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In vaccine design, antigens are often arrayed in a multivalent nanoparticle form, but in vivo mechanisms underlying the enhanced immunity elicited by such vaccines remain poorly understood. We compared the fates of two different heavily glycosylated HIV antigens, a gp120-derived mini-protein and a large, stabilized envelope trimer, in protein nanoparticle or “free” forms after primary immunization. Unlike monomeric antigens, nanoparticles were rapidly shuttled to the follicular dendritic cell (FDC) network and then concentrated in germinal centers in a complement-, mannose-binding lectin (MBL)–, and immunogen glycan–dependent manner. Loss of FDC localization in MBL-deficient mice or via immunogen deglycosylation significantly affected antibody responses. These findings identify an innate immune–mediated recognition pathway promoting antibody responses to particulate antigens, with broad implications for humoral immunity and vaccine design.

Monoclonal antibodies (mAbs) have been developed as therapeutics, especially for the treatment of cancer, inflammation, and infectious diseases. Because the glycosylation of mAbs in the Fc region influences their interaction with effector cells that kill antibody-targeted cells, and the current method of antibody production is relatively expensive, efforts have been directed toward the development of alternative expressing systems capable of large-scale production of mAbs with desirable glycoforms. In this study, we demonstrate that the mAb trastuzumab expressed in glycoengineered P. pastoris can be remodeled through deglycosylation by endoglycosidases identified from the Carbohydrate Active Enzymes database and through transglycosylation using glycans with a stable leaving group to generate a homogeneous antibody designed to optimize the effector functions. The 10 newly identified recombinant bacterial endoglycosidases are complementary to existing endoglycosidases (EndoA, EndoH, EndoS), two of which can even accept sialylated triand tetraantennary glycans as substrates.

The phenylpropanoid pathway in plants is responsible for the biosynthesis of a huge amount of secondary metabolites derived from phenylalanine and tyrosine. Both flavonoids and lignins are synthesized at the end of this very diverse metabolic pathway, as well as many intermediate molecules whose precise biological functions remain largely unknown. The diversity of these molecules can be further increased under the action of UDP-glycosyltransferases (UGTs) leading to the production of glycosylated hydroxycinnamates and related aldehydes, alcohols and esters. Glycosylation can change phenylpropanoid solubility, stability and toxic potential, as well as influencing compartmentalization and biological activity. (De)-glycosylation therefore represents an extremely important regulation point in phenylpropanoid homeostasis. In this article we review recent knowledge on the enzymes involved in regulating phenylpropanoid glycosylation status and availability in different subcellular compartments. We also examine the potential link between monolignol glycosylation and lignification by exploring co-expression of lignin biosynthesis genes and phenolic (de)glycosylation genes. Of the different biological roles linked with their particular chemical properties, phenylpropanoids are often correlated with the plant's stress management strategies that are also regulated by glycosylation. UGTs can for instance influence the resistance of plants during infection by microorganisms and be involved in the mechanisms related to environmental changes. The impact of flavonoid glycosylation on the color of flowers, leaves, seeds and fruits will also be discussed. Altogether this paper underlies the fact that glycosylation and deglycosylation are powerful mechanisms allowing plants to regulate phenylpropanoid localisation, availability and biological activity.

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.

Background/Objectives: Salivary gland tumors (SGTs) are a rare and histologically heterogeneous group of neoplasms that are challenging to diagnose due to phenotypic heterogeneity and overlapping histomorphological markers. Accurate diagnosis is required for clinical management, particularly in unusual subtypes. The objective of this study was to ascertain whether attenuated total reflectance–Fourier transform infrared (ATR-FTIR) spectroscopy, in combination with enzymatic deglycosylation, would be useful in SGT classification by detecting glycosylation-related metabolic variations. Methods: 155 tissue sections, consisting of 80 SGTs and 75 controls, were analyzed. ATR-FTIR spectroscopy was used to record the mid-infrared (MIR) spectra (4000–400 cm−1) of enzymatically untreated and deglycosylated samples. Spectral data were preprocessed and analyzed by principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). Enzymatic deglycosylation focused on sialic acid and fucose residues with α2-3,6,8 neuraminidase, α1-2,4,6 fucosidase O, and α1-3,4 fucosidase. Results: Tumor and control samples were discriminated with an OPLS-DA model, achieving an accuracy of 81.9% (78.7% for controls and 85.0% for tumors), especially in the glycosylation-relevant spectral range (850–1250 cm−1). Classification between benign and malignant tumors was more challenging, with an accuracy of 70.0% (72.5% for benign and 67.5% for malignant cases). Enzymatic deglycosylation resulted in detectable changes in the MIR spectra, confirming the contribution of glycosylation to tumor-specific signatures. Benign vs. malignant tumor discrimination was still poor and was not much enhanced in the sense of incorporating glycosylation-specific regions. Conclusions: ATR-FTIR spectroscopy coupled with enzymatic deglycosylation can distinguish tumor and control tissues based on glycan-associated spectral differences. Application of the technique to benign/malignant SGT discrimination is hampered by spectral overlap and tumor heterogeneity. Further research will be necessary to explore other clustering algorithms and larger and more homogeneous datasets for improved diagnostic accuracy.

