Earticle

Acute respiratory syndrome coronavirus SARS-CoV-2 causing COVID-19 has been rapidly spreading worldwide since it occurred in December 2019. SARS-CoV-2 infection occurs as the receptor-binding domain (RBD) of the coronavirus envelope spike protein binds to the angiotensin-converting enzyme 2 (ACE2) receptor. ACE2 is a cell membrane protein activated when it is cleaved and released from the cell surface. The soluble ACE2 can act as a decoy receptor to neutralize the SARS-CoV-2 virus, which is a potential therapy for COVID-19. In this study, the recombinant ACE2 protein was produced in a transient Nicotiana benthamiana plant expression system. The ACE2 was cloned to a fragment crystallizable (Fc) tagged with the KDEL sequence, endoplasmic reticulum (ER) retention signal (ACE2-FcK) in pEAQ-HT, the Cowpea mosaic virus (CPMV)-based transient plant expression vector. To design the recombinant protein structure of ACE2-FcK, the transmembrane region of ACE2 was removed using a transmembrane region prediction program. Four N-glycosylation sites recognized were identified using the N-glycosylation prediction program. Finally, Agrobacterium (LBA4404) carrying pEAQ-HT ACE2-FcK vector was applied for transient expression of ACE2-FcK in N. benthamiana. The proteins (100kDa) were successfully expressed and purified from leaves using protein A affinity chromatography. Additionally, suggested that the plant-derived ACE2-FcK has bioactivities as a therapeutic agent for coronavirus. Taken together, the ACE2-FcK protein can be transiently produced with an optimized expression time and leaf position post-infiltration in plant.

Enzyme replacement therapy for lysosomal storage diseases usually requires the recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time in this study, a strategy for in vitro mannosyl-phosphorylation of high-mannose type N-glycans has been established employing a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal or C-terminal deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn1477-935 with deletion of N-terminal 77 amino acids spanning transmembrane domain (47 amino acids) and part of stem region (30 amino acids), showed the highest mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn1477-935 was determined through the enzyme assays using the high-mannose type N-glycan (Man8GlcNAc2) as a substrate. The rMnn1477-935 was shown to mannosyl-phosphorylate the high-mannose type N-glycans (Man7-9GlcNAc2) on the recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. In addition, the majority of the resulting mannosyl-phosphorylated glycans were bis-form, which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. In vitro N-glycan mannosyl-phosphorylation reaction using rMnn1477-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of “Biobetter” therapeutic enzymes.

α-Synuclein has been proposed to play a key pathogen in Parkinson's disease (PD). Especially, the abnormal accumulation and aggregation of the misfolded α-Synuclein protein is the neuropathological hallmark of all α-Synucleinopathies, including Parkinson's disease. Netrins (netrin-1, netrin-3, and netrin-4) are secreted proteins, which are laminin-related and involved in axon guidance function and cell survival molecular pathway. Remarkably, only netrin-1 is highly expressed in healthy adult substantia nigra brain region and inversely correlates with the expression of α-Synuclein, which led us to investigate the impact of the molecular interaction between α-Synuclein and netrin-1 in dopaminergic neuron fate. Here we show that netrin-1 and α-Synuclein directly interact in pre-formed fibrils (PFFs) generation test and real time binding assay. Strikingly, we observed that the level of netrin-1 was significantly decreased in SNCA Tg mouse brain with dopaminergic cell death, which matched the analysis result from human PD patients database. Our finding suggest that netrin-1 deprivation triggers dopaminergic neuronal cell death with α-Synuclein aggregation during aging process and netrin-1 can be the promising therapeutic or diagnostic molecule in α-Synucleinopathies.

Early diagnosis and appropriate treatment can significantly lower the death rate from cancer, so the discovery of low-cost and high-efficiency cancer biomarkers for early diagnosis of cancer has been ongoing for a long time. However, most biomarkers based on quantitative differences are not limited to specific cancers and have inconsistent reproducibility, so they are used as an auxiliary form of diagnosis. Accordingly, we tried to discover biomarkers with improved diagnostic ability by considering not only quantitative differences but also qualitative differences, that is, changes in glycosylation. Protein X, which was confirmed to have a specific glycosylation structure in liver cancer patients through our previous study, was selected as a target protein, and its potential as a liver cancer biomarker was investigated through quantitative and qualitative analysis. In particular, to analyze the glycosylation pattern of protein X, an aglycosylated antibody-based lectin-coupled immunoassay was introduced and verified. As a result of analyzing the patient's clinical samples through this method, it was confirmed that the fucosylation pattern of protein X was similar to that of AFP, which the FDA approved as a biomarker. Therefore, the fucosylation of protein X is expected to have potential as a liver cancer biomarker.

