Bioorthogonal Labeling Reveals Different Expression of Glycans in Mouse Hippocampal Neuron Cultures during Their Development (original) (raw)
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Priming the Cellular Glycocalyx for Neural Development
ACS Chemical Neuroscience, 2014
Glycans are important contributors to the development and function of the nervous system with enormous potential as therapeutic targets. However, a general lack of tools for tailoring the presentation of specific glycan structures on the surfaces of cells has left them largely unexplored in the biomedical context. In this Viewpoint, we briefly summarize the distinct challenges and complexities of the Glycome. We also highlight an emerging concept of cell surface engineering using synthetic nanoscale mimetics of native glycoconjugates to harness some of the unique biology of glycans, with an eye toward advancing stem cell-based neuroregenerative therapies.
Analytical Chemistry, 2015
Tissue glyco-capture (TGC), a highly sensitive MScompatible method for extraction of glycans from tissue, was combined with structure-specific nano-LC/MS for sensitive and detailed profiling of the mouse brain glycome. Hundreds of glycan structures were directly detected by accurate mass MS and structurally elucidated by MS/MS, revealing the presence of novel glycan motifs such as antennary fucosylation, sulfation, and glucuronidation that are potentially associated with cellular signaling and adhesion. Microgram-level sensitivity enabled glycomic analysis of specific regions of the brain, as demonstrated on not only brain sections (with a onedimensional spatial resolution of 20 μm) but also isolated brain structures (e.g., the hippocampus). Reproducibility was extraordinarily high (R > 0.98) for both method and instrumental replicates. The pairing of TGC with structure-specific nano-LC/MS was found to be an exceptionally powerful platform for qualitative and quantitative exploration of the brain glycome.
Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry
Journal of Visualized Experiments, 2011
Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide-or alkyne-tagged monosaccharides 1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy 3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid 4 and N-acetylgalactosamine 5, 6 , in developing zebrafish and in other living organisms. Protocol 1. Egg Collection and Dechorionation 2. Microinjection with GDP-FucAl 3. BTTES-Cu(I)-catalyzed Click Chemistry Reaction 4. Imaging
Glycobiology, 2006
Biosynthesis of N-glycans varies significantly among tissues and is strictly regulated spatially and temporally within the tissue. The strict molecular mechanisms that are responsible for control of N-glycan synthesis remain largely unknown. We developed complementary deoxyribonucleic acid (cDNA) macroarray system and analyzed gene expression levels of more than 140 glycosyltransferases and glycosidases in the cerebral cortex from developing and adult mice. We also analyzed the relative amounts of major N-glycans present in the cerebral cortex and examined how the synthesis of N-glycans might be regulated through the expression of these genes. We demonstrated that the content of N-linked oligosaccharides dramatically changed during the course of brain development. Some of these changes could not be explained by alterations in the expression of the corresponding genes. For example, the amount of core fucosylated sugar chains in the early embryonic brain and the expression level of fucosyltransferase VIII, the only gene known to be responsible for core fucosylation, did not change proportionately. This result suggests that posttranscriptional regulation of this gene plays an important role in regulating its enzymatic activity. On the other hand, the amount of b1,3-galactose residue-containing sugar chains increased postnatally following an increase in the level of b1,3-galactosyltransferase messenger ribonucleic acid (mRNA). Furthermore, the amount of sugar chains with an outer fucose residue, containing LewisX-BA-2, correlated well with the expression of fusocyltransferase IX mRNA. These findings add to our understanding of the molecular mechanisms responsible for the regulation of N-glycan biosynthesis in the cerebral cortex.
Tools for Studying Glycans: Recent Advances in Chemoenzymatic Glycan Labeling
ACS Chemical Biology, 2017
The study of cellular glycosylation presents many challenges due, in large part, to the nontemplate driven nature of glycan biosynthesis and their structural complexity. Chemoenzymatic glycan labeling (CEGL) has emerged as a new technique to address the limitations of existing methods for glycan detection. CEGL combines glycosyltransferases and unnatural nucleotide sugar donors equipped with a bioorthogonal chemical tag to directly label specific glycan acceptor substrates in situ within biological samples. This article reviews the current CEGL strategies that are available to characterize cell-surface and intracellular glycans. Applications include imaging glycan expression status in live cells and tissue samples, proteomic analysis of glycoproteins, and target validation. Combined with genetic and biochemical tools, CEGL provides new opportunities to elucidate the functional roles of glycans in human health and disease.
