Articular cartilage corefucosylation regulates tissue resilience in osteoarthritis.

This study aimed to investigate the glycan structural changes that occur before histological degeneration in osteoarthritis (OA) and to determine the mechanism by which these glycan conformational changes affect cartilage degeneration. An OA model was established in rabbits using mannosidase injection, which reduced high-mannose type N-glycans and led to cartilage degeneration. Further analysis of glycome in human OA cartilage identified specific corefucosylated N-glycan expression patterns. Inhibition of N-glycan corefucosylation in mice resulted in unrecoverable cartilage degeneration, while cartilage-specific blocking of corefucosylation led to accelerated development of aging-associated and instability-induced OA models. We conclude that α1,6 fucosyltransferase is required postnatally to prevent preosteoarthritic deterioration of articular cartilage. These findings provide a novel definition of early OA and identify glyco-phenotypes of OA cartilage, which may distinguish individuals at higher risk of progression.

Tolerable glycometabolic stress boosts cancer cell resilience through altered N-glycosylation and Notch signaling activation.

Chronic metabolic stress paradoxically elicits pro-tumorigenic signals that facilitate cancer stem cell (CSC) development. Therefore, elucidating the metabolic sensing and signaling mechanisms governing cancer cell stemness can provide insights into ameliorating cancer relapse and therapeutic resistance. Here, we provide convincing evidence that chronic metabolic stress triggered by hyaluronan production augments CSC-like traits and chemoresistance by partially impairing nucleotide sugar metabolism, dolichol lipid-linked oligosaccharide (LLO) biosynthesis and N-glycan assembly. Notably, preconditioning with either low-dose tunicamycin or 2-deoxy-D-glucose, which partially interferes with LLO biosynthesis, reproduced the promoting effects of hyaluronan production on CSCs. Multi-omics revealed characteristic changes in N-glycan profiles and Notch signaling activation in cancer cells exposed to mild glycometabolic stress. Restoration of N-glycan assembly with glucosamine and mannose supplementation and Notch signaling blockade attenuated CSC-like properties and further enhanced the therapeutic efficacy of cisplatin. Therefore, our findings uncover a novel mechanism by which tolerable glycometabolic stress boosts cancer cell resilience through altered N-glycosylation and Notch signaling activation.

Focal-adhesion kinase regulates the sialylation of N-glycans via the PI4KIIα-PI4P pathway.

Sialylation is a terminal glycosylated modification of glycoproteins that regulates critical biological events such as cell adhesion and immune response. Our previous study showed that integrin α3ß1 plays a crucial role in regulating the sialylation of N-glycans. However, the underlying mechanism for the regulation remains unclear. This study investigated how sialylation is affected by focal adhesion kinase (FAK), which is a critical downstream signal molecule of integrin ß1. We established a stable FAK knockout (KO) cell line using the CRISPR/Cas9 system in HeLa cells. The results obtained from lectin blot, flow cytometric analysis, and MS showed that the sialylation levels were significantly decreased in the KO cells compared with that in wild-type (WT) cells. Moreover, phosphatidylinositol 4-phosphate (PI4P) expression levels were also reduced in the KO cells due to a decrease in the stability of phosphatidylinositol 4-kinase-IIα (PI4KIIα). Notably, the decreased levels of sialylation, PI4P, and the complex formation between GOLPH3 and ST3GAL4 or ST6GAL1, which are the main sialyltransferases for modification of N-glycans, were significantly restored by the re-expression of FAK. Furthermore, the decreased sialylation and phosphorylation of Akt and cell migration caused by FAK deficiency all were restored by overexpressing PI4KIIα, which suggests that PI4KIIα is one of the downstream molecules of FAK. These findings indicate that FAK regulates sialylation via the PI4P synthesis pathway and a novel mechanism is suggested for the integrin-FAK-PI4KIIα-GOLPH3-ST axis modulation of sialylation in N-glycans.

Simultaneous and sialic acid linkage-specific N- and O-linked glycan analysis by ester-to-amide derivatization.

