Search

Duck plague virus (DPV) causes the highly pathogenic duck plague, and the envelope glycoprotein I (gI), as one of the key virulence genes, has not yet had its critical virulence sites identified through screening. This study used reverse genetics technology to target the gI, specifically within the DPV genome. Four DPV mutants with gI N-glycosylation site mutations were designed and constructed, and these mutant strains were successfully rescued. Our results confirmed that three asparagine residues of gI (N69, N78, and N265) are N-glycosylation sites, and western blot analysis substantiated that glycosylation at each predicted N-glycosylation site was compromised. The deglycosylation of gI leads to the protein misfolding and subsequent retention in the endoplasmic reticulum (ER). The subsequent deglycosylated gI is carried into the Golgi apparatus (GM130) in the interaction of gE. Compared to the parental virus, the mutated virus shows a 66.3% reduction in intercellular transmission capability. In ducks, the deglycosylation of gI significantly reduces DPV replication in vivo, thereby weakening the virulence of DPV. This study represents the first successful creation of a weak DPV virus strain by specific mutation at the N-glycosylation site. The findings provide a foundational understanding of DPV pathogenesis and form the basis for developing live attenuated vaccines against the disease. [ABSTRACT FROM AUTHOR]

Copper plays a key role in host-pathogen interaction. We find that during Leishmania major infection, the parasite-harboring macrophage regulates its copper homeostasis pathway in a way to facilitate copper-mediated neutralization of the pathogen. Copper-ATPase ATP7A transports copper to amastigote-harboring phagolysosomes to induce stress on parasites. Leishmania in order to evade the copper stress, utilizes a variety of manipulative measures to lower the host-induced copper stress. It induces deglycosylation and degradation of host-ATP7A and downregulation of copper importer, CTR1 by cysteine oxidation. Additionally, Leishmania induces CTR1 endocytosis that arrests copper uptake. In mouse model of infection, we report an increase in systemic bioavailable copper in infected animals. Heart acts as the major organ for diverting its copper reserves to systemic circulation to fight-off infection by downregulating its CTR1. Our study explores reciprocal mechanism of manipulation of host copper homeostasis pathway by macrophage and Leishmania to gain respective advantages in host-pathogen interaction. Leishmania major alters host copper dynamics. Macrophages utilize copper via ATP7A to stress the pathogen, countered by Leishmania that reduces ATP7A and the copper importer CTR1. Infection elevates mouse systemic copper that aids in its defense. [ABSTRACT FROM AUTHOR]

Many large-scale studies revealed that exogenous erythropoietin, erythropoiesis-stimulating agents, have no renoprotective effects. We reported the renoprotective effects of endogenous erythropoietin production on renal function in ischemic reperfusion injury (IRI) of the kidney using the prolyl hydroxylase domain (PHD) inhibitor, Roxadustat. The purpose of this study was to investigate the effects of daprodustat on the progression of chronic renal failure. We retrospectively investigated the effects of daprodustat on the progression of chronic renal failure and renal anemia in patients with stages 3a-5 chronic kidney diseases (estimated glomerular filtration rate, eGFR < 60 mL/min/1.73 m2). The results show that daprodustat largely slowed the reduction in eGFR. The recovery of renal function was observed in some patients. Daprodustat is useful not only for renal anemia but also for the preservation of renal function. The renoprotective effect of daprodustat was small in patients with serum creatinine larger than 3–4 mg/dL because of low residual renal function. The appearance of renal anemia would be a sign of the time to start using daprodustat. [ABSTRACT FROM AUTHOR]

Resistance to radiotherapy is a major barrier during cancer treatment. Here using genome-scale CRISPR/Cas9 screening, we identify CD274 gene, which encodes PD-L1, to confer lung cancer cell resistance to ionizing radiation (IR). Depletion of endogenous PD-L1 delays the repair of IR-induced DNA double-strand breaks (DSBs) and PD-L1 loss downregulates non-homologous end joining (NHEJ) while overexpression of PD-L1 upregulates NHEJ. IR induces translocation of PD-L1 from the membrane into nucleus dependent on deglycosylation of PD-L1 at N219 and CMTM6 and leads to PD-L1 recruitment to DSBs foci. PD-L1 interacts with Ku in the nucleus and enhances Ku binding to DSB DNA. The interaction between the IgC domain of PD-L1 and the core domain of Ku is required for PD-L1 to accelerate NHEJ-mediated DSB repair and produce radioresistance. Thus, PD-L1, in addition to its immune inhibitory activity, acts as mechanistic driver for NHEJ-mediated DSB repair in cancer. Resistance to radiotherapy is a major barrier during cancer treatment. Here the authors find that radiation-induced DNA double-strand-breaks (DSBs) facilitate PD-L1 deglycosylation and translocation into the nucleus, leading to promotion of NHEJ-mediated DSB repair and lung cancer radioresistance. [ABSTRACT FROM AUTHOR]

