Enzymatic Glycosylation Research Articles

Discovery and Biosynthesis of Glycosylated Cycloheximide from a Millipede-Associated Actinomycete.

Chemical redundancy of microbial natural products (NPs) underscores the importance to exploit new resources of microorganisms. Insect-associated microbes are prolific but largely underexplored sources of diverse NPs. Herein, we discovered the new compound α-l-rhamnosyl-actiphenol (1) from a millipede-associated Streptomyces sp. ML6, which is the first glycosylated cycloheximide-class natural product. Interestingly, bioinformatics analysis of the ML6 genome revealed that the biosynthesis of 1 involves a cooperation between two gene clusters (chx and rml) located distantly on the genome of ML6. We also carried out in vitro enzymatic glycosylation of cycloheximide using an exotic promiscuous glycosyltransferase BsGT-1, which resulted in the production of an additional cycloheximide glycoside cycloheximide 7-O-β-d-glucoside (5). Although the antifungal and cytotoxic activities of the new compounds 1 and 5 were attenuated relative to those of cycloheximide, our work not only enriches the chemical repertoire of the cycloheximide family but also provides new insights into the structure-activity relationship optimization and ecological roles of cycloheximide.

Plant glycosyltransferases for expanding bioactive glycoside diversity.

Glycosylation is a successful strategy to alter the pharmacological properties of small molecules, and it has emerged as a unique approach to expand the chemical space of natural products that can be explored in drug discovery. Traditionally, most glycosylation events have been carried out chemically, often requiring many protection and deprotection steps to achieve a target molecule. Enzymatic glycosylation by glycosyltransferases could provide an alternative strategy for producing new glycosides. In particular, the glycosyltransferase family has greatly expanded in plants, representing a rich enzymatic resource to mine and expand the diversity of glycosides with novel bioactive properties. This article highlights previous and prospective uses for plant glycosyltransferases in generating bioactive glycosides and altering their pharmacological properties.

New insights into the pathogenicity of TMEM165 variants using structural modeling based on AlphaFold 2 predictions

TMEM165 is a Golgi protein playing a crucial role in Mn2+ transport, and whose mutations in patients are known to cause Congenital Disorders of Glycosylation. Some of those mutations affect the highly-conserved consensus motifs E-φ-G-D-[KR]-[TS] characterizing the CaCA2/UPF0016 family, presumably important for the transport of Mn2+ which is essential for the function of many Golgi glycosylation enzymes. Others, like the G>R304 mutation, are far away from these motifs in the sequence. Until recently, the classical membrane protein topology prediction methods were unable to provide a clear picture of the organization of TMEM165 inside the cell membrane, or to explain in a convincing manner the impact of patient and experimentally-generated mutations on the transporter function of TMEM165. In this study, AlphaFold 2 was used to build a TMEM165 model that was then refined by molecular dynamics simulation with membrane lipids and water. This model provides a realistic picture of the 3D protein scaffold formed from a two-fold repeat of three transmembrane helices/domains where the consensus motifs face each other to form a putative acidic cation-binding site at the cytosolic side of the protein. It sheds new light on the impact of mutations on the transporter function of TMEM165, found in patients and studied experimentally in vitro, formerly and within this study. More particularly and very interestingly, this model explains the impact of the G>R304 mutation on TMEM165’s function. These findings provide great confidence in the predicted TMEM165 model whose structural features are discussed in the study and compared to other structural and functional TMEM165 homologs from the CaCA2/UPF0016 family and the LysE superfamily.

The centrality of proteoglycan research: Expect the unexpected

The centrality of proteoglycan research: Expect the unexpected

Improved preparative enzymatic glycosylation of vancomycin aglycon and analogues

Modifications to the enzymatic glycosylation of vancomycin and its residue 4 thioamide analogue are detailed that significantly reduce the enzyme loading and amount of glycosyl donor needed for each glycosylation reaction, provide a streamlined synthesis and replacement for the synthetic UDP-vancosamine glycosyl donor to improve both access and storage stability, and permit a single-pot, two-step conversion of the aglycons to the fully glycosylated synthetic glycopeptides now conducted at higher concentrations. The improvements are exemplified with the two-step, one-pot glycosylation of [Ψ[C(=S)NH]Tpg4]vancomycin aglycon (92%) conducted on a 400 mg scale (2 mg–1 g scales) and vancomycin aglycon itself (5 mg scale, 84%).