To guarantee proper glycoprotein folding, eukaryotes evolved a highly conserved N-glycan-dependent endoplasmic reticulum quality control mechanism (ERQC). In the ER, glycoprotein conformations are sensed by UDP-glucose: glycoprotein glucosyltransferase (UGGT), which selectively re-glycosylates misfolded glycoproteins for re-binding to the folding chaperones. ER-resident α-1,2-mannosidase I (MNS1) trims one mannose residue from the B branch of core N-oligosaccharides, initiating the formation of the molecular signals that either lead folded proteins towards the Golgi apparatus, or direct misfolded glycoproteins towards degradation. In this study, we investigated the molecular assembly and function of N-glycan-dependent ERQC in the human fungal pathogen Cryptococcus neoformans, which has a unique N-glycosylation pathway lacking glucosyltransferases but carrying multiple mannosidases. We constructed and functionally analyzed the C. neoformans mutant strains lacking UGGT (uggtΔ), MNS1A (mns1AΔ) and MNS1B (mns1BΔ). The uggtΔ mutant displayed reduced growth rates with aggregated forms, increased stress sensitivity, and induced the unfolded protein response even in the absence of exogenous ER stress. The mns1AΔ and mns1BΔ showed altered N-glycan profiles, indicating that whereas MNS1A acts as the widely known ER-α1,2-mannosidase I, MNS1B appears to be a novel mannosidase involved in mannose processing in the Golgi. Simultaneous deletion of MNS1A and MNS1B (mns1AΔ1BΔ) displayed mild growth retardation under several stress conditions. Moreover, qRT-PCR analysis of UGGT, MNS1A and MNS1B mRNA levels revealed that these genes were up-regulated upon DTT treatment, indicating these genes are essential upon ER stress induced by the presence of misfolded proteins. Notably, the uggtΔ mutant and the mns1AΔ1BΔ double mutant strains commonly displayed defects in capsule formation, which might contribute to a decrease in survival inside phagocytic cells and to avirulence in a murine model. Altogether, our data demonstrates the evolutionary unique C. neoformans N-glycan-dependent ERQC plays critical roles in cellular fitness under adverse conditions of the host environment, required for full fungal pathogenicity.

The human fungal pathogen Cryptococcus neoformans assembles O-linked mannosyl glycans on its proteins in two types; Ktr3p-meidated major-O-glycans without xylose and Cap6-mediated minor-O-glycans without xylose, respectively1. The C. neoformans ktr3Δcap6Δ double mutant strain was constructed to block completely O-mannosylation extension in the Golgi. The ktr3Δcap6Δ strain accumulated the O-glycans carrying only a single mannose and displayed defective growth under several stress conditions, accompanied with completely attenuated virulence in a mouse model of cryptococcosis. By RNA-Seq-based transcriptome analysis, we investigated the effect of defective O-mannosylation on the stress-responsive signaling pathways associated with virulence. The comparative transcriptome analysis of the wild-type (WT) and ktr3Δcap6Δ cells under normal growth and tunicamycin (TM) treatment conditions, respectively, revealed the decreased induction of Mpk1p-and calcineurin-Crz1-mediated cell wall integrity (CWI) signaling pathways. Furthermore, we examined the possible change of physiochemical properties of cell surface in the ktr3Δcap6Δ cells, by profile analysis of N-linked glycans assembled on cell wall proteins, flow cytometry analysis of cell wall components, and filipin-staining analysis of erogosterol. Although no apparent difference in the total amount and overall length of N-glycans was observed, the N-glycan profile of ktr3Δ cap6Δ showed a marginal change with the decreased neutral glycan peaks and increased acidic glycan peaks compared to that of the WT strain. We also observed the slightly increased amount of chitin in ktr3Δ cap6Δ, which might reflect the compensation mechanism of defective CWI. Notably, the ktr3Δ cap6Δ cells showed defective trafficking of ergosterol, an immunoactive fungal molecule, from the ER to the plasma membrane. In conclusion, our results demonstrate the importance of extended O-glycan structure for cell wall integrity signaling and physiochemical properties of cell surface, which critically affect the survival in the host cell environments and the interaction with host cells of C. neoformans during infection process.

Overexpression of human epidermal growth factor receptor type 2 (HER2) protein is one of the prognostic factors to diagnose breast cancer, and HER2 is proto-oncogene located on chromosome 17, and overexpression of this oncogene plays an essential role in the growth and progression of certain violent types of breast cancer. This study demonstrated the expression of the anti-HER2 camelid single domain antibody VHH in tobacco plant. And we confirmed the function of recombinant VHH for the treatment of breast cancer. The anti-HER2 VHH was fused to human IgG Fc domain along with KDEL endoplasmic reticulum (ER) (VHH-FcK) in a gene expression cassette. The plant gene expression cassette was transferred to tobacco plants using Agrobacterium-mediated transformation. The transformants were screened by PCR and western blots. ELISA confirmed the binding activity of purified anti-HER2 VHH-FcK to HER2 positive breast cancer cell line BT-474. In conclusion, this study verified the possibility of anti-HER2 VHH-FcK as a therapeutic agent and suggests that the anti-breast cancer anti-HER2 VHH-FcK can be expressed, properly assembled and purified from plant expression system, which can be as an alternative system for antibody production.