Tumor cells can express the immune checkpoint protein programmed death-1 (PD-1), but how cancer cell-intrinsic PD-1 is regulated in response to cellular stresses remains largely unknown. Here, we uncover a unique mechanism by which the chemotherapy drug doxorubicin (Dox) regulates cancer cell-intrinsic PD-1. Dox upregulates PD-1 mRNA while reducing PD-1 protein levels in tumor cells. Although Dox shortens the PD-1 half-life, it fails to directly induce PD-1 degradation. Instead, we observe that Dox promotes the interaction between peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase (NGLY1) and PD-1, facilitating NGLY1-mediated PD-1 deglycosylation and destabilization. The maintenance of PD-1 sensitizes tumor cells to Dox-mediated antiproliferative effects. Our study unveils a regulatory mechanism of PD-1 in response to Dox and highlights a potential role of cancer cell-intrinsic PD-1 in Dox-mediated antitumor effects. [ABSTRACT FROM AUTHOR]

O-GlcNAcase (OGA) is the only human enzyme that catalyzes the hydrolysis (deglycosylation) of O-linked beta-N-acetylglucosaminylation (O-GlcNAcylation) from numerous protein substrates. OGA has broad implications in many challenging diseases including cancer. However, its role in cell malignancy remains mostly unclear. Here, we report that a cancer-derived point mutation on the OGA's noncatalytic stalk domain aberrantly modulates OGA interactome and substrate deglycosylation toward a specific set of proteins. Interestingly, our quantitative proteomic studies uncovered that the OGA stalk domain mutant preferentially deglycosylated protein substrates with +2 proline in the sequence relative to the O-GlcNAcylation site. One of the most dysregulated substrates is PDZ and LIM domain protein 7 (PDLIM7), which is associated with the tumor suppressor p53. We found that the aberrantly deglycosylated PDLIM7 suppressed p53 gene expression and accelerated p53 protein degradation by promoting the complex formation with E3 ubiquitin ligase MDM2. Moreover, deglycosylated PDLIM7 significantly up-regulated the actin-rich membrane protrusions on the cell surface, augmenting the cancer cell motility and aggressiveness. These findings revealed an important but previously unappreciated role of OGA's stalk domain in protein substrate recognition and functional modulation during malignant cell progression. [ABSTRACT FROM AUTHOR]

The stems and leaves of Panax notoginseng contain high saponins, but they are often discarded as agricultural waste. In this study, the predominant ginsenosides Rg1, Rc, and Rb2, presented in the stems and leaves of ginseng plants, were biotransformed into value-added rare ginsenosides F1, compound Mc1 (C-Mc1), and Rd2, respectively. A fungal strain YMS6 (Penicillium sp.) was screened from the soil as a biocatalyst with high selectivity for the deglycosylation of major ginsenosides. Under the optimal fermentation conditions, the yields of F1, C-Mc1, and Rd2 were 97.95, 68.64, and 79.58%, respectively. This study provides a new microbial resource for the selective conversion of protopanaxadiol-type and protopanaxatriol-type major saponins into rare ginsenosides via the whole-cell biotransformation and offers a solution for the better utilization of P. notoginseng waste. [ABSTRACT FROM AUTHOR]

IgMs are the first antibodies produced by the immune system upon encounter of a possible pathogen and are one of five antibody subclasses in humans. For IgG, the most intensively studied antibody class, the N‐linked glycosylation site located in the Fc‐domain is directly involved in high affinity binding to the respective receptors and initiation of corresponding immune response. IgM molecules have five N‐glycosylation sites and one N‐glycosylation site in the J‐chain, which can be incorporated in IgM or IgA molecules. There is only limited knowledge available concerning the function of these N‐glycosylations in IgMs. To address this question, we produced IgM molecules lacking a particular N‐glycosylation site and tested these variants as well as IgA molecules for binding to the known receptors: the polymeric immunoglobulin receptor (pIgR), the dual receptor for IgA and IgM, FcαμR, and the specific receptor for IgM, FcμR. The single glycosylation sites did not show an impact on expression and multimerization, except for variant N402Q, which could not be expressed. In SPR measurements, no major impact on the binding to the receptors by particular glycosylation sites could be detected. In cellular assays, deglycosylated variants showed some alterations in induction of CDC activity. Most strikingly, we observed also binding of IgA to the FcμR in the same affinity range as IgM, suggesting that this might have a physiological role. To further substantiate the binding of IgA to FcμR we used IgA from different origins and were able to confirm binding of IgA preparations to the FcμR. [ABSTRACT FROM AUTHOR]

Many large-scale studies show that exogenous erythropoietin, erythropoiesis-stimulating agents, lack any renoprotective effects. We investigated the effects of endogenous erythropoietin on renal function in kidney ischemic reperfusion injury (IRI) using the prolyl hydroxylase domain (PHD) inhibitor, Roxadustat (ROX). Four h of hypoxia (7% O2) and 4 h treatment by ROX prior to IRI did not improve renal function. In contrast, 24–72 h pretreatment by ROX significantly improved the decline of renal function caused by IRI. Hypoxia and 4 h ROX increased interstitial cells-derived Epo production by 75- and 6-fold, respectively, before IRI, and worked similarly to exogenous Epo. ROX treatment for 24–72 h increased Epo production during IRI by 9-fold. Immunohistochemistry revealed that 24 h ROX treatment induced Epo production in proximal and distal tubules and worked similarly to endogenous Epo. Our data show that tubular endogenous Epo production induced by 24–72 h ROX treatment results in renoprotection but peritubular exogenous Epo production by interstitial cells induced by hypoxia and 4 h ROX treatment did not. Stimulation of tubular, but not peritubular, Epo production may link to renoprotection. [ABSTRACT FROM AUTHOR]

