What comes next in glycobiology.

Glycans, with their variable compositions and highly dynamic conformations, vastly expand the heterogeneity of whatever factor or cell they are attached to. These properties make them crucial contributors to biological function and organismal health and also very difficult to study. That may be changing as we look to the future of glycobiology.

Construction of mouse cochlin mutants with different GAG-binding specificities and their use for immunohistochemistry.

Glycosaminoglycan (GAG) is a polysaccharide present on the cell surface as an extracellular matrix component, and is composed of repeating disaccharide units consisting of an amino sugar and uronic acid except in the case of the keratan sulfate. Sulfated GAGs, such as heparan sulfate, heparin, and chondroitin sulfate mediate signal transduction of growth factors, and their functions vary with the type and degree of sulfated modification. We have previously identified human and mouse cochlins as proteins that bind to sulfated GAGs. Here, we prepared a recombinant cochlin fused to human IgG-Fc or Protein A at the C-terminus as a detection and purification tag and investigated the ligand specificity of cochlin. We found that cochlin can be used as a specific probe for highly sulfated heparan sulfate and chondroitin sulfate E. We then used mutant analysis to identify the mechanism by which cochlin recognizes GAGs and developed a GAG detection system using cochlin. Interestingly, a mutant lacking the vWA2 domain bound to various types of GAGs. The N-terminal amino acid residues of cochlin contributed to its binding to heparin. Pathological specimens from human myocarditis patients were stained with a cochlin-Fc mutant. The results showed that both tryptase-positive and tryptase-negative mast cells were stained with this mutant. The identification of detailed modification patterns of GAGs is an important method to elucidate the molecular mechanisms of various diseases. The method developed for evaluating the expression of highly sulfated GAGs will help understand the biological and pathological importance of sulfated GAGs in the future.

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.

Functional analysis of glycosylation using Drosophila melanogaster.

The glycosylation of proteins and lipids has various essential roles in a diverse range of biological processes, including embryogenesis, organ development, neurogenesis, maintenance of homeostasis, immune response, and tumorigenesis. Drosophila melanogaster is one of the representative multicellular model organisms, which have many useful genetic manipulation tools; it is used in developmental biology as well as classical and molecular genetics. Glycobiology is not an exception and many studies using Drosophila have been performed in this field to clarify novel functions of glycans. Recently, genome-wide screening and functional analyses were performed in whole body, wings, eyes, neuromuscular junctions, and immune organs. Furthermore, detailed studies with Drosophila mutants of glycosyltransferases, nucleotide sugar transporters, and glycosidases revealed novel functions of N-linked glycans, glycosaminoglycans, glycolipids, and O-linked glycans including mucin type O-glycan, O-Fuc, O-Man, and O-GlcNAc. As many of these functions are common between Drosophila and humans, these mutants represent good models for human disease. In this review, recent studies of glycan functions using Drosophila are summarized.

E190V substitution of H6 hemagglutinin is one of key factors for binding to sulfated sialylated glycan receptor and infection to chickens.

Avian influenza viruses (AIVs) recognize sialic acid linked α2,3 to galactose (SAα2,3Gal) glycans as receptors. In this study, the interactions between hemagglutinins (HAs) of AIVs and sulfated SAα2,3Gal glycans were analyzed to clarify the molecular basis of interspecies transmission of AIVs from ducks to chickens. It was revealed that E190V and N192D substitutions of the HA increased the recovery of viruses derived from an H6 duck virus isolate, A/duck/Hong Kong/960/1980 (H6N2), in chickens. Recombinant HAs from an H6 chicken virus, A/chicken/Tainan/V156/1999 (H6N1), bound to sulfated SAα2,3Gal glycans, whereas the HAs from an H6 duck virus did not. Binding preference of mutant HAs revealed that an E190V substitution is critical for the recognition of sulfated SAα2,3Gal glycans. These results suggest that the binding of the HA from H6 AIVs to sulfated SAα2,3Gal glycans explains a part of mechanisms of interspecies transmission of AIVs from ducks to chickens.

Glucuronylated core 1 glycans are required for precise localization of neuromuscular junctions and normal formation of basement membranes on Drosophila muscles.

