Targeted Protein O-GlcNAcylation Using Bifunctional Small Molecules.

Protein O-linked ß-N-acetylglucosamine modification (O-GlcNAcylation) plays a crucial role in regulating essential cellular processes. The disruption of the homeostasis of O-GlcNAcylation has been linked to various human diseases, including cancer, diabetes, and neurodegeneration. However, there are limited chemical tools for protein- and site-specific O-GlcNAc modification, rendering the precise study of the O-GlcNAcylation challenging. To address this, we have developed heterobifunctional small molecules, named O-GlcNAcylation TArgeting Chimeras (OGTACs), which enable protein-specific O-GlcNAcylation in living cells. OGTACs promote O-GlcNAcylation of proteins such as BRD4, CK2α, and EZH2 in cellulo by recruiting FKBP12F36V-fused O-GlcNAc transferase (OGT), with temporal, magnitude, and reversible control. Overall, the OGTACs represent a promising approach for inducing protein-specific O-GlcNAcylation, thus enabling functional dissection and offering new directions for O-GlcNAc-targeting therapeutic development.

Efficient Escorting Strategy for Aggregation-Prone Notch EGF Repeats with Sparcl1.

Epidermal growth factor (EGF) repeats are present in various proteins and form well-defined structures with three disulfide bonds. One representative protein is the Notch receptor. Each EGF repeat contains unique atypical O-linked glycans, such as O-linked N-acetylglucosamine (O-GlcNAc). To generate a monoclonal antibody against the O-GlcNAc moiety in mouse Notch1, we expressed the recombinant C-terminal His6-tagged Notch1 EGF14-15 protein in HEK293T cells to prepare the immunogen. Most of the proteins were not secreted and showed higher molecular weight ladders in the cell lysate, suggesting protein aggregation. To overcome this issue, we fused Sparcl1 as an extracellular escorting tag to the N-terminus of Notch1 EGF14-15. The fusion protein was efficiently secreted extracellularly without protein aggregates in the lysates. Following PreScission protease treatment, Notch1 EGF14-15 was efficiently released from the escorting tag. Notch1 EGF14-15 prepared using this method was indeed O-GlcNAcylated. The optimal length of the escorting tag was determined by generating deletion mutants to improve the extracellular secretion of EGF14-15. Hence, a large amount of EGF14-15 was successfully prepared from the culture supernatant of HEK293T cells, which were otherwise prone to aggregation.

O-GlcNAc regulates anti-fibrotic genes in lung fibroblasts through EZH2.

Epigenetic modifications are involved in fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), and contribute to the silencing of anti-fibrotic genes. H3K27me3, a key repressive histone mark, is catalysed by the methyltransferase enhancer of Zeste homologue 2 (EZH2), which is regulated by the post-translational modification, O-linked N-Acetylglucosamine (O-GlcNAc). In this study, we explored the effects of O-GlcNAc and EZH2 on the expression of antifibrotic genes, cyclooxygenase-2 (Cox2) and Heme Oxygenase (Homx1). The expression of Cox2 and Hmox1 was examined in primary IPF or non-IPF lung fibroblasts with or without EZH2 inhibitor EZP6438, O-GlcNAc transferase (OGT) inhibitor (OSMI-1) or O-GlcNAcase (OGA) inhibitor (thiamet G). Non-IPF cells were also subjected to TGF-ß1 with or without OGT inhibition. The reduced expression of Cox2 and Hmox1 in IPF lung fibroblasts is restored by OGT inhibition. In non-IPF fibroblasts, TGF-ß1 treatment reduces Cox2 and Hmox1 expression, which was restored by OGT inhibition. ChIP assays demonstrated that the association of H3K27me3 is reduced at the Cox2 and Hmox1 promoter regions following OGT or EZH2 inhibition. EZH2 levels and stability were decreased by reducing O-GlcNAc. Our study provided a novel mechanism of O-GlcNAc modification in regulating anti-fibrotic genes in lung fibroblasts and in the pathogenesis of IPF.

