Investigation of the potential regulation of the UDP-glycosyltransferase genes on rice grain size and abiotic stress response.

Uridine diphosphate (UDP) glycosyltransferases (UGTs) are widely involved in various metabolic processes. In the present study, we performed a genome-wide survey and identified 199 Oryza sativa UGT genes (OsUGTs), which were classified into 17 groups. We showed that tandem duplication played a major role in the expansion of the OsUGT family, which experienced purifying selection during the evolution process. 163 OsUGTs were expressed in at least one of the six tested tissues, and were clustered into three groups according to their tissue expression profiles. By using the RFGB database, we identified different haplotypes of seven OsUGTs that were highly expressed in seeds, and showed significant differences in grain size among different haplotypes. Moreover, our results also uncovered differential responses of OsUGTs expression to abiotic stresses and hormone treatments, including drought, salt, cold, heat, ABA, JA and AUXIN. By using quantitative real-time PCR, we further confirmed the differential expression of nine selected OsUGTs under ABA, JA, salt, drought and cold treatments, among which OsUGT5 and OsUGT182 were induced by all these five treatments. Our results provide insight into the role of several UGT genes for physiological responses, which will facilitate to investigate their function in regulating rice development and abiotic stress responses.

Regulation of human GnT-IV family activity by the lectin domain.

N-Glycan branching critically regulates glycoprotein functions and is involved in various diseases. Among the glycosyltransferases involved in N-glycan branching is the human N-acetylglucosaminyltransferase-IV (GnT-IV) family, which has four members: GnT-IVa, GnT-IVb, GnT-IVc, and GnT-IVd. GnT-IVa and GnT-IVb have glycosyltransferase activity that generates the type-2 diabetes-related ß1,4-GlcNAc branch on the α1,3-Man arm of N-glycans, whereas GnT-IVc and GnT-IVd do not. Recently, this enzyme family was found to have a unique lectin domain in the C-terminal region, which is essential for enzyme activity toward glycoprotein substrates but not toward free N-glycans. Furthermore, interaction between the lectin domain of GnT-IV and N-glycan attached to GnT-IV enables self-regulation of GnT-IV activity, indicating that the lectin domain plays a unique and pivotal role in the regulation of GnT-IV activity. In this review, we summarize the GnT-IV family's biological functions, selectivity for glycoprotein substrates, and regulation of enzymatic activity, with a focus on its unique C-terminal lectin domain.

Predictive Production of a New Highly Soluble Glucoside, Corylin-7-O-ß-Glucoside with Potent Anti-inflammatory and Anti-melanoma Activities.

Computational tools can now facilitate screening precursors and selecting suitable biotransformation enzymes for producing new bioactive compounds. This study applied the data-mining approach to screen for candidate precursors of glycosyltransferases to produce new glucosides from 412 commercial natural compounds. Among five candidates, experimental results showed that only corylin could be glycosylated by the bacterial glycosyltransferase, BsUGT489. Analysis of interaction potential between candidates and glycosyltransferase by molecular docking tools also found that corylin was the only compatible substrate. The new glucoside was purified and confirmed to be corylin-7-O-ß-glucoside. The aqueous solubility of corylin-7-O-ß-glucoside was 14.2 times more than its precursor aglycone, corylin. Corylin-7-O-ß-glucoside retained anti-inflammatory activity in lipopolysaccharide-induced nitric oxide production of murine macrophage RAW 264.7 cells, with an IC50 value of 121.1 ± 9.5 µM. Further, corylin-7-O-ß-glucoside exhibited more potent anti-melanoma activity against murine B16 and human A2058 melanoma cells than corylin. Together, predictive studies facilitate the production of a new glucoside, corylin-7-O-ß-glucoside, which is highly soluble and possesses anti-inflammatory and anti-melanoma activities and therefore has promising future applications in pharmacology.

Development of a FUT8 Inhibitor with Cellular Inhibitory Properties.

