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.

BACKGROUND: The β1,6-GlcNAc branch in N-glycans, produced by a glycosyltransferase N-acetylglucosaminyltransferase V (GnT-V or MGAT5), is associated with cancer and autoimmune diseases.
METHODS: Here, we summarize the structure and activity regulation of GnT-V. We also describe the roles of the β1,6-GlcNAc branch on glycoproteins in cells and the phenotypes of Mgat5-deficient mice, focusing on cancer and the immune system.
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.
CONCLUSIONS: 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.

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.

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.
CONCLUSIONS: A prognostic model based on GTs was developed to forecast the response to immunotherapy, offering a novel approach to CRC management.

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.

Drought stress poses a significant threat to global agriculture, highlighting the urgent need to elucidate the molecular mechanisms underlying plant drought tolerance. The UDP-glycosyltransferase (UGT) gene family plays crucial roles in diverse biological processes in plants. In this study, we conducted a comprehensive analysis of the UGT gene family in wild barley EC_S1, focusing on gene characteristics, subcellular localization, phylogenetic relationships, and protein structure. A total of 175 UGT gene family members were identified, exhibiting diverse patterns in protein length, molecular weight, isoelectric point, hydrophilicity, and subcellular localization. Most genes are located at chromosome ends. Phylogenetic analysis grouped the UGT genes into seven clusters, with barley-specific group E. Expression analysis across barley tissues showed upregulation in roots and senescent leaves, implying diverse roles. Under drought stress, expression patterns varied, with drought-tolerant varieties showing fewer changes than sensitive ones. Clustering analysis revealed distinct expression patterns, suggesting regulatory functions in barley\'s drought response. As a case, the HvUGT1 was cloned. Overexpression of HvUGT1 in Arabidopsis enhanced drought tolerance, with increased water retention, reduced cell damage, and elevated flavonoid levels. Conversely, HvUGT1 silencing in wild barley decreased drought tolerance, accompanied by reduced antioxidant enzyme activity and flavonoid content. These results highlight HvUGT1\'s importance in enhancing plant drought tolerance, possibly through flavonoid-mediated ROS clearance. The research provides gene resources and valuable insights for the development of drought-resistant crops through targeted genetic manipulation strategies.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s12298-024-01487-w.

暂无摘要。

Cellulose and hemicellulose are the major structural β-glycan polysaccharides in cell walls of land plants. They are characterized by a backbone of β-(1,3)- and/or β-(1,4)-linked sugars such as glucose, mannose, or xylose. The backbones of these polymers are produced by processive glycosyltransferases (GTs) called synthases having multiple transmembrane domains anchoring them to the membrane. Thus, they are among the most difficult membrane proteins to test in vitro and to purify. Recently, we developed an in vitro GT-array (i-GTray) platform and showed that non-processive type II membrane GTs could be produced via cell-free system in a soluble and active form and tested in this platform. To determine whether i-GT-ray platform is adequate for the production and testing of β-glycan synthases, we tested five synthases involved in cellulose, xyloglucan, (gluco)mannan, and β-(1,3)(1,4)-mixed-linkage glucan synthesis. Our results revealed unsuspected features of these enzymes. For example, all these synthases could be produced in a soluble and active form and are active in the absence of detergent or membrane lipids, and none of them required a primer for initiation of synthesis. All synthases produced ethanol-insoluble products that were susceptible to the appropriate hydrolases (i.e., cellulase, lichenase, mannanase). Using this platform, we showed that AtCslC4 and AtXXT1 interact directly to form an active xyloglucan synthase that produced xylosylated cello-oligosaccharides (up to three xylosyl residues) when supplied with UDP-Glc and UDP-Xyl. i-GTray platform represents a simple and powerful functional genomics tool for discovery of new insights of synthase activities and can be adapted to other enzymes.

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.

UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.