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Glucosamine (GlcN) is an important bioactive substance that is widely used in medicine, dietary supplements, cosmetics, and other fields. The traditional method of producing GlcN is mainly through chitosan hydrolysis catalyzed by strong acid, but this process is usually accompanied by environmental pollution and high energy consumption. Therefore, the development of green and efficient production methods of glucosamine has become the focus of current research.In this study, N-acetylglucosamine (GlcNAc) was used as the substrate to facilitate the enzymatic synthesis of GlcN by deacetylase. Four deacetylases (TkDAc, PkDAc, PpDAc and AbDAc) were selected from marine thermophilic microorganisms, and Escherichia coli (E. coli) was used as the host for recombinant expression.The soluble expression of PpDAc was poor, so several groups of solubilizing labels were tried, and the results showed that the soluble expression of recombinant plasmid ArsC-PpDAc carrying pro-solubilization labels was greatly improved.The effects of temperature and pH on enzyme activity were investigated by single factor analysis. Kinetic parameters further revealed that ArsC-PpDAc exhibited the highest catalytic activity, with a Kcat/Km value of 7.29, and achieved a conversion rate of over 95 %. The condition of ArsC-PpDAc was optimized, and the results showed that ArsC-PpDAc showed good tolerance to organic solvents, and its catalytic activity was not significantly affected. [ABSTRACT FROM AUTHOR]

N-glycans with complex core chitobiose modifications are observed in various free-living and parasitic nematodes but are absent in mammals. Using Caenorhabditis elegans as a model, we demonstrated that the core N-acetylglucosamine (GlcNAc) residues are modified by three fucosyltransferases (FUTs), namely FUT-1, FUT-6, and FUT-8. Interestingly, FUT-6 can only fucosylate N-glycans lacking the a1,6-mannose upper arm, indicating that a specific a-mannosidase is required to generate substrates for subsequent FUT-6 activity. By analyzing the N-glycomes of aman-3 KOs using offline HPLC-MALDI-TOF MS/MS, we observed that the absence of aman-3 abolishes a1,3-fucosylation of the distal GlcNAc of N-glycans, which suggests that AMAN-3 is the relevant mannosidase on whose action FUT-6 depends. Enzymatic characterization of recombinant AMAN-3 and confocal microscopy studies using a knock-in strain (aman-3::eGFP) demonstrated a Golgi localization. In contrast to the classical Golgi a-mannosidase II (AMAN-2), AMAN-3 displayed a cobalt-dependent a1,6-mannosidase activity toward N-glycans. Using AMAN-3 and other C. elegans glycoenzymes, we were able to mimic nematode N-glycan biosynthesis in vitro by remodeling a fluorescein conjugated-glycan and generate a tri-fucosylated structure. In addition, using a high-content computer-assisted C. elegans analysis platform, we observed that aman-3 deficient worms display significant developmental delays, morphological, and behavioral alterations in comparison to the WT. Our data demonstrated that AMAN-3 is a Golgi a-mannosidase required for core fucosylation of the distal GlcNAc of N-glycans. This enzyme is essential for the formation of the unusual trifucosylated chitobiose modifications in nematodes, which may play important roles in nematode development and behavior. [ABSTRACT FROM AUTHOR]

Psoriasis (Ps) is a chronic inflammatory disorder marked by skin plaque formation, driven by immune dysregulation and genetic factors. Despite the available treatments, incidence of Ps is increasing in the dermatology patients. Novel strategies are crucial due to current treatment limitations. The interleukin 17 (IL-17) pathway is pivotal in Ps pathogenesis, however the expression of its putative target gene placenta expressed transcript 1 (Plet1) remains unstudied in Ps. Considering the potential anti-inflammatory properties of N-Acetylglucosamine (GlcNAc), our study explored its role in modulating Plet1 expression in an imiquimod (IMQ)-induced Ps mouse model. Our data demonstarted a significant reduction of inflammation and Psoriasis Area and Severity Index (PASI) scores, downregulation of growth factors (GFs), IL-17 A, and MAPK expression after GlcNAc treatment. In addition, GlcNAc treatment reduced neutrophils, monocyte-dendritic cells (Mo-DC) and conventional T cells (Tcons) while increasing monocyte-macrophages (Mo-Macs) and regulatory T cells (Tregs). GlcNAc treatment also downregulated Plet1 overexpression in psoriatic mouse skin and in vitro, reduced proliferation and apoptosis in IL-17 A stimulated human dermal fibroblasts (HDF), along with IL-17 A and TGF-β mRNA expression. Together, these data suggest that, GlcNAc interferes with downstream mechanisms in IL-17 pathway and downregulating Plet1 expression, presenting a promising strategy for Ps treatment. [ABSTRACT FROM AUTHOR]

Heparosan, an unsulfated polysaccharide, plays a pivotal role as a primary precursor in the biosynthesis of heparin—an influential anticoagulant with diverse therapeutic applications. To enhance heparosan production, the utilization of metabolic engineering in nonpathogenic microbial strains is emerging as a secure and promising strategy. In the investigation of heparosan production by recombinant Bacillus megaterium, a kinetic modeling approach was employed to explore the impact of initial substrate concentration and the supplementation of precursor sugars. The adapted logistic model was utilized to thoroughly analyze three vital parameters: the B. megaterium growth dynamics, sucrose utilization, and heparosan formation. It was noted that at an initial sucrose concentration of 30 g L−1 (S1), it caused an inhibitory effect on both cell growth and substrate utilization. Intriguingly, the inclusion of N‐acetylglucosamine (S2) resulted in a significant 1.6‐fold enhancement in heparosan concentration. In addressing the complexities of the dual substrate system involving S1 and S2, a multi‐substrate kinetic models, specifically the double Andrew's model was employed. This approach not only delved into the intricacies of dual substrate kinetics but also effectively described the relationships among the primary state variables. Consequently, these models not only provide a nuanced understanding of the system's behavior but also serve as a roadmap for optimizing the design and management of the heparosan production method. [ABSTRACT FROM AUTHOR]

N-acetylglucosamine (GlcNAc) is a versatile monosaccharide with broad applications in medicine, food, and cosmetics due to its antimicrobial, antioxidant, and anti-tumor properties. Efficient synthesis methods for GlcNAc remain a prominent research focus. O -GlcNAc modification, prevalent in nuclear and cytoplasmic proteins, is of particular interest, especially regarding the catalytic mechanism of the O -GlcNAcase (OGA) enzyme. Enzymatic processes using chitin as a substrate, yield GlcNAc-based oligosaccharides, offering diverse health benefits including antioxidant, anti-inflammatory, anti-tumor, and antimicrobial effects. This review examines GlcNAc preparation methods, O -GlcNAc modification mechanisms, and the applications of GlcNAc-based oligosaccharides in various fields. [ABSTRACT FROM AUTHOR]

The relationship between O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and mitosis is intertwined. Besides the numerous mitotic OGT substrates that have been identified, OGT itself is also a target of the mitotic machinery. Previously, our investigations have shown that Checkpoint kinase 1 (Chk1) phosphorylates OGT at Ser-20 to increase OGT levels during cytokinesis, suggesting that OGT levels oscillate as mitosis progresses. Herein we studied its underlying mechanism. We set out from an R17C mutation of OGT, which is a uterine carcinoma mutation in The Cancer Genome Atlas. We found that R17C abolishes the S20 phosphorylation of OGT, as it lies in the Chk1 phosphorylating consensus motif. Consistent with our previous report that pSer-20 is essential for OGT level increases during cytokinesis, we further demonstrate that the R17C mutation renders OGT less stable, decreases vimentin phosphorylation levels and results in cytokinesis defects. Based on bioinformatic predictions, pSer-20 renders OGT more likely to interact with 14-3-3 proteins, the phospho-binding signal adaptor/scaffold protein family. By screening the seven isoforms of 14-3-3 family, we show that 14-3-3e specifically associates with Ser-20-phosphorylated OGT. Moreover, we studied the R17C and S20A mutations in xenograft models and demonstrated that they both inhibit uterine carcinoma compared to wild-type OGT, probably due to less cellular reproduction. Our work is a sequel of our previous report on pS20 of OGT and is in line with the notion that OGT is intricately regulated by the mitotic network. [ABSTRACT FROM AUTHOR]

