Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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

DOI： 10.1111/febs.16185

PubMed

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

N-glycosylation is a fundamental post-translational protein modification in the endoplasmic reticulum of eukaryotic cells. The biosynthetic and catabolic flux of N-glycans in eukaryotic cells has long been analyzed by metabolic labeling using radiolabeled sugars. Here, we introduce a non-radiolabeling protocol for the isolation, structural determination, and quantification of N-glycan precursors, dolichol-linked oligosaccharides, and the related metabolites, including phosphorylated oligosaccharides and nucleotide sugars. Our protocol allows for capturing of the biosynthesis and degradation of N-glycan precursors at steady state. For complete details on the use and execution of this protocol, please refer to Harada et al. (2013), Harada et al. (2020), and Nakajima et al. (2013).

DOI： 10.1016/j.xpro.2021.100316

Open Access

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Extracellular vesicles (EVs), a generic term for any vesicles or particles that are released from cells, play an important role in modulating numerous biological and pathological events, including development, differentiation, aging, thrombus formation, immune responses, neurodegenerative diseases, and tumor progression. During the biogenesis of EVs, they encapsulate biologically active macromolecules (i.e., nucleotides and proteins) and transmit signals for delivering them to neighboring or cells that are located some distance away. In contrast, there are receptor molecules on the surface of EVs that function to mediate EV-to-cell and EV-to-matrix interactions. A growing body of evidence indicates that the EV surface is heavily modified with glycans, the function of which is to regulate the biogenesis and extracellular behaviors of EVs. In this chapter, we introduce the current status of our knowledge concerning EV glycosylation and discuss how it influences EV biology, highlighting the potential roles of EV glycans in clinical applications.

DOI： 10.1007/978-3-030-70115-4_6

PubMed

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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

DOI： 10.1016/j.celrep.2020.108261

Open Access

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Oligosaccharyltransferase (OST) is a multi-span membrane protein complex that catalyzes the addition of glycans to selected Asn residues within nascent polypeptides in the lumen of the endoplasmic reticulum. This process, termed N-glycosylation, is a fundamental post-translational protein modification that is involved in the quality control, trafficking of proteins, signal transduction, and cell-to-cell communication. Given these crucial roles, N-glycosylation is essential for homeostasis at the systemic and cellular levels, and a deficiency in genes that encode for OST subunits often results in the development of complex genetic disorders. A growing body of evidence has also demonstrated that the expression of OST subunits is cell context-dependent and is frequently altered in malignant cells, thus contributing to tumor cell survival and proliferation. Importantly, a recently developed inhibitor of OST has revealed this enzyme as a potential target for the treatment of incurable drug-resistant tumors. This review summarizes our current knowledge regarding the functions of OST in the light of health and tumor progression, and discusses perspectives on the clinical relevance of inhibiting OST as a tumor treatment.

DOI： 10.3390/ijms20236074

Open Access

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Tumor cells secrete membrane vesicles of various sizes, termed extracellular vesicles (EVs), which have gained increasing attention as potential tumor diagnostic markers. Tumor-derived EVs are enriched with high-mannose-type glycans. Here, we report the affinity isolation of EVs from human melanoma A375 cells by using high-mannose-type glycan-specific agglutinin from Oscillatoria Agardhii (OAA). Glycan analysis of melanoma EVs revealed the presence of high-mannose-type glycans with structural units preferred by OAA. We showed that in solution, OAA binds to melanoma EVs in a high-mannose-type glycan-dependent manner. Furthermore, OAA-immobilized beads were found to capture 60% of the particles and most proteinous components from melanoma EVs. Major EV glycoproteins that potentially interact with OAA were identified to be cluster of differentiation 109 (CD109), integrin α6 and a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10). In addition to melanoma EVs, OAA captured EVs from human lung cancer, glioblastoma and colon cancer cells, but not those from endothelial cells and fibroblasts. These results indicate that OAA-immobilized beads may serve as a novel platform for affinity-capture of tumor-derived EVs.

