The Human Glycome Atlas Project for cataloging all glycan-related omics data in human.

The Human Glycome Atlas (HGA) Project was launched in April 2023, spearheaded by three Japanese institutes: the Tokai National Higher Education and Research System, the National Institutes of Natural Sciences, and Soka University. This was the first time that a field in the life sciences was adopted by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) for a Large-scale Academic Frontiers Promotion Project. This project aims to construct a knowledgebase of human glycans and glycoproteins as a standard for the human glycome. A high-throughput pipeline for comprehensively analyzing 20,000 blood samples in its first five years is planned, at which time an access-controlled version of a human glycomics knowledgebase, called TOHSA, will be released. By the end of the final tenth year, TOHSA will provide a central resource linking human glycan data with other omics data including disease-related information.

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

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

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

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

Inhibitory machinery for the functional dystroglycan glycosylation.

Dystroglycan (DG), a muscular transmembrane protein, plays a critical role in transducing extracellular matrix-derived signals to the cytoskeleton and provides physical strength to skeletal muscle cell membranes. The extracellular domain of DG, α-DG, displays unique glycosylation patterns. Fully functional glycosylation is required for this domain to interact with components of extracellular matrices, including laminin. One of the unique sugar compositions found in such functional glycans on DG is two ribitol phosphates that are transferred by the sequential actions of fukutin (FKTN) and fukutin-related protein (FKRP), which use CDP-ribitol as a donor substrate. These are then further primed for matriglycan biosynthesis. A recent in vitro study reported that glycerol phosphate could be similarly added to α-DG by FKTN and FKRP if they used CDP-glycerol (CDP-Gro) as a donor substrate. However, the physiological relevance of these findings remains elusive. Imae et al. addressed the knowledge gap regarding whether CDP-Gro is present in mammals and how CDP-Gro is synthesized and functions in mammals.

Evidence that only EWS among the FET proteins acquires a low partitioning property for the hyperosmotic stress response by O-GlcNAc glycosylation on its low-complexity domain.

FET proteins (FUS, EWS, and TAF15) share a common domain organization, bind RNA/DNA, and perform similarly multifunctional roles in the regulation of gene expression. Of the FET proteins, however, only EWS appears to have a distinct property in the cellular stress response. Therefore, we focused on the relationship between hyperosmotic stress response and post-translational modifications of the FET proteins. We confirmed that the hyperosmotic stress-dependent translocation from the nucleus to the cytoplasm and the cellular granule formation of FET proteins, and that EWS is less likely to partition into cellular granules in the cytoplasm than FUS or TAF15. The domain involved in the less partitioning property of EWS was found to be its low-complexity domain (LCD). Chemoenzymatic labeling analysis of O-linked ß-N-acetylglucosamine (O-GlcNAc) residues revealed that O-GlcNAc glycosylation occurs frequently in the LCD of EWS. A correlation was observed between the glycosylation of EWS and the less partitioning property under the hyperosmotic stress. These results suggest that among the FET proteins, only EWS has acquired the unique property through O-GlcNAc glycosylation. The glycosylation may play an essential role in regulating physiological functions of EWS, such as transcriptional activity, in addition to the property in cellular stress response.

Secretory expression of mammalian NOTCH tandem epidermal growth factor-like repeats based on increased O-glycosylation.

The Notch pathway represents evolutionarily conserved intercellular signaling essential for cell-to-cell communication during development. Dysregulation of Notch signaling has been implicated in various diseases, and its control represents a potential cancer treatment strategy. Notch signaling is initiated by the interaction of NOTCH receptors with their ligands on neighboring cells. Therefore, the truncated NOTCH ectodomain, composed mainly of tandem repeats of epidermal growth factor-like (EGF) domains, serves as a decoy molecule that competes for ligand binding and thus inhibits ligand-dependent Notch signaling. Although full-length NOTCH EGF repeats exhibited potent Notch inhibitory activity, they were poorly produced in the transfected cells. This study evaluated the effect of EGF domain-modifying glycosyltransferases on the secretion of NOTCH EGF repeats. Our results in HEK293T cells revealed that, unlike the effect on endogenous NOTCH receptors, overexpressed EGF domain-specific O-GlcNAc transferase (EOGT) markedly enhanced the secretion of NOTCH1 EGF repeats in an enzyme activity-dependent manner. The co-expression of protein O-glucosyltransferase 1 further manifested the effect of EOGT. The resultant changes in O-glycosylation of NOTCH3 were evaluated by label-free glycopeptide quantification. This study provides an experimental strategy to efficiently generate NOTCH EGF repeats by manipulating the expression of glycosyltransferases that alter the O-glycosylation of EGF domains.