During the COVID-19 outbreak, numerous tools including protein-based vaccines have been developed. The methylotrophic yeast Pichia pastoris (synonymous to Komagataella phaffii) is an eukaryotic cost-effective and scalable system for recombinant protein production, with the advantages of an efficient secretion system and the protein folding assistance of the secretory pathway of eukaryotic cells. In a previous work, we compared the expression of SARS-CoV-2 Spike Receptor Binding Domain in P. pastoris with that in human cells. Although the size and glycosylation pattern was different between them, their protein structural and conformational features were indistinguishable. Nevertheless, since high mannose glycan extensions in proteins expressed by yeast may be the cause of a nonspecific immune recognition, we deglycosylated RBD in native conditions. This resulted in a highly pure, homogenous, properly folded and monomeric stable protein. This was confirmed by circular dichroism and tryptophan fluorescence spectra and by SEC-HPLC, which were similar to those of RBD proteins produced in yeast or human cells. Deglycosylated RBD was obtained at high yields in a single step, and it was efficient in distinguishing between SARS-CoV-2-negative and positive sera from patients. Moreover, when the deglycosylated variant was used as an immunogen, it elicited a humoral immune response ten times greater than the glycosylated form, producing antibodies with enhanced neutralizing power and eliciting a more robust cellular response. The proposed approach may be used to produce at a low cost, many antigens that require glycosylation to fold and express, but do not require glycans for recognition purposes. [ABSTRACT FROM AUTHOR]

Biological degradation of natural product glycosides involves, alongside hydrolysis, β-elimination for glycosidic bond cleavage. Here, we discover an O-glycoside β-eliminase (OGE) from Agrobacterium tumefaciens that converts the C3-oxidized O-β-d-glucoside of phloretin (a plant-derived flavonoid) into the aglycone and the 2-hydroxy-3-keto-glycal elimination product. While unrelated in sequence, OGE is structurally homologous to, and shows effectively the same Mn2+ active site as, the C-glycoside deglycosylating enzyme (CGE) from a human intestinal bacterium implicated in β-elimination of 3-keto C-β-d-glucosides. We show that CGE catalyzes β-elimination of 3-keto O- and C-β-d-glucosides while OGE is specific for the O-glycoside substrate. Substrate comparisons and mutagenesis for CGE uncover positioning of aglycone for protonic assistance by the enzyme as critically important for C-glycoside cleavage. Collectively, our study suggests convergent evolution of active site for β-elimination of 3-keto O-β-d-glucosides. C-Glycoside cleavage is a specialized feature of this active site which is elicited by substrate through finely tuned enzyme-aglycone interactions. Biological degradation of glycosides involves, alongside hydrolysis, β-elimination for glycosidic bond cleavage. Here, the authors report an O-glycoside β-eliminase from Agrobacterium tumefaciens that converts the C3-oxidized O-β-d-glucoside of phloretin into the aglycone and the 2-hydroxy-3-keto-d-glycal elimination product, and suggest convergent evolution of β-eliminase active sites for the cleavage of natural product 3-keto-O-glycosides. [ABSTRACT FROM AUTHOR]

Flagellin, the TLR5 agonist, shows potent adjuvant activities in diverse vaccines and immunotherapies. Vibrio vulnificus flagellin B expressed in eukaryotic cells (eFlaB) could not stimulate TLR5 signaling. Enzymatic deglycosylation restored eFlaB's TLR5 stimulating functionality, suggesting that glycosylation interferes with eFlaB binding to TLR5. Site-directed mutagenesis of N-glycosylation residues restored TLR5 stimulation and adjuvanticity. Collectively, deglycosylated eFlaB may provide a built-in adjuvant platform for eukaryotic-expressed antigens and nucleic acid vaccines. [ABSTRACT FROM AUTHOR]

Soluble adenylyl cyclase (sAC)‐derived cAMP regulates various cellular processes; however, the regulatory landscape mediating sAC protein levels remains underexplored. We consistently observed a 85 kD (sAC85) or 75 kD (sAC75) sAC protein band under glucose‐sufficient or glucose‐deprived states, respectively, in H69 cholangiocytes by immunoblotting. Deglycosylation by PNGase‐F demonstrated that both sAC75 and sAC85 are N‐linked glycosylated proteins with the same polypeptide backbone. Deglycosylation with Endo‐H further revealed that sAC75 and sAC85 carry distinct sugar chains. We observed release of N‐linked glycosylated sAC (sACEV) in extracellular vesicles under conditions that support intracellular sAC85 (glucose‐sufficient) as opposed to sAC75 (glucose‐deprived) conditions. Consistently, disrupting the vesicular machinery affects the maturation of intracellular sAC and inhibits the release of sACEV into extracellular vesicles. The intracellular turnover of sAC85 is extremely short (t1/2 ~30 min) and release of sACEV in the medium was detected within 3 h. Our observations support the maturation and trafficking in cholangiocytes of an N‐linked glycosylated sAC isoform that is rapidly released into extracellular vesicles. [ABSTRACT FROM AUTHOR]