DOI： 10.1016/j.ab.2019.06.001

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

We investigated the correlation between metastatic behaviors of tumor cells and asparagine-linked glycosylation (N-glycosylation) of tumor-derived extracellular vesicles (EVs). Three mouse melanoma B16 variants with distinct metastatic potentials show similar gene expression levels and enzymatic activities of glycosyltransferases involved in N-glycosylation. All melanoma variants and EVs have nearly identical profiles of de-sialylated N-glycans. The major de-sialylated N-glycan structures of cells and EVs are core-fucosylated, tetra-antennary N-glycans with β1,6-N-acetylglucosamine branches. A few N-glycans are extended by N-acetyllactosamine repeats. Sialylation of these N-glycans may generate cell-type-specific N-glycomes on EVs. Taken together, melanoma-derived EVs show high expression of tumor-associated N-glycans, and the core structure profile is inherited during multiple selection cycles of B16 melanomas and from tumor cells to EVs.

DOI： 10.1002/1873-3468.13377

Open Access

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Cells secrete heterogeneous populations of extracellular vesicles (EVs) via unknown mechanisms. EV biogenesis has been postulated to involve lipid-protein clusters, also known as membrane microdomains. METHODS: Membrane properties and heterogeneity of melanoma-derived EVs were analyzed by a detergent solubilization assay, sucrose density gradient ultracentrifugation and immunoprecipitation. EV secretion was modulated by RNA interference and pharmacological treatments. RESULTS: We identified two EV membranes (low-density exosomal detergent-insoluble membranes [EV-DIMs]; EV detergent-soluble membranes [EV-DSMs]) and discovered an abundant, novel type of high-density EV-DIMs. The high-density EV-DIMs accumulated the microdomain-resident protein flotillin-1, as well as a disintegrin and metalloproteinase domain containing protein 10 (Adam10), the hepatocyte growth factor receptor Met and its proteolytic fragments. Low-density EV-DIMs also contained flotillin-1. EV-DSMs were enriched with tetraspanin CD81, melanogenic enzymes and proteolytic fragments of Adam10. Intact and fragmented forms of Adam10, which resided in distinct membrane types, were secreted by different EVs. The fragmented form of Met was associated with DIMs much more efficiently than the intact form and they were secreted by distinct EVs. We identified that the endosomal sorting complexes required for transport machinery was indispensable for EV secretion of both mature and fragmented forms of Adam10 and Met. CONCLUSION: The findings of this study reveal the role of the interplay between membrane organization and sorting machineries in generating the heterogeneity of EVs. GENERAL SIGNIFICANCE: This study provides novel insights into important aspects of EV biogenesis.

DOI： 10.1016/j.bbagen.2019.01.015

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M115.685313

Open Access

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/glycob/cwv055

Web of Science

PubMed

Language：English  

DOI： 10.1007/s00018-015-1881-7

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

DOI： 10.3390/biom5020958

Open Access

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1073/pnas.1414593112

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2014.05.137

Web of Science

PubMed

Language：English   Publishing type：Part of collection (book)   Publisher：Springer Japan  

DOI： 10.1007/978-4-431-54240-7_9

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M113.486985

Open Access

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1073/pnas.1312187110

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M110.159517

Open Access

Web of Science

PubMed

Language：English  

DOI： 10.1073/pnas.0812489106

PubMed

Publishing type：Research paper (scientific journal)   Publisher：Gavin Publishers  

DOI： 10.29011/2574-7754.101995

Open Access

Language：English   Publishing type：Research paper (scientific journal)  