T antigen (Galß1-3GalNAcα1-Ser/Thr) is an evolutionary-conserved mucin-type core 1 glycan structure in animals synthesized by core 1 ß1,3-galactosyltransferase 1 (C1GalT1). Previous studies showed that T antigen produced by Drosophila C1GalT1 (dC1GalT1) was expressed in various tissues and dC1GalT1 loss in larvae led to various defects, including decreased number of circulating hemocytes, hyper-differentiation of hematopoietic stem cells in lymph glands, malformation of the central nervous system, mislocalization of neuromuscular junction (NMJ) boutons, and ultrastructural abnormalities in NMJs and muscle cells. Although glucuronylated T antigen (GlcAß1-3Galß1-3GalNAcα1-Ser/Thr) has been identified in Drosophila, the physiological function of this structure has not yet been clarified. In this study, for the first time, we unraveled biological roles of glucuronylated T antigen. Our data show that in Drosophila, glucuronylation of T antigen is predominantly carried out by Drosophila ß1,3-glucuronyltransferase-P (dGlcAT-P). We created dGlcAT-P null mutants and found that mutant larvae showed lower expression of glucuronylated T antigen on the muscles and at NMJs. Furthermore, mislocalization of NMJ boutons and a partial loss of the basement membrane components collagen IV (Col IV) and nidogen (Ndg) at the muscle 6/7 boundary were observed. Those two phenotypes were correlated and identical to previously described phenotypes in dC1GalT1 mutant larvae. In addition, dGlcAT-P null mutants exhibited fewer NMJ branches on muscles 6/7. Moreover, ultrastructural analysis revealed that basement membranes that lacked Col IV and Ndg were significantly deformed. We also found that the loss of dGlcAT-P expression caused ultrastructural defects in NMJ boutons. Finally, we showed a genetic interaction between dGlcAT-P and dC1GalT1. Therefore, these results demonstrate that glucuronylated core 1 glycans synthesized by dGlcAT-P are key modulators of NMJ bouton localization, basement membrane formation, and NMJ arborization on larval muscles.

Highly sulfated hyaluronic acid maintains human induced pluripotent stem cells under feeder-free and bFGF-free conditions.

Human induced pluripotent stem (hiPS) cells are attracting attention as a tool for regenerative medicine. However, several problems need to be overcome for their widespread and safe use, for example, the high cost of maintaining hiPS cells and the possibility of xenogeneic cell contamination in hiPS cell cultures. One of the main contributors to the high cost of maintaining hiPS cells is basic fibroblast growth factor (bFGF), which is essential for such cultures. Xenogeneic contamination can occur because of the use of mouse-derived feeder cells to culture hiPS cells. To overcome the problems of cell culture cost and xenogeneic contamination, we have developed a novel culture method in which the undifferentiated state and pluripotency of hiPS cells can be maintained under feeder-free and bFGF-free conditions. Our new approach involves the addition to the culture medium of highly sulfated hyaluronic acid (HA-HS), in which the hydroxyl groups of d-glucuronic acid (GlcA) and N-acetyl-d-glucosamine (GlcNAc) are chemically sulfated. HA-HS promotes bFGF signaling and maintains the undifferentiated state and pluripotency of hiPS cells under feeder-free and bFGF-free conditions. By contrast, non-sulfated hyaluronic acid and low sulfated hyaluronic acid do not maintain the undifferentiated state and pluripotency of hiPS cells. These results indicate that the maintenance of hiPS cells under feeder-free and bFGF-free conditions is an HA-HS specific effect. This study is the first to demonstrate the effects of sulfated hyaluronic acid on mammalian pluripotent stem cells, and provides a novel method for maintaining hiPS cells using HA-HS.

Cell Profiling Based on Sugar-Chain-Cell Binding Interaction and Its Application to Typing and Quality Verification of Cells.

Developing methods to determine cell type and cell state has been a significant challenge in the field of cancer diagnosis as well as in typing and quality verification for cultured cells. Herein, we report a cell profiling method based on binding interactions between cell-surface sugar-chain-binding proteins and sugar-chain-immobilized fluorescent nanoparticles (SFNPs), together with a method for cell typing and cell quality verification. Binding profiles of cells against sugar chains were analyzed by performing flow cytometry analysis with SFNPs. Discrimination analysis based on binding profiles could classify cell type and evaluate the quality of cultured cells. By applying our method to differentiated cells originating from conventional cell lines and also to mouse embryotic stem cells, we could detect the cells before and after differentiation. Our method can be utilized not only for the biofunctional analysis of cells but also for diagnosis of cancer cells and quality verification of cultured cells.

Mucin-type core 1 glycans regulate the localization of neuromuscular junctions and establishment of muscle cell architecture in Drosophila.