O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a common and important post-translational modification (PTM) linking O-linked β-N-acetylglucosamine (O-GlcNAc) to serine and threonine residues in proteins. Extensive research indicates its impact on target protein stability, activity, and interactions. O-linked N-acetylglucosamine transferase (OGT) is a critical enzyme that catalyzes O-GlcNAc modification, responsible for adding O-GlcNAc to proteins. OGT and O-GlcNAcylation are overexpressed in many tumors and closely associated with tumor growth, invasion, metabolism, drug resistance, and immune evasion. This review delineates the biochemical functions of OGT and summarizes its effects and mechanisms in tumors. Targeting OGT presents a promising novel approach for treating human malignancies. [ABSTRACT FROM AUTHOR]

O-linked N-acetylglucosamine protein modification (O-GlcNAcylation) is a dynamic post-translational modification (PTM) involving the covalent binding of serine and/or threonine residues, which regulates bone cell homeostasis. Reactive oxygen species (ROS) are increased due to oxidative stress in various pathological contexts related to bone remodeling, such as osteoporosis, arthritis, and bone fracture. Autophagy serves as a scavenger for ROS within bone marrow-derived mesenchymal stem cells, osteoclasts, and osteoblasts. However, oxidative stress-induced autophagy is affected by the metabolic status, leading to unfavorable clinical outcomes. O-GlcNAcylation can regulate the autophagy process both directly and indirectly through oxidative stress-related signaling pathways, ultimately improving bone remodeling. The present interventions for the bone remodeling process often focus on promoting osteogenesis or inhibiting osteoclast absorption, ignoring the effect of PTM on the overall process of bone remodeling. This review explores how O-GlcNAcylation synergizes with autophagy to exert multiple regulatory effects on bone remodeling under oxidative stress stimulation, indicating the application of O-GlcNAcylation as a new molecular target in the field of bone remodeling. [ABSTRACT FROM AUTHOR]

N‐acetylglucosamine (GlcNAc) is an important structural component of the cell wall chitin, N‐glycans, glycolipids, and GPI‐anchors in eukaryotes. GlcNAc kinase phosphorylates GlcNAc into GlcNAc‐6‐phosphate, a precursor of uridine diphosphate N‐acetylglucosamine (UDP‐GlcNAc) that serves as a substrate for glycan synthesis. Although GlcNAc kinase is found widely in organisms ranging from microorganisms to mammals, it has never been found in the model yeast Saccharomyces cerevisiae. Here, we demonstrate the presence of GlcNAc metabolism for UDP‐GlcNAc biosynthesis in S. cerevisiae through Ngk1, a GlcNAc kinase we discovered previously. The overexpression or deletion of Ngk1 in the presence of GlcNAc affected the amount of both UDP‐GlcNAc and chitin, suggesting that GlcNAc metabolism via Ngk1 promotes UDP‐GlcNAc synthesis. Our data suggest that the Ngk1‐mediated GlcNAc metabolism compensates for the hexosamine pathway, a known pathway for UDP‐GlcNAc synthesis. [ABSTRACT FROM AUTHOR]

The cell surface of Toxoplasma gondii is rich in glycoconjugates which hold diverse and vital functions in the lytic cycle of this obligate intracellular parasite. Additionally, the cyst wall of bradyzoites, that shields the persistent form responsible for chronic infection from the immune system, is heavily glycosylated. Formation of glycoconjugates relies on activated sugar nucleotides, such as uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). The glucosamine-phosphate-N-acetyltransferase (GNA1) generates N-acetylglucosamine-6-phosphate critical to produce UDP-GlcNAc. Here, we demonstrate that downregulation of T. gondii GNA1 results in a severe reduction of UDP-GlcNAc and a concomitant drop in glycosylphosphatidylinositols (GPIs), leading to impairment of the parasite's ability to invade and replicate in the host cell. Surprisingly, attempts to rescue this defect through exogenous GlcNAc supplementation fail to completely restore these vital functions. In depth metabolomic analyses elucidate diverse causes underlying the failed rescue: utilization of GlcNAc is inefficient under glucose-replete conditions and fails to restore UDP-GlcNAc levels in GNA1-depleted parasites. In contrast, GlcNAc-supplementation under glucose-deplete conditions fully restores UDP-GlcNAc levels but fails to rescue the defects associated with GNA1 depletion. Our results underscore the importance of glucosamine-6-phosphate acetylation in governing T. gondii replication and invasion and highlight the potential of the evolutionary divergent GNA1 in Apicomplexa as a target for the development of much-needed new therapeutic strategies. Author summary: Toxoplasma gondii, Plasmodium, and Cryptosporidium spp., pose serious threats to human health. T. gondii, an intracellular and opportunistic pathogen, effectively avoids the host immune defences by forming long-lasting tissue cysts. Finding potent drugs to eliminate these persisting parasites remains a challenge. The glucosamine-phosphate-N-acetyltransferase (GNA1) catalyses a critical key step in the production of activated sugar nucleotides to build glycoconjugates essential for various functions in the cell. In P. falciparum, this enzyme has been identified as a potential target for antimalarial drugs. In this study, we explored the importance of this pathway in T. gondii and discovered that these sugar-containing compounds play a vital role in the parasite's ability to invade and replicate in host cells–crucial processes for its survival and ability to cause disease. Intriguingly, unlike some organisms that can bypass the pathway, T. gondii relies critically on glucosamine-6-phosphate acetylation. This reliance sheds light on the parasite's distinct metabolic properties and highlights the pathway's potential as a target for new therapeutic strategies. [ABSTRACT FROM AUTHOR]

This study presents a novel approach in the realm of catalytic organic synthesis by integrating biomass catalytic conversion with organic synthesis techniques. Utilizing N-acetylglucosamine as the primary feedstock, the first phase of the research involves its catalytic transformation into 5-hydroxymethylfurfural (HMF). The subsequent phase employs a condensation reaction between HMF and 3,3-Dimethyl-2-butanone to synthesize a new compound, (E)-1-(5-(hydroxymethyl) furan-2-yl)-4,4-dimethylpent-1-en-3-one. This two-step process not only demonstrates the feasibility of converting biomass into valuable chemical precursors but also exemplifies the synthesis of novel compounds through green chemistry principles. The successful execution of this methodology offers fresh insights and opens new avenues for advancements in catalytic organic synthesis, emphasizing sustainability and efficiency. [ABSTRACT FROM AUTHOR]

Fatty acid synthesis has been extensively investigated as a therapeutic target in cancers, including colorectal cancer (CRC). Fatty acid synthase (FASN), a key enzyme of de novo lipid synthesis, is significantly upregulated in CRC, and therapeutic approaches of targeting this enzyme are currently being tested in multiple clinical trials. However, the mechanisms behind the pro-oncogenic action of FASN are still not completely understood. Here, for the first time, we show that overexpression of FASN increases the expression of glutamine–fructose-6-phosphate transaminase 1 (GFPT1) and O-linked N-acetylglucosamine transferase (OGT), enzymes involved in hexosamine metabolism, and the level of O-GlcNAcylation in vitro and in vivo. Consistently, expression of FASN significantly correlates with expression of GFPT1 and OGT in human CRC tissues. shRNA-mediated downregulation of GFPT1 and OGT inhibits cellular proliferation and the level of protein O-GlcNAcylation in vitro, and knockdown of GFPT1 leads to a significant decrease in tumor growth and metastasis in vivo. Pharmacological inhibition of GFPT1 and OGT leads to significant inhibition of cellular proliferation and colony formation in CRC cells. In summary, our results show that overexpression of FASN increases the expression of GFPT1 and OGT as well as the level of protein O-GlcNAcylation to promote progression of CRC; targeting the hexosamine biosynthesis pathway could be a therapeutic approach for this disease. [ABSTRACT FROM AUTHOR]

