysis revealed a significant movement of the nucleophilic glutamate residue during the reaction. Enzymatic assays indicated that, as expected, the acid/base glutamate residue is crucial for the glycosylation step and partly contributes to deglycosylation. Moreover, a conserved tyrosine residue in the −1 subsite, Tyr201, plays a determinant role in both the glycosylation and deglycosylation steps, since the GEI was trapped in the RBcel1_Y201F variant. The approach used to obtain the GEI presented here could easily be transposed to other retaining GHs in clan GH‐A. [ABSTRACT FROM AUTHOR]

During endoplasmic reticulum-associated degradation (ERAD), the cytoplasmic enzyme N-glycanase 1 (NGLY1) is proposed to remove N-glycans from misfolded N-glycoproteins after their retrotranslocation from the ER to the cytosol. We previously reported that NGLY1 regulates Drosophila BMP signaling in a tissue-specific manner (Galeone et al., 2017). Here, we establish the Drosophila Dpp and its mouse ortholog BMP4 as biologically relevant targets of NGLY1 and find, unexpectedly, that NGLY1-mediated deglycosylation of misfolded BMP4 is required for its retrotranslocation. Accumulation of misfolded BMP4 in the ER results in ER stress and prompts the ER recruitment of NGLY1. The ER-associated NGLY1 then deglycosylates misfolded BMP4 molecules to promote their retrotranslocation and proteasomal degradation, thereby allowing properly-folded BMP4 molecules to proceed through the secretory pathway and activate signaling in other cells. Our study redefines the role of NGLY1 during ERAD and suggests that impaired BMP4 signaling might underlie some of the NGLY1 deficiency patient phenotypes.

Abstract The detection of erythropoietin (Epo) protein by Western blotting has required pre‐purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis‐stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG‐bound epoetin β pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Severe hypoxia (7% O2, 4 hr) caused a 400‐fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney but not the liver is the main site of Epo production in control and severe hypoxia. Our method will make the tests for Epo doping and detection easy.

N-Glycanase 1 (NGLY1) is a cytosolic enzyme involved in removing N-linked glycans of misfolded N-glycoproteins and is considered to be a component of endoplasmic reticulum-associated degradation (ERAD). The 2012 identification of recessive NGLY1 mutations in a rare multisystem disorder has led to intense research efforts on the roles of NGLY1 in animal development and physiology, as well as the pathophysiology of NGLY1 deficiency. Here, we present a review of the NGLY1-deficient patient phenotypes, along with insights into the function of this gene from studies in rodent and invertebrate animal models, as well as cell culture and biochemical experiments. We will discuss critical processes affected by the loss of NGLY1, including proteasome bounce-back response, mitochondrial function and homeostasis, and bone morphogenetic protein (BMP) signaling. We will also cover the biologically relevant targets of NGLY1 and the genetic modifiers of NGLY1 deficiency phenotypes in animal models. Together, these discoveries and disease models have provided a number of avenues for preclinical testing of potential therapeutic approaches for this disease.

Anemia is a major complication of chronic renal failure. To treat this anemia, prolylhydroxylase domain enzyme (PHD) inhibitors as well as erythropoiesis-stimulating agents (ESAs) have been used. Although PHD inhibitors rapidly stimulate erythropoietin (Epo) production, the precise sites of Epo production following the administration of these drugs have not been identified. We developed a novel method for the detection of the Epo protein that employs deglycosylation-coupled Western blotting. With protein deglycosylation, tissue Epo contents can be quantified over an extremely wide range. Using this method, we examined the effects of the PHD inhibitor, Roxadustat (ROX), and severe hypoxia on Epo production in various tissues in rats. We observed that ROX increased Epo mRNA expression in both the kidneys and liver. However, Epo protein was detected in the kidneys but not in the liver. Epo protein was also detected in the salivary glands, spleen, epididymis and ovaries. However, both PHD inhibitors (ROX) and severe hypoxia increased the Epo protein abundance only in the kidneys. These data show that, while Epo is produced in many tissues, PHD inhibitors as well as severe hypoxia regulate Epo production only in the kidneys.