The O-GlcNAc Modification of Recombinant Tau Protein and Characterization of the O-GlcNAc Pattern for Functional Study.

The neuronal microtubule-associated tau protein is characterized in vivo by a large number of post-translational modifications along the entire primary sequence that modulates its function. The primary modification of tau is phosphorylation of serine/threonine or tyrosine residues that is involved in the regulation of microtubule binding and polymerization. In neurodegenerative disorders referred to as tauopathies including Alzheimer's disease, tau is abnormally hyperphosphorylated and forms fibrillar inclusions in neurons progressing throughout different brain area during the course of the disease. The O-ß-linked N-acetylglucosamine (O-GlcNAc) is another reversible post-translational modification of serine/threonine residues that is installed and removed by the unique O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA), respectively. This modification was described as a potential modulator of tau phosphorylation and functions in the physiopathology. Moreover, reducing protein O-GlcNAc levels in the brain upon treatment of tauopathy mouse models with an OGA inhibitor reveals a beneficial effect on tau pathology and neurodegeneration. However, whether the role of tau O-GlcNAcylation is responsible of the protective effect against tau toxicity remains to be determined. The production of O-GlcNAc modified recombinant tau protein is a valuable tool for the investigations of the impact of O-GlcNAcylation on tau functions, modulation of interactions with partners and crosstalk with other post-translational modifications, including but not restricted to phosphorylation. We describe here the in vitro O-GlcNAcylation of tau with recombinant OGT for which we provide an expression and purification protocol. The use of the O-GlcNAc tau protein in functional studies requires the analytical characterization of the O-GlcNAc pattern. Here, we describe a method for the O-GlcNAc modification of tau protein with recombinant OGT and the analytical characterization of the resulting O-GlcNAc pattern by a combination of methods for the overall characterization of tau O-GlcNAcylation by chemoenzymatic labeling and mass spectrometry, as well as the quantitative, site-specific pattern by NMR spectroscopy.

Bacterial Enzyme Assay for Mucin Glycan Degradation.

Bacterial sialidase and sulfoglycosidase may act on the acidic modifications of mucin O-glycans, producing sialic acid and 6-sulfated N-acetylglucosamine, respectively. Assays for these enzymes, using mucin as a substrate, are enabled by the detection and/or quantification of the free monosaccharides that are released by these enzymes. This chapter describes enzyme reactions with mucin, detection by thin-layer chromatography of sialic acid, and quantification of 6-sulfated N-acetylglucosamine by liquid chromatography-tandem mass spectrometry.

The role of O-GlcNAcylation in RNA polymerase II transcription.

Eukaryotic RNA polymerase II (RNAPII) is responsible for the transcription of the protein-coding genes in the cell. Enormous progress has been made in discovering the protein activities that are required for transcription to occur, but the effects of post-translational modifications (PTMs) on RNAPII transcriptional regulation are much less understood. Most of our understanding relates to the cyclin-dependent kinases (CDKs), which appear to act relatively early in transcription. However, it is becoming apparent that other PTMs play a crucial role in the transcriptional cycle, and it is doubtful that any sort of complete understanding of this regulation is attainable without understanding the spectra of PTMs that occur on the transcriptional machinery. Among these is O-GlcNAcylation. Recent experiments have shown that the O-GlcNAc PTM likely has a prominent role in transcription. This review will cover the role of the O-GlcNAcylation in RNAPII transcription during initiation, pausing, and elongation, which will hopefully be of interest to both O-GlcNAc and RNAPII transcription researchers.

Nitrogen assimilation by E. coli in the mammalian intestine.