Significant Roles of Notch O-Glycosylation in Cancer.

Notch signaling, which was initially identified in Drosophila wing morphogenesis, plays pivotal roles in cell development and differentiation. Optimal Notch pathway activity is essential for normal development and dysregulation of Notch signaling leads to various human diseases, including many types of cancers. In hematopoietic cancers, such as T-cell acute lymphoblastic leukemia, Notch plays an oncogenic role, while in acute myeloid leukemia, it has a tumor-suppressive role. In solid tumors, such as hepatocellular carcinoma and medulloblastoma, Notch may have either an oncogenic or tumor-suppressive role, depending on the context. Aberrant expression of Notch receptors or ligands can alter the ligand-dependent Notch signaling and changes in trafficking can lead to ligand-independent signaling. Defects in any of the two signaling pathways can lead to tumorigenesis and tumor progression. Strikingly, O-glycosylation is one such process that modulates ligand-receptor binding and trafficking. Three types of O-linked modifications on the extracellular epidermal growth factor-like (EGF) repeats of Notch receptors are observed, namely O-glucosylation, O-fucosylation, and O-N-acetylglucosamine (GlcNAc) modifications. In addition, O-GalNAc mucin-type O-glycosylation outside the EGF repeats also appears to occur in Notch receptors. In this review, we first briefly summarize the basics of Notch signaling, describe the latest information on O-glycosylation of Notch receptors classified on a structural basis, and finally describe the regulation of Notch signaling by O-glycosylation in cancer.

Glycoproteomics of NOTCH1 EGF repeat fragments overexpressed with different glycosyltransferases in HEK293T cells reveals insights into O-GlcNAcylation of NOTCH1.

O-GlcNAc modification of Notch receptors regulates Notch ligand interactions in a manner distinct from other forms of O-glycans on epidermal growth factor (EGF)-like repeats of Notch receptors. Although many proteins, besides Notch receptors, are expected to be O-GlcNAcylated by EGF domain-specific O-GlcNAc transferase (EOGT), only a small number of proteins have been reported to be modified in vivo, and elongated O-GlcNAc glycans have not been extensively explored. To extend our view of the specificity and variety of the glycan modification, we conducted a comprehensive analysis of O-GlcNAc glycans on NOTCH1 in mammals. Mass spectrometric analysis of NOTCH1 fragments expressed in HEK293T cells revealed that several EGF domains with putative O-GlcNAcylation sites were hardly modified with O-GlcNAc. Although amino acid residues before the modification site are preferentially occupied with aromatic residues, Phe and Tyr are preferable to Trp for the apparent modification with O-GlcNAc. Furthermore, a minor form of fucosylated O-GlcNAc glycans was detected in a subset of EGF domains. Fucosylation of O-GlcNAc glycans was enhanced by FUT1, FUT2, or FUT9 expression. The FUT9-dependent Lewis X epitope was confirmed by immunoblotting using an anti-Lewis X antibody. As expected from the similarity in the extended structures between O-Fuc and O-GlcNAc glycans, the Lexis X antigen was detected on NOTCH1 fragments co-expressed with L-Fringe, which mediates elongation of O-Fuc glycans. Our results refined the putative consensus sequence for the EOGT-dependent O-GlcNAc modification in mammals and revealed the structural diversity of functional Notch O-glycans.

Contribution of Glucosylceramide Synthase to the Proliferation of Mouse Osteoblasts.

