A highly specific antibody against the core fucose of the N-glycan in IgG identifies the pulmonary diseases and its regulation by CCL2.

Glycan structure is often modulated in disease or predisease 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 (noncore fucosylated) IgG was increased in the sera of the patients with lung cancer, chronic obstructive pulmonary disease, and interstitial pneumonia compared to healthy subjects. In a coculture 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 coculture. A dramatic alteration in gene expression profiles for cytokines, chemokines, and their receptors were also observed after coculturing, and we found that the identified C-C motif chemokine 2 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 C-C motif chemokine 2 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 (noncore fucosylated) IgG, which would be a novel biomarker for the diagnosis of patients with pulmonary diseases.

Metabolic clogging of mannose triggers dNTP loss and genomic instability in human cancer cells.

Mannose has anticancer activity that inhibits cell proliferation and enhances the efficacy of chemotherapy. How mannose exerts its anticancer activity, however, remains poorly understood. Here, using genetically engineered human cancer cells that permit the precise control of mannose metabolic flux, we demonstrate that the large influx of mannose exceeding its metabolic capacity induced metabolic remodeling, leading to the generation of slow-cycling cells with limited deoxyribonucleoside triphosphates (dNTPs). This metabolic remodeling impaired dormant origin firing required to rescue stalled forks by cisplatin, thus exacerbating replication stress. Importantly, pharmacological inhibition of de novo dNTP biosynthesis was sufficient to retard cell cycle progression, sensitize cells to cisplatin, and inhibit dormant origin firing, suggesting dNTP loss-induced genomic instability as a central mechanism for the anticancer activity of mannose.

In order to grow and divide, cells require a variety of sugars. Breaking down sugars provides energy for cells to proliferate and allows them to make more complex molecules, such as DNA. Although this principle also applies to cancer cells, a specific sugar called mannose not only inhibits cancer cell division but also makes them more sensitive to chemotherapy. These anticancer effects of mannose are particularly strong in cells lacking a protein known as MPI, which breaks down mannose. Evidence from honeybees suggests that a combination of mannose and low levels of MPI leads to a build-up of a modified form of mannose, called mannose-6-phosphate, within cells. As a result, pathways required to release energy from glucose become disrupted, proving lethal to these insects. However, it was not clear whether the same processes were responsible for the anticancer effects of mannose. To investigate, Harada et al. removed the gene that encodes the MPI protein in two types of human cancer cells. The experiments showed that mannose treatment was not lethal to these cells but overall slowed the cell cycle ­ a fundamental process for cell growth and division. More detailed biochemical experiments showed that cancer cells with excess mannose-6-phosphate could not produce the molecules required to make DNA. This prevented them from doubling their DNA ­ a necessary step for cell division ­ and responding to stress caused by chemotherapy. Harada et al. also noticed that cancer cells lacking MPI did not all react to mannose treatment in exactly the same way. Therefore, future work will address these diverse reactions, potentially providing an opportunity to use the mannose pathway to search for new cancer treatments.

Involvement of langerin in the protective function of a keratan sulfate-based disaccharide in an emphysema mouse model.

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.

N-acetylglucosaminyltransferase-V (GnT-V)-enriched small extracellular vesicles mediate N-glycan remodeling in recipient cells.

Small extracellular vesicles (sEVs) secreted from cancer cells play pivotal roles in cancer metastasis and malignancy by transferring biomolecules and conditioning future metastatic sites. Studies have elucidated structures and functions of glycans on sEVs; however, whether sEVs remodel glycans in recipient cells remains poorly understood. Here, we examined the enzyme activity of glycosyltransferases for complex N-glycan biosynthesis in cancer-derived sEVs and discovered that cancer-related glycosyltransferase, N-acetylglucosaminyltransferase-V (GnT-V, a.k.a. MGAT5), is selectively enriched in sEVs among various glycosyltransferases. GnT-V in sEVs is a cleaved form, and cleavage by SPPL3 protease is necessary for loading GnT-V in sEVs. Fractionation experiments and single-particle imaging further revealed that GnT-V was enriched in non-exosomal sEVs. Strikingly, we found that enzymatically active GnT-V in sEVs was transferred to recipient cells and the N-glycan structures of recipient cells were remodeled to express GnT-V-produced glycans. Our results suggest GnT-V-enriched sEVs' role in glycan remodeling in cancer metastasis.