Golgi Complex form and Function: A Potential Hub Role Also in Skeletal Muscle Pathologies?

A growing number of disorders has been associated with mutations in the components of the vesicular transport machinery. The early secretory pathway consists of Endoplasmic Reticulum, numerous vesicles, and the Golgi Complex (GC), which work together to modify and package proteins to deliver them to their destination. The GC is a hub organelle, crucial for organization of the other secretory pathway components. As a consequence, GC's form and function are key players in the pathogenesis of several disorders. Skeletal muscle (SKM) damage can be caused by defective protein modifications and traffic, as observed in some Limb girdle muscular dystrophies. Interestingly, in turn, muscle damage in Duchenne dystrophic SKM cells also includes the alteration of GC morphology. Based on the correlation between GC's form and function described in non-muscle diseases, we suggest a key role for this hub organelle also in the onset and progression of some SKM disorders. An altered GC could affect the secretory pathway via primary (e.g., mutation of a glycosylation enzyme), or secondary mechanisms (e.g., GC mis-localization in Duchenne muscles), which converge in SKM cell failure. This evidence induces considering the secretory pathway as a potential therapeutic target in the treatment of muscular dystrophies.

Global mapping of GalNAc-T isoform-specificities and O-glycosylation site-occupancy in a tissue-forming human cell line

Mucin-type-O-glycosylation on proteins is integrally involved in human health and disease and is coordinated by an enzyme family of 20 N-acetylgalactosaminyltransferases (GalNAc-Ts). Detailed knowledge on the biological effects of site-specific O-glycosylation is limited due to lack of information on specific glycosylation enzyme activities and O-glycosylation site-occupancies. Here we present a systematic analysis of the isoform-specific targets of all GalNAc-Ts expressed within a tissue-forming human skin cell line, and demonstrate biologically significant effects of O-glycan initiation on epithelial formation. We find over 300 unique glycosylation sites across a diverse set of proteins specifically regulated by one of the GalNAc-T isoforms, consistent with their impact on the tissue phenotypes. Notably, we discover a high variability in the O-glycosylation site-occupancy of 70 glycosylated regions of secreted proteins. These findings revisit the relevance of individual O-glycosylation sites in the proteome, and provide an approach to establish which sites drive biological functions.

Synthesis of O-Linked Glycoconjugates in the Nervous System.

Glycoproteins carrying O-linked N-acetylgalactosamine, N-acetylglucosamine, mannose, fucose, glucose, and xylose are found in the nervous system. Lipids are glycosylated by distinct glycosylation enzymes as well. Membrane lipid, ceramide, is modified by the addition of either glucose or galactose to form glycosphingolipid, galactosylceramide, or glucosylceramide. Recent careful analyses by MS have identified glucosylated lipids of cholesterol and phosphatidic acid. These O-linked carbohydrate residues are found primarily on the outer surface of the plasma membrane or in the extracellular space. Their expression is cell or tissue specific and developmentally regulated. Due to their structural diversity, they play important roles in a variety of biological processes such as membrane transport, metabolic stress responses, cell-cell interactions and so on. Discoveries of human diseases associated with glycosylation enzyme deficits have proved modification of lipids and proteins with carbohydrates play critical roles in human health and disease in the nervous systems.

Altered mannose metabolism in chronic stress and depression is rapidly reversed by vitamin B12.

GDP-Mannose Pyrophosphorylase B (GMPPB) is a key enzyme for glycosylation. Previous studies suggested a dysregulation of GMPBB and mannose in depression. Evidence, however, was sporadic and interventions to reverse these changes are unknown. Here, we show that GMPPB protein, but not RNA abundance is increased in the postmortem prefrontal cortex (PFC) of depressed patients and the chronic variable stress (CVS) mouse-model. This is accompanied by higher plasma mannose levels. Importantly, a single dose of intraperitoneally administered vitamin B12, which has previously been shown to rapidly reverse behavioral symptoms and molecular signatures of chronic stress in mice, normalized GMPPB plasma mannose levels and elevated GDP-mannose abundance. In summary, these data underline metabolic dysregulation in chronic stress and depression and provide further support for rapid effects of vitamin B12 on chronic stress.