Core fucosylation is catalyzed by α-1,6-fucosyltransferase (FUT8), which fucosylates the innermost GlcNAc of N-glycans. Given the association of FUT8 with various diseases including cancer, selective FUT8 inhibitors applicable to in vivo or cell-based systems are highly sought-after. Here, we report the discovery of a compound that selectively inhibits FUT8 in cell-based assays. High-throughput screening revealed a FUT8-inhibiting pharmacophore, and further structural optimization yielded an inhibitor with a KD of 49 nM. Notably, this binding occurs only in the presence of GDP (a product of the enzymatic reaction catalyzed by FUT8). Mechanistic studies suggested that this inhibitor generates a highly reactive naphthoquinone methide derivative at the binding site in FUT8, which subsequently reacts with FUT8. Furthermore, prodrug derivatization of this inhibitor improved its stability, enabling suppression of core fucose expression and subsequent EGFR and T-cell signaling in cell-based assays, paving the way for the development of drugs targeting core fucosylation.

Tetraphenylborate enhanced enzymatic production of γ-cyclodextrin: System construction and its mechanism.

γ-Cyclodextrin (γ-CD) is an attractive material among the natural cyclodextrins owing to its excellent properties. γ-CD is primarily produced from starch by γ-cyclodextrin glycosyltransferase (γ-CGTase) in a controlled system. However, difficulty in separation and low conversion rate leads to high production costs for γ-CD. In this study, γ-CGTase from Bacillus sp. G-825-6 STB17 was used in γ-CD production from cassava starch. With the introduction of sodium tetraphenylborate (NaBPh4), the total conversion rate was promoted from an initial 18.07 % to 50.49 % and the γ-CD ratio reached 78.81 % with a yield of 39.79 g/L. Furthermore, the mechanism was conducted via the determination of binding constant, which indicated that γ-CD exhibited much stronger binding strength with NaBPh4 than ß-CD. The reformation of water molecules and the chaotropic effect might be the main driving forces for the interaction. Additionally, the conformations of CD complexes were depicted by NMR and molecular docking. The results further verified different binding patterns between CDs and tetraphenylborate ions, which might be the primary reason for the specific binding. This system not only guides γ-CD production with an efficient and easy-to-remove production aid but also offers a new perspective on the selection of complexing agents in CD production.

Investigation of physiological roles of UDP-glycosyltransferase UGT76F2 in auxin homeostasis through the TAA-YUCCA auxin biosynthesis pathway.

Cellular auxin (indole-3-acetic acid, IAA) levels are coordinately regulated by IAA biosynthesis and inactivation. IAA is synthesized through sequential reactions by two enzymes, TAA1 and YUCCA, in a linear indole-3-pyruvic acid (IPA) pathway. TAA1 converts tryptophan to IPA, and YUCCA catalyzes the oxidative decarboxylation of IPA into IAA. Arabidopsis UDP-glycosyltransferase UGT76F2 (At3g55710) was previously reported to catalyze the glycosylation of IPA and consequently modulate IAA levels. We carefully analyzed the physiological roles of UGT76F2 and its close homolog UGT76F1 (At3g55700) in IAA homeostasis. We generated two independent ugt76f1 ugt76f2 double null Arabidopsis mutants (ugt76f1f2) with a 2.7 kb deletion, along with two independent ugt76f2 single null mutants by CRISPR/Cas9 gene editing technology. Surprisingly, these null mutants exhibited indistinguishable phenotypes from the wild-type seedlings under our laboratory conditions. Our results indicate that UGT76F1 and UGT76F2 do not play important roles in regulating IAA biosynthesis via the IPA glycosylation.

Structure and function of N-acetylglucosaminyltransferase V (GnT-V).