Summary: Production of recombinant pharmaceutical glycoproteins has been carried out in multiple expression systems. However, N‐glycosylation, which increases heterogeneity and raises safety concerns due to the presence of non‐human residues, is usually not controlled. The presence and composition of N‐glycans are also susceptible to affect protein stability, function and immunogenicity. To tackle these issues, we are developing glycoengineered Nicotiana tabacum Bright Yellow‐2 (BY‐2) cell lines through knock out and ectopic expression of genes involved in the N‐glycosylation pathway. Here, we report on the generation of BY‐2 cell lines producing deglycosylated proteins. To this end, endoglycosidase T was co‐expressed with an immunoglobulin G or glycoprotein B of human cytomegalovirus in BY‐2 cell lines producing only high mannose N‐glycans. Endoglycosidase T cleaves high mannose N‐glycans to generate single, asparagine‐linked, N‐acetylglucosamine residues. The N‐glycosylation profile of the secreted antibody was determined by mass spectrometry analysis. More than 90% of the N‐glycans at the conserved Asn297 site were deglycosylated. Likewise, extensive deglycosylation of glycoprotein B, which possesses 18 N‐glycosylation sites, was observed. N‐glycan composition of gB glycovariants was assessed by in vitro enzymatic mobility shift assay and proven to be consistent with the expected glycoforms. Comparison of IgG glycovariants by differential scanning fluorimetry revealed a significant impact of the N‐glycosylation pattern on the thermal stability. Production of deglycosylated pharmaceutical proteins in BY‐2 cells expands the set of glycoengineered BY‐2 cell lines. [ABSTRACT FROM AUTHOR]

Identifying new environmental resistance determinants is significant to combat rising antibiotic resistance. Herein we report the unexpected correlation of a lobophorin (LOB) resistance‐related glycosidase KijX with the host‐dependent chemical diversity of LOBs, by a process of glycosylation, deglycosylation and reglycosylation. KijX homologues are widespread among bacteria, archaea and fungi, and encode the same glycohydrolytic activity on LOBs. The crystal structure of AcvX (a KijX homologue) shows a similar fold to that of the glycoside hydrolase family 113 and a special negatively charged groove to accommodate and deglycosylate LOBs. Antagonistic assays indicate kijX as a defense weapon of actinomycetes to combat LOB producers in environment, reflecting an elegant coevolution relationship. Our study provides insight into the KijX‐related glycosidases as preexisting resistance determinants and represents an example of resistance genes accidentally integrated into natural product assembly. [ABSTRACT FROM AUTHOR]

Identifying new environmental resistance determinants is significant to combat rising antibiotic resistance. Herein we report the unexpected correlation of a lobophorin (LOB) resistance‐related glycosidase KijX with the host‐dependent chemical diversity of LOBs, by a process of glycosylation, deglycosylation and reglycosylation. KijX homologues are widespread among bacteria, archaea and fungi, and encode the same glycohydrolytic activity on LOBs. The crystal structure of AcvX (a KijX homologue) shows a similar fold to that of the glycoside hydrolase family 113 and a special negatively charged groove to accommodate and deglycosylate LOBs. Antagonistic assays indicate kijX as a defense weapon of actinomycetes to combat LOB producers in environment, reflecting an elegant coevolution relationship. Our study provides insight into the KijX‐related glycosidases as preexisting resistance determinants and represents an example of resistance genes accidentally integrated into natural product assembly. [ABSTRACT FROM AUTHOR]

Detection of erythropoietin (Epo) was difficult until a method was developed by the World Anti-Doping Agency (WADA). WADA recommended the Western blot technique using isoelectric focusing (IEF)-PAGE to show that natural Epo and injected erythropoiesis-stimulating agents (ESAs) appear in different pH areas. Next, they used sodium N-lauroylsarcosinate (SAR)-PAGE for better differentiation of pegylated proteins, such as epoetin β pegol. Although WADA has recommended the use of pre-purification of samples, we developed a simple Western blotting method without pre-purification of samples. Instead of pre-purification, we used deglycosylation of samples before SDS-PAGE. The double detection of glycosylated and deglycosylated Epo bands increases the reliability of the detection of Epo protein. All of the endogenous Epo and exogenous ESAs shift to 22 kDa, except for Peg-bound epoetin β pegol. All endogenous Epo and exogenous ESAs were detected as 22 kDa deglycosylated Epo by liquid chromatography/mass spectrum (LC/MS) analysis. The most important factor for the detection of Epo is the selection of the antibody against Epo. WADA recommended clone AE7A5, and we used sc-9620. Both antibodies are useful for the detection of Epo protein by Western blotting. [ABSTRACT FROM AUTHOR]