Characterization of O-glycans linked to serine or threonine residues in glycoproteins has mostly been achieved using chemical reaction approaches because there are no known O-glycan-specific endoglycosidases. Most O-glycans are modified with sialic acid residues at the non-reducing termini through various linkages. In this study, we developed a novel approach for sialic acid linkage-specific O-linked glycan analysis through lactone-driven ester-to-amide derivatization combined with non-reductive ß-elimination in the presence of hydroxylamine. O-glycans released by non-reductive ß-elimination were efficiently purified using glycoblotting via chemoselective ligation between carbohydrates and a hydrazide-functionalized polymer, followed by modification of methyl or ethyl ester groups of sialic acid residues on solid-phase. In-solution lactone-driven ester-to-amide derivatization of ethyl-esterified O-glycans was performed, and the resulting sialylated glycan isomers were discriminated by mass spectrometry. In combination with PNGase F digestion, we carried out simultaneous, quantitative, and sialic acid linkage-specific N- and O-linked glycan analyses of a model glycoprotein and human cartilage tissue. This novel glycomic approach will facilitate detailed characterization of biologically relevant sialylated N- and O-glycans on glycoproteins.

Exposure to brefeldin A induces unusual expression of hybrid- and complex-type free N-glycans in HepG2 cells.

This study determined the effect of brefeldin A (BFA) on the free N-glycomic profile of HepG2 cells to better understand the effect of blocking intracellular vesicle formation and transport of proteins from the endoplasmic reticulum to the Golgi apparatus. A series of exoglycosidase- and endoglycosidase-assisted analyses clarified the complex nature of altered glycomic profiles. A key feature of BFA-mediated alterations in Gn2-type glycans was the expression of unusual hybrid-, monoantennary- and complex-type free N-glycans (FNGs). BFA-mediated alterations in Gn1-type glycans were characterized by the expression of unusual hybrid- and monoantennary-FNGs, without significant expression of complex-type FNGs. A time course analysis revealed that sialylated hybrid- and complex-type Gn2-type FNGs were generated later than asialo-Gn2-type FNGs, and the expression profiles of Gn2-type FNGs and N-glycans were found to be similar, suggesting that the metabolic flux of FNGs is the same as that of protein-bound N-glycans. Subcellular glycomic analysis revealed that almost all FNGs were detected in the cytoplasmic extracts. Our data suggest that hybrid-, monoantennary- and complex-type Gn2-type FNGs were cleaved from glycoproteins in the cytosol by cytosolic PNGase, and subsequently digested by cytosolic endo-ß-N-acetylglucosaminidase (ENGase) to generate Gn1-type FNGs. The substrate specificity of ENGase explains the limited expression of complex Gn1 type FNGs.

Simultaneous determination of heparan sulfate, chondroitin/dermatan sulfates, and hyaluronan glycosaminoglycan disaccharides by high-performance liquid chromatography using a reverse-phase column with adamantyl groups.

Glycosaminoglycans (GAGs), which are one of the major components of proteoglycans, play a pivotal role in physiological processes such as signal transduction, cell adhesion, growth, and differentiation. Characterization of GAGs is challenging due to the tremendous structural diversity of heteropolysaccharides with numerous sulfate or carboxyl groups. In this present study, we examined the analysis of 2-aminobenzamide (2-AB) labeled GAG disaccharides by high-performance liquid chromatography (HPLC) using a reverse-phase (RP)-column with adamantyl groups. Under the analytical conditions, 17 types of 2-AB labeled GAG disaccharides derived from heparan sulfate, chondroitin/dermatan sulfates, and hyaluronan were sequentially separated in a single analysis. The analysis time was fast with high retention time reproducibility. Moreover, the RP-HPLC column with adamantyl groups allowed the quantification of GAGs in various biological samples, such as serum, cultured cells, and culture medium.

Comprehensive Glycan Analysis of Sphingolipids in Human Serum/Plasma.