T antigen (Galß1-3GalNAcα1-Ser/Thr), a core 1 mucin-type O-glycan structure, is synthesized by Drosophila core 1 ß1,3-galactosyltrasferase 1 (dC1GalT1) and is expressed in various tissues. We previously reported that dC1GalT1 synthesizes T antigen expressed in hemocytes, lymph glands, and the central nervous system (CNS) and that dC1GalT1 mutant larvae display decreased numbers of circulating hemocytes and excessive differentiation of hematopoietic stem cells in lymph glands. dC1GalT1 mutant larvae have also been shown to have morphological defects in the CNS. However, the functions of T antigen in other tissues remain largely unknown. In this study, we found that glycans contributed to the localization of neuromuscular junction (NMJ) boutons. In dC1GalT1 mutant larvae, NMJs were ectopically formed in the cleft between muscles 6 and 7 and connected with these two muscles. dC1GalT1 synthesized T antigen, which was expressed at NMJs. In addition, we determined the function of mucin-type O-glycans in muscle cells. In dC1GalT1 mutant muscles, myofibers and basement membranes were disorganized. Moreover, ultrastructural defects in NMJs and accumulation of large endosome-like structures within both NMJ boutons and muscle cells were observed in dC1GalT1 mutants. Taken together, these results demonstrated that mucin-type O-glycans synthesized by dC1GalT1 were involved in the localization of NMJ boutons, synaptogenesis of NMJs, establishment of muscle cell architecture, and endocytosis.

Glycans define the stemness of naïve and primed pluripotent stem cells.

Cell surface glycans are tissue-specific and developmentally regulated. They function as essential modulators in cell-cell interactions, cell-extracellular matrix interactions, and ligand-receptor interactions, binding to various ligands, including Wnt, fibroblast growth factors, and bone morphogenetic proteins. Embryonic stem (ES) cells, originally derived from the inner cell mass of blastocysts, have the essential characteristics of pluripotency and self-renewal. Recently, it has been proposed that mouse and human conventional ES cells are present in different developmental stages, namely pre-implantation blastocyst and post-implantation blastocyst stages, also called the naïve state and the primed state, respectively. They therefore require different extrinsic signals for the maintenance of self-renewal and pluripotency, and also appear to require different surface glycans. Understanding of molecular mechanisms involving glycans in self-renewal and pluripotency of ES cells is increasingly important for potential clinical applications, as well as for basic research. This review focuses on the roles of glycans in the two different states of pluripotent stem cells, namely the naïve state and the primed state, and the transition between these two states.

Reduction of T antigen causes loss of hematopoietic progenitors in Drosophila through the inhibition of filopodial extensions from the hematopoietic niche.

Hematopoietic stem cells (HSCs) are present in hematopoietic organs and differentiate into mature blood cells as required. Defective HSCs have been implicated in the human autoimmune disease Tn syndrome, which results from the failure of the core 1 ß1,3-galactosyltransferase 1 enzyme (C1ß3GalT1) to synthesize T antigen. In both mice and humans, a reduced level of T antigen is associated with a reduction in blood cell numbers. However, the precise roles of T antigen in hematopoiesis are unknown. Here, we show that the Drosophila T antigen, supplied by plasmatocytes, is essential for the regulation of HSCs. T antigen appears to be an essential factor in maintaining the extracellular environment to support filopodial extensions from niches that are responsible for transmitting signaling molecules to maintain the HSCs. In addition, our results revealed that the clotting factor, hemolectin, disrupted the hemolymph environment of C1ß3GalT1 mutants. This study identified a novel mucin function for the regulation of HSCs that may be conserved in other species.

O-GlcNAc is required for the survival of primed pluripotent stem cells and their reversion to the naïve state.

"Naïve" mouse embryonic stem cells (ESCs) are derived from pre-implantation embryos and possess pluripotency, the ability to differentiate into any cell type of the body. "Primed" mouse epiblast stem cells (EpiSCs) are also pluripotent but are derived from post-implantation embryos. ESC-derived EpiSCs (ESD-EpiSCs) are "primed" pluripotent stem cells and can revert to naïve reverted ESCs (rESCs). O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation) is a posttranslational modification in the cytoplasm and nucleus. O-GlcNAc is transferred to serine and threonine residues of proteins by O-GlcNAc transferase (Ogt) and removed from them by O-GlcNAcase (Oga). In naïve ESCs, O-GlcNAc contributes to maintain the undifferentiated state. In the transition from naïve state to primed state, Ogt maintains cell survival, whereas Oga has no function. However, the function of O-GlcNAc in primed ESD-EpiSCs and during the reversion from the primed state to naïve rESCs remains unclear. Here, we show that Ogt is required for the survival of primed ESD-EpiSCs. The expression of cytosolic Oga was significantly increased during induction from naïve ESCs to primed ESD-EpiSCs. Furthermore, both Ogt and Oga were required for the reversion from primed ESD-EpiSCs to naïve rESCs. These findings indicate that O-GlcNAcylation plays an important role in the survival of primed ESD-EpiSCs and in their reversion to naïve rESCs.