Bacterial pathogens have evolved intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Streptococcus pyogenes serotypes secrete two multi-modular endo-β-N-acetylglucosaminidases, EndoS and EndoS2, that specifically deglycosylate the conserved N-glycan at Asn297 on IgG Fc, disabling antibody-mediated effector functions. Amongst thousands of known carbohydrate-active enzymes, EndoS and EndoS2 represent just a handful of enzymes that are specific to the protein portion of the glycoprotein substrate, not just the glycan component. Here, we present the cryoEM structure of EndoS in complex with the IgG1 Fc fragment. In combination with small-angle X-ray scattering, alanine scanning mutagenesis, hydrolytic activity measurements, enzyme kinetics, nuclear magnetic resonance and molecular dynamics analyses, we establish the mechanisms of recognition and specific deglycosylation of IgG antibodies by EndoS and EndoS2. Our results provide a rational basis from which to engineer novel enzymes with antibody and glycan selectivity for clinical and biotechnological applications. Bacterial pathogens have evolved intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Here, the authors establish the mechanisms of recognition and specific deglycosylation of IgG antibodies by the multi-modular enzymes EndoS and EndoS2 [ABSTRACT FROM AUTHOR]

β-glucosidases are found in all domains of life and has the ability to hydrolyse the glycosidic bond between carbohydrates and non-carbohydrates residues making it a potent enzyme from an industrial and agricultural perspective. The aim of the current study was isolation, screening, and identification of bacterial strains having maximum β-glucosidase activity from soil sample of Jacobabad, Pakistan. The isolates were screened primarily, and efficient isolates were confirmed for β-glucosidase production through secondary screening. Among the different isolated strains, 1j2 was selected as the best β-glucosidase producer. The isolate showed 99% similarity with Bacillus stercoris after 16S rRNA sequencing. Different culture conditions were optimized for effective β-glucosidase production. Enhanced level of β-glucosidase was achieved at 40°C, pH 8 at 150 rpm in a β-glucosidase production medium supplemented with a combination of agricultural residues of wheat bran 0.25% (w/v) and orange peel 0.25% (w/v), using 5% inoculum. Bacillus stercoris produced a higher yield (2.89U/mg) of β-glucosidase under optimized conditions. Partial purification of β-glucosidase was achieved after 80% ammonium sulfate precipitation. The enzyme was purified and had shown maximum specific activity of 53.2 U/mg with molecular weight of approximately 36 kDa. In all the tested metals Cu2+ was observed to have inhibitory effect while Ca2+ had not affected the enzyme activity significantly. While organic solvents and surfactants had significantly reduced the β-glucosidase activity. Based on the afore mentioned abilities of purified β-glucosidase from Bacillus stercoris strain it could be a promising player for the industrial applications of lignocellulose hydrolysis found in agricultural residues for the cheap production of biofuels. [ABSTRACT FROM AUTHOR]

Gut alkaline phosphatases (AP) dephosphorylate the lipid moiety of endotoxin and other pathogen-associated-molecular patterns members, thus maintaining gut eubiosis and preventing metabolic endotoxemia. Early weaned pigs experience gut dysbiosis, enteric diseases and growth retardation in association with decreased intestinal AP functionality. However, the role of glycosylation in modulation of the weaned porcine gut AP functionality is unclear. Herein three different research approaches were taken to investigate how deglycosylation affected weaned porcine gut AP activity kinetics. In the first approach, weaned porcine jejunal AP isoform (IAP) was fractionated by the fast protein-liquid chromatography and purified IAP fractions were kinetically characterized to be the higher-affinity and lower-capacity glycosylated mature IAP (p < 0.05) in comparison with the lower-affinity and higher-capacity non-glycosylated pre-mature IAP. The second approach enzyme activity kinetic analyses showed that N-deglycosylation of AP by the peptide N-glycosidase-F enzyme reduced (p < 0.05) the IAP maximal activity in the jejunum and ileum and decreased AP affinity (p < 0.05) in the large intestine. In the third approach, the porcine IAP isoform-X1 (IAPX1) gene was overexpressed in the prokaryotic ClearColiBL21 (DE3) cell and the recombinant porcine IAPX1 was associated with reduced (p < 0.05) enzyme affinity and maximal enzyme activity. Therefore, levels of glycosylation can modulate plasticity of weaned porcine gut AP functionality towards maintaining gut microbiome and the whole-body physiological status. [ABSTRACT FROM AUTHOR]