Helicobacter pylori employs a general protein glycosylation system for the modification of outer membrane adhesins

ABSTRACT Helicobacter pylori infection is associated with the development of several gastric diseases including gastric cancer. To reach a long-term colonization in the host stomach, H. pylori employs multiple outer membrane adhesins for binding to the gastric mucosa. However, due to the redundancy of adhesins that complement the adhesive function of bacteria, targeting each individual adhesin alone usually achieves nonideal outcomes for preventing bacterial adhesion. Here, we report that key adhesins AlpA/B and BabA/B in H. pylori are modified by glycans and display a two-step molecular weight upshift pattern from the cytoplasm to the inner membrane and from the inner membrane to the outer membrane. Nevertheless, this upshift pattern is missing when the expression of some enzymes related to lipopolysaccharide (LPS) biosynthesis, including the LPS O-antigen assembly and ligation enzymes WecA, Wzk, and WaaL, is disrupted, indicating that the underlying mechanisms and the involved enzymes for the adhesin glycosylation are partially shared with the LPS biosynthesis. Loss of the adhesin glycosylation not only reduces the protease resistance and the stability of the tested adhesins but also changes the adhesin-binding ability. In addition, mutations in the LPS biosynthesis cause a significant reduction in bacterial adhesion in the in vitro cell-line model. The current findings reveal that H. pylori employs a general protein glycosylation system related to LPS biosynthesis for adhesin modification and its biological significance. The enzymes required for adhesin glycosylation rather than the adhesins themselves are potentially better drug targets for preventing or treating H. pylori infection.

Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages.

The fungal pathogen Histoplasma capsulatum (Hc) invades, replicates within, and destroys macrophages. To interrogate the molecular mechanisms underlying this interaction, we conducted a host-directed CRISPR-Cas9 screen and identified 361 genes that modify macrophage susceptibility to Hc infection, greatly expanding our understanding of host gene networks targeted by Hc. We identified pathways that have not been previously implicated in Hc interaction with macrophages, including the ragulator complex (involved in nutrient stress sensing), glycosylation enzymes, protein degradation machinery, mitochondrial respiration genes, solute transporters, and the ER membrane complex (EMC). The highest scoring protective hits included the complement C3a receptor (C3aR), a G-protein coupled receptor (GPCR) that recognizes the complement fragment C3a. Although it is known that complement components react with the fungal surface, leading to opsonization and release of small peptide fragments such as C3a, a role for C3aR in macrophage interactions with fungi has not been elucidated. We demonstrated that whereas C3aR is dispensable for macrophage phagocytosis of bacteria and latex beads, it is critical for optimal macrophage capture of pathogenic fungi, including Hc, the ubiquitous fungal pathogen Candida albicans, and the causative agent of Valley Fever Coccidioides posadasii. We showed that C3aR localizes to the early phagosome during Hc infection where it coordinates the formation of actin-rich membrane protrusions that promote Hc capture. We also showed that the EMC promotes surface expression of C3aR, likely explaining its identification in our screen. Taken together, our results provide new insight into host processes that affect Hc-macrophage interactions and uncover a novel and specific role for C3aR in macrophage recognition of fungi.

Generation 6 Polyamidoamine Dendrimer Provides an Ideal Nanoparticular Platform for Enzyme-assisted Synthesis of Glycopeptides Having Bulky and Complex Glycans

We demonstrate that generation 6 polyamidoamine dendrimer 7.2 nm in a diameter (MW = 58,048 Da) provides an ideal platform allowing for highly efficient enzymatic glycosylation of the peripheral bulky glycopeptidic substrates.

A nexus of lipid and O-Glcnac metabolism in physiology and disease.