BACKGROUND: The ß1,6-GlcNAc branch in N-glycans, produced by a glycosyltransferase N-acetylglucosaminyltransferase V (GnT-V or MGAT5), is associated with cancer and autoimmune diseases. SCOPE: Here, we summarize the structure and activity regulation of GnT-V. We also describe the roles of the ß1,6-GlcNAc branch on glycoproteins in cells and the phenotypes of Mgat5-deficient mice, focusing on cancer and the immune system. MAJOR CONCLUSIONS: GnT-V has a unique structure for substrate recognition, and its activity and function are regulated by shedding. The glycans produced by GnT-V play pivotal roles in the differentiation of neural cells, cancer malignancy and immunotherapy, and the development of autoimmune diseases by regulating the functions and cell surface residency of glycoproteins. GENERAL SIGNIFICANCE: Controlling the expression or activity of GnT-V could be a therapeutic option against cancer and autoimmune diseases. Future work should clarify how GnT-V selectively modifies the specific glycoproteins or N-glycosylation sites in vivo.

Identification of glycosyltransferase genes for diagnosis of T-cell mediated rejection and prediction of graft loss in kidney transplantation.

BACKGROUND: Glycosylation is a complex and fundamental metabolic biosynthetic process orchestrated by multiple glycosyltransferases (GT) and glycosidases enzymes. Functions of GT have been extensively examined in multiple human diseases. Our study investigated the potential role of GT genes in T-cell mediated rejection (TCMR) and possible prediction of graft loss of kidney transplantation. METHODS: We downloaded the microarray datasets and GT genes from the GEO and the HUGO Gene Nomenclature Committee (HGNC) databases, respectively. Differentially expressed GT genes (DE-GTGs) were obtained by differential expression and Venn analysis. A TCMR diagnostic model was developed based on the hub DE-GTGs using LASSO regression and XGboost machine learning algorithms. In addition, a predictive model for graft survival was constructed by univariate Cox and LASSO Cox regression analysis. RESULTS: We have obtained 15 DE-GTGs. Both GO and KEGG analyses showed that the DE-GTGs were mainly involved in the glycoprotein biosynthetic process. The TCMR diagnostic model exhibited high diagnostic potential with generally highly correlated accuracies [aera under the curve (AUC) of 0.83]. The immune characteristics analysis revealed that higher levels of immune cell infiltration and immune responses were observed in the high-risk group than in the low-risk group. In particular, the Kaplan-Meier survival analysis revealed that renal grafts in the high-risk group have poor prognostic outcomes than the low-risk group. The predictive AUC values of 1-, 2- and 3-year graft survival were 0.76, 0.81, and 0.70, respectively. CONCLUSION: Our results indicated that GT genes could be used for diagnosis of TCMR and prediction of graft loss in kidney transplantation. These results provide new perspectives and tools for diagnosing, treating and predicting kidney transplant-related diseases.

Combining an Enhanced Polyphosphate Kinase-Driven UDP-Glucose Regeneration System with the Screening of Key Glycosyltransferases for Efficient In Vitro Synthesis of Nucleoside Disaccharides.

Nucleoside disaccharides are essential glycosides that naturally occur in specific living organisms. This study developed an enhanced UDP-glucose regeneration system to facilitate the in vitro multienzyme synthesis of nucleoside disaccharides by integrating it with nucleoside-specific glycosyltransferases. The system utilizes maltodextrin and polyphosphate as cost-effective substrates for UDP-glucose supply, catalyzed by α-glucan phosphorylase (αGP) and UDP-glucose pyrophosphorylase (UGP). To address the low activity of known polyphosphate kinases (PPKs) in the UDP phosphorylation reaction, a sequence-driven screening identified RhPPK with high activity against UDP (>1000 U/mg). Computational design further led to the creation of a double mutant with a 2566-fold increase in thermostability at 50 °C. The enhanced UDP-glucose regeneration system increased the production rate of nucleoside disaccharide synthesis by 25-fold. In addition, our UDP-glucose regeneration system is expected to be applied to other glycosyl transfer reactions.

Functional characterization, structural basis, and protein engineering of a rare flavonoid 2'-O-glycosyltransferase from Scutellaria baicalensis.