Background and Objectives: Early studies indicate that red cell A and B antigens are attached primarily onto band 3 and GLUT1 on the erythrocyte membrane and little onto glycophorin A (GPA) and glycophorin B (GPB). But as GPA and band 3 form stable protein complexes and GPA is much more heavily glycosylated than band 3, this study re‐examined the association between ABO antigens and GPA/GPB. Materials and Methods: Band 3/GPA‐associated protein complexes were first immunoprecipitated, followed by differential enzymatic deglycosylation that removed sialic acids, N‐glycans and O‐glycans. Serological anti‐A (BIRMA 1) and anti‐B IgM (GAMA 110) could be used for western blot (WB); however, only the anti‐B IgM showed significant reactivity for the immunoprecipitates isolated by anti‐band 3. The expression of the B antigen in un‐deglycosylated and differentially deglycosylated band 3 immunoprecipitates was thus compared. Results: Besides attachment to band 3, red cell B antigen expressed substantially on GPA monomer and homodimer, GPA*GPB heterodimer, and GPB monomer and dimer via attachments through the N‐ and O‐glycans. Conclusion: Immunoprecipitation (IP), as a means of protein separation and concentration, was used in combination with a WB to differentiate glycosylation on different proteins and oligomers. This study implemented differential enzymatic deglycosylation during IP of the band 3 complexes. This combined approach allowed separate identification of the B antigen on GPA/GPB monomer and dimer and GPA*GPB heterodimer, and band 3 on the WB and verified non‐trivial expression of the B antigen on GPA and GPB on the erythrocyte surface. [ABSTRACT FROM AUTHOR]

DNA damages are regarded as having harmful effects on cell. The base excision repair mechanism combats these effects by removing damaged bases. The deglycosylation mechanism of excising damaged bases by DNA glycosylase and the state of the leaving base have been controversial. The enzymatic reaction of DNA glycosylase to remove the damaged bases involves not only the formation and breaking of chemical bonds, but also complex polarization effect and charge transfer, which cannot be accurately simulated by the QM/MM method combined with the fixed charge force field. This work has developed the ABEEM fluctuating polarizable force field combining with the QM method, that is (QM/MM[ABEEM]), to accurately simulate the proton transfer, charge transfer and the charge distribution. The piecewise function is used as the valence‐state electronegativity in the QM/MM (ABEEM) to realize the accurate fitting of the charge distribution in reaction. And the charge transfer is accurately simulated by the local charge conservation conditions. Four deglycosylation mechanisms including the monofunctional and difunctional mechanisms of four neutral and protonated cytosine derivatives are explored. It is confirmed that the monofunctional mechanism of Asp‐activated nucleophile water is a better deglycosylation mechanism and the base is protonated before the reaction occurs. Protonization of the base reduced the activation energy by 10.00–17.00 kcal/mol. Asp provides the necessary charge for the reaction, and DNA glycosylase preferentially cleaves ɛC. This work provides a theoretical basis for the research of excising damaged bases by DNA glycosylase. [ABSTRACT FROM AUTHOR]

A glycosylated heat stable trypsin chymotrypsin inhibitor was isolated from Cyamopsis tetragonoloba seeds. It is being reported for the first time that deglycosylation of the inhibitor chemically by Trifluoro methane sulfonic acid or enzymatically by salivary amylase or fungal diastase abrogates the inhibitory activity indicating that glycosylation was important for inhibitory activity of the protein. Treatment with salivary amylase decreases the inhibition in in vitro digestion experiment also. Germination of Cyamopsis tetragonoloba seeds results in reduction in inhibitory activity with concomitant increase in amylase activity. It is speculated that increase in amylase during germination decreases the protease inhibitory activity by deglycosylating the inhibitor. [ABSTRACT FROM AUTHOR]

This article is a Commentary on Lacchini et al. (2023), 239: 705–719. [ABSTRACT FROM AUTHOR]

NGLY1 deficiency is a rare in which affected individuals show developmental delay/intellectual disability in the mild to profound range, epilepsy, auditory neuropathy, abnormal liver function, complex hyperkinetic movement disorder and poor growth. Here, we present the follow-up results of hearing status in a patient with NGLY1 deficiency. [ABSTRACT FROM AUTHOR]

Sorsby fundus dystrophy (SFD) is an autosomal dominant macular disorder caused by mutations in tissue Inhibitor of the metalloproteinase-3 (TIMP3) gene with the onset of symptoms including choroidal neovascularization as early as the second decade of life. We have previously reported that wild-type TIMP3 is an endogenous angiogenesis inhibitor that inhibits Vascular Endothelial Growth Factor (VEGF)-mediated signaling in endothelial cells. In contrast, SFD-related S179C-TIMP3 when expressed in endothelial cells, does not have angiogenesis-inhibitory properties. To evaluate if this is a common feature of TIMP3 mutants associated with SFD, we examined and compared endothelial cells expressing S179C, Y191C and S204C TIMP3 mutants for their angiogenesis-inhibitory function. Western blot analysis, zymography and reverse zymography and migration assays were utilized to evaluate TIMP3 protein, Matrix Metalloproteinase (MMP) and MMP inhibitory activity, VEGF signaling and in vitro migration in endothelial cells expressing (VEGF receptor-2 (VEGFR-2) and wild-type TIMP3 or mutant-TIMP3. We demonstrate that mutant S179C, Y191C- and S204C-TIMP3 all show increased glycosylation and multimerization/aggregation of the TIMP3 protein. In addition, endothelial cells expressing TIMP3 mutations show increased angiogenic activities and elevated VEGFR-2. Removal of N-glycosylation by mutation of Asn184, the only potential N-glycosylation site in mutant TIMP3, resulted in increased aggregation of TIMP3, further upregulation of VEGFR-2, VEGF-induced phosphorylation of VEGFR2 and VEGF-mediated migration concomitant with reduced MMP inhibitory activity. These results suggest that even though mutant TIMP3 proteins are more glycosylated, post-translational deglycosylation may play a critical role in the aggregation of mutant TIMP3 and contribute to the pathogenesis of SFD. The identification of factors that might contribute to changes in the glycome of patients with SFD will be useful. Future studies will evaluate whether variations in the glycosylation of mutant TIMP3 proteins are contributing to the severity of the disease. [ABSTRACT FROM AUTHOR]