Although traditionally considered a glucose metabolism-associated modification, the O-linked β-N-Acetylglucosamine (O-GlcNAc) regulatory system interacts extensively with lipids and is required to maintain lipid homeostasis. The enzymes of O-GlcNAc cycling have molecular properties consistent with those expected of broad-spectrum environmental sensors. By direct protein-protein interactions and catalytic modification, O-GlcNAc cycling enzymes may provide both acute and long-term adaptation to stress and other environmental stimuli such as nutrient availability. Depending on the cell type, hyperlipidemia potentiates or depresses O-GlcNAc levels, sometimes biphasically, through a diversity of unique mechanisms that target UDP-GlcNAc synthesis and the availability, activity and substrate selectivity of the glycosylation enzymes, O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA). At the same time, OGT activity in multiple tissues has been implicated in the homeostatic regulation of systemic lipid uptake, storage and release. Hyperlipidemic patterns of O-GlcNAcylation in these cells are consistent with both transient physiological adaptation and feedback uninhibited obesogenic and metabolic dysregulation. In this review, we summarize the numerous interconnections between lipid and O-GlcNAc metabolism. These links provide insights into how the O-GlcNAc regulatory system may contribute to lipid-associated diseases including obesity and metabolic syndrome.

Pro-Survival Factor EDEM3 Confers Therapy Resistance in Prostate Cancer.

Prostate cancer is the most common cancer in men, and it is primarily driven by androgen steroid hormones. The glycosylation enzyme EDEM3 is controlled by androgen signalling and is important for prostate cancer viability. EDEM3 is a mannosidase that trims mannose from mis-folded glycoproteins, tagging them for degradation through endoplasmic reticulum-associated degradation. Here, we find that EDEM3 is upregulated in prostate cancer, and this is linked to poorer disease-free survival. Depletion of EDEM3 from prostate cancer cells induces an ER stress transcriptomic signature, and EDEM3 overexpression is cyto-protective against ER stressors. EDEM3 expression also positively correlates with genes involved in the unfolded protein response in prostate cancer patients, and its expression can be induced through exposure to radiation. Importantly, the overexpression of EDEM3 promotes radio-resistance in prostate cancer cells and radio-resistance can be reduced through depletion of EDEM3. Our data thus implicate increased levels of EDEM3 with a role in prostate cancer pathology and reveal a new therapeutic opportunity to sensitise prostate tumours to radiotherapy.

Characterization of regioselective glycosyltransferase of Rhizobium pusense JCM 16209T useful for resveratrol 4′-O-α-d-glucoside production

Enzymatic glycosylation is an industrially useful technique for improving the properties of compounds with hydroxy groups, and the biological activities of the resulting glycosides differ depending on the glycosylation position. Therefore, regioselective glycosyltransferases are required for precise synthesis of glycosides. We found that Rhizobium pusense JCM 16209T could catalyze the regioselective glycosylation of resveratrol. To identify the regioselective glycosyltransferase, two α-glucosidases of R.pusense JCM 16209T (RpG I and RpG II) were cloned and expressed in Escherichia coli. The molecular mass of purified recombinant RpG I and II was estimated to be 60kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RpG I showed strong glycosylation activity toward resveratrol with 4'-selectivity of 98.3%. The enzyme activity was maximized at pH 8.0 and 50°C, and enhanced in the presence of Cs+ and Li+ ions. The maximum molar yield of resveratrol 4'-O-α-glucoside from resveratrol reached 41.6% at 30min, and the concentration of the product was 2.08mmolL-1. Glycosylation activity was observed toward resveratrol as well as toward caffeic acid, ferulic acid, 6-gingerol, flavonoid, and isoflavonoid compounds with high regioselectivity, indicating that RpG I could glycosylate a wide range of substrates. To the best of our knowledge, there are few reports on microbial glycosyltransferases that are useful for regioselective glycosylation. This research could be the first step toward developing technologies for the precise synthesis of glycosides.

Enzymatic Biosynthesis of Simple Phenolic Glycosides as Potential Anti-Melanogenic Antioxidants.