Glycosylation is an important post-modification reaction in plant secondary metabolism, and contributes to structural diversity of bioactive natural products. In plants, glycosylation is usually catalyzed by UDP-glycosyltransferases. Flavonoid 2'-O-glycosides are rare glycosides. However, no UGTs have been reported, thus far, to specifically catalyze 2'-O-glycosylation of flavonoids. In this work, UGT71AP2 was identified from the medicinal plant Scutellaria baicalensis as the first flavonoid 2'-O-glycosyltransferase. It could preferentially transfer a glycosyl moiety to 2'-hydroxy of at least nine flavonoids to yield six new compounds. Some of the 2'-O-glycosides showed noticeable inhibitory activities against cyclooxygenase 2. The crystal structure of UGT71AP2 (2.15 Å) was solved, and mechanisms of its regio-selectivity was interpreted by pK a calculations, molecular docking, MD simulation, MM/GBSA binding free energy, QM/MM, and hydrogen‒deuterium exchange mass spectrometry analysis. Through structure-guided rational design, we obtained the L138T/V179D/M180T mutant with remarkably enhanced regio-selectivity (the ratio of 7-O-glycosylation byproducts decreased from 48% to 4%) and catalytic efficiency of 2'-O-glycosylation (k cat/K m, 0.23 L/(s·µmol), 12-fold higher than the native). Moreover, UGT71AP2 also possesses moderate UDP-dependent de-glycosylation activity, and is a dual function glycosyltransferase. This work provides an efficient biocatalyst and sets a good example for protein engineering to optimize enzyme catalytic features through rational design.

Bulk and single-cell RNA sequencing analyses coupled with multiple machine learning to develop a glycosyltransferase associated signature in colorectal cancer.

BACKGROUND: This study aims to identify key glycosyltransferases (GTs) in colorectal cancer (CRC) and establish a robust prognostic signature derived from GTs. METHODS: Utilizing the AUCell, UCell, singscore, ssgsea, and AddModuleScore algorithms, along with correlation analysis, we redefined genes related to GTs in CRC at the single-cell RNA level. To improve risk model accuracy, univariate Cox and lasso regression were employed to discover a more clinically subset of GTs in CRC. Subsequently, the efficacy of seven machine learning algorithms for CRC prognosis was assessed, focusing on survival outcomes through nested cross-validation. The model was then validated across four independent external cohorts, exploring variations in the tumor microenvironment (TME), response to immunotherapy, mutational profiles, and pathways of each risk group. Importantly, we identified potential therapeutic agents targeting patients categorized into the high-GARS group. RESULTS: In our research, we classified CRC patients into distinct subgroups, each exhibiting variations in prognosis, clinical characteristics, pathway enrichments, immune infiltration, and immune checkpoint genes expression. Additionally, we established a Glycosyltransferase-Associated Risk Signature (GARS) based on machine learning. GARS surpasses traditional clinicopathological features in both prognostic power and survival prediction accuracy, and it correlates with higher malignancy levels, providing valuable insights into CRC patients. Furthermore, we explored the association between the risk score and the efficacy of immunotherapy. CONCLUSION: A prognostic model based on GTs was developed to forecast the response to immunotherapy, offering a novel approach to CRC management.

Transcriptome analysis reveals activation of detoxification and defense mechanisms in smoke-exposed Merlot grape (Vitis vinifera) berries.

A significant consequence of climate change is the rising incidence of wildfires. When wildfires occur close to wine grape (Vitis vinifera) production areas, smoke-derived volatile phenolic compounds can be taken up by the grape berries, negatively affecting the flavor and aroma profile of the resulting wine and compromising the production value of entire vineyards. Evidence for the permeation of smoke-associated compounds into grape berries has been provided through metabolomics; however, the basis for grapevines' response to smoke at the gene expression level has not been investigated in detail. To address this knowledge gap, we employed time-course RNA sequencing to observe gene expression-level changes in grape berries in response to smoke exposure. Significant increases in gene expression (and enrichment of gene ontologies) associated with detoxification of reactive compounds, maintenance of redox homeostasis, and cell wall fortification were observed in response to smoke. These findings suggest that the accumulation of volatile phenols from smoke exposure activates mechanisms that render smoke-derived compounds less reactive while simultaneously fortifying intracellular defense mechanisms. The results of this work lend a better understanding of the molecular basis for grapevines' response to smoke and provide insight into the origins of smoke-taint-associated flavor and aroma attributes in wine produced from smoke-exposed grapes.