The present study was conducted to qualitatively and quantitatively elucidate dynamic changes of ginsenosides in ginseng pulp steamed under different temperatures (100 or 120 °C) for different durations (1–6 h) through UPLC-QTOF-MS/MS and HPLC with the aid of as numerous as 18 authentic standards of ginsenosides. Results show that levels of eight polar ginsenosides (i.e., Rg1, Re, Rb1, Rc, Rb2, Rb3, F1, and Rd) declined but those of 10 less-polar ginsenosides [i.e., Rf, Rg2, 20(S)-Rh1, 20(R)-Rg2, F4, 20(S)-Rg3, 20(R)-Rg3, PPT, Rg5, and 20(R)-Rh2] elevated with increases of both steaming temperature and duration; the optimum steaming conditions for achieving the highest total ginsenosides were 100 °C for 1 h. Particular, 20(R)-Rg3, a representative less-polar ginsenoside with high bioactivity such as potent anti-cancer effect, increased sharply but Re, the most abundant polar ginsenoside in fresh ginseng pulp, decreased dramatically. More importantly, ginsenoside species enhanced from 18 to 42 after steaming, mainly due to transformation of polar into less-polar ginsenosides. Furthermore, four malonyl-ginsenosides were detected in fresh ginseng pulps and ten acetyl-ginsenosides were formed during steaming, demonstrating that demalonylation and acetylation of ginsenosides were the dominant underling mechanisms for transformation of polar into less-polar ginsenosides. [ABSTRACT FROM AUTHOR]

Glycoproteins, proteins that are co- and posttranslationally modified by sugars (glycans), have significant roles in pathophysiology of many different diseases. One of the main steps in sample preparation for free N-glycan analysis is deglycosylation or glycan removal. The aim of this study was to compare different peptide N-glycosidase F (PNGase F) enzymes (Rapid PNGase F and two recombinant versions) for deglycosylation of total human plasma glycoproteins and different amounts of human immunoglobulin G (IgG). Deglycosylation with different PNGase F enzymes resulted in different IgG and plasma N-glycosylation hydrophilic interaction liquid chromatography ultra-performance liquid chromatography profiles. Additionally, one recombinant version of PNGase F is more efficient in deglycosylation of complex N-glycans compared with Rapid PNGase F and recombinant version of PNGase F from a different manufacturer. In terms of chromatographic peak intensities and coefficient of variation %Area values, all tested versions of PNGase F enzymes were very reproducible and on the similar level when used in optimal conditions. However, care should be taken in terms of which enzyme is used with which protocol, particularly when scaling up. [ABSTRACT FROM AUTHOR]

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method to study the structural dynamics of proteins. However, glycoproteins represent a challenge to the traditional HDX-MS workflow for determining the deuterium uptake of the protein segments that contain the glycan. We have recently demonstrated the utility of the glycosidase PNGase A to enable HDX-MS analysis of N-glycosylated protein regions. Here, we have investigated the use of the acidic glycosidase PNGase H+, which has a pH optimum at 2.6, to efficiently deglycosylate N-linked glycosylated peptides during HDX-MS analysis of glycoproteins. Our results show that PNGase H+ retains high deglycosylation activity at HDX quench conditions. When used in an HDX-MS workflow, PNGase H+ allowed the extraction of HDX data from all five glycosylated regions of the serpin α1-antichymotrypsin. We demonstrate that PNGase A and PNGase H+ are capable of similar deglycosylation performance during HDX-MS analysis of α1-antichymotrypsin and the IgG1 antibody trastuzumab (TZ). However, PNGase H+ provides broader specificity and greater tolerance to the disulfide-bond reducing agent TCEP, while PNGase A offers advantages in terms of commercial availability and purity. Overall, our findings demonstrate the unique features of PNGase H+ for improving conformational analysis of glycoproteins by HDX-MS, in particular, challenging glycoproteins containing both glycosylations and disulfide bonds. [ABSTRACT FROM AUTHOR]