Simple phenolics (SPs) and their glycosides have recently gained much attention as functional skin-care resources for their anti-melanogenic and antioxidant activities. Enzymatic glycosylation of SP aglycone make it feasible to create SP glycosides with updated bioactive potentials. Herein, a glycosyltransferase (GT)-encoding gene was cloned from the fosmid libraries of Streptomyces tenjimariensis ATCC 31603 using GT-specific degenerate PCR followed by in silico analyses. The recombinant StSPGT was able to flexibly catalyze the transfer of two glycosyl moieties towards two SP acceptors, (hydroxyphenyl-2-propanol [HPP2] and hydroxyphenyl-3-propanol [HPP3]), generating stereospecific α-anomeric glycosides as follows: HPP2-O-α-glucoside, HPP2-O-α-2″-deoxyglucoside, HPP3-O-α-glucoside and HPP3-O-α-2″-deoxyglucoside. This enzyme seems not only to prefer UDP-glucose and HPP2 as a favorable glycosyl donor and acceptor, respectively but also differentiates the positional difference of the hydroxyl function as acceptor catalytic sites. Paired in vitro and in vivo antioxidant assays represented SPs and their corresponding glycosides as convincing antioxidants in a time- and concentration-dependent manner by scavenging DPPH radicals and intracellular ROS. Even compared to the conventional agents, HPP2 and glycoside analogs displayed improved tyrosinase inhibitory activity in vitro and still suppressed in vivo melanogenesis. Both HPP2 glycosides are further likely to exert the best inhibitory activity against elastase, eventually highlighting these glycosides with enhanced anti-melanogenic and antioxidant activities as promising anti-wrinkle hits.

In silico analysis of the human milk oligosaccharide glycome reveals key enzymes of their biosynthesis

Human milk oligosaccharides (HMOs) form the third most abundant component of human milk and are known to convey several benefits to the neonate, including protection from viral and bacterial pathogens, training of the immune system, and influencing the gut microbiome. As HMO production during lactation is driven by enzymes that are common to other glycosylation processes, we adapted a model of mucin-type GalNAc-linked glycosylation enzymes to act on free lactose. We identified a subset of 11 enzyme activities that can account for 206 of 226 distinct HMOs isolated from human milk and constructed a biosynthetic reaction network that identifies 5 new core HMO structures. A comparison of monosaccharide compositions demonstrated that the model was able to discriminate between two possible groups of intermediates between major subnetworks, and to assign possible structures to several previously uncharacterised HMOs. The effect of enzyme knockouts is presented, identifying β-1,4-galactosyltransferase and β-1,3-N-acetylglucosaminyltransferase as key enzyme activities involved in the generation of the observed HMO glycosylation patterns. The model also provides a synthesis chassis for the most common HMOs found in lactating mothers.

An Integrated Mass Spectrometry-Based Glycomics-Driven Glycoproteomics Analytical Platform to Functionally Characterize Glycosylation Inhibitors.

Cancer progression is linked to aberrant protein glycosylation due to the overexpression of several glycosylation enzymes. These enzymes are underexploited as potential anticancer drug targets and the development of rapid-screening methods and identification of glycosylation inhibitors are highly sought. An integrated bioinformatics and mass spectrometry-based glycomics-driven glycoproteomics analysis pipeline was performed to identify an N-glycan inhibitor against lung cancer cells. Combined network pharmacology and in silico screening approaches were used to identify a potential inhibitor, pictilisib, against several glycosylation-related proteins, such as Alpha1-6FucT, GlcNAcT-V, and Alpha2,6-ST-I. A glycomics assay of lung cancer cells treated with pictilisib showed a significant reduction in the fucosylation and sialylation of N-glycans, with an increase in high mannose-type glycans. Proteomics analysis and in vitro assays also showed significant upregulation of the proteins involved in apoptosis and cell adhesion, and the downregulation of proteins involved in cell cycle regulation, mRNA processing, and protein translation. Site-specific glycoproteomics analysis further showed that glycoproteins with reduced fucosylation and sialylation were involved in apoptosis, cell adhesion, DNA damage repair, and chemical response processes. To determine how the alterations in N-glycosylation impact glycoprotein dynamics, modeling of changes in glycan interactions of the ITGA5–ITGB1 (Integrin alpha 5-Integrin beta-1) complex revealed specific glycosites at the interface of these proteins that, when highly fucosylated and sialylated, such as in untreated A549 cells, form greater hydrogen bonding interactions compared to the high mannose-types in pictilisib-treated A549 cells. This study highlights the use of mass spectrometry to identify a potential glycosylation inhibitor and assessment of its impact on cell surface glycoprotein abundance and protein–protein interaction.