Glycosphingolipids (GSLs) are glycolipids with ceramide and carbohydrate head groups that play an important role in numerous biological processes. Previously, we performed GSL-glycan analysis of various cell lines and virus-infected cells using a glycoblotting approach. Recently, we developed several methods for sialic acid linkage-specific chemical modification to distinguish sialylated glycan isomers by mass spectrometry. In this chapter, we describe a method for analyzing GSL-glycans in human serum/plasma using glycoblotting combined with aminolysis-SALSA (sialic acid linkage-specific alkylamidation) and lactone-driven ester-to-amide derivatization (LEAD)-SALSA for comprehensive and detailed structural glycomics.

Toolbox Accelerating Glycomics (TAG): Improving Large-Scale Serum Glycomics and Refinement to Identify SALSA-Modified and Rare Glycans.

Glycans are involved in many fundamental cellular processes such as growth, differentiation, and morphogenesis. However, their broad structural diversity makes analysis difficult. Glycomics via mass spectrometry has focused on the composition of glycans, but informatics analysis has not kept pace with the development of instrumentation and measurement techniques. We developed Toolbox Accelerating Glycomics (TAG), in which glycans can be added manually to the glycan list that can be freely designed with labels and sialic acid modifications, and fast processing is possible. In the present work, we improved TAG for large-scale analysis such as cohort analysis of serum samples. The sialic acid linkage-specific alkylamidation (SALSA) method converts differences in linkages such as α2,3- and α2,6-linkages of sialic acids into differences in mass. Glycans modified by SALSA and several structures discovered in recent years were added to the glycan list. A routine to generate calibration curves has been implemented to explore quantitation. These improvements are based on redefinitions of residues and glycans in the TAG List to incorporate information on glycans that could not be attributed because it was not assumed in the previous version of TAG. These functions were verified through analysis of purchased sera and 74 spectra with linearity at the level of R2 > 0.8 with 81 estimated glycan structures obtained including some candidate of rare glycans such as those with the N,N'-diacetyllactosediamine structure, suggesting they can be applied to large-scale analyses.

Glycosphingolipid GM3 prevents albuminuria and podocytopathy induced by anti-nephrin antibody.

Podocytopathy, which is characterized by injury to podocytes, frequently causes proteinuria or nephrotic syndrome. There is currently a paucity of effective therapeutic drugs to treat proteinuric kidney disease. Recent research suggests the possibility that glycosphingolipid GM3 maintains podocyte function by acting on various molecules including nephrin, but its mechanism of action remains unknown. Here, various analyses were performed to examine the potential relationship between GM3 and nephrin, and the function of GM3 in podocytes using podocytopathy mice, GM3 synthase gene knockout mice, and nephrin injury cells. Reduced amounts of GM3 and nephrin were observed in podocytopathy mice. Intriguingly, this reduction of GM3 and nephrin, as well as albuminuria, were inhibited by administration of valproic acid. However, when the same experiment was performed using GM3 synthase gene knockout mice, valproic acid administration did not inhibit albuminuria. Equivalent results were obtained in model cells. These findings indicate that GM3 acts with nephrin in a collaborative manner in the cell membrane. Taken together, elevated levels of GM3 stabilize nephrin, which is a key molecule of the slit diaphragm, by enhancing the environment of the cell membrane and preventing albuminuria. This study provides novel insight into new drug discovery, which may offer a new therapy for kidney disease with albuminuria.

Establishment of the removal method of undifferentiated induced pluripotent stem cells coexisting with chondrocytes using R-17F antibody.

Aim: Tumorigenicity of residual undifferentiated induced pluripotent stem cells (iPSCs) is a major concern. The purpose of this study was to investigate the optimal conditions for removal of iPSCs using R-17F antibody, which recognizes specific glycosphingolipids glycans on undifferentiated iPSCs and exhibits selective cytotoxicity to iPSCs. Materials & methods: After adding of R-17F and secondary antibody to co-cultured iPSCs and chondrocytes, residual iPSCs were quantitatively evaluated by iPS specific glycome analysis. Results: Undifferentiated iPSCs were sufficiently removed using R-17F in combination with an equal amount of a secondary antibody. Furthermore, teratomas were not observed upon transplantation of co-cultured cells pretreated under the same conditions into testes of immunodeficient mice. Conclusion: This removal method incorporating R-17F may be useful for regenerative medicine using iPSCs.