The analysis of glycoproteins and the comparison of protein N‐glycosylation from different eukaryotic origins require unbiased and robust analytical workflows. The structural and functional analysis of vertebrate protein N‐glycosylation currently depends extensively on bacterial peptide‐N4‐(N‐acetyl‐β‐glucosaminyl) asparagine amidases (PNGases), which are indispensable enzymatic tools in releasing asparagine‐linked oligosaccharides (N‐glycans) from glycoproteins. So far, only limited PNGase candidates are available for N‐glycans analysis, and particularly the analysis of plant and invertebrate N‐glycans is hampered by the lack of suitable PNGases. Furthermore, liquid chromatography–mass spectrometry (LC–MS) workflows, such as hydrogen deuterium exchange mass spectrometry (HDX‐MS), require a highly efficient enzymatic release of N‐glycans at low pH values to facilitate the comprehensive structural analysis of glycoproteins. Herein, we describe a previously unstudied superacidic bacterial N‐glycanase (PNGase H+) originating from the soil bacterium Rudaea cellulosilytica (Rc), which has significantly improved enzymatic properties compared to previously described PNGase H+ variants. Active and soluble recombinant PNGase Rc was expressed at a higher protein level (3.8‐fold) and with higher specific activity (~56% increase) compared to the currently used PNGase H+ variant from Dyella japonicum (Dj). Recombinant PNGase Rc was able to deglycosylate the glycoproteins horseradish peroxidase and bovine lactoferrin significantly faster than PNGase Dj (10 min vs. 6 h). The versatility of PNGase Rc was demonstrated by releasing N‐glycans from a diverse array of samples such as peach fruit, king trumpet mushroom, mouse serum, and the soil nematode Caenorhabditis elegans. The presence of only two disulfide bonds shown in the AlphaFold protein model (so far all other superacidic PNGases possess more disulfide bonds) could be corroborated by intact mass‐ and peptide mapping analysis and provides a possible explanation for the improved recombinant expression yield of PNGase Rc. [ABSTRACT FROM AUTHOR]

Taxifolin is known to have multiple biological functions. It has been widely used as a multifunctional food additive, and consequently, the global demand for taxifolin is increasing. The main method for taxifolin production is an extraction from larch wood, but the global resources of larch are limited. Astilbin, taxifolin-3-o-rhamnoside, is abundant in many plants and much more readily available, meaning taxifolin can be obtained by deglycosylation of astilbin. In this study, a fungal strain, Aspergillus fumigatus SQH4, was isolated from an enrichment culture of Smilax glabra rhizome to achieve the deglycosylation reaction. A culture of SQH4, adjusted to pH 6.5, with 5 g/L astilbin achieved a yield of taxifolin of 91.3% after biotransformation for 14 h at 35 °C. These findings offer an alternative method for the production of taxifolin. [ABSTRACT FROM AUTHOR]

aimen People's Hospital Affiliated to Nantong University, Haimen, 226100, People's Republic of China; 3The School of Public Health, Nanjing Medical University, Nanjing, 210000, People's Republic of China*These authors contributed equally to this workCorrespondence: Meilin Wang; Chaoying Liu, Email [email protected] ; [email protected] Purpose: The aim of this study was to explore the clinical significance of deglycosylated PD-L1 level and its correlation with EGFR and ALK mutation in lung adenocarcinoma. Materials and Methods: We estimated the intensity of both native and deglycosylated PD-L1 signals using a 28– 8 antibody on lung adenocarcinoma tissue microarray sections. We analyzed the difference in the H-score between tumor and paratumor tissues, as well as that before and after deglycosylation. Correlations between EGFR or ALK status and PD-L1 expression were analyzed. We also evaluated the differences among survival curves. Results: The expression level of PD-L1 in lung adenocarcinoma tissues was significantly higher than that in paratumor tissues (P< 0.0001). Deglycosylation significantly enhanced the detection of PD-L1 in tumor tissues (P< 0.0001). There was no statistical significance between the signal intensity of deglycosylated PD-L1 and the survival of patients (P=0.9099). However, the response to deglycosylation of PD-L1 was significantly correlated with the survival of patients with stage N1-N3 (P=0.0435) and stage T3-T4 (P=0.0366) and male patients (P=0.0258). A statistical trend was found in the correlation between the response to deglycosylation of PD-L1 and the survival of patients with grade II–III plus grade III (P=0.0973). Correlation between EGFR or ALK status and the expression of PD-L1 was not found (P> 0.05). Conclusion: PD-L1 deglycosylation enhances the detection of PD-L1 when utilizing a 28– 8 antibody. Moreover, the response to deglycosylation of PD-L1 may predict the survival of certain patients with lung adenocarcinoma. [ABSTRACT FROM AUTHOR]