Recombinant antibody proteins can be applied to treat many various cancers. They are primarily produced using mammalian, insect, and bacteria cell culture systems. However, plant expression systems have been currently developed to produce antibodies. In particular, production of recombinant therapeutic antibodies using plant expression systems have several advantages, which are lack of human pathogenic agents, efficient production cost, and easy large scale up. In this study, we generated transgenic Nicotiana tabacum plant expressing both anti-colorectal cancer large single chain (LSC) CO17-1AK and anti-human epidermal growth factor receptor 2 (HER2) VHH-FcK monoclonal antibodies (mAbs) by cross-pollinating with plants expressing LSC CO17-1AK and anti-HER2 VHH-FcK, respectively. Plants expressing both LSC CO17-1AK and anti-HER2 VHH-FcK were screened with polymerase chain reaction and Western blot analyses. Enzyme-linked immunosorbent assay (ELISA) confirmed the binding of the LSC CO17-1AK and anti-HER2 VHH-FcK to the HER2 and the GA733 proteins, respectively. The result of this study indicated that both mAb LSC CO17-1AK and anti-HER2 VHH-FcK produced in plant have their specific binding activities against HER2 and the GA733 antigens similar to their parental antibodies, respectively.

As a part of the Chromosome-centric Human Proteome Project (C-HPP), we have developed a few algorithms for accurate identification of missing proteins, alternative splicing variants, single amino acid variants, and characterization of function unannotated proteins. We have found missing proteins, novel and known ASVs, and SAAVs using LC-MS/MS data from human brain and olfactory epithelial tissue, where we validated their existence using synthetic peptides. We used the latest released neXtProt database (2022-02) which incorporates PeptideAtlas human (2022-01 build) and MassIVE, where they contains 2 and 7 new sample types (e.g. normal muscle, cancer from other female reproductive organ), respectively. According to the neXtProt database, the number of missing proteins in chromosome 11 shows a decreasing pattern. The development of genomic and transcriptomic sequencing techniques make the number of protein variants in chromosome 11 tremendously increase. For the 73 uPE1 in chromosome 11, we have studied the function annotaion of CCDC90B (NX_Q9GZT6), SMAP (NX_O00193), and C11orf52 (NX_Q96A22).

Alginic acid, which is widely used in various industries like food, cosmetics, biomedical, and pharmaceuticals, is an unbranched polysaccharide derived from matrix and cell walls of brown algae. It has a backbone of 1, 4-glycosidic bonds with two conformational residues arranged randomly, β-D-mannuronic acid (M) and α-L-guluronic acid (G). Recently, alginic acid has been found to exhibit physiological activities such as anti-inflammatory, antioxidant, and cholesterol lowering, drawing attention again as a functional biomaterial. However, there are still few studies on the relationship between the structural characteristics of alginic acid and the biological function. This is because, unlike other polysaccharides, alginic acid does not have a repeating unit and has a heterogeneous structure. Here, we have developed an analytical strategy for characterization of alginic acid using LC/MS/MS. First, the average molecular weight and molecular weight range of alginic acid were determined via SEC-MALS. To facilitate the structural analysis, alginic acid was acid hydrolyzed to small oligomers, which are further purified by PGC-SPE. The acid-hydrolyzed alginic acid was chromatographically separated on a PGC column suitable for isomer separation and analyzed by MS/MS. This analytical strategy enabled us to obtain M/G ratio, degree of polymerization, molecular weight, modification, impurities, and quantitative information. In addition, comparative analysis was performed on various alginic acid products to characterize the structure of alginic acid for each product. Ultimately, it can be used for quality index data of raw materials for the production of high value-added alginic acid products derived from seaweeds by examining the correlation between the structural characteristics and biological functions of alginic acid.

In recent years, liquid biopsy has been noted for its outstanding advantages over tissue biopsy including ease of sampling, effective monitoring, and longitudinal assessment of treatment dynamics. In particular, Cerebrospinal fluid(CSF), a body fluid that directly interacts with the extracellular fluid of the brain and central nervous system, is a representative body fluid used for liquid biopsy and is becoming a potential source for neurological study. Mucopolysaccharidosis is a fatal neurodegenerative lysosomal storage disease caused by lysosomal β-gal deficiency and of galactose-containing substrates accumulate in various tissues such as brain and liver. Therefore, continuous monitoring of changes in the expression of galactose substrates in the brain is important for the diagnosis and treatment of disease. However, the low protein content of the biofluid makes this difficult. Here, we developed a highly sensitive, absolute quantitation platform for N-glycans containing terminal galactose(A2G2/A2G2F) to determine mucopolysaccharidosis using liquid biopsy of mouse CSF within 2hours. N-glycans labeled with InstantPC were quantified using hydrophilic interaction chromatography(HILIC) coupled with multiple response monitoring(MRM)-MS. In particular, A2G2/A2G2F glycans are present in less than 5% even in very small amount of mouse CSF, but it is possible to quantify glycans in 300nL mouse CSF at the femtomole level. Indeed, we were able to achieve high reproducibility within 15% of CV in experimental/instrumental replicates in the concentration range of 0.1 to 500pg/μL with quantitative linearity. Our glycan quantification platform can be potentially applied for diagnosis and monitoring of patients for neurodegenerative diseases at trace amounts of CSF (about 5μL) in a much more convenient and non-invasive manner.