Glycosylation of 6-gingerol and unusual spontaneous deglucosylation of two novel intermediates to form 6-shogaol-4'-O-ß-glucoside by bacterial glycosyltransferase.

6-Gingerol is a major phenolic compound within ginger (Zingiber officinale), often used in healthcare; however, its lower bioavailability is partly due to its poor solubility. Four bacterial glycosyltransferases (GTs) were tested to glycosylate 6-gingerol into soluble gingerol glucosides. BsUGT489 was a suitable GT to biotransform 6-gingerol into five significant products, which could be identified via nucleic magnetic resonance and mass spectrometry as 6-gingerol-4',5-O-ß-diglucoside (1), 6-gingerol-4'-O-ß-glucoside (2), 6-gingerol-5-O-ß-glucoside (3), 6-shogaol-4'-O-ß-glucoside (4), and 6-shogaol (5). The enzyme kinetics of BsUGT489 showed substrate inhibition toward 6-gingerol for producing two glucosides. The kinetic parameters were determined as KM (110 µM), kcat (862 min-1), and KI (571 µM) for the production of 6-gingerol-4'-O-ß-glucoside (2) and KM (104 µM), kcat (889 min-1), and KI (545 µM) for the production of 6-gingerol-5-O-ß-glucoside (3). The aqueous solubility of the three 6-gingerol glucosides, compound (1) to (3), was greatly improved. However, 6-shogaol-4'-O-ß-glucoside (4) was found to be a product biotransformed from 6-shogaol (5). This study first confirmed that the glucose moiety at the C-5 position of both 6-gingerol-4',5-O-ß-diglucoside (1) and 6-gingerol-5-O-ß-glucoside (3) caused spontaneous deglucosylation through ß-elimination to form 6-shogaol-4'-O-ß-glucoside (4) and 6-shogaol (5), respectively. Moreover, the GTs could glycosylate 6-shogaol to form 6-shogaol-4'-O-ß-glucoside (4). The assays showed 6-shogaol-4'-O-ß-glucoside (4) had higher anti-inflammatory activity (IC50 value of 10.3 ± 0.2 µM) than 6-gingerol. The 6-gingerol-5-O-ß-glucoside (3) possessed 346-fold higher solubility than 6-shogaol, in which the highly soluble glucoside is a potential prodrug of 6-shogaol via spontaneous deglucosylation. This unusual deglucosylation plays a vital role in influencing the anti-inflammatory activity. IMPORTANCE: Both 6-gingerols and 6-shogaol possess multiple bioactivities. However, their poor solubility limits their application. The present study used bacterial GTs to catalyze the glycosylation of 6-gingerol, and the resulting gingerol glycosides were found to be new compounds with improved solubility and anti-inflammatory activity. In addition, two of the 6-gingerol glucosides were found to undergo spontaneous deglucosylation to form 6-shogaol or 6-shogaol glucosides. The unique spontaneous deglucosylation property of the new 6-gingerol glucosides makes them a good candidate for the prodrug of 6-shogaol.

Oxygen-dependent regulation of F-box proteins in Toxoplasma gondii is mediated by Skp1 glycosylation.

A dynamic proteome is required for cellular adaption to changing environments including levels of O2, and the SKP1/CULLIN-1/F-box protein/RBX1 (SCF) family of E3 ubiquitin ligases contributes importantly to proteasome-mediated degradation. We examine, in the apicomplexan parasite Toxoplasma gondii, the influence on the interactome of SKP1 by its novel glycan attached to a hydroxyproline generated by PHYa, the likely ortholog of the HIFα PHD2 oxygen-sensor of human host cells. Strikingly, the representation of several putative F-box proteins (FBPs) is substantially reduced in PHYaΔ parasites grown in fibroblasts. One, FBXO13, is a predicted lysyl hydroxylase related to the human JmjD6 oncogene except for its F-box domain. The abundance of FBXO13, epitope-tagged at its genetic locus, was reduced in PHYaΔ parasites thus explaining its diminished presence in the SKP1 interactome. A similar effect was observed for FBXO14, a cytoplasmic protein of unknown function that may have co-evolved with PHYa in apicomplexans. Similar findings in glycosylation-mutant cells, rescue by proteasomal inhibitors, and unchanged transcript levels, suggested the involvement of the SCF in their degradation. The effect was selective, because FBXO1 was not affected by loss of PHYa. These findings are physiologically significant because the effects were phenocopied in parasites reared at 0.5% O2. Modest impact on steady-state SKP1 modification levels suggests that effects are mediated during a lag phase in hydroxylation of nascent SKP1. The dependence of FBP abundance on O2-dependent SKP1 modification likely contributes to the reduced virulence of PHYaΔ parasites owing to impaired ability to sense O2 as an environmental signal.