Comprehensive Cellular Glycan Profiling of Glycoproteins and Glycosphingolipids by Glycoblotting and BEP Methods.

The glycocalyx is a layer of glycans that covers the surface of every cell. Glycans are covalently attached to proteins and lipids, and are classified into subclasses such as N-linked glycans, glycosaminoglycans, glycosphingolipid-glycans, free oligosaccharides, and O-linked glycans according to their biosynthetic pathways. These complex glycans affect various biological and pathological processes, such as cell growth, differentiation, and adhesion. During infection, bacteria and viruses often use glycans to recognize and attack host cells. In this chapter, we describe detailed protocols to prepare glycans, and perform comprehensive cellular glycomic analysis using glycoblotting and ß-elimination with pyrazolone methods.

Evaluation of the context of downstream N- and free N-glycomic alterations induced by swainsonine in HepG2 cells.

Swainsonine (SWA), a potent inhibitor of class II α-mannosidases, is present in a number of plant species worldwide and causes severe toxicosis in livestock grazing these plants. The mechanisms underlying SWA-induced animal poisoning are not fully understood. In this study, we analyzed the alterations that occur in N- and free N-glycomic upon addition of SWA to HepG2 cells to understand better SWA-induced glycomic alterations. After SWA addition, we observed the appearance of SWA-specific glycomic alterations, such as unique fucosylated hybrid-type and fucosylated M5 (M5F) N-glycans, and a remarkable increase in all classes of Gn1 FNGs. Further analysis of the context of these glycomic alterations showed that (fucosylated) hybrid type N-glycans were not the precursors of these Gn1 FNGs and vice versa. Time course analysis revealed the dynamic nature of glycomic alterations upon exposure of SWA and suggested that accumulation of free N-glycans occurred earlier than that of hybrid-type N-glycans. Hybrid-type N-glycans, of which most were uniquely core fucosylated, tended to increase slowly over time, as was observed for M5F N-glycans. Inhibition of swainsonine-induced unique fucosylation of hybrid N-glycans and M5 by coaddition of 2-fluorofucose caused significant increases in paucimannose- and fucosylated paucimannose-type N-glycans, as well as paucimannose-type free N-glycans. The results not only revealed the gross glycomic alterations in HepG2 cells induced by swainsonine, but also provide information on the global interrelationships between glycomic alterations.

Comprehensive and Comparative Structural Glycome Analysis in Mouse Epiblast-like Cells.

Glycosylation is one of the most abundant posttranslational modifications and is involved in a wide range of cellular processes. Glycome diversity in mammals is generated by the action of over 200 distinct glycosyltransferases and related enzymes. Nevertheless, glycosylation dynamics are tightly coordinated to allow proper organismal development. Here, using mouse embryonic stem cells (mESCs) and mouse epiblast-like cells (mEpiLCs) as model systems, we describe a robust protocol that allows comprehensive and comparative structural analysis of the glycome.

Comprehensive Glycomic Approach Reveals Novel Low-Molecular-Weight Blood Group-Specific Glycans in Serum and Cerebrospinal Fluid.

ABO blood antigens on the human red blood cell membrane as well as different cells in various human tissues have been thoroughly studied. Anti-A and -B antibodies of IgM are present in serum/plasma, but blood group-specific glyco-antigens have not been extensively described. In this study, we performed comprehensive and quantitative serum glycomic analyses of various glycoconjugates and free oligosaccharides in all blood groups. Our comprehensive glycomic approach revealed that blood group-specific antigens in serum/plasma are predominantly present on glycosphingolipids on lipoproteins rather than glycoproteins. Expression of the ABO antigens on glycosphingolipids depends not only on blood type but also on secretor status. Blood group-specific glycans in serum/plasma were classified as type I, whereas those on RBCs had different structures including hexose and hexosamine residues. Analysis of free oligosaccharides revealed that low-molecular-weight blood group-specific glycans, commonly containing lacto-N-difucotetraose, were expressed in serum/plasma according to blood group. Furthermore, comprehensive glycomic analysis in human cerebrospinal fluid showed that many kinds of free oligosaccharides were highly expressed, and low-molecular-weight blood group-specific glycans, which existed in plasma from the same individuals, were present. Our findings provide the first evidence for low-molecular-weight blood group-specific glycans in both serum/plasma and cerebrospinal fluid.