Immunohistochemical (IHC) predictive quantitation of PDL1 expression is obligatory in many cancer entities with improved response to immune checkpoint inhibition in PDL1-positive subgroups. With recent demonstration of increased positivity rates after enzymatic deglycosylation in breast cancer specimens, a comparative analysis with two different antibodies and extended controls was performed in a cohort of head and neck squamous cell cancer samples (HNSCC). Formalin-fixed paraffin-embedded tissue from HNSCC specimens was used for initial on-slide method optimization based on the PNGase F assay. SDS-PAGE and immunoblotting with the PDL1 antibody 28–8 was performed to evaluate deglycosylation efficiency. A tissue micro array of n = 527 tissue cores of 181 patients with HNSCC was used to determine the effects of deglycosylation on staining pattern and intensity with PDL1 antibodies 28–8 and E1L3N. Successful on-slide deglycosylation with PNGase F was confirmed by immunoblot but varied across replicates. Using E1L3N (intracellular binding domain, most probably not glycosylated), mean signal intensity as well as the fraction of PDL1 positive cells was increased by deglycosylation. Opposite effects were observed with 28–8 (extracellular binding domain, glycosylated). Deglycosylation reduces diagnostic performance of the PDL1 antibody 28–8. In contrast, effects for E1L3N are complex and probably involve reduction of off-target binding leading to specifically improved signal intensity. However, enzymatic deglycosylation adds further variance to IHC. [ABSTRACT FROM AUTHOR]

Glycosidic bond of C-glycosides is difficult to be broken due to its chemical stability. Screening specific microbe from microbiota is a practical way to deglycosylate these compounds. In this study, a new strain W974-1 which is capable of cleaving C-glycosidic bonds was isolated from human gut microbiota by spread plate method. It deglycosylates flavonoid 8-C-glycosides such as orientin and vitexin to their aglycones with the enzymes secreted outside the bacterial cells. This strain was identified as Enterococcus avium by 16S rDNA sequencing, physiological and biochemical characterization. [ABSTRACT FROM AUTHOR]

Aim: The 20(S)-ginsenoside Rh2 (Rh2) is being developed as a new antitumor drug. However, to date, little is known about the kinetics of its deglycosylation metabolite (protopanoxadiol) (PPD) following Rh2 administration. The aim of this work was to 1) simultaneously characterise the pharmacokinetics of Rh2 and PPD following intravenous and oral Rh2 administration, 2) develop and validate a mechanism-based pharmacokinetic model to describe the deglycosylation kinetics and 3) predict the percentage of Rh2 entering the systemic circulation in PPD form. Methods: Plasma samples were collected from rats after the I.V. or P.O. administration of Rh2. The plasma Rh2 and PPD concentrations were determined using HPLC-MS. The transformation from Rh2 to PPD, its absorption, and elimination were integrated into the mechanism based pharmacokinetic model to describe the pharmacokinetics of Rh2 and PPD simultaneously at 10 mg/kg. The concentration data collected following a 20 mg/kg dose of Rh2 was used for model validation. Results: Following Rh2 administration, PPD exhibited high exposure and atypical double peaks. The model described the abnormal kinetics well and was further validated using external data. A total of 11% of the administered Rh2 was predicted to be transformed into PPD and enter the systemic circulation after I.V. administration, and a total of 20% of Rh2 was predicted to be absorbed into the systemic circulation in PPD form after P.O. administration of Rh2. Conclusion: The developed model provides a useful tool to quantitatively study the deglycosylation kinetics of Rh2 and thus, provides a valuable resource for future pharmacokinetic studies of glycosides with similar deglycosylation metabolism. [ABSTRACT FROM AUTHOR]

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]

BACKGROUND: Flaxseed and soybean are an important source of lignans and flavonoids. Previously, the metabolism of isoflavones and lignans from soybean and flaxseed extracts by the microbiota of adult individuals (n = 14) and infants (n = 23) was analyzed. Thus, the present study aimed to examine the metabolism of flavones, flavanones and flavonols, as well as the production of phenolic acids, by the intestinal microbiota of these individuals. RESULTS: Concentrations of aglycones of flavonoids, such as herbacetin, quercetin, quercetagetin, myricetin, kaempferol, apigenin and luteolin, increased for most of individuals as a consequence of deglycosylation reactions. On the other hand, a diminution in the antioxidant activity and phenolic compound concentration and an increase in the concentration of 3,4‐dihydroxyphenylacetic acid, 2‐(4‐hydroxyphenyl)‐propionic acid, protocatechuic acid and catechol was also observed. CONCLUSION: The present study found that deglycosylation reactions were the main reactions and accelerated the formation of more bioavailable flavonoids, with greater biological activity, in most of the individuals. However, other reactions also occurred, including the total or partial catabolism of flavonoids. © 2021 Society of Chemical Industry. [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]

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]

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]

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]

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]

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]