The clinical severity of multiple sclerosis (MS), an autoimmune disorder of the central nervous system, is thought to be determined by environmental and genetic factors that have not yet been identified. In a recent genome-wide association study (GWAS), a single nucleotide polymorphism (SNP), rs10191329, has been associated with MS severity in two large independent cohorts of patients. Different approaches were followed by the authors to prioritize the genes that are transcriptionally regulated by such an SNP. It was concluded that the identified SNP regulates a group of proximal genes involved in brain resilience and cognitive abilities rather than immunity. Here, by conducting an alternative strategy for gene prioritization, we reached the opposite conclusion. According to our re-analysis, the main target of rs10191329 is N-Acetylglucosamine Kinase (NAGK), a metabolic gene recently shown to exert major immune functions via the regulation of the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) pathway. To gain more insights into the immunometabolic functions of NAGK, we analyzed the currently known list of NAGK protein partners. We observed that NAGK integrates a dense network of human proteins that are involved in glucose metabolism and are highly expressed by classical monocytes. Our findings hold potentially major implications for the understanding of MS pathophysiology. [ABSTRACT FROM AUTHOR]

The application of N-acetylglucosamine (GlcNAc) and melatonin (Mel) in agriculture could be a promising avenue for improving crop resilience and productivity, especially under challenging environmental conditions. In the current study, we treated the cucumber plant with GlcNAc and Mel solely and combinedly under salt stress (150 mM) then studied photosynthetic attributes using the transient OJIP fluorescence method. The results showed that the combination of GlcNAc × Mel significantly improved the plant morphological attributes, such as root and shoot biomass, and also improved chlorophyll and photosynthetic components. The mineral elements such as K, Mg, Ca, and P were significantly elevated, whereas a lower influx of Na was observed in GlcNAc × Mel treated cucumber shoots. A significant reduction in abscisic acid was observed, which was validated by the reduction in proline content and the increase in stomatal conductance (Gs), transpiration rate (E), and substomatal CO2 concentration (Ci). Furthermore, the activities of antioxidants such as polyphenol and flavonoid were considerably improved, resulting in a decrease in SOD and CAT with GlcNAc × Mel treatment. In addition, GlcNAc × Mel treatment dropped levels of the toxic radical Malondialdehyde (MDA) and elevated amino acids in cucumber shoots. These findings suggest that the combination of GlcNAc × Mel could be an effective elicitor for modeling plant metabolism to confer stress tolerance in crops. [ABSTRACT FROM AUTHOR]

Candida albicans, an opportunistic fungal pathogen, is able to switch between two distinct cell types: white and opaque. While white‐to‐opaque switching is typically repressed by the a1/α2 heterodimer in MTLa/α cells, it was recently reported that switching can also occur in some natural MTLa/α strains under certain environmental conditions. However, the regulatory program governing white‐opaque switching in MTLa/α cells is not fully understood. Here, we collected 90 clinical isolates of C. albicans, 16 of which possess the ability to form opaque colonies. Among the known regulators implicated in white‐opaque switching, only OFI1 exhibited significantly higher expression in these 16 strains compared to the reference strain SC5314. Importantly, ectopic expression of OFI1 in both clinical isolates and laboratory strains promoted switching frequency even in the absence of N‐acetylglucosamine and high CO2, the optimal condition for white‐to‐opaque switching in MTLa/α strains. Deleting OFI1 resulted in a reduction in opaque‐formation frequency and the stability of the opaque cell in MTLa/α cells. Ofi1 binds to the promoters of WOR1 and WOR3 to induce their expression, which facilitates white‐to‐opaque switching. Ofi1 is conserved across the CTG species. Altogether, our study reported the identification of a transcription factor Ofi1 as the critical regulator that promotes white‐to‐opaque switching in natural MTLa/α isolates of C. albicans. [ABSTRACT FROM AUTHOR]

Cells continuously fine-tune signaling pathway proteins to match nutrient and stress levels in their local environment by modifying intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc) sugars, an essential process for cell survival and growth. The small size of these monosaccharide modifications poses a challenge for functional determination, but the chemistry and biology communities have together created a collection of precision tools to study these dynamic sugars. This review presents the major themes by which O-GlcNAc influences signaling pathway proteins, including G-protein coupled receptors, growth factor signaling, mitogen-activated protein kinase (MAPK) pathways, lipid sensing, and cytokine signaling pathways. Along the way, we describe in detail key chemical biology tools that have been developed and applied to determine specific O-GlcNAc roles in these pathways. These tools include metabolic labeling, O-GlcNAc-enhancing RNA aptamers, fluorescent biosensors, proximity labeling tools, nanobody targeting tools, O-GlcNAc cycling inhibitors, light-activated systems, chemoenzymatic labeling, and nutrient reporter assays. An emergent feature of this signaling pathway meta-analysis is the intricate interplay be-tween O-GlcNAc modifications across different signaling systems, underscoring the importance of O-GlcNAc in regulating cellular processes. We highlight the significance of O-GlcNAc in signaling and the role of chemical and biochemical tools in unraveling distinct glycobiological regulatory mechanisms. Collectively, our field has determined effective strategies to probe O-GlcNAc roles in biology. At the same time, this survey of what we do not yet know presents a clear roadmap for the field to use these powerful chemical tools to explore cross-pathway O-GlcNAc interactions in signaling and other major biological pathways. [ABSTRACT FROM AUTHOR]

Chitin deacetylases (CDAs) emerge as a valuable tool to produce chitosans with a nonrandom distribution of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) units. We hypothesized before that CDAs tend to bind certain sequences within the substrate matching their subsite preferences for either GlcNAc or GlcN units. Thus, they deacetylate or N-acetylate their substrates at nonrandom positions. To understand the molecular basis of these preferences, we analyzed the binding site of a CDA from Pestalotiopsis sp. (PesCDA) using a detailed activity screening of a site-saturation mutagenesis library. In addition, molecular dynamics simulations were conducted to get an in-depth view of crucial interactions along the binding site. Besides elucidating the function of several amino acids, we were able to show that only 3 residues are responsible for the highly specific binding of PesCDA to oligomeric substrates. The preference to bind a GlcNAc unit at subsite −2 and −1 can mainly be attributed to N75 and H199, respectively. Whereas an exchange of N75 at subsite −2 eliminates enzyme activity, H199 can be substituted with tyrosine to increase the GlcN acceptance at subsite −1. This change in substrate preference not only increases enzyme activity on certain substrates and changes composition of oligomeric products but also significantly changes the pattern of acetylation (PA) when N-acetylating polyglucosamine. Consequently, we could clearly show how subsite preferences influence the PA of chitosans produced with CDAs. Chitin deacetylases are a valuable tool for producing custom chitosans with non-random N-acetylglucosamine and glucosamine units. Analysis of a chitin deacetylase active site reveals residues crucial for substrate binding, allowing engineering to drastically change the acetylation pattern of a chitosan product. [ABSTRACT FROM AUTHOR]

In various life forms, fucose-containing glycans play vital roles in immune recognition, developmental processes, plant immunity, and host-microbe interactions. Together with glucose, galactose, N-acetylglucosamine, and sialic acid, fucose is a significant component of human milk oligosaccharides (HMOs). Fucosylated HMOs benefit infants by acting as prebiotics, preventing pathogen attachment, and potentially protecting against infections, including HIV. Although the need for fucosylated derivatives is clear, their availability is limited. Therefore, synthesis methods for various fucosylated oligosaccharides are explored, employing enzymatic approaches and α-L-fucosidases. This work aimed to characterise α-L-fucosidases identified in an alpaca faeces metagenome. Based on bioinformatic analyses, they were confirmed as members of the GH29A subfamily. The recombinant α-L-fucosidases were expressed in Escherichia coli and showed hydrolytic activity towards p-nitrophenyl-α-L-fucopyranoside and 2′-fucosyllactose. Furthermore, the enzymes' biochemical properties and kinetic characteristics were also determined. All four α-L-fucosidases could catalyse transfucosylation using a broad diversity of fucosyl acceptor substrates, including lactose, maltotriose, L-serine, and L-threonine. The results contribute insights into the potential use of α-L-fucosidases for synthesising fucosylated amino acids. [ABSTRACT FROM AUTHOR]