N-glycan branching enzymes involved in cancer, Alzheimer's disease and COPD and future perspectives.

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.

The ulcerative colitis-associated gene FUT8 regulates the quantity and quality of secreted mucins.

Mucins are the main macrocomponents of the mucus layer that protects the digestive tract from pathogens. Fucosylation of mucins increases mucus viscoelasticity and its resistance to shear stress. These properties are altered in patients with ulcerative colitis (UC), which is marked by a chronic inflammation of the distal part of the colon. Here, we show that levels of Fucosyltransferase 8 (FUT8) and specific mucins are increased in the distal inflamed colon of UC patients. Recapitulating this FUT8 overexpression in mucin-producing HT29-18N2 colonic cell line increases delivery of MUC1 to the plasma membrane and extracellular release of MUC2 and MUC5AC. Mucins secreted by FUT8 overexpressing cells are more resistant to removal from the cell surface than mucins secreted by FUT8-depleted cells (FUT8 KD). FUT8 KD causes intracellular accumulation of MUC1 and alters the ratio of secreted MUC2 to MUC5AC. These data fit well with the Fut8-/- mice phenotype, which are protected from UC. Fut8-/- mice exhibit a thinner proximal colon mucus layer with an altered ratio of neutral to acidic mucins. Together, our data reveal that FUT8 modifies the biophysical properties of mucus by controlling levels of cell surface MUC1 and quantity and quality of secreted MUC2 and MUC5AC. We suggest that these changes in mucus viscoelasticity likely facilitate bacterial-epithelial interactions leading to inflammation and UC progression.

The expression of keratan sulfate in malignant melanoma enhances the adhesion and invasion activity of melanoma cells.

Mammals express a wide variety of glycans that include N-glycans, O-glycans, proteoglycans, glycolipids, etc. Glycan expression can modulate the cellular functions, and hence is strongly involved in the onset and progression of numerous diseases. Here, we report the relevance of the ectopic expression of keratan sulfate (KS) glycan chains in human malignant melanomas. Using a human melanoma cell line, we found that the KS enhanced the invasiveness of the cells but caused no change in the growth rate of the cells. The phosphorylation of paxillin, a focal adhesion-associated adaptor protein, was strong at the region where KS was expressed in the melanoma tissues, indicating that KS stimulated the phosphorylation of paxillin. We also observed that KS enhanced the adhesion of melanoma cells and this was accompanied by a greatly increased level of phosphorylation of paxillin. These data suggest that the expression of KS contributes to the development of malignant phenotypes such as strong cell adhesion and the invasiveness of melanoma cells.

Identification of distinct N-glycosylation patterns on extracellular vesicles from small-cell and non-small-cell lung cancer cells.

Asparagine-linked glycosylation (N-glycosylation) of proteins in the cancer secretome has been gaining increasing attention as a potential biomarker for cancer detection and diagnosis. Small extracellular vesicles (sEVs) constitute a large part of the cancer secretome, yet little is known about whether their N-glycosylation status reflects known cancer characteristics. Here, we investigated the N-glycosylation of sEVs released from small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC) cells. We found that the N-glycans of SCLC-sEVs were characterized by the presence of structural units also found in the brain N-glycome, while NSCLC-sEVs were dominated by typical lung-type N-glycans with NSCLC-associated core fucosylation. In addition, lectin-assisted N-glycoproteomics of SCLC-sEVs and NSCLC-sEVs revealed that integrin αV was commonly expressed in sEVs of both cancer cell types, while the epithelium-specific integrin α6ß4 heterodimer was selectively expressed in NSCLC-sEVs. Importantly, N-glycomics of the immunopurified integrin α6 from NSCLC-sEVs identified NSCLC-type N-glycans on this integrin subunit. Thus, we conclude that protein N-glycosylation in lung cancer sEVs may potentially reflect the histology of lung cancers.

Glycan quality control in and out of the endoplasmic reticulum of mammalian cells.

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.

Extracellular Vesicles and Glycosylation.

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.

