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Enzymatic production of nucleotide sugars on a multigram scale presents a challenge, as only a few processes have been reported for large-scale nucleotide sugar production. They rely primarily on batch synthesis and employ exceptional amounts of enzymes. This study introduces a novel approach for the multigram-scale production of nucleotide sugars with a continuous fed-batch membrane reactor. We successfully synthesized five main nucleotide sugars: UDP-Gal, UDP-GalNAc, UDP-GlcA, GDP-Man, and CMP-Neu5Ac on a multigram scale. Efficient biocatalyst utilization results in high performance, including space-time yield (STY, g*L-1h-1), total turnover number (TTN, g product per g enzyme), and an efficient product formation rate (g/h) suitable for industrially relevant bioprocesses. The established continuous-fed batch reactor system produced up to 8.2 g CMP-Neu5Ac in three consecutive productions in less than 15 h with satisfying TTNs of 91 gProduct/gEnzyme. Continuous production of UDP-GlcA over 28 h resulted in a final product amount of 14.8 g and TTN of 493 gP/gE. This process enables the production of nucleotide sugars with stable product formation, requiring minimal technical equipment for multigram quantities of nucleotide sugars at the laboratory scale. Notably, the system exhibited robustness and flexibility, allowing its application to various enzymatic nucleotide sugar synthesis cascades.

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Sialylation during N-glycosylation plays an important role in the half-life of therapeutic glycoproteins in vivo and has sparked interest in the production of therapeutic proteins using recombinant Chinese hamster ovary (rCHO) cells. To improve the sialylation of therapeutic proteins, we examined the effect of sialyllactose supplementation on sialylation of Fc-fusion glycoproteins produced in rCHO cells. Two enzymatically-synthesized sialyllactoses, 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), were administered separately to two rCHO cell lines producing the same Fc-fusion glycoprotein derived from DUKX-B11 and DG44, respectively. Two sialyllactoses successfully increased sialylation of Fc-fusion glycoprotein in both cell lines, as evidenced by isoform distribution, sialylated N-glycan formation, and sialic acid content. Increased sialylation by adding sialyllactose was likely the result of increased amount of intracellular CMP-sialic acid (CMP-SA), the direct nucleotide sugar for sialylation. Furthermore, the degree of sialylation enhanced by sialyllactoses was slightly effective or nearly similar compared with the addition of N-acetylmannosamine (ManNAc), a representative nucleotide sugar precursor, to increase sialylation of glycoproteins. The effectiveness of sialyllactose was also confirmed using three commercially available CHO cell culture media. Taken together, these results suggest that enzymatically-synthesized sialyllactose represents a promising candidate for culture media supplementation to increase sialylation of glycoproteins in rCHO cell culture.

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Nucleotide sugars (NS) fulfil important roles in all living organisms and in humans, related defects result in severe clinical syndromes. NS can be seen as the "activated" sugars used for biosynthesis of a wide range of glycoconjugates and serve as substrates themselves for the synthesis of other nucleotide sugars. NS analysis is complicated by the presence of multiple stereoisomers without diagnostic transition ions, therefore requiring separation by liquid chromatography. In this paper, we explored weak anion-exchange/reversed-phase chromatography on a hybrid column for the separation of 17 nucleotide sugars that can occur in humans. A robust and reproducible method was established with intra- and inter-day coefficients of variation below 10% and a linear range spanning three orders of magnitude. Application to patient fibroblasts with genetic defects in mannose-1-phosphate guanylyltransferase beta, CDP-L-ribitol pyrophosphorylase A, and UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase showed abnormal levels of guanosine-5'-diphosphate-α-D-mannose (GDP-Man), cytidine-5'-diphosphate-L-ribitol (CDP-ribitol), and cytidine-5'-monophosphate-N-acetyl-ß-D-neuraminic acid (CMP-Neu5Ac), respectively, in consonance with expectations based on the diagnosis. In conclusion, a novel, semi-quantitative method was established for the analysis of nucleotide sugars that can be applied to diagnose several genetic glycosylation disorders in fibroblasts and beyond.