Nitrogen is an essential element for all living organisms, including Escherichia coli. Potential nitrogen sources are abundant in the intestine, but knowledge of those used specifically by E. coli to colonize remains limited. Here, we sought to determine the specific nitrogen sources used by E. coli to colonize the streptomycin-treated mouse intestine. We began by investigating whether nitrogen is limiting in the intestine. The NtrBC two-component system upregulates approximately 100 genes in response to nitrogen limitation. We showed that NtrBC is crucial for E. coli colonization, although most genes of the NtrBC regulon are not induced, which indicates that nitrogen is not limiting in the intestine. RNA-seq identified upregulated genes in colonized E. coli involved in transport and catabolism of seven amino acids, dipeptides and tripeptides, purines, pyrimidines, urea, and ethanolamine. Competitive colonization experiments revealed that L-serine, N-acetylneuraminic acid, N-acetylglucosamine, and di- and tripeptides serve as nitrogen sources for E. coli in the intestine. Furthermore, the colonization defect of a L-serine deaminase mutant was rescued by excess nitrogen in the drinking water but not by an excess of carbon and energy, demonstrating that L-serine serves primarily as a nitrogen source. Similar rescue experiments showed that N-acetylneuraminic acid serves as both a carbon and nitrogen source. To a minor extent, aspartate and ammonia also serve as nitrogen sources. Overall, these findings demonstrate that E. coli utilizes multiple nitrogen sources for successful colonization of the mouse intestine, the most important of which is L-serine. IMPORTANCE: While much is known about the carbon and energy sources that are used by E. coli to colonize the mammalian intestine, very little is known about the sources of nitrogen. Interrogation of colonized E. coli by RNA-seq revealed that nitrogen is not limiting, indicating an abundance of nitrogen sources in the intestine. Pathways for assimilation of nitrogen from several amino acids, dipeptides and tripeptides, purines, pyrimidines, urea, and ethanolamine were induced in mice. Competitive colonization assays confirmed that mutants lacking catabolic pathways for L-serine, N-acetylneuraminic acid, N-acetylglucosamine, and di- and tripeptides had colonization defects. Rescue experiments in mice showed that L-serine serves primarily as a nitrogen source, whereas N-acetylneuraminic acid provides both carbon and nitrogen. Of the many nitrogen assimilation mutants tested, the largest colonization defect was for an L-serine deaminase mutant, which demonstrates L-serine is the most important nitrogen source for colonized E. coli.

Functional significance of O-linked N-acetylglucosamine protein modification in regulating autophagy.

Autophagy is a core molecular pathway that preserves cellular and organismal homeostasis. Being susceptible to nutrient availability and stress, eukaryotic cells recycle or degrade internal components via membrane transport pathways to provide sustainable biological molecules and energy sources. The dysregulation of this highly conserved physiological process has been strongly linked to human disease. Post-translational modification, a mechanism that regulates protein function, plays a crucial role in autophagy regulation. O-linked N-acetylglucosamine protein modification (O-GlcNAcylation), a monosaccharide post-translational modification of intracellular proteins, is essential in nutritional and stress regulatory mechanisms. O-GlcNAcylation has emerged as an essential regulatory mechanism of autophagy. It regulates autophagy throughout its lifetime by targeting the core components of the autophagy regulatory network. This review provides an overview of the O-GlcNAcylation of autophagy-associated proteins and their regulation and function in the autophagy pathway. Therefore, this article may contribute to further understanding of the role of O-GlcNAc-regulated autophagy and provide new perspectives for the treatment of human diseases.

Characterization of galactosyltransferase and sialyltransferase genes mediating the elongation of the extracellular O-GlcNAc glycans.

O-GlcNAc is a unique post-translational modification found in cytoplasmic, nuclear, and mitochondrial proteins. In a limited number of extracellular proteins, O-GlcNAc modifications occur through the action of EOGT, which specifically modifies subsets of epidermal growth factor-like (EGF) domain-containing proteins such as Notch receptors. The abnormalities due to EOGT mutations in mice and humans and the increased EOGT expression in several cancers signify the importance of EOGT pathophysiology and extracellular O-GlcNAc. Unlike intracellular O-GlcNAc monosaccharides, extracellular O-GlcNAc extends to form elongated glycan structures. However, the enzymes involved in the O-GlcNAc glycan extension have not yet been reported. In our study, we comprehensively screened potential galactosyltransferase and sialyltransferase genes related to the canonical O-GlcNAc glycan pathway and revealed the essential roles of B4GALT1 and ST3GAL4 in O-GlcNAc glycan elongation in human HEK293 cells. These findings were confirmed by sequential glycosylation of Drosophila EGF20 in vitro by EOGT, ß4GalT-1, and ST3Gal-IV. Thus, the findings from our study throw light on the specific glycosyltransferases that mediate O-GlcNAc glycan elongation in human HEK293 cells.