Peptide Sequence Mapping around Bisecting GlcNAc-Bearing N-Glycans in Mouse Brain.

N-glycosylation is essential for many biological processes in mammals. A variety of N-glycan structures exist, of which, the formation of bisecting N-acetylglucosamine (GlcNAc) is catalyzed by N-acetylglucosaminyltransferase-III (GnT-III, encoded by the Mgat3 gene). We previously identified various bisecting GlcNAc-modified proteins involved in Alzheimer's disease and cancer. However, the mechanisms by which GnT-III acts on the target proteins are unknown. Here, we performed comparative glycoproteomic analyses using brain membranes of wild type (WT) and Mgat3-deficient mice. Target glycoproteins of GnT-III were enriched with E4-phytohemagglutinin (PHA) lectin, which recognizes bisecting GlcNAc, and analyzed by liquid chromatograph-mass spectrometry. We identified 32 N-glycosylation sites (Asn-Xaa-Ser/Thr, Xaa ≠ Pro) that were modified with bisecting GlcNAc. Sequence alignment of identified N-glycosylation sites that displayed bisecting GlcNAc suggested that GnT-III does not recognize a specific primary amino acid sequence. The molecular modeling of GluA1 as one of the good cell surface substrates for GnT-III in the brain, indicated that GnT-III acts on N-glycosylation sites located in a highly flexible and mobile loop of GluA1. These results suggest that the action of GnT-III is partially affected by the tertiary structure of target proteins, which can accommodate bisecting GlcNAc that generates a bulky flipped-back conformation of the modified glycans.

Protocol for analyzing the biosynthesis and degradation of N-glycan precursors in mammalian cells.

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).

Keratan sulfate-based glycomimetics using Langerin as a target for COPD: lessons from studies on Fut8 and core fucose.

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.

Rab11-mediated post-Golgi transport of the sialyltransferase ST3GAL4 suggests a new mechanism for regulating glycosylation.

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.

True significance of N-acetylglucosaminyltransferases GnT-III, V and α1,6 fucosyltransferase in epithelial-mesenchymal transition and cancer.

It is well known that numerous cancer-related changes occur in glycans that are attached to glycoproteins, glycolipids and proteoglycans on the cell surface and these changes in structure and the expression of the glycans are largely regulated by glycosyl-transferases, glycosidases, nucleotide sugars and their related genes. Such structural changes in glycans on cell surface proteins may accelerate the progression, invasion and metastasis of cancer cells. Among the over 200 known glycosyltransferases and related genes, ß 1,6 N-acetylglucosaminyltransferase V (GnT-V) (the MGAT5 gene) and α 1,6 fucosyltransferase (FUT8) (the FUT8 gene) are representative enzymes in this respect because changes in glycans caused by these genes appear to be related to cancer metastasis and invasion in vitro as well as in vivo, and a number of reports on these genes in related to epithelial-mesenchymal transition (EMT) have also appeared. Another enzyme, one of the N-glycan branching enzymes, ß1,4 N-acetylglucosaminyltransferase III (GnT-III) (the MGAT3 gene) has been reported to suppress EMT. However, there are intermediate states between EMT and mesenchymal-epithelial transition (MET) and some of these genes have been implicated in both EMT and MET and are also probably in an intermediate state. Therefore, it would be difficult to clearly define which specific glycosyltransferase is involved in EMT or MET or an intermediate state. The significance of EMT and N-glycan branching glycosyltransferases needs to be reconsidered and the inhibition of their corresponding genes would also be desirable in therapeutics. This review mainly focuses on GnT-III, GnT-V and FUT8, major players as N-glycan branching enzymes in cancer in relation to EMT programs, and also discusses the catalytic mechanisms of GnT-V and FUT8 whose crystal structures have now been obtained.

Glycometabolic Regulation of the Biogenesis of Small Extracellular Vesicles.

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.

Oligosaccharyltransferase: A Gatekeeper of Health and Tumor Progression.

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.

Application of high-mannose-type glycan-specific lectin from Oscillatoria Agardhii for affinity isolation of tumor-derived extracellular vesicles.

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.

N-glycome inheritance from cells to extracellular vesicles in B16 melanomas.

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 ß1,6-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.