BACKGROUND/AIM: Glycosphingolipids are known to be involved in bone metabolism. However, their roles and regulatory mechanisms in osteoblast proliferation are largely unknown. In this study, we examined the effects of inhibitors of glucosylceramide synthase (GCS), which is responsible for the generation of all glycosphingolipids, on osteoblast proliferation. MATERIALS AND METHODS: We analyzed the expression of glycosphingolipids and cell growth in MC3T3-E1 mouse osteoblast cells treated with the GCS inhibitors miglustat, D-PDMP and D-PPMP. We also conducted microarray analysis and RNA interference to identify genes involved in cell growth regulated by GCS. RESULTS: Glycosphingolipids GD1a and Gb4 expressed in MC3T3-E1 cells, were suppressed by GCS inhibitors. Furthermore, the proliferation of MC3T3-E1 cells was suppressed by the inhibitors. Using microarray analysis, we predicted nine genes (Fndc1, Acta2, Igfbp5, Cox6a2, Cth, Mymk, Angptl6, Mab21l2, and Igsf10) suppressed by all three inhibitors. Furthermore, partial silencing of Angptl6 by RNA interference reduced MC3T3-E1 cell growth. CONCLUSION: These results show that GCS regulates proliferation through Angptl6 in osteoblasts.

St8sia1-deficiency in mice alters tumor environments of gliomas, leading to reduced disease severity.

Ganglioside GD3/GD2 are over-expressed in various neuroectoderm-derived tumors. Previous studies indicated that GD3 is involved in the enhancement of cancer properties such as rapid growth and increased invasiveness. However, little is known about the functions of GD3/GD2 in glioma cells and glioma microenvironments. To clarify the functions of GD3/GD2 in gliomas, we used a mouse glioma model based on the RCAS/Gtv-a system. At first, we compared the gliomas size between wild-type (WT) and GD3 synthase (GD3S) knockout (KO) mice, showing a less malignant histology and slower tumor growth in GD3S-KO mice than in WT mice. Immunohistochemistry of glioma sections from WT and GD3S-KO mice revealed that reactive microglia/macrophages showed different localization patterns between the two genetic types of mice. CD68+ cells were more frequently stained inside glioma tissues of GD3S-KO mice, while they were stained mainly around glioma tissues in WT mice. The number of CD68+ cells markedly increased in tumor tissues of GD3S-KO mice at 2 weeks after injection of transfectant DF-1 cells. Furthermore, CD68+ cells in GD3S(-/-) glioma tissues expressed higher levels of inducible nitric oxide synthase. We observed higher expression levels of pro-inflammatory cytokine genes in primary-cultured glioma cells of WT mice than in GD3S-KO mice. DNA microarray data also revealed differential expression levels of various cytokines and chemokines in glioma tissues between WT and GD3S-KO mice. These results suggest that expression of GD3S allows glioma cells to promote polarization of microglia/macrophages towards M2-like phenotypes by modulating the expression levels of chemokines and cytokines.

Current Views on the Roles of O-Glycosylation in Controlling Notch-Ligand Interactions.

The 100th anniversary of Notch discovery in Drosophila has recently passed. The Notch is evolutionarily conserved from Drosophila to humans. The discovery of human-specific Notch genes has led to a better understanding of Notch signaling in development and diseases and will continue to stimulate further research in the future. Notch receptors are responsible for cell-to-cell signaling. They are activated by cell-surface ligands located on adjacent cells. Notch activation plays an important role in determining the fate of cells, and dysregulation of Notch signaling results in numerous human diseases. Notch receptors are primarily activated by ligand binding. Many studies in various fields including genetics, developmental biology, biochemistry, and structural biology conducted over the past two decades have revealed that the activation of the Notch receptor is regulated by unique glycan modifications. Such modifications include O-fucose, O-glucose, and O-N-acetylglucosamine (GlcNAc) on epidermal growth factor-like (EGF) repeats located consecutively in the extracellular domain of Notch receptors. Being fine-tuned by glycans is an important property of Notch receptors. In this review article, we summarize the latest findings on the regulation of Notch activation by glycosylation and discuss future challenges.

Bioinformatics and Functional Analyses Implicate Potential Roles for EOGT and L-fringe in Pancreatic Cancers.

Notch signaling receptors, ligands, and their downstream target genes are dysregulated in pancreatic ductal adenocarcinoma (PDAC), suggesting a role of Notch signaling in pancreatic tumor development and progression. However, dysregulation of Notch signaling by post-translational modification of Notch receptors remains poorly understood. Here, we analyzed the Notch-modifying glycosyltransferase involved in the regulation of the ligand-dependent Notch signaling pathway. Bioinformatic analysis revealed that the expression of epidermal growth factor (EGF) domain-specific O-linked N-acetylglucosamine (EOGT) and Lunatic fringe (LFNG) positively correlates with a subset of Notch signaling genes in PDAC. The lack of EOGT or LFNG expression inhibited the proliferation and migration of Panc-1 cells, as observed by the inhibition of Notch activation. EOGT expression is significantly increased in the basal subtype, and low expression of both EOGT and LFNG predicts better overall survival in PDAC patients. These results imply potential roles for EOGT- and LFNG-dependent Notch signaling in PDAC.