O-GlcNAcylation is a posttranslational modification where N-acetylglucosamine (O-GlcNAc) is attached and detached from a serine/threonine position by two enzymes: O-GlcNAc transferase and O-GlcNAcase. In addition to roles in diabetes and cancer, recent pharmacological and genetic studies have revealed that O-GlcNAcylation is involved in neuronal function, specifically synaptic transmission. Global alteration of the O-GlcNAc level does not affect basal synaptic transmission while the effect on synaptic plasticity is unclear. Although synaptic proteins that are O-GlcNAcylated are gradually being discovered, the mechanism of how O-GlcNAcylated synaptic protein modulate synaptic transmission has only been reported on CREB, synapsin, and GluA2 subunit of AMPAR. Future research enabling the manipulation of O-GlcNAcylation in individual synaptic proteins should reveal hidden aspects of O-GlcNAcylated synaptic proteins as modulators of synaptic transmission. [ABSTRACT FROM AUTHOR]

Oligosaccharides produced by degradation of hyaluronan (HA) can be applied in functional foods. In this study, Variovorax sp. J25 with high secreted HA lyase activity (16.0 U/mL), was isolated from soil, and its properties were investigated. The activities toward chondroitin sulfates A and C (CS-A and CS-C) were much lower, at 0.439 and 0.325 U/mL, respectively. The molecular weight of the purified HA lyase was around 73 kDa. The optimal reaction pH and temperature were approximately 6.7 and 50°C, respectively. The amounts of sugar produced from HA, CS-A, and CS-C were 550, 175, and 271 mg/g after reaction for 24 h, respectively. The degradation of CS-A and CS-C proceeded gradually. The major product from HA was disaccharide [ΔUA–GlcNAc], and tetrasaccharide [(ΔUA–GlcNAc)2] was also detected (GlcNAc: N-acetylglucosamine, ΔUA: 4-deoxy-α-L-threo-hex-4-enepyranosyluronic acid). In the degradation of CS-A and CS-C, disaccharide [ΔUA–GalNAc-OSO3H] was released (GalNAc: N-acetylgalactosamine). [ABSTRACT FROM AUTHOR]

3‐Acetamido‐5‐acetylfuran (3A5AF) is a potential platform compound for the production of nitrogen‐containing pharmaceuticals and chemicals. 3A5AF can be obtained by dehydration of chitin or its monomer, N‐acetylglucosamine (NAG). Here, we examined the use of solid catalysts for the dehydration of NAG to 3A5AF to achieve a more economical process that uses a recyclable catalyst. NAG was dehydrated using various solid catalysts in the presence of NaCl and N,N‐dimethyl acetamide as solvent at 433 K. The yield of 3A5AF with the solid catalysts decreased in the following order: Al‐exchanged montmorillonite>H‐ZSM‐5 (SiO2/Al2O3=40)>H‐montmorillonite (K‐10)>Amberlyst15>H‐ZSM‐5 (SiO2/Al2O3=300)>TiO2>γ‐Al2O3>ZrO2>SiO2 ⋅ MgO>Na‐montmorillonite. The highest yield of 3A5AF (14 %) was obtained with the Al‐exchanged montmorillonite. The montmorillonite catalysts were characterized by using inductively coupled plasma optical emission spectroscopy, energy‐dispersive X‐ray spectroscopy, N2 adsorption, Fourier‐transformed infrared spectroscopy, X‐ray diffraction, and 27Al magic‐angle spinning nuclear magnetic resonance spectroscopy (MAS‐NMR). In addition, a combined catalyst of Al‐exchanged montmorillonite and Cl− from synthetic hydrotalcite was found to be an active and recyclable solid catalyst for NAG dehydration to 3A5AF. [ABSTRACT FROM AUTHOR]

Staphylococcus epidermidis expresses glycerol phosphate wall teichoic acid (WTA), but some health care-associated methicillin-resistant S. epidermidis (HA-MRSE) clones produce a second, ribitol phosphate (RboP) WTA, resembling that of the aggressive pathogen Staphylococcus aureus. RboP-WTA promotes HA-MRSE persistence and virulence in bloodstream infections. We report here that the TarM enzyme of HA-MRSE [TarM(Se)] glycosylates RboP-WTAwith glucose, instead of N-acetylglucosamine (GlcNAc) by TarM(Sa) in S. aureus. Replacement of GlcNAc with glucose in RboP-WTA impairs HA-MRSE detection by human immunoglobulin G, which may contribute to the immune-evasion capacities of many invasive S. epidermidis. Crystal structures of complexes with uridine diphosphate glucose (UDP-glucose), and with UDP and glycosylated poly(RboP), reveal the binding mode and glycosylation mechanism of this enzyme and explain why TarM(Se) and TarM(Sa) link different sugars to poly(RboP). These structural data provide evidence that TarM(Se) is a processive WTA glycosyltransferase. Our study will support the targeted inhibition of TarM enzymes, and the development of RboP-WTA targeting vaccines and phage therapies. [ABSTRACT FROM AUTHOR]

Chitosan is a linear biopolymer composed of D-glucosamine and N-acetylglucosamine units. The percentage of D-glucosamine in the polymeric chain can vary from one sample to another and is expressed as the degree of deacetylation (DDA). Since this parameter has an impact on many properties, its determination is often critical, and potentiometric titration is a common analytical technique to measure the DDA. Cross-linking with glutaraldehyde is one of the most explored modifications of chitosan; however, the determination of the DDA for the resulting reticulated chitosan resins can be challenging. In this paper, we report a new, rapid, and efficient method to determine the DDA of glutaraldehyde-cross-linked chitosan resins via HPLC. This method relies on the use of 2,4-dinitrophenylhydrazine (DNPH) as a derivatizing agent to measure the level of reticulation of the polymer (LR) after the reticulation step. In this study, we prepare three calibration curves (with an R2 value over 0.92) for three series of reticulated polymers covering a large range of reticulation levels to demonstrate that a correlation can be established between the LR established via HPLC and the DDA obtained via titration. The polymers are derived from three different chitosan starting materials. These standard calibration curves are now used on a routine basis in our lab, and the HPLC method has allowed us to change our DDA analysis time from 20 h to 5 min. [ABSTRACT FROM AUTHOR]