O-GlcNAcylation is one of the post-translational modification that occurs at Ser/Thr residues of a protein by O-GlcNAc transferase (OGT), but the O-GlcNAcylation is regulated by O-GlcNAcase (OGA). The O-GlcNAcylation is involved in various cellular processes and diseases including diabetes, cancer and Alzheimer’s disease. Herein, we suggest a new method for identifying O-GlcNAcylated proteins including classification of O-GlcNAc and O-GalNAc with the same molecular weight. Using the fractionated raw files of LC-MS/MS from T24 cell lines, we developed an identification algorithm coded by python 3.9 as follows: 1.Selection of spectra with oxonium ions in ms2 file extracted from raw MS data obtained using LC-MS/MS; 2.Spectral matching with 20 ppm against a tryptic and O-GlcNAcylated peptide database generated from proteins identified using IP2 proteomic search result; 3.In the candidates, matching between tandem MS with b/y and c/z ion list and experimental HCD, ETD spectra, respectively; 4.Selection of the best scored O-GlcNAcylated peptide from the candidated peptides calculated by scoring system; 5.Classification of O-GlcNAc and O-GalNAc based on their corresponding fragment ions; 6.Evaluating a model using ROC curve comparing search results with manual validation. In addition, we also performed relative quantitative analysis in conjunction with the TMT Quantification method. In the glycoscience field, our study could be applied to various human disease treatments by identifying O-GlcNAcylated proteins and by engaging in cell signaling after regulating the function of them.

We have developed an Identification and Quantification by GlycoProteome Analyzer (IQ-GPA) for high throughput analysis of N- and O-glycoproteome. Herein, we introduce the web version of IQ-GPA for a database search and algorithmic suite with tandem mass spectrometry of HCD/CID/ETD (EThcD) in a shotgun experiment. We have designed to easily handle high-throughput glycoproteomic data with graphical user interfaces, and users can instantly access to a range of Glycan Database Builder as well as N and O-GPA modules using Microsoft Azure cloud platforms. With spectra files and the glycopeptide database of interest, the modules identify and quantify intact N- and O-linked glycopeptides in the complex samples, which requires the following steps: 1) DB Builder Module generates a GPA database using the detailed parameters after select working directory. 2) Analysis Module requires a spectrum file and a GPA-Database to analyze N- and O- glycopeptides with user specific parameters. 3) GPA Report Module shows the results and statistical charts on the website. In addition, IQ-GPA contains a module for the classification of N-glycopeptides as core- and outer-fucosylated types using the artificial intelligence ( https://www.iqgpa.org ).

A system was developed to successfully biosynthesize glucoside derivatives of therapeutic and relevant N-linked compounds in the generally regarded as safe (GRAS) Corynebacterium glutamicum. Using glucose, as the sole carbon source, YdhE, which is a promiscuous glycosyltransferase from Bacillus lichenformis and has been introduced in the Corynebacterium glutamicum cell factory system, was able to biosynthesize most of the N-linked compounds to their rare glucosides. Optimal conditions such as IPTG concentrations and induction time, Temperature, Substrate and glucose concentration, and Production time on the In Vivo conversion were successfully developed. This yielded almost 85% of glucosylated butyl-4-aminobenzoate glucoside after only 10 hours post substrate induction. The developed system uses recombinant Corynebacterium glutamicum to achieve In Vivo glucosylation using cheap glucose as the main source of UDP-sugar moieties. Hence making it feasible for industrial-scale biosynthesis of glucoside derivatives by bypassing the demand and use of the more expensive UDP-glucose cofactor that is used in most work that involves glucosylation.

A highly efficient biocatalytic system was constructed to make bioactive resveratrol poly-glucosides in a chain by using sucrose as a cheap source of UDP-glucose donors with the help of amylosucrase, DgAS from Deinococcus geothermalis. The system is superior as the reaction is very fast, direct, high rate of conversion of substrates to products, high thermostable, and regio-stereospecific with very effective in action at 40°C. Successfully our system can convert around 98% resveratrol to its resveratrol glucosides in a short time. The amylosucrase DgAS not only added glucose in multiple hydroxyl positions but also conjugates glucose in the chain as a result highly soluble glucosylated products are formed. Because of the highest thermostability and specific action among all known amylosucrases (ASases), DgAS has proven its importance for the transglucosylation activities in different polyphenols on an industrial scale. Therefore, our system can be used industrially for the cost-effective production of resveratrol glucosides.