SIGNIFICANCE: Reduction-oxidation (redox) regulation is an important biological phenomenon that provides a balance between antioxidants and the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) under pathophysiological conditions. Structural and functional changes in glycans are also important as post-translational modifications of proteins. The integration of glycobiology and redox biology, called Glyco-redox has provided new insights into the mechanisms of epithelial-mesenchymal transition (EMT)/mesenchymal-epithelial transition (MET), cancer, and various diseases including Alzheimer's disease (AD), chronic obstructive lung disease (COPD), type 2 diabetes, interstitial pneumonitis, and ulcerative colitis (UC), . RECENT ADVANCES: Glycans are biosynthesized by specific glycosyltransferases and each glycosyltransferase is either directly or indirectly regulated by oxidative stress and redox regulation. A typical example of Glyco-redox is the role of N-glycan referred to as core fucose in superoxide dismutase 3 (SOD3). This glycan was found to be involved in the growth inhibition of cancer cell lines. CRITICAL ISSUES: The significance of Glyco-redox in EMT/MET, cancer and various diseases was found in major N-glycan branching glycosyltransferases GnT-III, GnT-IV, GnT-V, VI, GnT-IX, Fut8, and ST6Gal1. Herein, we summarize previous reports on the target proteins and how this relates to oxidative stress. We also discuss the products of these processes and their significance to cancer and various diseases.

DOI： 10.1089/ars.2024.0774

PubMed

Language：English   Publishing type：Research paper (other academic)   Publisher：(一社)日本癌学会  

Language：Japanese   Publishing type：Research paper (other academic)   Publisher：日本分子腫瘍マーカー研究会  

DOI： 10.11241/jsmtmr.39.31

Open Access

Language：English   Publishing type：Research paper (scientific journal)  

Glycan structure is often modulated in disease or pre-disease states, suggesting that such changes might serve as biomarkers. Here, we generated a monoclonal antibody (mAb) against the core fucose of the N-glycan in human IgG. Notably, this mAb can be used in Western blotting and ELISA. ELISA using this mAb revealed a low level of the core fucose of the N-glycan in IgG, suggesting that the level of acore fucosylated (non-core fucosylated) IgG was increased in the sera of the patients with lung cancer, COPD, and interstitial pneumonia (IP) compared to healthy subjects. In a co-culture analysis using human lung adenocarcinoma A549 cells and antibody-secreting B cells, the downregulation of the FUT8 (α1,6 fucosyltransferase) gene and a low level of core fucose of the N-glycan in IgG in antibody-secreting B cells were observed after co-culture. A dramatic alteration in gene expression profiles for cytokines, chemokines, and their receptors were also observed after co-culturing, and we found that the identified C-C motif chemokine 2 (CCL2) was partially involved in the downregulation of the FUT8 gene and the low level of core fucose of the N-glycan in IgG in antibody-secreting B cells. We also developed a latex turbidimetric immunoassay using this mAb. These results suggest that communication with CCL2 between lung cells and antibody-secreting B cells downregulate the level of core fucose of the N-glycan in IgG, i.e., the increased level of acore fucosylated (non-core fucosylated) IgG, which would be a novel biomarker for the diagnosis of patients with pulmonary diseases.

DOI： 10.1016/j.jbc.2023.105365

Open Access

PubMed

Language：Japanese   Publishing type：Research paper (other academic)   Publisher：日本分子腫瘍マーカー研究会  

Language：English   Publishing type：Research paper (scientific journal)  