Background: In the demyelinating disease multiple sclerosis (MS), chronic-active brain inflammation, remyelination failure and neurodegeneration remain major issues despite immunotherapy. While B cell depletion and blockade/sequestration of T and B cells potently reduces episodic relapses, they act peripherally to allow persistence of chronic-active brain inflammation and progressive neurological dysfunction. N-acetyglucosamine (GlcNAc) is a triple modulator of inflammation, myelination and neurodegeneration. GlcNAc promotes biosynthesis of Asn (N)-linked-glycans, which interact with galectins to co-regulate the clustering/signaling/endocytosis of multiple glycoproteins simultaneously. In mice, GlcNAc crosses the blood brain barrier to raise N-glycan branching, suppress inflammatory demyelination by T and B cells and trigger stem/progenitor cell mediated myelin repair. MS clinical severity, demyelination lesion size and neurodegeneration inversely associate with a marker of endogenous GlcNAc, while in healthy humans, age-associated increases in endogenous GlcNAc promote T cell senescence. Objectives and methods: An open label dose-escalation mechanistic trial of oral GlcNAc at 6 g (n = 18) and 12 g (n = 16) for 4 weeks was performed in MS patients on glatiramer acetate and not in relapse from March 2016 to December 2019 to assess changes in serum GlcNAc, lymphocyte N-glycosylation and inflammatory markers. Post-hoc analysis examined changes in serum neurofilament light chain (sNfL) as well as neurological disability via the Expanded Disability Status Scale (EDSS). Results: Prior to GlcNAc therapy, high serum levels of the inflammatory cytokines IFNγ, IL-17 and IL-6 associated with reduced baseline levels of a marker of endogenous serum GlcNAc. Oral GlcNAc therapy was safe, raised serum levels and modulated N-glycan branching in lymphocytes. Glatiramer acetate reduces TH1, TH17 and B cell activity as well as sNfL, yet the addition of oral GlcNAc dose-dependently lowered serum IFNγ, IL-17, IL-6 and NfL. Oral GlcANc also dose-dependently reduced serum levels of the anti-inflammatory cytokine IL-10, which is increased in the brain of MS patients. 30% of treated patients displayed confirmed improvement in neurological disability, with an average EDSS score decrease of 0.52 points. Conclusions: Oral GlcNAc inhibits inflammation and neurodegeneration markers in MS patients despite concurrent immunomodulation by glatiramer acetate. Blinded studies are required to investigate GlcNAc's potential to control residual brain inflammation, myelin repair and neurodegeneration in MS. [ABSTRACT FROM AUTHOR]

Summary: Establishment of symbiosis between plants and arbuscular mycorrhizal (AM) fungi depends on fungal chitooligosaccharides (COs) and lipo‐chitooligosaccharides (LCOs). The latter are also produced by nitrogen‐fixing rhizobia to induce nodules on leguminous roots. However, host enzymes regulating structure and levels of these signals remain largely unknown.Here, we analyzed the expression of a β‐N‐acetylhexosaminidase gene of Medicago truncatula (MtHEXO2) and biochemically characterized the enzyme. Mutant analysis was performed to study the role of MtHEXO2 during symbiosis.We found that expression of MtHEXO2 is associated with AM symbiosis and nodulation. MtHEXO2 expression in the rhizodermis was upregulated in response to applied chitotetraose, chitoheptaose, and LCOs. M. truncatula mutants deficient in symbiotic signaling did not show induction of MtHEXO2. Subcellular localization analysis indicated that MtHEXO2 is an extracellular protein. Biochemical analysis showed that recombinant MtHEXO2 does not cleave LCOs but can degrade COs into N‐acetylglucosamine (GlcNAc). Hexo2 mutants exhibited reduced colonization by AM fungi; however, nodulation was not affected in hexo2 mutants.In conclusion, we identified an enzyme, which inactivates COs and promotes the AM symbiosis. We hypothesize that GlcNAc produced by MtHEXO2 may function as a secondary symbiotic signal. [ABSTRACT FROM AUTHOR]

Members of the Vibrionaceae family are predominantly fast-growing and halophilic microorganisms that have captured the attention of researchers owing to their potential applications in rapid biotechnology. Among them, Vibrio alginolyticus FA2 is a particularly noteworthy halophilic bacterium that exhibits superior growth capability. It has the potential to serve as a biotechnological platform for sustainable and eco-friendly open fermentation with seawater. To evaluate this hypothesis, we integrated the N-acetylglucosamine (GlcNAc) pathway into V. alginolyticus FA2. Seven nag genes were knocked out to obstruct the utilization of GlcNAc, and then 16 exogenous gna1s co-expressing with EcglmS were introduced to strengthen the flux of GlcNAc pathway, respectively. To further enhance GlcNAc production, we fine-tuned promoter strength of the two genes and inactivated two genes alsS and alsD to prevent the production of acetoin. Furthermore, unsterile open fermentation was carried out using simulated seawater and a chemically defined medium, resulting in the production of 9.2 g/L GlcNAc in 14 h. This is the first report for de-novo synthesizing GlcNAc with a Vibrio strain, facilitated by an unsterile open fermentation process employing seawater as a substitute for fresh water. This development establishes a basis for production of diverse valuable chemicals using Vibrio strains and provides insights into biomanufacture. [ABSTRACT FROM AUTHOR]

Human milk oligosaccharides (HMOs) are very distinctive components in human milk and are beneficial for infant health. Lacto-N-biose I (LNB) is the core structural unit of HMOs, which could be used for the synthesis of other HMOs. In this study, an ATP-free in vitro synthetic enzymatic biosystem contained four thermostable enzymes (alpha-glucan phosphorylase from Thermococcus kodakarensis, UDP-glucose-hexose-1-phosphate uridylyltransferase from Thermotoga maritima, UDP-glucose 4-epimerase from T. maritima, lacto-N-biose phosphorylase from Clostridium thermobutyricum) were constructed for the biosynthesis of LNB from starch and Nacetylglucosamine (GlcNAc). Under the optimal conditions, 0.75 g/L and 2.23 g/L LNB was produced from 1.1 g/ L and 4.4 g/L GlcNAc and excess starch, with the molar yield of 39% and 29% based on the GlcNAc concentration, respectively, confirming the feasibility of this in vitro synthetic enzymatic biosystem for LNB synthesis and shedding light on the biosynthesis of other HMOs using LNB as the core structural unit from low cost polysaccharides. [ABSTRACT FROM AUTHOR]

Soft-template carbonized mesopores were developed for the purpose of enriching urinary exosomal glycans through organic–organic self-assembly using block copolymers and resol precursors. With a high surface area of 229 m2 g−1, a small pore size of 3.1 nm, and a significant amount of carbon that specifically interacts with oligosaccharides in glycans, this carbonized mesopore material exhibits high selectivity and low limits of detection (5 ng μL−1) towards glycans. Our analysis of complex urine samples from healthy volunteers and bladder carcinoma patients successfully profiled 48 and 56 exosomal glycans, respectively, and 16 of them were significantly changed. Moreover, one upregulated bisecting N-acetylglucosamine (GlcNAc)-type glycan with core fucose, two upregulated and two downregulated terminal-sialylated glycans were revealed to be linked to bladder carcinoma. This approach is of significant importance for understanding diseases that arise from protein glycosylation mutations, and it may contribute to the development of novel diagnostic and therapeutic strategies for bladder carcinoma. [ABSTRACT FROM AUTHOR]

N-acetylglucosamine kinase (NAGK) has been identified as an anchor protein that facilitates neurodevelopment with its non-canonical structural role. Similarly, small nuclear ribonucleoprotein polypeptide N (SNRPN) regulates neurodevelopment and cognitive ability. In our previous study, we revealed the interaction between NAGK and SNRPN in the neuron. However, the precise role in neurodevelopment is elusive. In this study, we investigate the role of NAGK and SNRPN in the axodendritic development of neurons. NAGK and SNRPN interaction is significantly increased in neurons at the crucial stages of neurodevelopment. Furthermore, overexpression of the NAGK and SNRPN proteins increases axodendritic branching and neuronal complexity, whereas the knockdown inhibits neurodevelopment. We also observe the interaction of NAGK and SNRPN with the dynein light-chain roadblock type 1 (DYNLRB1) protein variably during neurodevelopment, revealing the microtubule-associated delivery of the complex. Interestingly, NAGK and SNRPN proteins rescued impaired axodendritic development in an SNRPN depletion model of Prader–Willi syndrome (PWS) patient-derived induced pluripotent stem cell neurons. Taken together, these findings are crucial in developing therapeutic approaches for neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Adipose tissues accumulate excess energy as fat and heavily influence metabolic homeostasis. O-linked N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation), which involves the addition of N-acetylglucosamine to proteins by O-GlcNAc transferase (Ogt), modulates multiple cellular processes. However, little is known about the role of O-GlcNAcylation in adipose tissues during body weight gain due to overnutrition. Here, we report on O-GlcNAcylation in mice with high-fat diet (HFD)-induced obesity. Mice with knockout of Ogt in adipose tissue achieved using adiponectin promoter-driven Cre recombinase (Ogt-FKO) gained less body weight than control mice under HFD. Surprisingly, Ogt-FKO mice exhibited glucose intolerance and insulin resistance, despite their reduced body weight gain, as well as decreased expression of de novo lipogenesis genes and increased expression of inflammatory genes, resulting in fibrosis at 24 weeks of age. Primary cultured adipocytes derived from Ogt-FKO mice showed decreased lipid accumulation. Both primary cultured adipocytes and 3T3-L1 adipocytes treated with OGT inhibitor showed increased secretion of free fatty acids. Medium derived from these adipocytes stimulated inflammatory genes in RAW 264.7 macrophages, suggesting that cell-to-cell communication via free fatty acids might be a cause of adipose inflammation in Ogt-FKO mice. In conclusion, O-GlcNAcylation is important for healthy adipose expansion in mice. Glucose flux into adipose tissues may be a signal to store excess energy as fat. [ABSTRACT FROM AUTHOR]