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Azido sugars hold great promise as substrates in numerous click-chemistry applications. However, the synthesis of activated azido sugars is limited by cost and complexity. Conventional chemical activation methods are intricate and time-consuming. In response, we have developed a process for the large-scale production of UDP-6-azido-GalNAc through enzymatic nucleotide sugar synthesis on a gram scale. Our optimization strategies encompassed refining the process parameters of an enzyme cascade featuring NahK from Bifidobacterium longum and AGX1 from Homo sapiens. Using the repetitive-batch-mode technology, we synthesized up to 2.1 g of UDP-6-azido-GalNAc, achieving yields up to 97 % in five consecutive batch cycles using a single enzyme batch. The synthesis process demonstrated to have total turnover numbers (TTNs) between 4.4-4.8 g of product per gram of enzyme (gP/gE) and STYs ranging from 1.7-2.4 g per liter per hour (g*L-1*h-1). By purification of a product solution pool containing 2.6 g (4.1 mmol) UDP-6-azido-GalNAc, 2.1 g (2,122.1 mg) UDP-6-azido-GalNAc (sodium salt) with a purity of 99.96 % (HPLC) were obtained. The overall recovery after purification was 81 % (3.32 mmol). Our work establishes a robust production platform for the gram-scale synthesis of unnatural nucleotide sugars, opening new avenues for applications in glycan engineering.

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Glycosylation of biomolecules can greatly alter their physicochemical properties, cellular recognition, subcellular localization, and immunogenicity. Glycosylation reactions rely on the stepwise addition of sugars using nucleotide diphosphate (NDP)-sugars. Making these substrates readily available will greatly accelerate the characterization of new glycosylation reactions, elucidation of their underlying regulation mechanisms, and production of glycosylated molecules. In this work, we engineered Saccharomyces cerevisiae to heterologously express nucleotide sugar synthases to access a wide variety of uridine diphosphate (UDP)-sugars from simple starting materials (i.e., glucose and galactose). Specifically, activated glucose, uridine diphosphate d-glucose (UDP-d-Glc), can be converted to UDP-d-glucuronic acid (UDP-d-GlcA), UDP-d-xylose (UDP-d-Xyl), UDP-d-apiose (UDP-d-Api), UDP-d-fucose (UDP-d-Fuc), UDP-l-rhamnose (UDP-l-Rha), UDP-l-arabinopyranose (UDP-l-Arap), and UDP-l-arabinofuranose (UDP-l-Araf) using the corresponding nucleotide sugar synthases of plant and microbial origins. We also expressed genes encoding the salvage pathway to directly activate free sugars to achieve the biosynthesis of UDP-l-Arap and UDP-l-Araf. We observed strong inhibition of UDP-d-Glc 6-dehydrogenase (UGD) by the downstream product UDP-d-Xyl, which we circumvented using an induction system (Tet-On) to delay the production of UDP-d-Xyl to maintain the upstream UDP-sugar pool. Finally, we performed a time-course study using strains containing the biosynthetic pathways to produce five non-native UDP-sugars to elucidate their time-dependent interconversion and the role of UDP-d-Xyl in regulating UDP-sugar metabolism. These engineered yeast strains are a robust platform to (i) functionally characterize sugar synthases in vivo, (ii) biosynthesize a diverse selection of UDP-sugars, (iii) examine the regulation of intracellular UDP-sugar interconversions, and (iv) produce glycosylated secondary metabolites and proteins.

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The occurrence of autophagy in recombinant Chinese hamster ovary (rCHO) cell culture has attracted attention due to its effects on therapeutic protein production. Given the significance of glycosylation in therapeutic proteins, this study examined the effects of autophagy-inhibiting chemicals on sialylation of Fc-fusion glycoproteins in rCHO cells. Three chemical autophagy inhibitors known to inhibit different stages were separately treated with two rCHO cell lines that produce the same Fc-fusion glycoprotein derived from DUKX-B11 and DG44. All autophagy inhibitors significantly decreased the sialylation of Fc-fusion glycoprotein in both cell lines. The decrease in sialylation of Fc-fusion glycoprotein is unlikely to be attributed to the release of intracellular enzymes, given the high cell viability and low activity of extracellular sialidases. Interestingly, the five intracellular nucleotide sugars remained abundant in cells treated with autophagy inhibitors. In the mRNA expression profiles of 27 N-glycosylation-related genes using the NanoString nCounter system, no significant differences in gene expression were noted. With the positive effect of supplementing nucleotide sugar precursors on sialylation, attempts were made to enhance the levels of intracellular nucleotide sugars by supplying these precursors. The addition of nucleotide sugar precursors to cultures treated with inhibitors successfully enhanced the sialylation of Fc-fusion glycoproteins compared to the control culture. This was particularly evident under mild stress conditions and not under relatively severe stress conditions, which were characterized by a high decrease in sialylation. These results suggest that inhibiting autophagy in rCHO cell culture decreases sialylation of Fc-fusion glycoprotein by constraining the availability of intracellular nucleotide sugars. KEY POINTS: •  The autophagy inhibition in rCHO cell culture leads to a significant reduction in the sialylation of Fc-fusion glycoprotein. •  The pool of five intracellular nucleotide sugars remained highly abundant in cells treated with autophagy inhibitors. •  Supplementation of nucleotide sugar precursors effectively restores decreased sialylation, particularly under mild stress conditions but not in relatively severe stress conditions.