• Mouse IgG (mIgG) subclasses cross-react with human Fc gamma receptors (hFcγRs). • Exact affinities were determined for the full spectrum of mIgG-hFcγR interactions. • Antibody deglycosylation decreases mIgG binding to hFcγRs. • Antibody deglycosylation only eliminates mIgG2b binding to hFcγRs. Immunoglobulin G (IgG) antibodies are important for protection against pathogens and exert effector functions through binding to IgG-Fc receptors (FcγRs) on myeloid and natural killer cells, resulting in destruction of opsonized target cells. Despite interspecies differences, IgG subclasses and FcγRs show substantial similarities and functional conservation between mammals. Accordingly, binding of human IgG (hIgG) to mouse FcγRs (mFcγRs) has been utilized to study effector functions of hIgG in mice. In other applications, such as immunostaining with mouse IgG monoclonal antibodies (mAbs), these cross-reactivities are undesired and prone to misinterpretation. Despite this drawback, the binding of mouse IgG (mIgG) subclasses to human FcγR (hFcγR) classes has never been fully documented. Here, we report detailed and quantifiable characterization of binding affinities for all mIgG subclasses to hFcγRs, including functional polymorphic variants. mIgG subclasses show the strongest binding to hFcγRIa, with relative affinities mIgG2a = mIgG2c > mIgG3 >> mIgG2b, and no binding by mIgG1. hFcγRIIa/b showed general low reactivities to all mIgG (mIgG1> mIgG2a/c > mIgG2b), with no reactivity to mIgG3. A particularly high affinity was observed for mIgG1 to the hFcγRIIa-R131 polymorphic variant. hFcγRIIIa showed lower binding (mIgG2a/c > mIgG3), slightly favouring binding to the hFcγRIIIa-V158 over the F158 polymorphic variant. No binding was observed of mIgG to hFcγRIIIb. Deglycosylation of mIgG1 did not abrogate binding to hFcγRIIa-R131, nor did deglycosylation of mIgG2a/c and mIgG3 prevent hFcγRIa binding. Importantly, deglycosylation of the least cross-reactive mIgG subclass, mIgG2b, abrogated reactivity to all hFcγRs. Together, these data document for the first time the full spectrum of cross-reactivities of mouse IgG to human FcγRs. [ABSTRACT FROM AUTHOR]

H5N1 subtype avian influenza virus (AIV) with a deletion of 20 amino acids at residues 49–68 in the stalk region of neuraminidase (NA) became a major epidemic virus. To determine the effect of truncation or deglycosylation of the NA stalk on virulence, we used site-directed mutagenesis to insert 20 amino acids in the short-stalk virus A/mallard/Huadong/S/2005 (SY) to recover the long-stalk virus (rSNA+). A series of short-stalk or deglycosylated-stalk viruses were also constructed basing on the long-stalk virus, and then the characteristics and pathogenicity of the resulting viruses were evaluated. The results showed that most of the short-stalk or deglycosylated-stalk viruses had smaller plaques, and increased thermal and low-pH stability, and a decreased neuraminidase activity when compared with the virus rSNA+. In a mallard ducks challenge study, most of the short-stalk or deglycosylated-stalk viruses showed increased pathological lesions and virus titers in the organ tissues and increased virus shedding in the oropharynx and cloaca when compared with the rSNA+ virus, while most of the short-stalk viruses, especially rSNA-20, showed higher pathogenicity than the deglycosylated-stalk virus. In addition, the short-stalk viruses showed a significantly upregulated expression of the immune-related factors in the lungs of the infected mallard ducks, including IFN-α, Mx1, and IL-8. The results suggested that NA stalk truncation or deglycosylation increases the pathogenicity of H5N1 subtype AIV in mallard ducks, which will provide a pre-warning for prevention and control of H5N1 subtype avian influenza in the waterfowl. [ABSTRACT FROM AUTHOR]

1. Bupleuri Radix (BR) is a herbal medicine traditionally used orally in oriental countries, which inevitably comes into contact with the intestinal microbiota. However, whether gut microbiota contribute to the biotransformation of BR, and/or the formation of pharmacologically active compounds remains unknown. 2. In this study, the main saikosaponins (SAPs) of Bupleurum (including saikosaponin a, b1, b2, c, d, f, h) and BR extract (BRE) were individually incubated with human fecal suspensions (HFS), and metabolic time courses of SAPs and their metabolites by human gut bacteria were systematically characterized. 3. Deglycosylation and dehydration were the main metabolic pathways identified for SAPs including newly investigated saikosaponin f (SSf) and saikosaponin h (SSh); dehydration had not been reported previously. A total of 19 dehydrated and deglycosylated metabolites of SAPs were detected and characterized, and 10 of them were newly identified. Moreover, SAPs of BRE were found to be deglycosylated to prosaikogenins. In addition, 13 metabolic pathways related to human gut microbiota were identified for phytochemicals of BRE except for SAPs. Gut microbiota may play a significant role in the biotransformation of BR in humans. [ABSTRACT FROM AUTHOR]