Phenotype-based clustering of glycosylation-related genes by RNAi-mediated gene silencing.

Glycan structures are synthesized by a series of reactions conducted by glycosylation-related (GR) proteins such as glycosyltransferases, glycan-modifying enzymes, and nucleotide-sugar transporters. For example, the common core region of glycosaminoglycans (GAGs) is sequentially synthesized by peptide-O-xylosyltransferase, ß1,4-galactosyltransferase I, ß1,3-galactosyltransferase II, and ß1,3-glucuronyltransferase. This raises the possibility that functional impairment of GR proteins involved in synthesis of the same glycan might result in the same phenotypic abnormality. To examine this possibility, comprehensive silencing of genes encoding GR and proteoglycan core proteins was conducted in Drosophila. Drosophila GR candidate genes (125) were classified into five functional groups for synthesis of GAGs, N-linked, O-linked, Notch-related, and unknown glycans. Spatiotemporally regulated silencing caused a range of malformed phenotypes that fell into three types: extra veins, thick veins, and depigmentation. The clustered phenotypes reflected the biosynthetic pathways of GAGs, Fringe-dependent glycan on Notch, and glycans placed at or near nonreducing ends (herein termed terminal domains of glycans). Based on the phenotypic clustering, CG33145 was predicted to be involved in formation of terminal domains. Our further analysis showed that CG33145 exhibited galactosyltransferase activity in synthesis of terminal N-linked glycans. Phenotypic clustering, therefore, has potential for the functional prediction of novel GR genes.

Frequent glycan structure mining of influenza virus data revealed a sulfated glycan motif that increased viral infection.

MOTIVATION: It is well known influenza viruses recognize and bind terminal sialic acid (SA) on glycans that are found on the cell surface. In this work, we used a data mining technique to analyze the glycan array data of influenza viruses to find novel glycan structures other than SA that may be involved in viral infection. RESULTS: In addition to SA structures noted previously, we noted the sulfated structures in the mining results. For verification, we overexpressed the sulfotransferase that is involved in synthesizing these structures, and we performed a viral infection experiment to assess changes in infection in these cells. In our results, we found that there is a 70-fold increase in these cells compared with the control. Thus, we have found a novel pattern in glycan structures that may be involved in viral infection. AVAILABILITY AND IMPLEMENTATION: The Glycan Miner Tool is available from the RINGS resource at http://www.rings.t.soka.ac.jp.

Identification of ß1,3-galactosyltransferases responsible for biosynthesis of insect complex-type N-glycans containing a T-antigen unit in the honeybee.

Honeybees (Apis mellifera) produce unique complex-type N-glycans bearing a Galß1-3GalNAc (T-antigen) unit, and honeybee-specific N-glycans are linked to royal jelly glycoproteins. In this study, we identified two novel honeybee ß1,3-galactosyltransferase (ß1,3-GalT) genes responsible for biosynthesis of the T-antigen in insect N-glycans. The products of the two putative ß1,3-GalT genes (ß1,3-GalT1 and ß1,3-GalT2), which were expressed in Sf21 insect cells, transferred galactose (Gal) residues to GalNAc2GlcNAc2Man3GlcNAc2-PA to form the Galß1-3GalNAc unit, indicating that the identified genes were involved in biosynthesis of the ß1-3 Gal-containing N-glycan. Therefore, using biochemistry and molecular biology techniques, we revealed a unique N-glycan biosynthesis mechanism in the cephalic region of honeybees, which has not previously been found in other animal or plant cells.

Immuno-electron microscopy of primary cell cultures from genetically modified animals in liquid by atmospheric scanning electron microscopy.