Caffeine-induced protein kinase A activation restores cognitive deficits induced by sleep deprivation by regulating O-GlcNAc cycling in adult zebrafish.

Sleep deprivation (SD) is widely acknowledged as a significant risk factor for cognitive impairment. In this study, intraperitoneal caffeine administration significantly ameliorated the learning and memory (L/M) deficits induced by SD and reduced aggressive behaviors in adult zebrafish. SD led to a reduction in protein kinase A (PKA) phosphorylation, phosphorylated-cAMP response element-binding protein (p-CREB), and c-Fos expression in zebrafish brain. Notably, these alterations were effectively reversed by caffeine. In addition, caffeine mitigated neuroinflammation induced by SD, as evident from suppression of the SD-mediated increase in glial fibrillary acidic protein (GFAP) and nuclear factor-κB (NF-κB) activation. Caffeine restored normal O-GlcNAcylation and O-GlcNAc transferase (OGT) levels while reversing the increased expression of O-GlcNAcase (OGA) in zebrafish brain after SD. Intriguingly, rolipram, a selective phosphodiesterase 4 (PDE4) inhibitor, effectively mitigated cognitive deficits, restored p-CREB and c-Fos levels, and attenuated the increase in GFAP in brain induced by SD. In addition, rolipram reversed the decrease in O-GlcNAcylation and OGT expression as well as elevation of OGA expression following SD. Treatment with H89, a PKA inhibitor, significantly impaired the L/M functions of zebrafish compared with the control group, inducing a decrease in O-GlcNAcylation and OGT expression and, conversely, an increase in OGA expression. The H89-induced changes in O-GlcNAc cycling and L/M dysfunction were effectively reversed by glucosamine treatment. H89 suppressed, whereas caffeine and rolipram promoted O-GlcNAc cycling in Neuro2a cells. Our collective findings underscore the interplay between PKA signaling and O-GlcNAc cycling in the regulation of cognitive function in the brain, offering potential therapeutic targets for cognitive deficits associated with SD.NEW & NOTEWORTHY Our observation highlights the intricate interplay between cAMP/PKA signaling and O-GlcNAc cycling, unveiling a novel mechanism that potentially governs the regulation of learning and memory functions. The dynamic interplay between these two pathways provides a novel and nuanced perspective on the molecular foundation of learning and memory regulation. These insights open avenues for the development of targeted interventions to treat conditions that impact cognitive function, including SD.

Targeting Breast Cancer with N-Acetyl-D-Glucosamine: Integrating Machine Learning and Cellular Assays for Promising Results.

BACKGROUND: Breast cancer is a common cancer with high mortality rates. Early diagnosis is crucial for reducing the prognosis and mortality rates. Therefore, the development of alternative treatment options is necessary. OBJECTIVE: This study aimed to investigate the inhibitory effect of N-acetyl-D-glucosamine (D-GlcNAc) on breast cancer using a machine learning method. The findings were further confirmed through assays on breast cancer cell lines. METHODS: MCF-7 and 4T1 cell lines (ATCC) were cultured in the presence and absence of varying concentrations of D-GlcNAc (0.5 mM, 1 mM, 2 mM, and 4 mM) for 72 hours. A xenograft mouse model for breast cancer was established by injecting 4T1 cells into mammary glands. D-GlcNAc (2 mM) was administered intraperitoneally to mice daily for 28 days, and histopathological effects were evaluated at pre-tumoral and post-tumoral stages. RESULTS: Treatment with 2 mM and 4 mM D-GlcNAc significantly decreased cell proliferation rates in MCF-7 and 4T1 cell lines and increased Fas expression. The number of apoptotic cells was significantly higher than untreated cell cultures (p < 0.01 - p < 0.0001). D-GlcNAc administration also considerably reduced tumour size, mitosis, and angiogenesis in the post-treatment group compared to the control breast cancer group (p < 0.01 - p < 0.0001). Additionally, molecular docking/dynamic analysis revealed a high binding affinity of D-GlcNAc to the marker protein HER2, which is involved in tumour progression and cell signalling. CONCLUSION: Our study demonstrated the positive effect of D-GlcNAc administration on breast cancer cells, leading to increased apoptosis and Fas expression in the malignant phenotype. The binding affinity of D-GlcNAc to HER2 suggests a potential mechanism of action. These findings contribute to understanding D-GlcNAc as a potential anti-tumour agent for breast cancer treatment.