Chitin, the world's second most abundant biopolymer after cellulose, is composed of β-1,4-N-acetylglucosamine (GlcNAc) residues. It is the key structural component of many organisms, including crustaceans, mollusks, marine invertebrates, algae, fungi, insects, and nematodes. There has been a significant increase in the generation of chitinous waste from seafood businesses, resulting in a big amount of scrap. Although several organisms, such as plants, crustaceans, insects, nematodes, and animals, produce chitinases, microorganisms are promising candidates and a sustainable option that mediates chitin degradation. Fungi are the dominant group of chitinase producers among microorganisms. In fungi, chitinases are involved in morphogenesis, cell division, autolysis, chitin acquisition for nutritional purposes, and mycoparasitism. Many efficient chitinolytic fungi with potential applications have been identified in a variety of environments, including soil, water, marine wastes, and plants. The current review highlights the key sources of chitinolytic fungi and the characterization of fungal chitinases. It also discusses the applications of fungal chitinases and the cloning of fungal chitinase genes. [ABSTRACT FROM AUTHOR]

Background: Changes in protein glycosylation are widely observed in tumor cells. N-glycan branching through adding β1,6-linked N-acetylglucosamine (β1,6-GlcNAc) to an α1,6-linked mannose, which is catalyzed by the N-acetylglucosaminyltransferase V (MGAT5 or GnT-V), is one of the most frequently observed tumor-associated glycan structure formed. Increased levels of this branching structure play a pro-tumoral role in various ways, for example, through the stabilization of growth factor receptors, the destabilization of intercellular adhesion, or the acquisition of a migratory phenotype. Conclusion: In this review, we provide an updated and comprehensive summary of the physiological and pathophysiological roles of MGAT5 and β1,6-GlcNAc branched N-glycans, including their regulatory mechanisms. Specific emphasis is given to the role of MGAT5 and β1,6-GlcNAc branched N-glycans in cellular mechanisms that contribute to the development and progression of solid tumors. We also provide insight into possible future clinical implications, such as the use of MGAT5 as a prognostic biomarker. [ABSTRACT FROM AUTHOR]

This study focused on the CDMBI-mediated synthesis of GlcNAc oxazoline. Through our research, we uncovered and characterized a previously unreported by-product. We proposed a mechanism for its formation and developed a new strategy to prevent its occurrence. These findings represent an important step towards gaining a deeper understanding of this reaction and may provide a new avenue for optimizing the oxazoline synthesis. [ABSTRACT FROM AUTHOR]

The conversion of chitin enables the utilisation of naturally-fixed nitrogen in addition to carbon toward establishing a sustainable carbon and nitrogen cycle. Chitin is an abundant biomass, 100 Gt per year, but most chitin-containing waste is discarded due to its recalcitrant properties. This feature article summarises the challenges and our work on chitin conversion to N-acetylglucosamine and oligomers with fascinating applications. Afterwards, we introduce recent progress on the chemical transformation of N-acetylglucosamine, followed by a discussion of future perspectives based on current status and findings. [ABSTRACT FROM AUTHOR]

Parageobacillus thermoglucosidasius is a thermophilic bacterium characterized by rapid growth, low nutrient requirements, and amenability to genetic manipulation. These characteristics along with its ability to ferment a broad range of carbohydrates make P. thermoglucosidasius a potential workhorse in whole-cell biocatalysis. The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) catalyzes the transport and phosphorylation of carbohydrates and sugar derivatives in bacteria, making it important for their physiological characterization. In this study, the role of PTS elements on the catabolism of PTS and non-PTS substrates was investigated for P. thermoglucosidasius DSM 2542. Knockout of the common enzyme I, part of all PTSs, showed that arbutin, cellobiose, fructose, glucose, glycerol, mannitol, mannose, N-acetylglucosamine, N-acetylmuramic acid, sorbitol, salicin, sucrose, and trehalose were PTS-dependent on translocation and coupled to phosphorylation. The role of each putative PTS was investigated and six PTS-deletion variants could not grow on arbutin, mannitol, N-acetylglucosamine, sorbitol, and trehalose as the main carbon source, or showed diminished growth on N-acetylmuramic acid. We concluded that PTS is a pivotal factor in the sugar metabolism of P. thermoglucosidasius and established six PTS variants important for the translocation of specific carbohydrates. This study lays the groundwork for engineering efforts with P. thermoglucosidasius towards efficient utilization of diverse carbon substrates for whole-cell biocatalysis. [ABSTRACT FROM AUTHOR]

Background: The Trichosporonaceae family comprises a large number of basidiomycetes widely distributed in nature. Some of its members, especially Trichosporon asahii, have the ability to cause human infections. This ability is related to a series of virulence factors, which include lytic enzymes production, biofilm formation, resistance to oxidising agents, melanin and glucuronoxylomannan in the cell wall, metabolic plasticity and phenotypic switching. The last two are poorly addressed within human pathogenic Trichosporonaceae. Objective: These factors were herein studied to contribute with the knowledge of these emerging pathogens and to uncover mechanisms that would explain the higher frequency of T. asahii in human infections. Methods: We included 79 clinical isolates phenotypically identified as Trichosporon spp. and performed their molecular identification. Lactate and N‐acetyl glucosamine were the carbon sources of metabolic plasticity studies. Morphologically altered colonies after subcultures and incubation at 37°C indicated phenotypic switching. Results and Conclusion: The predominant species was T. asahii (n = 65), followed by Trichosporon inkin (n = 4), Apiotrichum montevideense (n = 3), Trichosporon japonicum (n = 2), Trichosporon faecale (n = 2), Cutaneotrichosporon debeurmannianum (n = 1), Trichosporon ovoides (n = 1) and Cutaneotrichosporon arboriforme (n = 1). T. asahii isolates had statistically higher growth on lactate and N‐acetylglucosamine and on glucose during the first 72 h of culture. T. asahii, T. inkin and T. japonicum isolates were able to perform phenotypic switching. These results expand the virulence knowledge of Trichosporonaceae members and point for a role for metabolic plasticity and phenotypic switching on the trichosporonosis pathogenesis. [ABSTRACT FROM AUTHOR]

Summary: Various methods can be applied for the purification of oligosaccharides such as galactooligosaccharides (GOS), fructooligosaccharides (FOS) or heterooligosaccharides (HOS). In the present work, two methods (microbial treatment and nanofiltration (NF)) for purification of an enzymatically synthesised HOS mixture were investigated and compared. With the microbial fermentation, the monosaccharides including glucose, galactose and N‐acetylglucosamine (GlcNAc) could be completely removed whereas lactose was only partially consumed. However, ethanol and acetate were produced as the result of microbial fermentation, which may be considered as an additional problem. The purification yield and the purity of the resulting HOS mixture were 79.4 and 59.3%, respectively. Applying NF with a molecular weight cut‐off (MWCO) of 400 Dalton, the yield and the purity of the HOS mixture were 78.0 and 54.9%, respectively. The resulting product mixture (retentate) obtained after nanofiltration is significantly more concentrated than that of fermentation (257 vs. 106 g L−1 of HOS), however, GlcNAc could not be removed. [ABSTRACT FROM AUTHOR]