The use of decellularized xenogeneic heart valves might offer a solution to overcome the issue of human valve shortage. The aim of this study was to revise decellularization protocols in combination with enzymatic deglycosylation, in order to reduce the immunogenicity of porcine pulmonary heart valves, in means of cells, carbohydrates, and, primarily, Galα1‐3Gal (α‐Gal) epitope removal. In particular, the valves were decellularized with sodium dodecylsulfate/sodium deoxycholate (SDS/SD), Triton X‐100 + SDS (Tx + SDS), or Trypsin + Triton X‐100 (Tryp + Tx) followed by enzymatic digestion with PNGaseF, Endoglycosidase H, or O‐glycosidase combined with Neuraminidase. Results showed that decellularization alone reduced carbohydrate structures only to a limited extent, and it did not result in an α‐Gal free scaffold. Nevertheless, decellularization with Tryp + Tx represented the most effective decellularization protocol in means of carbohydrates reduction. Overall, carbohydrates and α‐Gal removal could strongly be improved by applying PNGaseF, in particular in combination with Tryp + Tx treatment, contrary to Endoglycosidase H and O‐glycosidase treatments. Furthermore, decellularization with PNGaseF did not affect biomechanical stability, in comparison with decellularization alone, as shown by burst pressure and uniaxial tensile tests. In conclusion, valves decellularized with Tryp + Tx and PNGaseF resulted in prostheses with potentially reduced immunogenicity and maintained mechanical stability. [ABSTRACT FROM AUTHOR]

Deglycosylation has previously been suggested to modulate the biological activities of various phytochemicals. The current study was designed to evaluate the effects of deglycosylation on antioxidant potential of crude methanolic extracts (CMEs) of four medicinal plants: Adhatoda vasica, Skimmia laureola, Olea ferruginea, and Cymbopogon citratus. The CMEs were prepared in 80% methanol and their antioxidant activity was measured using DPPH (diphenyl picryl hydrazyl) photometric assay. The resultant IC50 values were 82 μg/mL for O. ferruginea, 127 μg/mL for S. laureola, 169 μg/mL for C. citrates, 170 μg/mL for A. vasica, and 3.1 μg/mL for ascorbic acid, which was used as a standard. Glycosidase enzyme was extracted from Medicago sativa (alfalfa) seeds and assayed chromogenically using X-gal as its substrate. The glycosidase enzyme successfully deglycosylated CMEs of all the four plants. These degylosylated extracts were also analyzed for their antioxidant activities. New IC50 values were 52 μg/mL for O. ferruginea, 71 μg/mL for S. laureola, 103 μg/mL for A. vasica, and 111 μg/mL for C. citratus. These observations show a significant increase in the antioxidant potential of all four CMEs as a result of their deglycosylation. All results were analyzed statistically and significant difference (p = 0) was established between CMEs and deglycosylated CMEs of all the four selected plants tested separately. [ABSTRACT FROM AUTHOR]

A human intestinal bacterium strain related to Dorea species, PUE, can metabolize the isoflavone C-glucoside puerarin (daidzein 8-C-glucoside) to daidzein and glucose. We reported previously that 3"-oxo-puerarin is an essential reaction intermediate in enzymatic puerarin degradation, and we characterized a bacterial enzyme, the DgpB-DgpC complex, that cleaved the C-glycosidic bond in 3"-oxo-puerarin. However, the exact enzyme catalyzing the oxidation of the C-3" hydroxyl in puerarin has not been identified. In this study, we demonstrated that recombinant DgpA, a Gfo/Idh/MocA family oxidoreductase, catalyzed puerarin oxidation in the presence of 3-oxo-glucose as the hydride acceptor. In the redox reaction, NAD(H) functioned as the cofactor, which bound tightly but noncovalently to DgpA. Kinetics analysis of DgpA revealed that the reaction proceeded via a ping-pong mechanism. Enzymatic C-deglycosylation of puerarin was achieved by a combination of recombinant DgpA, the DgpB-DgpC complex, and 3-oxo-glucose. In addition, the metabolite derived from the sugar moiety in the 3"-oxo-puerarin-cleaving reaction catalyzed by the DgpB-DgpC complex was characterized as 1,5-anhydro-d-erythro-hex-1-en-3-ulose, suggesting that the C-glycosidic linkage is cleaved through a β-elimination-like mechanism. [ABSTRACT FROM AUTHOR]