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Plants possess an innate ability to generate vast amounts of sugar and produce a range of sugar-derived compounds that can be utilized for applications in industry, health, and agriculture. Nucleotide sugars lie at the unique intersection of primary and specialized metabolism, enabling the biosynthesis of numerous molecules ranging from small glycosides to complex polysaccharides. Plants are tolerant to perturbations to their balance of nucleotide sugars, allowing for the overproduction of endogenous nucleotide sugars to push flux towards a particular product without necessitating the re-engineering of upstream pathways. Pathways to produce even non-native nucleotide sugars may be introduced to synthesize entirely novel products. Heterologously expressed glycosyltransferases capable of unique sugar chemistries can further widen the synthetic repertoire of a plant, and transporters can increase the amount of nucleotide sugars available to glycosyltransferases. In this opinion piece, we examine recent successes and potential future uses of engineered nucleotide sugar biosynthetic, transport, and utilization pathways to improve the production of target compounds. Additionally, we highlight current efforts to engineer glycosyltransferases. Ultimately, the robust nature of plant sugar biochemistry renders plants a powerful chassis for the production of target glycoconjugates and glycans.

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Nucleotide sugars, the glycosyl donors in the biosynthesis of carbohydrates, are critical ingredients in the growth and development of all living organisms. A variety of nucleotide sugars simultaneously exist in biological samples. They, however, have only minor structural differences, which make them extremely difficult to discriminate. In this work, a phenylboronic acid (PBA)-modified Mycobacterium smegmatis porin A (MspA) hetero-octamer was applied to sense nucleotide sugars. Five representative nucleotide sugars, including guanosine diphosphate mannose (GDP-Man), adenosine diphosphate glucose (ADP-Glc), uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), uridine diphosphate glucose (UDP-Glc), and uridine diphosphate glucoronic acid (UDP-GlcA), were successfully distinguished. A custom machine learning algorithm was also employed to automatically identify events, reporting a general accuracy of 99.4%. This sensing strategy provides a rapid, direct, and accurate method for identifying different nucleotide sugars. However, single-molecule identification of nucleotide sugars has never been previously reported, to the best of our knowledge.

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A promiscuous CDP-tyvelose 2-epimerase (TyvE) from Thermodesulfatator atlanticus (TaTyvE) belonging to the nucleotide sugar active short-chain dehydrogenase/reductase superfamily (NS-SDRs) was recently discovered. TaTyvE performs the slow conversion of NDP-glucose (NDP-Glc) to NDP-mannose (NDP-Man). Here, we present the sequence fingerprints that are indicative of the conversion of UDP-Glc to UDP-Man in TyvE-like enzymes based on the heptagonal box motifs. Our data-mining approach led to the identification of 11 additional TyvE-like enzymes for the conversion of UDP-Glc to UDP-Man. We characterized the top two wild-type candidates, which show a 15- and 20-fold improved catalytic efficiency, respectively, on UDP-Glc compared to TaTyvE. In addition, we present a quadruple variant of one of the identified enzymes with a 70-fold improved catalytic efficiency on UDP-Glc compared to TaTyvE. These findings could help the design of new nucleotide production pathways starting from a cheap sugar substrate like glucose or sucrose.