Ganglioside GD2 Enhances the Malignant Phenotypes of Melanoma Cells by Cooperating with Integrins.

Gangliosides have been considered to modulate cell signals in the microdomain of the cell membrane, lipid/rafts, or glycolipid-enriched microdomain/rafts (GEM/rafts). In particular, cancer-associated gangliosides were reported to enhance the malignant properties of cancer cells. In fact, GD2-positive (GD2+) cells showed increased proliferation, invasion, and adhesion, compared with GD2-negative (GD2-) cells. However, the precise mechanisms by which gangliosides regulate cell signaling in GEM/rafts are not well understood. In order to analyze the roles of ganglioside GD2 in the malignant properties of melanoma cells, we searched for GD2-associating molecules on the cell membrane using the enzyme-mediated activation of radical sources combined with mass spectrometry, and integrin ß1 was identified as a representative GD2-associating molecule. Then, we showed the physical association of GD2 and integrin ß1 by immunoprecipitation/immunoblotting. Close localization was also shown by immuno-cytostaining and the proximity ligation assay. During cell adhesion, GD2+ cells showed multiple phospho-tyrosine bands, i.e., the epithelial growth factor receptor and focal adhesion kinase. The knockdown of integrin ß1 revealed that the increased malignant phenotypes in GD2+ cells were clearly cancelled. Furthermore, the phosphor-tyrosine bands detected during the adhesion of GD2+ cells almost completely disappeared after the knockdown of integrin ß1. Finally, immunoblotting to examine the intracellular distribution of integrins during cell adhesion revealed that large amounts of integrin ß1 were localized in GEM/raft fractions in GD2+ cells before and just after cell adhesion, with the majority being localized in the non-raft fractions in GD2- cells. All these results suggest that GD2 and integrin ß1 cooperate in GEM/rafts, leading to enhanced malignant phenotypes of melanomas.

Majority of alpha2,6-sialylated glycans in the adult mouse brain exist in O-glycans: SALSA-MS analysis for knockout mice of alpha2,6-sialyltransferase genes.

Sialic acids are unique sugars with negative charge and exert various biological functions such as regulation of immune systems, maintenance of nerve tissues and expression of malignant properties of cancers. Alpha 2,6 sialylated N-glycans, one of representative sialylation forms, are synthesized by St6gal1 or St6gal2 gene products in humans and mice. Previously, it has been reported that St6gal1 gene is ubiquitously expressed in almost all tissues. On the other hand, St6gal2 gene is expressed mainly in the embryonic and perinatal stages of brain tissues. However, roles of St6gal2 gene have not been clarified. Expression profiles of N-glycans with terminal α2,6 sialic acid generated by St6gal gene products in the brain have never been directly studied. Using conventional lectin blotting and novel sialic acid linkage-specific alkylamidationmass spectrometry method (SALSA-MS), we investigated the function and expression of St6gal genes and profiles of their products in the adult mouse brain by establishing KO mice lacking St6gal1 gene, St6gal2 gene, or both of them (double knockout). Consequently, α2,6-sialylated N-glycans were scarcely detected in adult mouse brain tissues, and a majority of α2,6-sialylated glycans found in the mouse brain were O-linked glycans. The majority of these α2,6-sialylated O-glycans were shown to be disialyl-T antigen and sialyl-(6)T antigen by mass spectrometry analysis. Moreover, it was revealed that a few α2,6-sialylated N-glycans were produced by the action of St6gal1 gene, despite both St6gal1 and St6gal2 genes being expressed in the adult mouse brain. In the future, where and how sialylated O-linked glycoproteins function in the brain tissue remains to be clarified.

Glycoproteomic analysis identifies cryptdin-related sequence 1 as O-glycosylated protein modified with α1,2-fucose in the small intestine.