Exposure of Keratinocytes to Candida Albicans in the Context of Atopic Milieu Induces Changes in the Surface Glycosylation Pattern of Small Extracellular Vesicles to Enhance Their Propensity to Interact With Inhibitory Siglec Receptors.

Candida albicans (C. albicans) infection is a potential complication in the individuals with atopic dermatitis (AD) and can affect clinical course of the disease. Here, using primary keratinocytes we determined that atopic milieu promotes changes in the interaction of small extracellular vesicles (sEVs) with dendritic cells and that this is further enhanced by the presence of C. albicans. sEV uptake is largely dependent on the expression of glycans on their surface; modelling of the protein interactions indicated that recognition of this pathogen through C. albicans-relevant pattern recognition receptors (PRRs) is linked to several glycosylation enzymes which may in turn affect the expression of sEV glycans. Here, significant changes in the surface glycosylation pattern, as determined by lectin array, could be observed in sEVs upon a combined exposure of keratinocytes to AD cytokines and C. albicans. This included enhanced expression of multiple types of glycans, for which several dendritic cell receptors could be proposed as binding partners. Blocking experiments showed predominant involvement of the inhibitory Siglec-7 and -9 receptors in the sEV-cell interaction and the engagement of sialic acid-containing carbohydrate moieties on the surface of sEVs. This pointed on ST6 β-Galactoside α-2,6-Sialyltransferase 1 (ST6GAL1) and Core 1 β,3-Galactosyltransferase 1 (C1GALT1) as potential enzymes involved in the process of remodelling of the sEV surface glycans upon C. albicans exposure. Our results suggest that, in combination with atopic dermatitis milieu, C. albicans promotes alterations in the glycosylation pattern of keratinocyte-derived sEVs to interact with inhibitory Siglecs on antigen presenting cells. Hence, a strategy aiming at this pathway to enhance antifungal responses and restrict pathogen spread could offer novel therapeutic options for skin candidiasis in AD.

Beta-Cell Golgi Stress Response to Lipotoxicity and Glucolipotoxicity: A Preliminary Study of a Potential Mechanism of Beta-Cell Failure in Posttransplant Diabetes and Intraportal Islet Transplant.

Lipotoxicity and glucolipotoxicity are among the mostimportanttriggers of beta-cell failure in patients with type 2 and posttransplant diabetes. Because the Golgi apparatus is a vital organelle in secretory cells like beta cells, its behavior under stress conditions determines the cell's functional capacity. To mimic lipotoxicity and glucolipotoxicity as metabolic stresses for beta-cell failure, rat insulinoma INS-1E cells were treated with palmitic acid, glucose, or both. Cells were cultured in the presence of 5.0, 16.7, or 33 mM glucose with or without 0.5 mM palmitic acid for 8, 16, 24, and 48 hours. Incubation in the presence of any of the 3 concentrations of glucose with 0.5 mM palmitic acid provided glucolipotoxicity. In addition to the endoplasmic reticulum stress marker (Hspa5), we evaluated changes in Golgi function under experimental metabolic stresses. In doing this, we measured expression levels of the genes coding Golgi structural proteins (Acbd3,Golga2, and Arf1), Golgi glycosylation enzymes sialyltransferaz10 and sialyltransferase 1 (St3gal1), and Golgi stress mediators (Creb3 and Arf4). Golgi responded to lipotoxicity and glucolipotoxicity by increasing the expression of St3gal1 (P = .05 in both conditions) and Creb3 (P = .022 and P = .01, respectively). The Arf4 gene transcript also increased in glucolipotoxic media (P = .03). Glucotoxicity alone did not induce a change in the transcript levels of Creb3 and Arf4. Lipotoxicity and glucolipotoxicity induced Creb3 and Arf4 expression, which are important Golgi stress response mediators leading to apoptosis. This preliminary study showed that the Golgi stress response is important in lipotoxic and glucolipotoxic conditions in terms of beta-cell failure. Solving the mystery of intracellular molecular mechanisms leading to beta-cell dysfunction is crucial to understanding the pathophysiology of posttransplant diabetes and most probably the failure of intraportal islet transplants in the long term.