O-GlcNAc signaling increases neuron regeneration through one-carbon metabolism in Caenorhabditis elegans.

Cellular metabolism plays an essential role in the regrowth and regeneration of a neuron following physical injury. Yet, our knowledge of the specific metabolic pathways that are beneficial to neuron regeneration remains sparse. Previously, we have shown that modulation of O-linked ß-N-acetylglucosamine (O-GlcNAc) signaling, a ubiquitous post-translational modification that acts as a cellular nutrient sensor, can significantly enhance in vivo neuron regeneration. Here, we define the specific metabolic pathway by which O-GlcNAc transferase (ogt-1) loss of function mediates increased regenerative outgrowth. Performing in vivo laser axotomy and measuring subsequent regeneration of individual neurons in C. elegans, we find that glycolysis, serine synthesis pathway (SSP), one-carbon metabolism (OCM), and the downstream transsulfuration metabolic pathway (TSP) are all essential in this process. The regenerative effects of ogt-1 mutation are abrogated by genetic and/or pharmacological disruption of OCM and the SSP linking OCM to glycolysis. Testing downstream branches of this pathway, we find that enhanced regeneration is dependent only on the vitamin B12 independent shunt pathway. These results are further supported by RNA sequencing that reveals dramatic transcriptional changes by the ogt-1 mutation, in the genes involved in glycolysis, OCM, TSP, and ATP metabolism. Strikingly, the beneficial effects of the ogt-1 mutation can be recapitulated by simple metabolic supplementation of the OCM metabolite methionine in wild-type animals. Taken together, these data unearth the metabolic pathways involved in the increased regenerative capacity of a damaged neuron in ogt-1 animals and highlight the therapeutic possibilities of OCM and its related pathways in the treatment of neuronal injury.

Targeting O-GlcNAcylation in cancer therapeutic resistance: The sugar Saga continues.

O-linked-N-acetylglucosaminylation (O-GlcNAcylation), a dynamic post-translational modification (PTM), holds profound implications in controlling various cellular processes such as cell signaling, metabolism, and epigenetic regulation that influence cancer progression and therapeutic resistance. From the therapeutic perspective, O-GlcNAc modulates drug efflux, targeting and metabolism. By integrating signals from glucose, lipid, amino acid, and nucleotide metabolic pathways, O-GlcNAc acts as a nutrient sensor and transmits signals to exerts its function on genome stability, epithelial-mesenchymal transition (EMT), cell stemness, cell apoptosis, autophagy, cell cycle. O-GlcNAc also attends to tumor microenvironment (TME) and the immune response. At present, several strategies aiming at targeting O-GlcNAcylation are under mostly preclinical evaluation, where the newly developed O-GlcNAcylation inhibitors markedly enhance therapeutic efficacy. Here we systematically outline the mechanisms through which O-GlcNAcylation influences therapy resistance and deliberate on the prospects and challenges associated with targeting O-GlcNAcylation in future cancer treatments.

Review: Protein O-GlcNAcylation regulates DNA damage response: A novel target for cancer therapy.