The modification of galactose with α1,2-fucose is involved in symbiosis with intestinal bacteria and elimination of pathogenic bacteria. It is postulated that α1,2-fucosylated mucin secreted from goblet cells is involved in defending an organism against infections, but the detailed molecular mechanisms are yet to be elucidated. It was previously reported that Paneth cells of the small intestine were positive for UEA-1 lectin staining. However, glycoproteins in Paneth cells carrying α1,2-fucose have not yet been identified. Glycoproteomic analysis of ileal lysates identified 3212 O-linked and 2962 N-linked glycopeptides. In particular, cryptdin-related sequence 1 (CRS1) expressed in Paneth cells was found to be α1,2-fucosylated. Unlike other antimicrobial α-defensin proteins, CRS1 contains unique Thr residues, which are modified with O-glycans, with 3HexNAc2Hex1Fuc1NeuAc being the main glycoform. Identification of α1,2-fucose on the O-glycans of CRS1 expressed in Paneth cells will pave the way for a mechanistic understanding of α1,2-fucose-dependent symbiosis with intestinal bacteria and elimination of pathogenic bacteria in the intestine.

Differential Labeling of Glycoproteins with Alkynyl Fucose Analogs.

Fucosylated glycans critically regulate the physiological functions of proteins and cells. Alterations in levels of fucosylated glycans are associated with various diseases. For detection and functional modulation of fucosylated glycans, chemical biology approaches using fucose (Fuc) analogs are useful. However, little is known about how efficiently each unnatural Fuc analog is utilized by enzymes in the biosynthetic pathway of fucosylated glycans. We show here that three clickable Fuc analogs with similar but distinct structures labeled cellular glycans with different efficiency and protein specificity. For instance, 6-alkynyl (Alk)-Fuc modified O-Fuc glycans much more efficiently than 7-Alk-Fuc. The level of GDP-6-Alk-Fuc produced in cells was also higher than that of GDP-7-Alk-Fuc. Comprehensive in vitro fucosyltransferase assays revealed that 7-Alk-Fuc is commonly tolerated by most fucosyltransferases. Surprisingly, both protein O-fucosyltransferases (POFUTs) could transfer all Fuc analogs in vitro, likely because POFUT structures have a larger space around their Fuc binding sites. These findings demonstrate that labeling and detection of fucosylated glycans with Fuc analogs depend on multiple cellular steps, including conversion to GDP form, transport into the ER or Golgi, and utilization by each fucosyltransferase, providing insights into design of novel sugar analogs for specific detection of target glycans or inhibition of their functions.

Xylosyl Extension of O-Glucose Glycans on the Extracellular Domain of NOTCH1 and NOTCH2 Regulates Notch Cell Surface Trafficking.

Biochemical and genetic studies have indicated that O-linked glycosylation such as O-glucose (Glc), fucose (Fuc), and N-acetylglucosamine (GlcNAc) is critical for Notch signaling; however, it is not fully understood how O-glycans regulate the Notch receptor function. Notch receptors are type-I transmembrane proteins with large extracellular domains (ECD), containing 29-36 epidermal growth factor-like (EGF) repeats. Here, we analyzed O-Glc glycans on NOTCH1 and NOTCH2 expressed in HEK293T cells using an Orbitrap Fusion mass spectrometer and successfully revealed the structures and stoichiometries of all 17 EGF repeats of NOTCH1 with the O-Glc consensus sequence (C1-X-S-X-(P/A)-C2), and 16 out of 17 EGF repeats of NOTCH2 with the same consensus sequence. High levels of O-Glc attachment and xylosyl elongation were detected on most NOTCH1 and NOTCH2 EGF repeats. When both glucoside xylosyltransferases, GXYLT1 and GXYLT2, responsible for the xylosyl elongation of O-glucose, were genetically deleted, the expression of endogenous NOTCH1 and NOTCH2 on the surface of HEK293T cells did not change, but the cell surface expression of overexpressed NOTCH1 and NOTCH2 decreased compared with that in the wild type cells. In vitro secretion assays consistently showed a reduced secretion of both the NOTCH1 and NOTCH2 ECDs in GXYLT1 and GXYLT2 double knockout cells compared with the wild type cells, suggesting a significant role of the elongation of O-Glc glycans on the Notch ECDs in the quality control of Notch receptors.

N-Glycans on EGF domain-specific O-GlcNAc transferase (EOGT) facilitate EOGT maturation and peripheral endoplasmic reticulum localization.