Enhanced antimicrobial activity against oral bacteria Actinomyces viscous by cinnamaldehyde emulsion microencapsulated with cyclodextrin glycosyltransferase-catalyzed products.

Actinomyces viscous (A. viscous) is well documented as a major cariogenic bacterium in the oral cavity and needs to be inhibited and removed timely. Essential oils (EOs) are recognized as secure antibacterial agents for treating oral diseases, but their volatility and insolubility limit their application. In this study, cinnamaldehyde was screened as the optimum EO for inhibiting the A. viscous growth by a micro-agar dilution method and microencapsulated by cyclodextrin glycosyltransferase (CGTase)-catalyzed products. The antibacterial effects against A. viscous were investigated and compared with the free cinnamaldehyde. Antibacterial diameter, antibacterial efficiency and stability, and time-kill curve results revealed that the cinnamaldehyde emulsion had better antibacterial properties. 1 MIC of the cinnamaldehyde emulsion had an inhibitory zone of 9.92 nm, a 100 % inhibition rate when acting for 2 min or 5 min, and still maintained the same inhibitory effect for 2 years. The extracellular environment showed more pH decrease, conductivity increase, and protein leakage, suggesting damage to the cell membrane. Microstructure and flow cytometric analysis further revealed that the CGTase-catalyzed products induced more changes in the A. viscous membrane integrity. Based on the results, CGTase-catalyzed products can be used as a potential substance for encapsulating EOs for treating oral bacteria.

Advances in glycosyltransferase-mediated glycodiversification of small molecules.

Currently, numerous glycosides have been synthesized and used in clinical applications, neutraceuticals, cosmetics, and food processing. Structurally, a glycoside is composed of aglycone attaching to one or several sugar moieties so-called glycone. It is found that biochemical or biopharmaceutical properties of glycoside are mainly determined by its sugar part and thereby alternation of this glycone resulting in novel structure and characteristics as well. The use of traditional production methods of glycosides such as direct extraction and purification from plants, animals, or microorganisms is very challenging (laborious, time-consuming, technique, high price, low yield, etc.). Alternatively, the use of enzymatic methods for the biosynthesis of glycosides has become a highly promising tool. Particularly, the diverse structure of glycosides can be obtained using the promiscuous catalytic activity of glycosyltransferases (GT) mined from bioresources (plants, fungi, microorganisms, etc.). In addition, the exploration of GT catalytic promiscuity toward diverse aglycones, and glycones has indeed been interesting and played a key role in the production of novel glycosides. This review described the recent advances in glycosyltransferase-mediated glycodiversification of small molecules (flavonoids, steroids, terpenoids, etc.). Mostly, references were collected from 2014 to 2023.

Cloning and Direct Evolution of a Novel 7-O-Glycosyltransferase from Cucurbita moschata and Its Application in the Efficient Biocatalytic Synthesis of Luteolin-7-O-glucoside.