The DNA damage response (DDR) safeguards the stable genetic information inheritance by orchestrating a complex protein network in response to DNA damage. However, this mechanism can often hamper the effectiveness of radiotherapy and DNA-damaging chemotherapy in destroying tumor cells, causing cancer resistance. Inhibiting DDR can significantly improve tumor cell sensitivity to radiotherapy and DNA-damaging chemotherapy. Thus, DDR can be a potential target for cancer treatment. Post-translational modifications (PTMs) of DDR-associated proteins profoundly affect their activity and function by covalently attaching new functional groups. O-GlcNAcylation (O-linked-N-acetylglucosaminylation) is an emerging PTM associated with adding and removing O-linked N-acetylglucosamine to serine and threonine residues of proteins. It acts as a dual sensor for nutrients and stress in the cell and is sensitive to DNA damage. However, the explanation behind the specific role of O-GlcNAcylation in the DDR remains remains to be elucidated. To illustrate the complex relationship between O-GlcNAcylation and DDR, this review systematically describes the role of O-GlcNAcylation in DNA repair, cell cycle, and chromatin. We also discuss the defects of current strategies for targeting O-GlcNAcylation-regulated DDR in cancer therapy and suggest potential directions to address them.

O-GlcNAcylation and Its Roles in Neurodegenerative Diseases.

 As a non-classical post-translational modification, O-linked ß-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) is widely found in human organ systems, particularly in our brains, and is indispensable for healthy cell biology. With the increasing age of the global population, the incidence of neurodegenerative diseases is increasing, too. The common characteristic of these disorders is the aggregation of abnormal proteins in the brain. Current research has found that O-GlcNAcylation dysregulation is involved in misfolding or aggregation of these abnormal proteins to mediate disease progression, but the specific mechanism has not been defined. This paper reviews recent studies on O-GlcNAcylation's roles in several neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, Machado-Joseph's disease, and giant axonal neuropathy, and shows that O-GlcNAcylation, as glucose metabolism sensor, mediating synaptic function, participating in oxidative stress response and signaling pathway conduction, directly or indirectly regulates characteristic pathological protein toxicity and affects disease progression. The existing results suggest that targeting O-GlcNAcylation will provide new ideas for clinical diagnosis, prevention, and treatment of neurodegenerative diseases.

Emerging roles of O-GlcNAcylation in protein trafficking and secretion.

The emerging roles of O-GlcNAcylation, a distinctive post-translational modification, are increasingly recognized for their involvement in the intricate processes of protein trafficking and secretion. This modification exerts its influence on both conventional and unconventional secretory pathways. Under healthy and stress conditions, such as during diseases, it orchestrates the transport of proteins within cells, ensuring timely delivery to their intended destinations. O-GlcNAcylation occurs on key factors, like coat protein complexes (COPI and COPII), clathrin, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), and GRASP55 (Golgi reassembly stacking protein of 55 kDa) that control vesicle budding and fusion in anterograde and retrograde trafficking and unconventional secretion. The understanding of O-GlcNAcylation offers valuable insights into its critical functions in cellular physiology and the progression of diseases, including neurodegeneration, cancer, and metabolic disorders. In this review, we summarize and discuss the latest findings elucidating the involvement of O-GlcNAc in protein trafficking and its significance in various human disorders.

Studying the O-GlcNAcome of human placentas using banked tissue samples.

O-GlcNAcylation is a dynamic modulator of signaling pathways, equal in magnitude to the widely studied phosphorylation. With the rapid development of tools for its detection at the single protein level, the O-GlcNAc modification rapidly emerged as a novel diagnostic and therapeutic target in human diseases. Yet, mapping the human O-GlcNAcome in various tissues is essential for generating relevant biomarkers. In this study, we used human banked tissue as a sample source to identify O-GlcNAcylated protein targets relevant to human diseases. Using human term placentas, we propose (1) a method to clean frozen banked tissue of blood proteins; (2) an optimized protocol for the enrichment of O-GlcNAcylated proteins using immunoaffinity purification; and (3) a bioinformatic workflow to identify the most promising O-GlcNAc targets. As a proof-of-concept, we used 45 mg of banked placental samples from two pregnancies to generate intracellular protein extracts depleted of blood protein. Then, antibody-based O-GlcNAc enrichment on denatured samples yielded over 2000 unique HexNAc PSMs and 900 unique sites using 300 µg of protein lysate. Due to efficient sample cleanup, we also captured 82 HexNAc proteins with high placental expression. Finally, we provide a bioinformatic tool (CytOVS) to sort the HexNAc proteins based on their cellular localization and extract the most promising O-GlcNAc targets to explore further. To conclude, we provide a simple 3-step workflow to generate a manageable list of O-GlcNAc proteins from human tissue and improve our understanding of O-GlcNAcylation's role in health and diseases.