Epidermal growth factor (EGF) domain-specific O-GlcNAc transferase (EOGT) is an endoplasmic reticulum (ER)-resident protein that modifies EGF repeats of Notch receptors and thereby regulates Delta-like ligand-mediated Notch signaling. Several EOGT mutations that may affect putative N-glycosylation consensus sites are recorded in the cancer database, but the presence and function of N-glycans in EOGT have not yet been characterized. Here, we identified N-glycosylation sites in mouse EOGT and elucidated their molecular functions. Three predicted N-glycosylation consensus sequences on EOGT are highly conserved among mammalian species. Within these sites, we found that Asn-263 and Asn-354, but not Asn-493, are modified with N-glycans. Lectin blotting, endoglycosidase H digestion, and MS analysis revealed that both residues are modified with oligomannose N-glycans. Loss of an individual N-glycan on EOGT did not affect its endoplasmic reticulum (ER) localization, enzyme activity, and ability to O-GlcNAcylate Notch1 in HEK293T cells. However, simultaneous substitution of both N-glycosylation sites affected both EOGT maturation and expression levels without an apparent change in enzymatic activity, suggesting that N-glycosylation at a single site is sufficient for EOGT maturation and expression. Accordingly, a decrease in O-GlcNAc stoichiometry was observed in Notch1 co-expressed with an N263Q/N354Q variant compared with WT EOGT. Moreover, the N263Q/N354Q variant exhibited altered subcellular distribution within the ER in HEK293T cells, indicating that N-glycosylation of EOGT is required for its ER localization at the cell periphery. These results suggest critical roles of N-glycans in sustaining O-GlcNAc transferase function both by maintaining EOGT levels and by ensuring its proper subcellular localization in the ER.

Aminoglycosides are efficient reagents to induce readthrough of premature termination codon in mutant B4GALNT1 genes found in families of hereditary spastic paraplegia.

The readthrough of premature termination codon (PTC) by ribosome sometimes produces full-length proteins. We previously reported a readthrough of PTC of glycosyltransferase gene B4GALNT1 with hereditary spastic paraplegia (HSP). Here we featured the readthrough of B4GALNT1 of two mutants, M4 and M2 with PTC by immunoblotting and flow cytometry after transfection of B4GALNT1 cDNAs into cells. Immunoblotting showed a faint band of full-length mutant protein of M4 but not M2 at a similar position with that of wild-type B4GALNT1. AGC sequences at immediately before and after the PTC in M4 were critical for the readthrough. Treatment of cells transfected with mutant M4 cDNA with aminoglycosides resulted in increased readthrough of PTC. Furthermore, treatment of transfectants of mutant M2 cDNA with G418 also resulted in the induction of readthrough of PTC. Both M4 and M2 cDNA transfectants showed increased/induced bands in immunoblotting and GM2 expression in a dose-dependent manner of aminoglycosides. Results of mass spectrometry supported this effect. Here, we showed for the first time the induction and/or enhancement of the readthrough of PTCs of B4GALNT1 by aminoglycoside treatment, suggesting that aminoglycosides are efficient for patients with HSP caused by PTC of B4GALNT1, in which gradual neurological disorders emerged with aging.

Contribution of extracellular O-GlcNAc to the stability of folded epidermal growth factor-like domains and Notch1 trafficking.

The Notch signaling pathway is highly conserved and essential in animal development and tissue homeostasis. Regulation of Notch signaling is a crucial process for human health. Ligands initiate a signal cascade by binding to Notch receptors expressed on the neighboring cell. Notch receptors interact with ligands through their epidermal growth factor-like repeats (EGF repeats). Most EGF repeats are modified by O-glycosylation with residues, such as O-linked N-acetylglucosamine (O-GlcNAc), O-fucose, and O-glucose. A recent study revealed the distinct roles of these O-glycans in ligand binding, processing, and trafficking of Notch receptors. In particular, O-GlcNAc glycans are essential for Delta-like (DLL) ligand-mediated Notch signaling. In this study, we showed that O-GlcNAc promotes Notch1 trafficking to the cell surfaces under the condition that O-fucose and O-glucose are removed from consecutive EGF repeats of Notch1. Through in vitro experiments, we showed that O-GlcNAc mediates the stability of EGF domains in the same manner as O-fucose and O-glucose. Thus, O-GlcNAc on EGF domains possesses a shared function in the stability of EGF domains and Notch1 trafficking.