Luteolin-7-O-glucoside(L7G), a glycosylation product of luteolin, is present in a variety of foods, vegetables, and medicinal herbs and is commonly used in dietary supplements due to its health benefits. Meanwhile, luteolin-7-O-glucoside is an indicator component for the quality control of honeysuckle in the pharmacopoeia. However, its low content in plants has hindered its use in animal pharmacological studies and clinical practice. In this study, a novel 7-O-glycosyltransferase CmGT from Cucurbita moschata was cloned, which could efficiently convert luteolin into luteolin-7-O-glucoside under optimal conditions (40 °C and pH 8.5). To further improve the catalytic efficiency of CmGT, a 3D structure of CmGT was constructed, and directed evolution was performed. The mutant CmGT-S16A-T80W was obtained by using alanine scanning and iterative saturation mutagenesis. This mutant exhibited a kcat/Km value of 772 s-1·M-1, which was 3.16-fold of the wild-type enzyme CmGT. Finally, by introducing a soluble tag and UDPG synthesis pathway, the strain BXC was able to convert 1.25 g/L of luteolin into 1.91 g/L of luteolin-7-O-glucoside under optimal conditions, achieving a molar conversion rate of 96% and a space-time yield of 27.08 mg/L/h. This study provides an efficient method for the biosynthesis of luteolin-7-O-glucoside, which holds broad application prospects in the food and pharmaceutical industry.

Role of glycosylation-related gene MGAT1 in pancreatic ductal adenocarcinoma.

Background: pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with a very poor prognosis and a complex tumor microenvironment, which plays a key role in tumor progression and treatment resistance. Glycosylation plays an important role in processes such as cell signaling, immune response and protein stability. Materials and methods: single-cell RNA sequencing data and spatial transcriptome data were obtained from GSE197177 and GSE224411, respectively, and RNA-seq data and survival information were obtained from UCSC Xena and TCGA. Multiple transcriptomic data were comprehensively analyzed to explore the role of glycosylation processes in tumor progression, and functional experiments were performed to assess the effects of MGAT1 overexpression on PDAC cell proliferation and migration. Results: In PDAC tumor samples, the glycosylation level of macrophages was significantly higher than that of normal samples. MGAT1 was identified as a key glycosylation-related gene, and its high expression was associated with better patient prognosis. Overexpression of MGAT1 significantly inhibited the proliferation and migration of PDAC cells and affected intercellular interactions in the tumor microenvironment. Conclusion: MGAT1 plays an important role in PDAC by regulating glycosylation levels in macrophages, influencing tumor progression and improving prognosis.MGAT1 is a potential therapeutic target for PDAC and further studies are needed to develop targeted therapeutic strategies against MGAT1 to improve clinical outcomes.

In vivo mapping of the mouse Galnt3-specific O-glycoproteome.

The UDP-N-acetylgalactosamine polypeptide:N-acetylgalactosaminyltransferase (GalNAc-T) family of enzymes initiates O-linked glycosylation by catalyzing the addition of the first GalNAc sugar to serine or threonine on proteins destined to be membrane-bound or secreted. Defects in individual isoforms of the GalNAc-T family can lead to certain congenital disorders of glycosylation (CDG). The polypeptide N-acetylgalactosaminyltransferase 3 (GALNT)3-CDG, is caused by mutations in GALNT3, resulting in hyperphosphatemic familial tumoral calcinosis due to impaired glycosylation of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23) within osteocytes of the bone. Patients with hyperphosphatemia present altered bone density, abnormal tooth structure, and calcified masses throughout the body. It is therefore important to identify all potential substrates of GalNAc-T3 throughout the body to understand the complex disease phenotypes. Here, we compared the Galnt3-/- mouse model, which partially phenocopies GALNT3-CDG, with WT mice and used a multicomponent approach using chemoenzymatic conditions, a product-dependent method constructed using EThcD triggered scans in a mass spectrometry workflow, quantitative O-glycoproteomics, and global proteomics to identify 663 Galnt3-specific O-glycosites from 269 glycoproteins across multiple tissues. Consistent with the mouse and human phenotypes, functional networks of glycoproteins that contain GalNAc-T3-specific O-glycosites involved in skeletal morphology, mineral level maintenance, and hemostasis were identified. This library of in vivo GalNAc-T3-specific substrate proteins and O-glycosites will serve as a valuable resource to understand the functional implications of O-glycosylation and to unravel the underlying causes of complex human GALNT3-CDG phenotypes.