Saccharapolyspora spinosa is the producer of the environmentally friendly insecticide spinosad, which is a combination of spinosyn A and D. spinosyn is a tetracyclic polyketide aglycone to which is attached a neutral saccharide substituent (2,3,4-O-trimethyla-L-rhamnosyl) on the C-9 hydroxyl group and an aminosugar moiety (β D-forosaminyl) on the C-17 hydroxyl group). Both the rhamnose and forosamine moieties are essential for the insecticidal activity of spinosyns. We construct a vector; pIBR-SPN, cloned the sugar genes to it and transformed to Sa. spinosa. The best strain Sa. spinosa MUV pIBR-SPN FR produced 1,896.77 mg/L of spinosad, 17 folds higher than parent strain.

Human milk oligosaccharides (HMOs) have been proven to be a key ingredient found in breast milk and beneficial to infant health. Structurally, the fucose-containing oligosaccharides are major components, with a proportion of approximately 50–80% in the HMOs. In particular, 2-fucosyllactose (2’-FL) and 3-fucosyllactose (3-FL) are 2 major components of the abundant HMO component. Importantly, 2’-FL is approved for use in infant formula for improving probiotic functions by the FDA of the USA and the European Food Safety Authority, while 3-FL can be used as a prebiotic to improve the growth of beneficial microorganisms. Over the last decade, various approaches have been conducted for fucosyllactose (FL) production, including chemical synthesis, enzymatic conversion, and microbial fermentation. Most studies were performed in Escherichia coli due to the simple culture conditions and availability of various genetic tools. Besides E. coli, Bacillus subtilis and Saccharomyces cerevisiae have also been selected as the hosts for the production of FL due to their generally regarded safe (GRAS) status. As a type of GRAS strain, Corynebacterium glutamicum has been widely used in the biosynthesis of food-grade, high-value-added products, including lysine, glutamic acid, violacein and resveratrol. In this study, the biosynthetic enzymes of FL were condon-optimized and heterologously expressed in engineered C. glutamicum for the production of FL. The trace amount production of FL was confirmed by TLC, HPLC in this study.

Xenotransplantation using porcine organs is a potential solution to bridge the gap between donors and recipients, but difference in glycosylation between humans and porcine limit successful clinical trials. Recent studies have shown that representative non-human glycan moieties such as galactose-α1,3-galactose(α-gal), NeuGc, and SDa antigens act as epitopes triggering antibody-mediated rejection (AMR). NeuGc leads to several inflammatory disorders and α-gal is known to cause more severe hypersensitivity reactions. Furthermore, it is essential to identify the glycan antigens adorned on the surface because endothelial cells interact first with the recipient's immune component during the xenotransplantation process. Despite the emphasis on the importance of glycans in the immune system, studies on comprehensive characterization of glycosylation including the structure of xeno-glycan antigens in porcine, a representative xenotransplantation model, are insufficient. Here, we performed overall glycan profiling and structural analysis of glycans with non-human moieties in porcine aorta endothelial cells. We could identify various glycan isomers and heterogeneity characteristics by accurate masses, retention times, LC/MS/MS, and exoglycosidase digestion. The major components were di-NeuAc-sialylated glycans in endothelial cells. In particular, non-human glycans, which can cause problems during transplantation, were present in a small amount less than 10%. NeuGc-sialylated glycan ratios in cell are around 4%, A glycosylated unique form in which sialic acid attached to antennal N-acetyl glucosamine (HexNAc) is identified in aorta endothelial cells. The α-gal containing glycans were confirmed by not only MS/MS spectra but also α-gal glycan removal using galactosidase that breaks gal α1-3 linkages at the terminal in LC/MS chromatogram. Our data could be the reference for monitoring changes in glycan antigens in glycoengineering models for xenotransplantation and providing information for mammalian glycome studies.

Rapid eye movement (REM) sleep is associated with learning and memory (L/M) functions. Here, we demonstrate that REM sleep deprivation (REMSD)-induced impairment of contextual fear memory and spatial memory in mouse is linked to a downregulation in hexosamine biosynthetic pathway (HBP)/O-GlcNAc flux in mouse brain. In mice exposed to REMSD, O-GlcNAcylation proteins and O-GlcNAc transferase (OGT) were decreased while O-GlcNAcase was increased compared to control mouse brain. Foot shock fear conditioning (FC) induced activation of protein kinase A (PKA) and cAMP response element binding protein (CREB), which were significantly inhibited in brains of the REMSD group. Intriguingly, increasing O-GlcNAc cycling with glucosamine (GlcN) or OGA inhibitor, Thiamet G, restored defects in L/M functions and FC-induced PKA/CREB activation induced by REMSD. On behavior test, GlcN restored the contextual fear memory and spatial memory impairment induced by REMSD. Furthermore, Thiamet G restored the REMSD-induced decrease in dendritic spine density and learning and memory impairment. Suppression of O-GlcNAcylation by the glutamine fructose-6-phosphate amidotransferase (GFAT) inhibitor, 6‐diazo‐5‐oxo‐l‐norleucine (DON), or OGT inhibitor, OSMI-1, impaired memory function and inhibited FC-induced PKA/CREB activation. To our knowledge, this is the first study to provide comprehensive evidence of dynamic O-GlcNAcylation changes during the L/M process in mice and defects in this pathway in the brain of REM sleep-deprived mice. Our collective results highlight HBP/O-GlcNAc cycling as a novel molecular link between sleep and cognitive function.