SUMMARY: As an essential enzyme in the uridine diphosphate (UDP)‐GlcNAc biosynthesis pathway, the significant role of N‐acetylglucosamine phosphate mutase (AGM) remains unknown in plants. In the present study, a functional plant AGM (AtAGM) was identified from Arabidopsis thaliana. AtAGM catalyzes the isomerization of GlcNAc‐1‐P and GlcNAc‐6‐P, and has broad catalytic activity on different phosphohexoses. UDP‐GlcNAc contents were significantly decreased in AtAGM T‐DNA insertional mutants, which caused temperature‐dependent growth defects in seedlings and vigorous growth in adult plants. Further analysis revealed that protein O‐GlcNAcylation but not N‐glycosylation was dramatically impaired in Atagm mutants due to UDP‐GlcNAc shortage. Combined with the results from O‐GlcNAcylation or N‐glycosylation deficient mutants, and O‐GlcNAcase inhibitor all suggested that protein O‐GlcNAcylation impairment mainly leads to the phenotypic variations of Atagm plants. In conclusion, based on the essential role in UDP‐GlcNAc biosynthesis, AtAGM is important for plant growth mainly via protein O‐GlcNAcylation‐level regulation. [ABSTRACT FROM AUTHOR]

O-GlcNAc transferase (OGT) is an essential glycosylating enzyme that catalyzes the addition of N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. The enzyme glycosylates a broad range of peptide sequences and the prediction of glycosylation sites has proven challenging. The lack of an experimentally verified set of polypeptide sequences that are not glycosylated by OGT has made prediction of legitimate glycosylation sites more difficult. Here, we tested a number of intrinsically disordered protein regions as substrates of OGT to establish a set of sequences that are not glycosylated by OGT. The negative data set suggests an amino acid compositional bias for OGT targets. This compositional bias was validated by modifying the amino acid composition of the protein fused in sarcoma (FUS) to enhance glycosylation. NMR experiments demonstrate that the tetratricopeptide repeat region of OGT can bind FUS and that glycosylation-promoting mutations enhance binding. These results provide evidence that the tetratricopeptide repeat region recognizes disordered segments of substrates with particular compositions to promote glycosylation, providing insight into the broad specificity of OGT. [ABSTRACT FROM AUTHOR]

Candida albicans , a significant commensal fungus in the human gut, causes a wide spectrum of opportunistic infections. In a recent study, Yang et al. revealed the importance of a host-associated gut signal, GlcNAc, in C. albicans and described its significant role towards achieving a successful commensal–virulence trade-off program in the human body. [ABSTRACT FROM AUTHOR]

N-acetyl-D-glucosamine (GlcNAc) is a major component of bacterial cell walls. Many organisms recycle GlcNAc from the cell wall or metabolize environmental GlcNAc. The first step in GlcNAc metabolism is phosphorylation to GlcNAc-6-phosphate. In bacteria, the ROK family kinase N-acetylglucosamine kinase (NagK) performs this activity. Although ROK kinases have been studied extensively, no ternary complex showing the two substrates has yet been observed. Here, we solved the structure of NagK from the human pathogen Plesiomonas shigelloides in complex with GlcNAc and the ATP analog AMP-PNP. Surprisingly, PsNagK showed distinct conformational changes associated with the binding of each substrate. Consistent with this, the enzyme showed a sequential random enzyme mechanism. This indicates that the enzyme acts as a coordinated unit responding to each interaction. Our molecular dynamics modeling of catalytic ion binding confirmed the location of the essential catalytic metal. Additionally, site-directed mutagenesis confirmed the catalytic base and that the metal-coordinating residue is essential. Together, this study provides the most comprehensive insight into the activity of a ROK kinase. [ABSTRACT FROM AUTHOR]

Microbial production of hyaluronic acid (HA) is an area of research that has been gaining attention in recent years due to the increasing demand for this biopolymer for several industrial applications. Hyaluronic acid is a linear, non-sulfated glycosaminoglycan that is widely distributed in nature and is mainly composed of repeating units of N-acetylglucosamine and glucuronic acid. It has a wide and unique range of properties such as viscoelasticity, lubrication, and hydration, which makes it an attractive material for several industrial applications such as cosmetics, pharmaceuticals, and medical devices. This review presents and discusses the available fermentation strategies to produce hyaluronic acid. [ABSTRACT FROM AUTHOR]

Nanomedicine can represent a new strategy to treat several types of diseases such as those with inflammatory aetiology. Through this strategy, it is possible to obtain nanoparticles with controlled shape, size, and eventually surface charge. Moreover, the use of molecules in nanoform may allow more effective delivery into the diseased cells and tissues, reducing toxicity and side effects of the used compounds. The aim of the present manuscript was the evaluation of the effects of N-acetylglucosamine in nanoform (GlcNAc NP) in an in vitro model of osteoarthritis (OA). Human primary chondrocytes were treated with Tumor Necrosis Factor (TNF)-α to simulate a low-grade inflammation and then treated with both GlcNAc and GlcNAc NP, in order to find the lowest concentrations able to counteract the inflammatory state of the cells and ensure a chondroprotective action. The findings showed that GlcNAc NP was able to decrease the pro-inflammatory mediators, IL-6 and IL-8, which are among the main effectors of inflammation; moreover, the nanoparticles downregulated the production of metalloprotease enzymes. GlcNAc NP was effective at a very low concentration compared to GlcNAc in its native form. Furthermore, GlcNAc NP stimulated an increase in collagen type II synthesis. In conclusion, the GlcNAc in nanoform showed better performance than GlcNAc, at concentrations lower than those reached in the joints after oral administration to patients of 1.5 g/die of glucosamine. [ABSTRACT FROM AUTHOR]

Background: Filamentous fungi are extensively exploited as important enzyme producers due to the superior secretory capability. However, the complexity of their secretomes greatly impairs the titer and purity of heterologous enzymes. Meanwhile, high-efficient evaluation and production of bulk enzymes, such as biomass-degrading enzymes, necessitate constructing powerful expression systems for bio-refinery applications. Results: A novel sucrose-inducible expression system based on the host strain Aspergillus niger ATCC 20611 and the β-fructofuranosidase promoter (PfopA) was constructed. A. niger ATCC 20611 preferentially utilized sucrose for rapid growth and β-fructofuranosidase production. Its secretory background was relatively clean because β-fructofuranosidase, the key enzyme responsible for sucrose utilization, was essentially not secreted into the medium and the extracellular protease activity was low. Furthermore, the PfopA promoter showed a sucrose concentration-dependent induction pattern and was not subject to glucose repression. Moreover, the strength of PfopA was 7.68-fold higher than that of the commonly used glyceraldehyde-3-phosphate dehydrogenase promoter (PgpdA) with enhanced green fluorescence protein (EGFP) as a reporter. Thus, A. niger ATCC 20611 coupled with the PfopA promoter was used as an expression system to express a β-glucosidase gene (bgla) from A. niger C112, allowing the production of β-glucosidase at a titer of 17.84 U/mL. The crude β-glucosidase preparation could remarkably improve glucose yield in the saccharification of pretreated corncob residues when added to the cellulase mixture of Trichoderma reesei QM9414. The efficacy of this expression system was further demonstrated by co-expressing the T. reesei-derived chitinase Chi46 and β-N-acetylglucosaminidase Nag1 to obtain an efficient chitin-degrading enzyme cocktail, which could achieve the production of N-acetyl-D-glucosamine from colloidal chitin with a conversion ratio of 91.83%. Besides, the purity of the above-secreted biomass-degrading enzymes in the crude culture supernatant was over 86%. Conclusions: This PfopA-driven expression system expands the genetic toolbox of A. niger and broadens the application field of the traditional fructo-oligosaccharides-producing strain A. niger ATCC 20611, advancing it to become a high-performing enzyme-producing cell factory. In particular, the sucrose-inducible expression system possessed the capacity to produce biomass-degrading enzymes at a high level and evade endogenous protein interference, providing a potential purification-free enzyme production platform for bio-refinery applications. [ABSTRACT FROM AUTHOR]