Background: NGLY1-related congenital disorder of deglycosylation (NGLY1-CDDG) is a multisystemic neurodevelopmental disorder in which affected individuals show developmental delay, epilepsy, intellectual disability, abnormal liver function, and poor growth. This study presents a 10-month-old female infant with elevated liver transaminases, developmental delay, epilepsy (subclinical seizures), and constipation who possesses two compound heterozygous mutations in NGLY1. Case presentation: The proband was admitted to the Department of Gastroenterology, Children's Hospital of Soochow University, with elevated liver transaminases. She had a history of intrauterine growth retardation and exhibited elevated transaminases, global developmental delay, seizures and light constipation during early infancy. Whole-exome sequencing (WES) and Sanger sequencing revealed two compound heterozygous mutations in NGLY1 that had been inherited in an autosomal recessive manner from her parents. One was a termination mutation, c.1168C > T (p.R390*), and the other was a missense mutation, c.1156G > T (p.D386Y). NGLY1-CDDG is a rare disorder, with a few dozen cases. The two mutations of this proband has not been previously identified. Conclusions: This study investigated a Chinese proband with NGLY1-CDDG born from healthy parents who was studied using WES and Sanger sequencing to identify the causative mutations. We identified two novel compound heterozygous mutations in NGLY1, c.1168C > T (p.R390*)/c.1156G > T (p.D386Y), which are probably causative of disease. [ABSTRACT FROM AUTHOR]

The detection of erythropoietin (Epo) protein by Western blotting has required pre‐purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis‐stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG‐bound epoetin β pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Severe hypoxia (7% O2, 4 hr) caused a 400‐fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney but not the liver is the main site of Epo production in control and severe hypoxia. Our method will make the tests for Epo doping and detection easy. [ABSTRACT FROM AUTHOR]

This study evaluated the influence of different fluidized‐bed drying temperatures (20, 60, and 100 °C) on the cooking properties, in vitro starch digestibility, and phenolic bioaccessibility of black rice. The results indicated that the formation of fissures in the grains dried at or above 60 °C reduced the physical integrity of the grains after cooking, increasing the starch digestion and the rehydration ratio, and reduced the cooking time, the hardness and adhesiveness. Due to the higher digestibility of grains dried at higher temperatures, an increase in the bioaccessibility of ferulic acid, which was previously associated with the polysaccharides, was observed. Caffeic acid was the only phenolic compound whose levels decreased when the drying temperature increased. At high temperatures and in the gastric phase, cyanidin chalcones were formed due to the deglycosylation of cyanidin‐3‐O‐glucoside. Practical Application: The results of this study provide information to the food industry about the effects of different fluidized‐bed drying temperatures on the rice structure after cooking and that, consequently, affect the availability of bioactive compounds after digestion and the glycemic index of black rice. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

An enzyme mining strategy was applied to discover a novel β -Glucosidase (Bgps) from Phycicoccus sp. Soil748 2, which first demonstrate the production of CA by CMG. The Bgps showed the higher substrate concentration (80 g/L) and higher catalytic efficiency, indicating a great advantage on CA industrial production by enzyme catalysis. • Enzyme mining was conducted based on skeleton and glycosyl similarity. • A novel β -glucosidase was screened to prepare cycloastragenol. • The optimal substrate concentration of glucosidase was up to 80 g/L. • Mild deglyosylation of cycloastragenol-6- O - β -D-glucoside was first realized. Cycloastragenol (CA), the genuine sapogenin of astragaloside from Astragalus membranaceus , exhibits diverse pharmaceutical activities. Recently, the efficient production of CA has received considerable attention due to rapidly increasing market demands. In this study, enzyme mining was conducted, based on skeleton and glycosyl similarity, to explore an efficient β -glucosidase for CA preparation. A novel β -glucosidase from Phycicoccus sp. Soil748 (Bgps) was discovered, possessing the efficient conversion rate for cycloastragenol-6- O - β -D-glucoside (CMG) into CA. The optimum temperature and pH value of Bgps were determined as 45 °C and 7.0. The results of kinetic analysis suggested that Bgps catalyzed deglycosylation of CMG more efficiently than other substrates. Furthermore, the optimal substrate concentration of Bgps was up to 80 mg/mL with the conversion rate as 99.2%, suggesting its potential application in CA industrial production by biotransformation. [ABSTRACT FROM AUTHOR]