Chronic obstructive pulmonary disease (COPD), which includes emphysema and chronic bronchitis, is now the third cause of death worldwide, and COVID-19 infection has been reported as an exacerbation factor of them. In this study, we report that the intratracheal administration of the keratan sulfate-based disaccharide L4 mitigates the symptoms of elastase-induced emphysema in a mouse model. To know the molecular mechanisms, we performed a functional analysis of a C-type lectin receptor, langerin, a molecule that binds L4. Using mouse BMDCs (bone marrow-derived dendritic cells) as langerin-expressing cells, we observed the downregulation of IL-6 and TNFa and the upregulation of IL-10 after incubation with L4. We also identified CapG (a macrophage-capping protein) as a possible molecule that binds langerin by immunoprecipitation combined with a mass spectrometry analysis. We identified a portion of the CapG that was localized in the nucleus and binds to the promoter region of IL-6 and the TNFa gene in BMDCs, suggesting that CapG suppresses the gene expression of IL-6 and TNFa as an inhibitory transcriptional factor. To examine the effects of L4 in vivo, we also generated langerin-knockout mice by means of genome editing technology. In an emphysema mouse model, the administration of L4 did not mitigate the symptoms of emphysema as well as the inflammatory state of the lung in the langerin-knockout mice. These data suggest that the anti-inflammatory effect of L4 through the langerin-CapG axis represents a potential therapeutic target for the treatment of emphysema and COPD.

DOI： 10.1016/j.jbc.2023.105052

Open Access

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Over the past 3 decades, our group has been involved in studies related to the biosynthesis of N-glycan branching and related glycosyltransferases and have purified most of these Golgi-derived enzymes to homogeneity using classical purification methods and cloned the cDNA of GnT-III, IV, V, VI and Fut8 except GnT-IX(Vb) which was obtained by homology cloning. Based primarily on our data, we briefly summarize the significance of three major enzymes and discuss perspectives for future studies on the occasion of Ernesto's 90th birthday celebration.

DOI： 10.1016/j.bbrc.2022.09.027

PubMed

Language：Japanese   Publisher：(公社)日本生化学会  

Language：Japanese   Publisher：(公社)日本生化学会  

Language：English   Publisher：(一社)日本癌学会  

Language：English   Publisher：(一社)日本癌学会  

Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/1346-8138.16506

Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/1346-8138.16506

Publishing type：Research paper (scientific journal)  

DOI： 10.3390/ijms22168579

Open Access

Scopus

Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.mam.2020.100905

Language：English   Publishing type：Research paper (scientific journal)  

Glycosylation represents one of the most abundant posttranslational modification of proteins. Glycosylation products are diverse and are regulated by the cooperative action of various glycosyltransferases, glycosidases, substrates thereof: nucleoside sugars and their transporters, and chaperons. In this article, we focus on a glycosyltransferase, α1,6-fucosyltransferase (Fut8) and its product, the core fucose structure on N-glycans, and summarize the potential protective functions of this structure against emphysema and chronic obstructive pulmonary disease (COPD). Studies of FUT8 and its enzymatic product, core fucose, are becoming an emerging area of interest in various fields of research including inflammation, cancer and therapeutics. This article discusses what we can learn from studies of Fut8 and core fucose by using knockout mice or in vitro studies that were conducted by our group as well as other groups. We also include a discussion of the potential protective functions of the keratan sulfate (KS) disaccharide, namely L4, against emphysema and COPD as a glycomimetic. Glycomimetics using glycan analogs is one of the more promising therapeutics that compensate for the usual therapeutic strategy that involves targeting the genome and the proteome. These typical glycans using KS derivatives as glycomimetics, will likely become a clue to the development of novel and effective therapeutic strategies.