Baicalein and wogonin, two health-promoting flavonoid aglycones in Scutellaria baicalensis Georgi roots, have better bioactivity and bioavailability than their glycoside precursors (baicalin and wogonoside). The search for novel microorganisms for the fermentation of S. baicalensis roots to improve the yields of baicalein and wogonin is of great interest in the food and nutraceutical industries. In this study, a novel endophytic fungus ( Penicillium rubens ) capable of producing cellulase (0.82 ± 0.12 U/mL) and β-glucosidase (4.55 ± 0.97 U/mL) was used to ferment S. baicalensis roots to obtain high yields of baicalein and wogonin. Semi-solid-state fermentation with homogeneous fungal cultures was found to be the appropriate strategy. Under the optimal fermentation parameters (temperature 30 °C, inoculation dose of fungal cultures 4 mL/g, and time 60 h), the yields of baicalein and wogonin (46.95 ± 4.98 mg/g DW and 31.41 ± 9.36 mg/g DW) from S. baicalensis roots were tremendously increased by 16.13-fold and 15.47-fold over control, respectively. Results of industrial enzyme bioprocessing and scanning electron microscope indicated that the remarkable increase in aglycone product yields could be due to the synergistic effects of cellulase and β-glucosidase produced by P. rubens on cell wall hydrolysis and glycoside deglycosylation. In addition, extracts of S. baicalensis roots fermented by P. rubens showed a significant increase in antioxidant and antimicrobial activities. Overall, the semi-solid-state fermentation of S. baicalensis roots using the novel P. rubens was an effective strategy that could yield high quantities of baicalein and wogonin, which had promising potential in the food and nutraceutical industries. Graphical Abstract

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.

Development of specific IgE antibodies to the oligosaccharide galactose-α-1, 3-galactose (α-gal) following tick bites has been shown to be the source of red meat allergy. In this study, we investigated the presence of α-gal in four tick species: the lone-star tick ( ), the Gulf-Coast tick ( ), the American dog tick ( ), and the black-legged tick ( ) by using a combination of immunoproteomic approach and, carbohydrate analysis. Anti-α-gal antibodies identified α-gal in the salivary glands of both and , while and appeared to lack the carbohydrate. PNGase F treatment confirmed the deglycosylation of N-linked α-gal-containing proteins in tick salivary glands. Immunolocalization of α-gal moieties to the salivary secretory vesicles of the salivary acini also confirmed the secretory nature of α-gal-containing antigens in ticks. ticks were fed on human blood (lacks α-gal) using a silicone membrane system to determine the source of the α-gal. N-linked glycan analysis revealed that and have α-gal in their saliva and salivary glands, but contains no detectable quantity. Consistent with the glycan analysis, salivary samples from and stimulated activation of basophils primed with plasma from α-gal allergic subjects. Together, these data support the idea that bites from certain tick species may specifically create a risk for the development of α-gal-specific IgE and hypersensitivity reactions in humans. Alpha-Gal syndrome challenges the current food allergy paradigm and broadens opportunities for future research.

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.

O-linked N -acetylglucosamine ( O -GlcNAc) is an essential and dynamic post-translational modification that is presented on thousands of nucleocytoplasmic proteins. Interrogating the role of O -GlcNAc on a single target protein is crucial, yet challenging to perform in cells. Herein, we developed a nanobody-fused split O -GlcNAcase (OGA) as an O -GlcNAc eraser for selective deglycosylation of a target protein in cells. After systematic cellular optimization, we identified a split OGA with reduced inherent deglycosidase activity that selectively removed O -GlcNAc from the desired target protein when directed by a nanobody. We demonstrate the generality of the nanobody-fused split OGA using four nanobodies against five target proteins and use the system to study the impact of O -GlcNAc on the transcription factors c-Jun and c-Fos. The nanobody-directed O -GlcNAc eraser provides a new strategy for the functional evaluation and engineering of O -GlcNAc via the selective removal of O -GlcNAc from individual proteins directly in cells. Fusion of a split form of the protein O -GlcNAcase with nanobodies enables the targeted removal of O -GlcNAc protein modifications, providing a tool for probing the functional roles of specific O -GlcNAc modifications in a cellular context.