A defined glycosylation regulatory network modulates total glycome dynamics during pluripotency state transition.

Embryonic stem cells (ESCs) and epiblast-like cells (EpiLCs) recapitulate in vitro the epiblast first cell lineage decision, allowing characterization of the molecular mechanisms underlying pluripotent state transition. Here, we performed a comprehensive and comparative analysis of total glycomes of mouse ESCs and EpiLCs, revealing that overall glycosylation undergoes dramatic changes from early stages of development. Remarkably, we showed for the first time the presence of a developmentally regulated network orchestrating glycosylation changes and identified polycomb repressive complex 2 (PRC2) as a key component involved in this process. Collectively, our findings provide novel insights into the naïve-to-primed pluripotent state transition and advance the understanding of glycosylation complex regulation during early mouse embryonic development.

Alterations of Glycosphingolipid Glycans and Chondrogenic Markers during Differentiation of Human Induced Pluripotent Stem Cells into Chondrocytes.

Due to the limited intrinsic healing potential of cartilage, injury to this tissue may lead to osteoarthritis. Human induced pluripotent stem cells (iPSCs), which can be differentiated into chondrocytes, are a promising source of cells for cartilage regenerative therapy. Currently, however, the methods for evaluating chondrogenic differentiation of iPSCs are very limited; the main techniques are based on the detection of chondrogenic genes and histological analysis of the extracellular matrix. The cell surface is coated with glycocalyx, a layer of glycoconjugates including glycosphingolipids (GSLs) and glycoproteins. The glycans in glycoconjugates play important roles in biological events, and their expression and structure vary widely depending on cell types and conditions. In this study, we performed a quantitative GSL-glycan analysis of human iPSCs, iPSC-derived mesenchymal stem cell like cells (iPS-MSC like cells), iPS-MSC-derived chondrocytes (iPS-MSC-CDs), bone marrow-derived mesenchymal stem cells (BMSCs), and BMSC-derived chondrocytes (BMSC-CDs) using glycoblotting technology. We found that GSL-glycan profiles differed among cell types, and that the GSL-glycome underwent a characteristic alteration during the process of chondrogenic differentiation. Furthermore, we analyzed the GSL-glycome of normal human cartilage and found that it was quite similar to that of iPS-MSC-CDs. This is the first study to evaluate GSL-glycan structures on human iPS-derived cartilaginous particles under micromass culture conditions and those of normal human cartilage. Our results indicate that GSL-glycome analysis is useful for evaluating target cell differentiation and can thus support safe regenerative medicine.

Toolbox Accelerating Glycomics (TAG): Glycan Annotation from MALDI-TOF MS Spectra and Mapping Expression Variation to Biosynthetic Pathways.

Glycans present extraordinary structural diversity commensurate with their involvement in numerous fundamental cellular processes including growth, differentiation, and morphogenesis. Unlike linear DNA and protein sequences, glycans have heterogeneous structures that differ in composition, branching, linkage, and anomericity. These differences pose a challenge to developing useful software for glycomic analysis. To overcome this problem, we developed the novel Toolbox Accelerating Glycomics (TAG) program. TAG consists of three units: 'TAG List' creates a glycan list that is used for database searching in TAG Expression; 'TAG Expression' automatically annotates and quantifies glycan signals and draws graphs; and 'TAG Pathway' maps the obtained expression information to biosynthetic pathways. Herein, we discuss the concepts, outline the TAG process, and demonstrate its potential using glycomic expression profile data from Chinese hamster ovary (CHO) cells and mutants lacking a functional Npc1 gene (Npc1 knockout (KO) CHO cells). TAG not only drastically reduced the amount of time and labor needed for glycomic analysis but also detected and quantified more glycans than manual analysis. Although this study was limited to the analysis of N-glycans and free oligosaccharides, the glycomic platform will be expanded to facilitate the analysis of O-glycans and glycans of glycosphingolipids.