Synthetic assembly of α-O-linked-type GlcNAc using polymer chemistry affords sugar clusters, which effectively bind to lectins.

Fischer's glycoside synthesis was applied to linker precursor alcohols of two different lengths having appropriate alkane chains to obtain the corresponding α-glycoside and it was found to be applicable with moderate yields. Water-soluble glycomonomers were systematically prepared from N-acetyl-d-glucosamine (GlcNAc) by introducing two kinds of alcohols having different methylene lengths. Typical radical polymerizations of the glycomonomers with acrylamide as a modulator for control of the distance between carbohydrate residues in water in the presence of ammonium persulfate (APS)-N,N,N',N'-tetramethylethylenediamine (TEMED) gave a series of glycopolymers with various α-glycoside-type GlcNAc residue densities. Fluorometric analysis of the interaction of wheat germ agglutinin (WGA) with the glycopolymers was performed and the results showed unique binding specificities based on structural differences.

Development of a two-enzyme system in Aspergillus niger for efficient production of N-acetyl-ß-D-glucosamine from powdery chitin.

A chitinase (PbChi70) from Paenibacillus barengoltzii was engineered by directed evolution to enhance its hydrolysis efficiency towards powder chitin. Through two rounds of screening, a mutant (mPbChi70) with a maximum specific activity of 73.21 U/mg was obtained, which is by far the highest value ever reported. The mutant gene was further transformed into Aspergillus niger FBL-B (ΔglaA) which could secrete high level of endogenously ß-N-acetylglucosaminidase (GlcNAcase), thus a two-enzyme expression system was constructed. The highest chitinase activity of 61.33 U/mL with GlcNAcase activity of 353.1 U/mL was obtained in a 5-L fermentor by high-cell density fermentation. The chitin-degrading enzyme cocktail was used for the bioconversion of GlcNAc from powder chitin directly, and the highest conversion ratio reached high up to 71.9 % (w/w) with GlcNAc purity ≥95 % (w/w). This study may provide an excellent chitinase as well as a double enzyme cocktail system for efficient biological conversion of chitin materials.

Revealing the conformational dynamics of UDP-GlcNAc recognition by O-GlcNAc transferase via Markov state model.

The O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is a critical post-translational modification and closely linked to various physiological and pathological conditions. The O-GlcNAc transferase (OGT) functions as the only glycosyltransferase of O-GlcNAc glycosylation by transferring GlcNAc from UDP-GlcNAc to serine or threonine residues on protein substrates. The interaction mode of UDP-GlcNAc against OGT has been preliminarily revealed by the crystal structures, yet an atomic-level comprehension for the conformational dynamics of the recognition process remains elusive. Here, we construct the Markov state model based on extensive all-atom molecular dynamics (MD) simulations with an aggregated simulation time of ∼9 µs, and reveal that the UDP-GlcNAc recognition process by OGT encompasses four key metastable states, occurring within an estimated timescale of ∼10 µs. During UDP-GlcNAc recognition process, we find the pyrophosphate moiety (P2O52-) initially anchors to the active pocket via salt bridge and hydrogen bonds, facilitating subsequent binding of the uridine and GlcNAc moieties. Furthermore, the functional roles of K842 involved in the salt bridge with P2O52- were evaluated through extra mutant MD simulations. Overall, our study provides valuable insights into the UDP-GlcNAc recognition mechanism by OGT, which could further aid in mechanistic studies of O-GlcNAc glycosylation and drug development targeting on OGT.