DOI： 10.1042/BST20200780

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Glycosylation, the most common posttranslational modification of proteins, is a stepwise process that relies on tight regulation of subcellular glycosyltransferase location to control the addition of each monosaccharide. Glycosyltransferases primarily reside and function in the endoplasmic reticulum (ER) and the Golgi apparatus; whether and how they traffic beyond the-Golgi, how this trafficking is controlled, and how it impacts glycosylation remain unclear. Our previous work identified a connection between N-glycosylation and Rab11, a key player in the post-Golgi transport that connects recycling endosomes and other compartments. To learn more about the specific role of Rab11, we knocked down Rab11 in HeLa cells. Our findings indicate that Rab11 knockdown results in a dramatic enhancement in the sialylation of N-glycans. Structural analyses of glycans using lectins and LC-MS revealed that α2,3-sialylation is selectively enhanced, suggesting that an α2,3-sialyltransferase that catalyzes the sialyation of glycoproteins is activated or upregulated as the result of Rab11 knockdown. ST3GAL4 is the major α2,3-sialyltransferase that acts on N-glycans; we demonstrated that the localization of ST3GAL4, but not the levels of its mRNA, protein, or donor substrate, was altered by Rab11 depletion. In knockdown cells, ST3GAL4 is densely distributed in the trans-Golgi network, compared with the wider distribution in the Golgi and in other peripheral puncta in control cells, whereas the α2,6-sialyltransferase ST6GAL1 is predominantly localized to the Golgi regardless of Rab11 knockdown. This indicates that Rab11 may negatively regulate α2,3-sialylation by transporting ST3GAL4 to post-Golgi compartments (PGCs) which is a novel mechanism of glycosyltransferase regulation.

DOI： 10.1016/j.jbc.2021.100354

Open Access

PubMed

Publishing type：Part of collection (book)   Publisher：Elsevier  

DOI： 10.1016/b978-0-12-819475-1.00030-4

Language：English   Publishing type：Research paper (scientific journal)  

Uric acid (UA) therapy may prevent early ischemic worsening after acute stroke in thrombolysis patients. The aim of this study was to examine the influence of UA on the thrombolytic efficacy of alteplase in human blood samples by measuring thrombolysis under flow conditions using a newly developed microchip-based flow-chamber assay. Human blood samples from healthy volunteers were exposed to UA, alteplase, or a combination of UA and alteplase. Whole blood and platelet-rich plasma were perfused over a collagen- and thromboplastin-coated microchip, and capillary occlusion was monitored with a video microscope and flow-pressure sensor. The area under the curve (extent of thrombogenesis or thrombolysis) at 30 minutes was 92% lower in the UA-alteplase-treated group compared with the alteplase-treated group. D-dimers were measured to evaluate these effects in human platelet-poor plasma samples. Although hydrogen peroxide significantly decreased the elevation of D-dimers by alteplase, UA significantly inhibited the effect of hydrogen peroxide. Meanwhile, rat models of thromboembolic cerebral ischemia were treated with either alteplase or UA-alteplase combination therapy. Compared with alteplase alone, the combination therapy reduced the infarct volume and inhibited haemorrhagic transformation. UA enhances alteplase-mediated thrombolysis, potentially by preventing oxidative stress, which inhibits fibrinolysis by alteplase in thrombi.

DOI： 10.1038/s41598-018-34220-1

Open Access

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science  

DOI： 10.1371/journal.pgen.1006696

Open Access

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1155/2017/6873281

Open Access

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.pone.0151891

Open Access

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1080/09168451.2015.1072464

Web of Science

PubMed

Language：English  

Web of Science

Language：English  

DOI： 10.1016/j.bbrc.2014.05.075

Web of Science

PubMed

Language：Japanese   Publisher：FCCA(Forum: Carbohydrates Coming of Age)  

DOI： 10.4052/tigg.26.33

Open Access

CiNii Research

Other Link： https://jlc.jst.go.jp/DN/JALC/10027903024?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M113.464503

Open Access

Scopus

PubMed

J-GLOBAL

J-GLOBAL

Language：English   Publisher：(一社)日本癌学会  

Language：English   Publisher：(一社)日本癌学会  

Language：English   Publishing type：Research paper, summary (international conference)  

DOI： 10.1096/fasebj.2020.34.s1.03741

Web of Science

Language：English   Publisher：(一社)日本癌学会  

Language：Japanese  

CiNii Research

Language：English   Publishing type：Book review, literature introduction, etc.  

DOI： 10.4052/tigg.1512.1E

Web of Science

Language：English   Publishing type：Research paper, summary (international conference)  

Web of Science

Language：English  

DOI： 10.4052/tigg.25.91

Web of Science