C-Glycosides, a special type of glycoside, are frequently distributed in many kinds of medicinal plants, such as puerarin and mangiferin, showing various and significant bioactivities. C-Glycosides are usually characterized by the C–C bond that forms between the anomeric carbon of sugar moieties and the carbon atom of aglycon, which is usually resistant against acidic hydrolysis and enzymatic treatments. Interestingly, C-glycosides could be cleaved by several intestinal bacteria, but whether the enzymatic cleavage of C–C glycosidic bond is reduction or hydrolysis has been controversial; furthermore, whether existence of a "C-glycosidase" directly catalyzing the cleavage is not clear. Here we review research advances about the discovery and mechanism of intestinal bacteria in enzymatic cleavage of C–C glycosidic bond with an emphasis on the identification of enzymes manipulation the deglycosylation. Finally, we give a brief conclusion about the mechanism of C-glycoside deglycosylation and perspectives for future study in this field. [ABSTRACT FROM AUTHOR]

Objective: The objective is to study the effect of individual N-glycosylation sequon on Suc2 activity and stability, and improve inulin conversion capacity and ethanol production of yeast by manipulating the key N-glycosylation sequon in Suc2. Results: Based on previous reported data, it could be deduced that both the activity and thermal stability of Suc2 are probably influenced by some key N-glycosylation sequons. Thus, totally 13 N-glycosylation residues of Suc2 were individually deglycosylated by site-directed mutagenesis. Fermentation results indicated that deglycosylation at N4, N78 and N146 sequons improved Suc2 activity and ethanol production of host strains, whereas deglycosylation at N45 showed an opposite effect. Carbohydrate depletion of Suc2 N4 especially endowed the host strain with better ethanol fermentation performance from inulin when the strain was cultured under the higher temperature for 48 h, indicating that deglycosylation of N4 might improve the thermal stability of the Suc2. Conclusions: Carbohydrate chains at N4, N45, N78 and N146 played an important role in modulating Suc2 activity and inulin catabolism of the S. cerevisiae strain. These key N-deglycosylation sequons should become potential targets for rational engineering of S. cerevisiae strains to enhance the ethanol production from inulin. [ABSTRACT FROM AUTHOR]

Coumestrol (CMS), one of the soybean isoflavonoids which contains several benefits for maintaining skin function including antiageing properties. In this study, we evaluated various explant sources and plant genotypes to determine competent soybean adventitious root materials for the mass production of CMS, and investigated their skin care efficacies to be used as a novel cosmetic ingredient. Adventitious roots were directly induced from in vitro seedling derived from the mature seeds, extracts were prepared and refluxed for enzymatic deglycosylation. In vitro cell cytotoxicity was evaluated using normal human dermal fibroblast and murine B16 melanoma cells after treatment with increasing concentrations of methanol soybean adventitious roots extracts for 72 h. Finally, in vitro cell assays on HDF cells were performed to evaluate the effect of the soybean adventitious roots extracts in collagen production. The root induction frequency and biomass productivity were significantly affected by plant genotypes, explant sources, the type of auxin used and its concentration. The total CMS production (per 1 L medium) after 4 weeks of culture in a bulb-type bubble bioreactor (3 L capacity) was the highest in the adventitious roots induced from the radicles of Glycine max, 'Sinhwakong'. Different strengths of Murashige and Skoog (MS) medium were tested to develop culture protocols and the highest total CMS production (per 1 L medium) was observed at 1/2 MS. The content of coumestrin, the glycoside form of CMS, was higher than that of CMS in the roots cultured in 1/2 MS medium for 4 weeks in a bioreactor. The final content of CMS in the ethanol extract after enzymatic deglycosylation was 81.3-fold higher than non-enzymatic deglycosylation. Almost all the coumestrin in the roots were converted to CMS. Further, the enriched CMS root did not exhibit any cell cytotoxicity in normal human dermal fibroblast (HDF) and murine B16 melanoma cells (B16) for 72 h. In addition, in vitro collagen production assay on HDF cells showed that the enriched CMS root increased the collagen production compared to the coumestrol, daidzein, and non-enzyme-treated sample. Thus, enriched CMS root could be potential ingredient for the cosmetic applications [ABSTRACT FROM AUTHOR]