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O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a ubiquitous and dynamic non-canonical glycosylation of intracellular proteins. Several branches of metabolism converge at the hexosamine biosynthetic pathway (HBP) to produce the substrate for protein O-GlcNAcylation, the uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Availability of UDP-GlcNAc is considered a key regulator of O-GlcNAcylation. Yet UDP-GlcNAc concentrations are rarely reported in studies exploring the HBP and O-GlcNAcylation, most likely because the methods to measure it are restricted to specialized chromatographic procedures. Here, we introduce an enzymatic method to quantify cellular and tissue UDP-GlcNAc. The method is based on O-GlcNAcylation of a substrate peptide by O-linked N-acetylglucosamine transferase (OGT) and subsequent immunodetection of the modification. The assay can be performed in dot-blot or microplate format. We apply it to quantify UDP-GlcNAc concentrations in several mouse tissues and cell lines. Furthermore, we show how changes in UDP-GlcNAc levels correlate with O-GlcNAcylation and the expression of OGT and O-GlcNAcase (OGA).

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Glycans are the most abundant biopolymers on earth and are constituents of glycoproteins, glycolipids, and proteoglycans with multiple biological functions. The availability of different complex glycan structures is of major interest in biotechnology and basic research of biological systems. High complexity, establishment of general and ubiquitous synthesis techniques, as well as sophisticated analytics, are major challenges in the development of glycan synthesis strategies. Enzymatic glycan synthesis with Leloir-glycosyltransferases is an attractive alternative to chemical synthesis as it can achieve quantitative regio- and stereoselective glycosylation in a single step. Various strategies for synthesis of a wide variety of different glycan structures has already be established and will exemplarily be discussed in the scope of this review. However, the application of enzymatic glycan synthesis in an automated system has high demands on the equipment, techniques, and methods. Different automation approaches have already been shown. However, while these techniques have been applied for several glycans, only a few strategies are able to conserve the full potential of enzymatic glycan synthesis during the process - economical and enzyme technological recycling of enzymes is still rare. In this review, we show the major challenges towards Automated Enzymatic Glycan Synthesis (AEGS). First, we discuss examples for immobilization of glycans or glycosyltransferases as an important prerequisite for the embedment and implementation in an enzyme reactor. Next, improvement of bioreactors towards automation will be described. Finally, analysis and monitoring of the synthesis process are discussed. Furthermore, automation processes and cycle design are highlighted. Accordingly, the transition of recent approaches towards a universal automated glycan synthesis platform will be projected. To this end, this review aims to describe essential key features for AEGS, evaluate the current state-of-the-art and give thought- encouraging impulses towards future full automated enzymatic glycan synthesis.

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Nucleotide sugars play an elementary role in nature as building blocks of glycans, polysaccharides, and glycoconjugates used in the pharmaceutical, cosmetics, and food industries. As substrates of Leloir-glycosyltransferases, nucleotide sugars are essential for chemoenzymatic in vitro syntheses. However, high costs and the limited availability of nucleotide sugars prevent applications of biocatalytic cascades on a large industrial scale. Therefore, the focus is increasingly on nucleotide sugar synthesis strategies to make significant application processes feasible. The chemical synthesis of nucleotide sugars and their derivatives is well established, but the yields of these processes are usually low. Enzyme catalysis offers a suitable alternative here, and in the last 30 years, many synthesis routes for nucleotide sugars have been discovered and used for production. However, many of the published procedures shy away from assessing the practicability of their processes. With this review, we give an insight into the development of the (chemo)enzymatic nucleotide sugar synthesis pathways of the last years and present an assessment of critical process parameters such as total turnover number (TTN), space-time yield (STY), and enzyme loading.

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A novel series of pyridine, cytosine, and uracil thioglycoside analogs (4a-i, 9a,b, and 13a,b, respectively) and their corresponding phosphoramidates (6a-I, 10a,b, and 14a,b, respectively) were synthesized and assessed for their antiviral inhibitory activities in a dual-pathogen screening protocol against SARS-CoV-2 and influenza A virus (IAV). MTT cytotoxicity (TC50) and plaque reduction assays were used to explore inhibition and cytotoxicity percentage values for H5N1 influenza virus strain and the half-maximal cytotoxic concentration (CC50) and inhibitory concentration (IC50) for SARS-CoV-2 virus. Most of the tested compounds demonstrated dose-dependent inhibition behavior. Both cytosine thioglycoside phosphoramidates 10a and 10b exhibited the most potent profiles with 83% and 86% inhibition at 0.25 µM concentration against H5N1 and IC50 values of 12.16 µM, 14.9 µM against SARS-CoV-2, respectively. Moreover, compounds 10a and 10b have been shown to have the highest selectivity index (SI) among all the tested compounds against SARS-CoV-2 with 28.2 and 26.9 values, respectively.