Glucosamine (GlcN) is the amino sugar that increases the protein O-GlcNAcylation level by going into the hexosamine biosynthesis pathway (HBP pathway). Previous studies have reported that GlcN regulates autophagy in various organs including the brain, cartilage, and retina. However, its regulating mechanism and role in the liver are still unclear. Here, we observed glucosamine induces autophagy in human hepatocytes (HepG2). Moreover, LC3, one of the most important factors of autophagy, was upregulated by GlcN at the transcriptional and protein levels. We hypothesized that the HBP pathway is involved in GlcN-induced autophagy. Therefore, examined with inhibition of two enzymes, O-GlcNacase and O-GlcNAc transferase, which have an opposite role in the HBP pathway. As a result, the autophagy process was regulated by O-GlcNAcylation levels. To provide the role of GlcN in the liver autophagy process, we conducted the GlcN effect on the fatty liver, known autophagy disrupted disease. Palmitic acid (PA) induced autophagy inhibition and fat accumulation was relieved by GlcN in HepG2 cells. In addition, high-fat diet-induced fatty liver was restored by GlcN through autophagy in mice. Our results indicate that GlcN increased autophagy flux and through this, lipid accumulation can be restored both in vivo and in vitro.

Malignant melanoma, the out-of-growth of abnormal cells in the epidermis, is one of the most aggressive and invasive form of skin cancers which threatens human life expectancy in the world. A ganglioside, a molecule composed of a glycosphingolipid, has crucial roles involving in cell-cell interaction, cell growth, proliferation, inflammation, differentiation, and tumorigenesis. Meanwhile, 7,8-Dihydroxyflavone (7,8-DHF), also well known as tropoflavin and a small flavonoid, is natural polyphenolic compounds found in several vegetables, fruits, and tree leaves. Although 7,8-DHF is considered that potentially inhibited cancer growth, proliferation, invasion, and metastasis, the relationship between its effects on malignant melanoma and ganglioside expression is not fully understand yet. Accordingly, this study was to investigate the anti-cancer effects of 7,8-DHF based on the relationship between protein-protein interaction and ganglioside on human skin cancer. After treatment with 7,8-DHF, we found that abnormal cell elongation and morphological change in the proliferation and growth on melanoma cancer cells. 7,8-DHF directly induced nucleus fragmentation and changed the level of pro- and anti-apoptotic proteins. In our study, we confirmed the mitochondrial membrane permeabilization and mitochondrial ROS production. Also, we found that 7,8-DHF downregulated the expression level of ganglioside GD3 by high performance thin-layer chromatography analysis. This phytochemical was found to be a physiologically active substance that plays an important role in melanogenesis and has anti-cancer efficacy by inhibiting the expression and activity of ganglioside. Taken together, it might be an important evidence for treating malignant melanoma to figure out the molecular mechanisms.

Malignant melanoma is highly resistant to conventional treatments and is one of the most aggressive types of skin cancer. Conventional cancer treatments are limited due to drug resistance, tumor selectivity, and toxicities. Therefore, new treatments with fewer side effects and excellent effects should be developed. A ganglioside, a molecule composed of a glycosphingolipid, has crucial roles involved in cell-cell interaction, cell growth, proliferation, inflammation, differentiation, and tumorigenesis. Meanwhile, Quercetin (Qu) is considered that potentially inhibit cancer growth, proliferation, invasion, and metastasis, the relationship between its effects on malignant melanoma and ganglioside expression has not been clear. The aim of this study was to investigate the anti-cancer effects of Qu based on the relationship between protein structure and ganglioside on human skin cancer. In this study, quercetin, a flavonoid drug, did not induce toxicity in the human epithelial cell line HaCaT, but Its was abnormal cell elongation and morphological change in the proliferation and growth of skin cancer. Moreover, Its was treated so that cells in the G1/G0 phase cannot proceed to the subsequent S phase, which induces cell cycle arrest. The proliferation rate of cell migration was a certain range of proliferation was inhibited, it can be strongly assumed that this result is directly related to the expression of the various proteins analyzed above. Additionally, we discovered ganglioside decreased in melanoma with quercetin treatment. Taken together, it will be important evidence and present their potential as future therapeutic agents for treating carcinogenesis to figure out the molecular relationship between quercetin, ganglioside, and an anti-target protein of melanoma.