High-throughput immuno-electron microscopy is required to capture the protein-protein interactions realizing physiological functions. Atmospheric scanning electron microscopy (ASEM) allows in situ correlative light and electron microscopy of samples in liquid in an open atmospheric environment. Cells are cultured in a few milliliters of medium directly in the ASEM dish, which can be coated and transferred to an incubator as required. Here, cells were imaged by optical or fluorescence microscopy, and at high resolution by gold-labeled immuno-ASEM, sometimes with additional metal staining. Axonal partitioning of neurons was correlated with specific cytoskeletal structures, including microtubules, using primary-culture neurons from wild type Drosophila, and the involvement of ankyrin in the formation of the intra-axonal segmentation boundary was studied using neurons from an ankyrin-deficient mutant. Rubella virus replication producing anti-double-stranded RNA was captured at the host cell's plasma membrane. Fas receptosome formation was associated with clathrin internalization near the surface of primitive endoderm cells. Positively charged Nanogold clearly revealed the cell outlines of primitive endoderm cells, and the cell division of lactic acid bacteria. Based on these experiments, ASEM promises to allow the study of protein interactions in various complexes in a natural environment of aqueous liquid in the near future.

Integrated Systems Analysis Deciphers Transcriptome and Glycoproteome Links in Alzheimer's Disease.

Glycosylation is increasingly recognized as a potential therapeutic target in Alzheimer's disease. In recent years, evidence of Alzheimer's disease-specific glycoproteins has been established. However, the mechanisms underlying their dysregulation, including tissue- and cell-type specificity, are not fully understood. We aimed to explore the upstream regulators of aberrant glycosylation by integrating multiple data sources using a glycogenomics approach. We identified dysregulation of the glycosyltransferase PLOD3 in oligodendrocytes as an upstream regulator of cerebral vessels and found that it is involved in COL4A5 synthesis, which is strongly correlated with amyloid fiber formation. Furthermore, COL4A5 has been suggested to interact with astrocytes via extracellular matrix receptors as a ligand. This study suggests directions for new therapeutic strategies for Alzheimer's disease targeting glycosyltransferases.

The transition of mouse pluripotent stem cells from the naïve to the primed state requires Fas signaling through 3-O sulfated heparan sulfate structures recognized by the HS4C3 antibody.

The characteristics of pluripotent embryonic stem cells of human and mouse are different. The properties of human embryonic stem cells (hESCs) are similar to those of mouse epiblast stem cells (mEpiSCs), which are in a later developmental pluripotency state, the so-called "primed state" compared to mouse embryonic stem cells (mESCs) which are in a naïve state. As a result of the properties of the primed state, hESCs proliferate slowly, cannot survive as single cells, and can only be transfected with genes at low efficiency. Generating hESCs in the naïve state is necessary to overcome these problems and allow their application in regenerative medicine. Therefore, clarifying the mechanism of the transition between the naïve and primed states in pluripotent stem cells is important for the establishment of stable methods of generating naïve state hESCs. However, the signaling pathways which contribute to the transition between the naïve and primed states are still unclear. In this study, we carried out induction from mESCs to mEpiSC-like cells (mEpiSCLCs), and observed an increase in the activation of Fas signaling during the induction. The expression of Fgf5, an epiblast marker, was diminished by inhibition of Fas signaling using the caspase-8 and -3 blocking peptides, IETD and DEVD, respectively. Furthermore, during the induction, we observed increased expression of 3-O sulfated heparan sulfate (HS) structures synthesized by HS 3-O-sulfotransferase (3OST), which are recognized by the HS4C3 antibody (HS4C3-binding epitope). Knockdown of 3OST-5 reduced Fas signaling and the potential for the transition to mEpiSCLCs. This indicates that the HS4C3-binding epitope is necessary for the transition to the primed state. We propose that Fas signaling through the HS4C3-binding epitope contributes to the transition from the naïve state to the primed state.

Identification of genes required for neural-specific glycosylation using functional genomics.

Glycosylation plays crucial regulatory roles in various biological processes such as development, immunity, and neural functions. For example, α1,3-fucosylation, the addition of a fucose moiety abundant in Drosophila neural cells, is essential for neural development, function, and behavior. However, it remains largely unknown how neural-specific α1,3-fucosylation is regulated. In the present study, we searched for genes involved in the glycosylation of a neural-specific protein using a Drosophila RNAi library. We obtained 109 genes affecting glycosylation that clustered into nine functional groups. Among them, members of the RNA regulation group were enriched by a secondary screen that identified genes specifically regulating α1,3-fucosylation. Further analyses revealed that an RNA-binding protein, second mitotic wave missing (Swm), upregulates expression of the neural-specific glycosyltransferase FucTA and facilitates its mRNA export from the nucleus. This first large-scale genetic screen for glycosylation-related genes has revealed novel regulation of fucTA mRNA in neural cells.

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.