Mucin-type O-glycosylation controls pluripotency in mouse embryonic stem cells via Wnt receptor endocytosis.

Mouse embryonic stem cells (ESCs) can differentiate into a range of cell types during development, and this pluripotency is regulated by various extrinsic and intrinsic factors. Mucin-type O-glycosylation has been suggested to be a potential factor in the control of ESC pluripotency, and is characterized by the addition of N-acetylgalactosamine (GalNAc) to serine or threonine residues of membrane-anchored proteins and secreted proteins. To date, the relationship between mucin-type O-glycosylation and signaling in ESCs remains undefined. Here, we identify the elongation pathway via C1GalT1 that synthesizes T antigen (Galß1-3GalNAc) as the most prominent among mucin-type O-glycosylation modifications in ESCs. Moreover, we show that mucin-type O-glycosylation on the Wnt signaling receptor frizzled-5 (Fzd5) regulates its endocytosis via galectin-3 binding to T antigen, and that reduction of T antigen results in the exit of the ESCs from pluripotency via canonical Wnt signaling activation. Our findings reveal a novel regulatory mechanism that modulates Wnt signaling and, consequently, ESC pluripotency.This article has an associated First Person interview with the first author of the paper.

Lactone-Driven Ester-to-Amide Derivatization for Sialic Acid Linkage-Specific Alkylamidation.

Sialic acid attached to nonreducing ends of glycan chains via different linkages is associated with specific interactions and physiological events. Linkage-specific derivatization of sialic acid is of great interest for distinguishing sialic acids by mass spectrometry, specifically for events governed by sialyl linkage types. In the present study, we demonstrate that α-2,3/8-sialyl linkage-specific amidation of esterified sialyloligosaccharides can be achieved via an intramolecular lactone. The method of lactone-driven ester-to-amide derivatization for sialic acid linkage-specific alkylamidation, termed LEAD-SALSA, employs in-solution ester-to-amide conversion to directly generate stable and sialyl linkage-specific glycan amides from their ester form by mixing with a preferred amine, resulting in the easy assignments of sialyl linkages by comparing the signals of esterified and amidated glycan. Using this approach, we demonstrate the accumulation of altered N-glycans in cardiac muscle tissue during mouse aging. Furthermore, we find that the stability of lactone is important for ester-to-amide conversion based on experiments and density functional theory calculations of reaction energies for lactone formation. By using energy differences of lactone formation, the LEAD-SALSA method can be used not only for the sialyl linkage-specific derivatization but also for distinguishing the branching structure of galactose linked to sialic acid. This simplified and direct sialylglycan discrimination will facilitate important studies on sialylated glycoconjugates.

Tri-antennary tri-sialylated mono-fucosylated glycan of alpha-1 antitrypsin as a non-invasive biomarker for non-alcoholic steatohepatitis: a novel glycobiomarker for non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a progressive form of non-alcoholic fatty liver disease (NAFLD) that may lead to liver cirrhosis or hepatocellular carcinoma. Here, we examined the diagnostic utility of tri-antennary tri-sialylated mono-fucosylated glycan of alpha-1 antitrypsin (AAT-A3F), a non-invasive glycobiomarker identified in a previous study of NASH diagnosis. This study included 131 biopsy-proven Japanese patients with NAFLD. We evaluated the utility of AAT-A3F in NASH diagnosis, and conducted genetic analysis to analyse the mechanism of AAT-A3F elevation in NASH. Serum AAT-A3F concentrations were significantly higher in NASH patients than in NAFL patients, and in patients with fibrosis, lobular inflammation, and ballooning. Hepatic FUT6 gene expression was significantly higher in NASH than in NAFL. IL-6 expression levels were significantly higher in NASH than in NAFL and showed a positive correlation with FUT6 expression levels. The serum-AAT-A3F levels strongly correlated with hepatic FUT6 expression levels. AAT-A3F levels increased with fibrosis, pathological inflammation, and ballooning in patients with NAFLD and may be useful for non-invasive diagnosis of NASH from the early stages of fibrosis.