Repeated sublethal hypoxia exposure is reported to accelerate brain inflammation, and to affect the initiation and progression of cognitive dysfunction. Forskolin, an adenylate cyclase activator, is well-known as an inducer of the cAMP/PKA/CREB signaling. It has been shown that forskolin protects against various neuronal complications and induces long term memory; however, the underlying mechanism remains unclear. In this study, we showed that intraperitoneal administration of 200ng/g forskolin for 5 days on zebrafish notably recovered hypoxia-induced social interaction impairment and learning memory deficit. Forskolin suppressed hypoxia-induced neuroinflammation indicated by the decrease of NF-kB signaling and GFAP expression. On mouse neuroblastoma cell line, forskolin notably rescued hypoxia-induced cell death and dysfunction. The hypoxia model either in vivo or in vitro exerted a downregulation of PKA/CREB signaling and its downstream, c-Fos and BDNF expression, which was restored by forskolin. As previous studies proposed that hypoxia-induced hypo-O-GlcNAcylation could be an important causal factor for cognitive defect, we further investigated whether the HBP/O-GlcNAcylation cycling was changed by forskolin. Intriguingly, we observed that following forskolin treatment, OGT protein level (enzyme controls the addition of GlcNAc group to target proteins) induced considerably, which was accompanied by the upregulated O-GlcNAcylation level. Under forskolin treatment, O-GlcNAcylation flux on hypoxia-exposed zebrafish was reserved in line with the recovery of other inflammation and learning/memory markers. Next, we studied further about potential regulation of O-GlcNAcylation on the neuroprotective effect of forskolin. Zebrafish were treated with 500ng/g 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of GFAT1 (enzyme that catalyzes the reaction from fructose-6-P to glucosamine-6-P), to downregulate the O-GlcNAcylation of proteins. It was observed that DON treatment led to learning/memory impairment, which was rescued by forskolin. Moreover, forskolin conserved hypo-O-GlcNAcylation and neuroinflammation following DON administration. Further experiments are being conducted to examine how HBP/O-GlcNAc flux is involved in the beneficial effect of forskolin on cognitive function.

Traumatic brain injury (TBI) is known as a functional corruption of the brain caused by a bump or blast to the head when an object hits suddenly or pierces the skull and penetrates brain tissues. There is an urgent need of understanding the pathophysiology following TBI because the morbidity and mortality rates worldwide caused by TBI have remained high throughout decades. In this study, we used a novel model of adult zebrafish to study the molecular and physiological changes following TBI. Using T-maze, on the 3rd-day post-stab-lesion injury (dpi), learning and memory deficits and poor social response were observed. Results from mirror biting and novel tank tests also provided us insight into their defective mobility, explorational, and territorial instincts. Neurogenesis impairment and severe brain inflammation were also altered following TBI, especially at 3 dpi. Upon injury, a notable decrease of O-GlcNAc level was remarked at 3dpi. Deregulation of O-GlcNAcylation which led to impaired glucose metabolism was informed to contribute to the pathologies of neurodegenerative diseases. Glucosamine (GlcN), a natural compound found in cartilage, is known as a derivative of the pathway that can increase O-GlcNAcylation by bypassing the reaction catalyzed by a rate liming enzyme GFAT. To further investigate the correlation, after TBI exposure, zebrafish had been recovered 3 days in 0.1g/L glucosamine (GlcN) diluted in water. Under the treatment of GlcN, the inflammation level reduced significantly, indicated by the decrease of Nuclear factor-kappa B and Glial fibrillary acidic protein expression in the telencephalon. TBI-induced downregulation of the cAMP signaling pathway and its relevant proteins in synaptic plasticity were rescued by GlcN. O-GlcNAc deficiency observed previously, as well as L/M capability were retrieved. GlcN also elevated the number of new cells regenerations and neuronal differentiation. As a result, regulation of brain O-GlcNAcylation may hold a potential role in TBI’s recuperation.

단백질의 S-nitrosylation (SNO)와 O-GlcNAcylation은 중요한 Post-Translational Modification이다. 그러나 이들의 생물학적 연관성은 명확하게 밝혀진바 없다. 본 연구에서는 열충격에 의한 SNO와 O-GlcNAcylation간의 상관관계를 파악하고자 한다. Ex vivo상의 마우스 조직과 in vitro상의 세포에 열을 가하여 전반적인 SNO와 O-GlcNAcylation을 Biotin-switch assay (BSA)와 RL2 immunoblot을 통해 분석한 결과 Heat-shock에 의해 시간별로 SNO는 감소(hypo-S-nitrosylation) 하고 O-GlcNAcylation은 증가(hyper-O-GlcNAcylation)한다. 또한, SNO-OGT, SNO-Hsp70, SNO-Hsp90, SNO-Akt, SNO-actin을 포함한 내인성 SNO-단백질의 S-denitrosylation(SdeNO)이 일어난다. 특히 SNO-OGT의 SdeNO는 OGT의 활성을 높이는 역할을 한다. 이러한 결과는 heat-shock이 SNO-OGT의 SdeNO를 유발하여 OGT의 활성을 높이고, 결과적으로 hyper-O-GlcNAcylation을 유발한다는 것을 시사한다.