The enzyme catalysing the removal of N‐linked glycans from misfolded glycoproteins in the cytosol is an evolutionary well‐conserved glycanase called Peptide:N‐glycanase (PNGase; NGLY1 in humans). NGLY1 hydrolyses the amide bond between an Asn and the proximal N‐acetylglucosamine (GlcNAc) of the attached N‐glycan, thereby converting that particular Asn to Asp. Loss of NGLY1 due to heterozygous‐inactivating mutations cause a rare congenital disorder with a multisystemic clinical phenotype. Since the first case report in 2012, extensive research aiming at understanding the pathophysiology of the disease, revealed several crucial biological functions and pathways that involve NGLY1. Here, we highlight the research progress of the past decade. A special emphasis is given to the important role that NGLY1 plays in the transactivation of the transcription factor (NFE2L1; NRF1) that regulates many processes of cellular homeostasis, including proteasome bounce back and oxidative stress response. [ABSTRACT FROM AUTHOR]

Earlier studies have shown that the outer layers of the conidial and mycelial cell walls of Aspergillus fumigatus are different. In this work, we analyzed the polysaccharidome of the resting conidial cell wall and observed major differences within the mycelium cell wall. Mainly, the conidia cell wall was characterized by (i) a smaller amount of α-(1,3)-glucan and chitin; (ii) a larger amount of β-(1,3)-glucan, which was divided into alkali-insoluble and water-soluble fractions, and (iii) the existence of a specific mannan with side chains containing galactopyranose, glucose, and N-acetylglucosamine residues. An analysis of A. fumigatus cell wall gene mutants suggested that members of the fungal GH-72 transglycosylase family play a crucial role in the conidia cell wall β-(1,3)-glucan organization and that α-(1,6)-mannosyltransferases of GT-32 and GT-62 families are essential to the polymerization of the conidium-associated cell wall mannan. This specific mannan and the well-known galactomannan follow two independent biosynthetic pathways. [ABSTRACT FROM AUTHOR]

Nisin resistance development is one of food safety challenges posed by Listeria monocytogenes , an important foodborne pathogen that causes human listeriosis. The GtcA flippase enzyme is functionally crucial in two separate pathways that glycosylate cell envelope wall teichoic acids (WTA) with N -acetylglucosamine (NAG) and lipoteichoic acids (LTA) with galactose, respectively. This study investigated phenotypic roles and molecular mechanisms underlying GtcA involvement in L. monocytogenes nisin resistance. A GtcA A65V mutation was linked with increased nisin resistance in a food processing environment associated L. monocytogenes strain. Examination of nisin stress survival and growth phenotypes among L. monocytogenes gtcA mutants in different genetic backgrounds showed that GtcA function promoted sensitivity and loss of its function through genetic deletion (Δ gtcA) and a natural GtcA A65V mutation increased nisin resistance. Individual contributions of GtcA WTA NAG and LTA galactose glycosylation functions to nisin resistance modulation were examined through nisin sensitivity analysis of genetic deletion mutants and L. monocytogenes strains complemented using functionally altered GtcA mutants. This revealed WTA NAG glycosylation to be the main functional mechanism that determines GtcA dependent nisin phenotypic sensitization. An examination for mechanisms underlying GtcA involvement in nisin sensitivity revealed that the loss of GtcA function induces changes in the cell envelope carbohydrate composition profiles reducing cell surface hydrophobicity. Overall, our results showed that cell envelope WTA NAG glycosylation promotes nisin susceptibility through facilitation of hydrophobic interactions between nisin and the Listeria cell envelope. There may be practical implications from our observations since nisin resistance could be gained in food associated L. monocytogenes strains that develop phage resistance through acquisition of mutations in genes that cause loss of cell envelope WTA NAG modifications. • GtcA induced cell envelope glycosylation sensitizes L. monocytogenes to nisin. • Loss of GtcA function increases L. monocytogenes nisin resistance. • GtcA impact on nisin sensitivity largely depends on cell envelope WTA NAG glycosylation. • WTA NAG devoid cell envelope reduce L. monocytogenes cell surface hydrophobicity. • Nisin resistance induced through reduced hydrophobicity interaction with Listeria cells. [ABSTRACT FROM AUTHOR]

MurK is a MurNAc- and GlcNAc-specific amino sugar kinase, phosphorylates MurNAc and GlcNAc at the 6-hydroxyl group in an ATP-dependent manner, and contributes to the recovery of both amino sugars during the cell wall turnover in Clostridium acetobutylicum. Herein, we determined the crystal structures of MurK in complex with MurNAc, GlcNAc, and glucose, respectively. MurK represents the V-shaped fold, which is divided into a small N-terminal domain and a large C-terminal domain. The catalytic pocket is located within the deep cavity between the two domains of the MurK monomer. We mapped the significant enzyme-substrate interactions, identified key residues involved in the catalytic activity of MurK, and found that residues Asp77 and Arg78 from the β4-α2-loop confer structural flexibilities to specifically accommodate GlcNAc and MurNAc, respectively. Moreover, structural comparison revealed that MurK adopts closed-active conformation induced by the N -acetyl moiety from GlcNAc/MurNAc, rather than closed-inactive conformation induced by glucose, to carry out its catalytic reaction. Taken together, our study provides structural and functional insights into the molecular mechanism of MurK for the phosphorylation of both MurNAc and GlcNAc, sugar substrate specificity, and conformational changes upon sugar substrate binding. [ABSTRACT FROM AUTHOR]

Background: The level of linked N-acetylglucosamine (O-GlcNAc) has been proved to be a sensor of cell state, but its relationship with hyperoxia-induced alveolar type 2 epithelial cells injure and bronchopulmonary dysplasia (BPD) has not been clarified. In this study, we evaluated if these effects ultimately led to functional damage in hyperoxia-induced alveolar cells. Methods: We treated RLE-6TN cells at 85% hyperoxia for 0, 24 and 48 h with Thiamet G (TG), an OGA inhibitor; OSMI-1 (OS), an OGT inhibitor; or with UDP-GlcNAc, which is involved in synthesis of O-GlcNAc as a donor. The metabolic rerouting, cell viability and apoptosis resulting from the changes in O-GlcNAc glycosyltransferase levels were evaluated in RLE-6TN cells after hyperoxia exposure. We constructed rat Park2 overexpression and knockdown plasmmids for in vitro verification and Co-immunoprecipitation corroborated the binding of Parkin and O-GlcNAc. Finally, we assessed morphological detection in neonatal BPD rats with TG and OS treatment. Results: We found a decrease in O-GlcNAc content and levels of its metabolic enzymes in RLE-6TN cells under hyperoxia. However, the inhibition of OGT function with OSMI-1 ameliorated hyperoxia-induced lung epithelial cell injury, enhanced cell metabolism and viability, reduced apoptosis, and accelerated the cell proliferation. Mitochondrial homeostasis was affected by O-GlcNAc and regulated Parkin. Conclusion: The results revealed that the decreased O-GlcNAc levels and increased O-GlcNAcylation of Parkin might cause hyperoxia-induced alveolar type II cells injurys. [ABSTRACT FROM AUTHOR]

A late-stage diversification strategy for synthesizing ynamides has been developed. This strategy was enabled by the copper-catalyzed direct electrophilic diynylation of sulfonamides with a novel triisopropylsilyl diynyl benziodoxolone, deprotection, and the late-stage chemoselective copper-catalyzed azide–alkyne cycloaddition sequence, which yields various complex molecule-derived ynamides with pyrene, amino acid, nucleoside, and N-acetylglucosamine as substituents. [ABSTRACT FROM AUTHOR]