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Hyaluronan is a versatile macromolecule capable of an exceptional range of functions from cushioning and hydration to dynamic signaling in development and disease. Because of its critical roles, hyaluronan production is regulated at multiple levels including epigenetic, transcriptional, and posttranslational control of the three hyaluronan synthase (HAS) enzymes. Precursor availability can dictate the rate and amount of hyaluronan synthesized and shed by the cells producing it. However, the nucleotide-activated sugar substrates for hyaluronan synthesis by HAS also participate in exquisitely fine-tuned cross-talking pathways that intersect with glycosaminoglycan production and central carbohydrate metabolism. Multiple UDP-sugars have alternative metabolic fates and exhibit coordinated and reciprocal allosteric control of enzymes within their biosynthetic pathways to preserve appropriate precursor ratios for accurate partitioning among downstream products, while also sensing and maintaining energy homeostasis. Since the dysregulation of nucleotide sugar and hyaluronan synthesis is associated with multiple pathologies, these pathways offer opportunities for therapeutic intervention. Recent structures of several key rate-limiting enzymes in the UDP-sugar synthesis pathways have offered new insights to the overall regulation of hyaluronan production by precursor fate decisions. The details of UDP-sugar control and the structural basis for underlying mechanisms are discussed in this review.

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Nucleotide sugar (NS) dehydratases play a central role in the biosynthesis of deoxy and amino sugars, which are involved in a variety of biological functions in all domains of life. Bacteria are true masters of deoxy sugar biosynthesis as they can produce a wide range of highly specialized monosaccharides. Indeed, deoxy and amino sugars play important roles in the virulence of gram-positive and gram-negative pathogenic species and are additionally involved in the biosynthesis of diverse macrolide antibiotics. The biosynthesis of deoxy sugars relies on the activity of NS dehydratases, which can be subdivided into three groups based on their structure and reaction mechanism. The best-characterized NS dehydratases are the 4,6-dehydratases that, together with the 5,6-dehydratases, belong to the NS-short-chain dehydrogenase/reductase superfamily. The other two groups are the less abundant 2,3-dehydratases that belong to the Nudix hydrolase superfamily and 3-dehydratases, which are related to aspartame aminotransferases. 4,6-Dehydratases catalyze the first step in all deoxy sugar biosynthesis pathways, converting nucleoside diphosphate hexoses to nucleoside diphosphate-4-keto-6-deoxy hexoses, which in turn are further deoxygenated by the 2,3- and 3-dehydratases to form dideoxy and trideoxy sugars. In this review, we give an overview of the NS dehydratases focusing on the comparison of their structure and reaction mechanisms, thereby highlighting common features, and investigating differences between closely related members of the same superfamilies.

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The endoplasmic reticulum (ER) is equipped with multiple quality control systems (QCS) that are necessary for shaping the glycoproteome of eukaryotic cells. These systems facilitate the productive folding of glycoproteins, eliminate defective products, and function as effectors to evoke cellular signaling in response to various cellular stresses. These ER functions largely depend on glycans, which contain sugar-based codes that, when needed, function to recruit carbohydrate-binding proteins that determine the fate of glycoproteins. To ensure their functionality, the biosynthesis of such glycans is therefore strictly monitored by a system that selectively degrades structurally defective glycans before adding them to proteins. This system, which is referred to as the glycan QCS, serves as a mechanism to reduce the risk of abnormal glycosylation under conditions where glycan biosynthesis is genetically or metabolically stalled. On the other hand, glycan QCS increases the risk of global hypoglycosylation by limiting glycan availability, which can lead to protein misfolding and the activation of unfolded protein response to maintaining cell viability or to initiate cell death programs. This review summarizes the current state of our knowledge of the mechanisms underlying glycan QCS in mammals and its physiological and pathological roles in embryogenesis, tumor progression, and congenital disorders associated with abnormal glycosylation.