Many plant polysaccharide have immunomodulatory and anti-tumour activities. In the present study, water-soluble polysaccharides were isolated from an edible plant seed mainly by hot water extraction, 75% ethanol precipitation, and DEAE-cellulose column chromatography and tentatively named as PSP-F1. The PSP-F1 was shown to be a mixture of three major polysaccharides with molecular mass of approximately 1,200, 640, and 300 kDa, respectively, as estimated by size-exclusion HPLC analysis. The HPAEC-PAD analysis for neutral sugar composition and showed that PSP-F1 consists of arabinose (Ara) and xylose (Xyl), as the major neutral sugars, and other minor neutral sugars such as rhamnose (Rha), galactose (Gal), and glucose (Glc). HPLC analysis for uronic acids showed that galacturonic acid (GalA) presents as the major uronic acid and glucuronic acid (GlcA) was the minor one. Together with the neutral monosaccharides and uronic acids composition, the FT-IR and NMR analysis suggested that PSP-F1 appears to be a mixture of pectin type and heteroxylan polysaccharides. The results showed that the PSP-F1 (0-100 μg/ml) significantly induced the secretion of pro-inflammatory cytokines TNF-α and IL-6 and upregulation of MAPKs (ERK, JNK and p38) phosphorylation. The secretion of various pro-inflammatory cytokines from macrophages is mainly caused by MAPK cascade activation. These results demonstrated that the PSP-F1 isolated from a plant seed is a mixture of pectin type and heteroxylan polysaccharides, which are typical for plant cell wall polysaccharides, and that it effectively stimulates macrophages to secrete pro-inflammatory cytokines, such as TNF-α and IL-6, by upregulation of MAPKs phosphorylation. The results of this study suggests that PSP-F1 may be potential immunostimulator for health benefits and as a pharmaceutical component.

For an enhanced understanding of the biological mechanisms of human disease, it is essential to investigate protein functions. In a previous study, we developed a prediction method of gene ontology (GO) terms by the I-TASSER/COFACTOR result, and we applied this to uPE1 in chromosome 11. Furthermore, to validate the bioinformatics prediction of C11orf52, we utilized affinity purification and mass spectrometry to identify interacting partners of C11orf52. Using immunoprecipitation methods with three different peptide tags (Myc, Flag, and 2B8) in HEK 293T cell lines, we identified 79 candidate proteins that are expected to interact with C11orf52. The results of a pathway analysis of the GO and STRING database with candidate proteins showed that C11orf52 could be related to signaling receptor binding, cell−cell adhesion, and ribosome biogenesis. Then, we selected three partner candidates of DSG1, JUP, and PTPN11 for verification of the interaction with C11orf52 and confirmed them by colocalization at the cell−cell junctions by coimmunofluorescence experiments. In fact, cell-cell interactions and cell adhesion could be affected by glycosylation of adhesive molecules. Here, we focused on changes in protein expressions and glycosylation patterns caused by overexpression of C11orf52. Differentially expressed proetins and glycosylations were identified with TMT-based quantitative global and glyco- pretomic analysis and it will help to clarify how C11orf52 works in cells.

Mushroom lectins harboring carbohydrate binding specificity are powerful tools to detect glycoconjugates with N-linked or O-linked glycans for diagnosis. However, conventional preparation of diverse lectins and chemical labeling of target proteins remains a bottleneck. Here, we report a cell-free platform system for a recombinant fluorescent lectin by Escherichia coli extract in ExiProgen. Codon-optimized genes encoding the eight different types of mushroom lectins were successfully expressed as green fluorescent protein-fused forms in one-pot cell-free protein synthesis. Although the production yields of the recombinant proteins were depended on their coding genes, the resulting recombinant fluorescent lectins are apparent homo-multicomplex proteins with different molecular sizes and pI values. In hemagglutination inhibition assay, the lectins demonstrated agglutination activities towards various glycoconjugates. Moreover, GFP-fused lectins can be appliable to detect glycan binding specifies on using a glycan microarray. The glycan binding specificities of the flurescent lectins are also useful to analyze glycan epitopes expressed in different cancer cell lines. Taken together, these results provide an efficient and optimized procedure for the high-throughput synthesis and screening of lectin coding gene products based on in vitro production of the recombinant fluoresecnt lectins.

Human mesenchymal stromal cells (MSCs) are excellent candidates for regenerative medicine due to its pluripotency and paracrine functions. However, MSCs grown under two-dimensional (2D) culture conditions are losing their stemness and immunomodulatory function due to totally different culture environment from MSC niche in the human body. Here, we evaluated the way of improving ‘MSC-ness’ using in vivo like culture condition composed of functional polymers. In our results, functional polymer based three-dimensional (3D) culture increased the expression of stemness marker including Oct4 and Nanog in MSCs. Also, COX2 and HO1 which are related to immunomodulatory function of MSCs were increased. Moreover, MSCs on 3D culture elevated the secretion of PGE2 and highly suppressed TNFa from LPS stimulated splenocytes. When MSCs were injected intravenously into mouse neuro-inflammation model, MSCs on 3D significantly reduced the expression of Iba1, a microglia marker, and GFAP, an astrocyte marker, compared to MSCs for both acute and chronic inflammation. In addition, blood IL-10, an anti-inflammatory cytokine, was also increased in MSCs on 3D injected mice. Taken together, these results demonstrated that in vivo mimetic 3D culture provides MSCs beneficial microenvironment improving immunomodulatory function, eventually suggesting that MSC can be utilized as an effective source for cell therapy.