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Galactose toxicity (Gal-Tox) is a widespread phenomenon ranging from Escherichia coli to mammals and plants. In plants, the predominant pathway for the conversion of galactose into UDP-galactose (UDP-Gal) and UDP-glucose is catalyzed by the enzymes galactokinase, UDP-sugar pyrophosphorylase (USP) and UDP-galactose 4-epimerase. Galactose is a major component of cell wall polymers, glycolipids and glycoproteins; therefore, it becomes surprising that exogenous addition of galactose leads to drastic root phenotypes including cessation of primary root growth and induction of lateral root formation. Currently, little is known about galactose-mediated toxicity in plants. In this study, we investigated the role of galactose-containing metabolites like galactose-1-phosphate (Gal-1P) and UDP-Gal in Gal-Tox. Recently published data from mouse models suggest that a reduction of the Gal-1P level via an mRNA-based therapy helps to overcome Gal-Tox. To test this hypothesis in plants, we created Arabidopsis thaliana lines overexpressing USP from Pisum sativum. USP enzyme assays confirmed a threefold higher enzyme activity in the overexpression lines leading to a significant reduction of the Gal-1P level in roots. Interestingly, the overexpression lines are phenotypically more sensitive to the exogenous addition of galactose (0.5 mmol L-1 Gal). Nucleotide sugar analysis via high-performance liquid chromatography-mass spectrometry revealed highly elevated UDP-Gal levels in roots of seedlings grown on 1.5 mmol L-1 galactose versus 1.5 mmol L-1 sucrose. Analysis of plant cell wall glycans by comprehensive microarray polymer profiling showed a high abundance of antibody binding recognizing arabinogalactanproteins and extensins under Gal-feeding conditions, indicating that glycoproteins are a major target for elevated UDP-Gal levels in plants.

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Regulation of proteoglycan and glycosaminoglycan synthesis is critical throughout development, and to maintain normal adult functions in wound healing and the immune system, among others. It has become increasingly clear that these processes are also under tight metabolic control and that availability of carbohydrate and amino acid metabolite precursors has a role in the control of proteoglycan and glycosaminoglycan turnover. The enzyme uridine diphosphate (UDP)-glucose dehydrogenase (UGDH) produces UDP-glucuronate, an essential precursor for new glycosaminoglycan synthesis that is tightly controlled at multiple levels. Here, we review the cellular mechanisms that regulate UGDH expression, discuss the structural features of the enzyme, and use the structures to provide a context for recent studies that link post-translational modifications and allosteric modulators of UGDH to its function in downstream pathways.

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Glycoconjugates have great potential to improve human health in a multitude of different ways and fields. Prominent examples are human milk oligosaccharides and glycosaminoglycans. The typical choice for the production of homogeneous glycoconjugates is enzymatic synthesis. Through the availability of expression and purification protocols, recombinant Leloir glycosyltransferases are widely applied as catalysts for the synthesis of a wide range of glycoconjugates. Extensive utilization of these enzymes also depends on the availability of activated sugars as building blocks. Multi-enzyme cascades have proven a versatile technique to synthesize and in situ regenerate nucleotide sugar.In this chapter, the functions and mechanisms of Leloir glycosyltransferases are revisited, and the advantage of prokaryotic sources and production systems is discussed. Moreover, in vivo and in vitro pathways for the synthesis of nucleotide sugar are reviewed. In the second part, recent and prominent examples of the application of Leloir glycosyltransferase are given, i.e., the synthesis of glycosaminoglycans, glycoconjugate vaccines, and human milk oligosaccharides as well as the re-glycosylation of biopharmaceuticals, and the status of automated glycan assembly is revisited.

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The biogenesis of small extracellular vesicles (sEVs) is regulated by multiple molecular machineries generating considerably heterogeneous vesicle populations, including exosomes and non-exosomal vesicles, with distinct cargo molecules. However, the role of carbohydrate metabolism in generating such vesicle heterogeneity remains largely elusive. Here, we discover that 2-deoxyglucose (2-DG), a well-known glycolysis inhibitor, suppresses the secretion of non-exosomal vesicles by impairing asparagine-linked glycosylation (N-glycosylation) in mouse melanoma cells. Mechanistically, 2-DG is metabolically incorporated into N-glycan precursors, causing precursor degradation and partial hypoglycosylation. N-glycosylation blockade by Stt3a silencing is sufficient to inhibit non-exosomal vesicle secretion. In contrast, N-glycosylation blockade barely influences exosomal secretion of tetraspanin proteins. Functionally, N-glycosylation at specific sites of the hepatocyte growth factor receptor, a cargo protein of non-exosomal vesicles, facilitates its sorting into vesicles. These results uncover a link between N-glycosylation and unconventional vesicle secretion and suggest that N-glycosylation facilitates sEV biogenesis through cargo protein sorting.

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