European Journal of Biochemistry, 1998
Oligosaccharides expressed on cell surface and extracellular matrix glycoconjugates are potentially of crucial importance in determining many cell interactions. The complexity of cellular organisation of the brain and suggested involvement of N-glycosylation in neural development, make this an ideal system to study the potential role of glycosylation in tissue development, maintenance and function. Neural tissues are known to contain some highly unusual glycan structures but the structures expressed in neural tissue have not as yet been studied systematically. As a first initiative to assess the type of N-glycosylation occurring in neural tissue, we have characterised all of the major neutral N-linked oligosaccharides expressed in adult rat using a combination of matrix-assisted laser-desorption ionisation mass spectrometry, exoglycosidase sequencing combined with normal-phase HPLC, and two-dimensional HPLC mapping. Oligomannosidic glycans, Man (9Ϫ5)GlcNAc2, constituted approximately 15% of the total brain N-glycan pool. The other neutral N-glycan components consisted of a series of diantennary structures (6.5%), (2,6)branched triantennary glycans (1 %) and hybrid structures (3%). Both the complex and hybrid N-glycans were characterised by the presence of outer-arm A(1,3)-fucosylation (forming the Lewis x determinant), A(1,6)-core fucosylation and a bisecting GlcNAc residue. Some of these are unusual or novel structures not having been reported elsewhere. A large proportion of the diantennary N-glycans either lacked Gal residues entirely or were unsubstituted on one Man residue of the trimannosyl core, notably the Man A(1,3)-arm. This isomeric form is indicative of the action of a novel β-hexosaminidase activity and suggests a modification in the classical biosynthetic pathway for N-linked oligosaccharides. Furthermore, expression of large amounts of oligomannosidic glycans is not usually associated with tissue glycoproteins and suggests a possible involvement of these structures in neural cell interactions.
eLife, 2018
Proper brain development relies highly on protein N-glycosylation to sustain neuronal migration, axon guidance and synaptic physiology. Impairing the N-glycosylation pathway at early steps produces broad neurological symptoms identified in congenital disorders of glycosylation. However, little is known about the molecular mechanisms underlying these defects. We generated a cerebellum specific knockout mouse for , a gene involved in the initiation of N-glycosylation. In addition to motor coordination defects and abnormal granule cell development, deletion causes mild N-glycosylation impairment without significantly altering ER homeostasis. Using proteomic approaches, we identified that loss affects a subset of glycoproteins with high N-glycans multiplicity per protein and decreased protein abundance or N-glycosylation level. As IgSF-CAM adhesion proteins are critical for neuron adhesion and highly N-glycosylated, we observed impaired IgSF-CAM-mediated neurite outgrowth and axon guida...
European Journal of Biochemistry, 2001
Polysialylation of the neural cell adhesion molecule (N-CAM) is known to destabilize cell-cell adhesion and to promote plasticity in cell-cell interactions. To gain more insights into the molecular mechanisms regulating the selective expression of polysialic acid on distinct glycan chains, the underlying core structures of polysialylated N-CAM glycans from newborn mouse brain were examined. Starting from low picomolar amounts of oligosaccharides, a multistep approach was used that was based on various mass spectrometric techniques with minimized sample consumption. Evidence could be provided that polysialylated murine N-CAM glycans comprise diantennary, triantennary and tetraantennary core structures carrying, in part, type-1 N-acetyllactosamine antennae, sulfate groups linked to terminal galactose or subterminal N-acetylglucosamine residues and, as a characteristic feature, a sulfated glucuronic acid unit which was bound exclusively to C3 of terminal galactose in Mana3-linked type-2 antennae. Hence, our results reveal that part of the murine N-CAM carbohydrates are modified within a single oligosaccharide by polysialic acid plus a HSO 3-GlcA-moiety, which is likely to represent a HNK1-epitope. As HNK1-carbohydrates are also known to modulate cell-cell interactions, the simultaneous presence of both carbohydrate epitopes may reflect a new mechanism involved in the fine-tuning of N-CAM functions.
Monitoring Dynamic Glycosylation in Vivo Using Supersensitive Click Chemistry
Bioconjugate Chemistry, 2014
To monitor the kinetics of biological processes that take place within the minute time scale, simple and fast analytical methods are required. In this article, we present our discovery of an azide with an internal Cu(I)-chelating motif that enabled the development of the fastest protocol for Cu(I)catalyzed azide−alkyne cycloaddition (CuAAC) to date, and its application toward following the dynamic process of glycan biosynthesis. We discovered that an electron-donating picolyl azide boosted the efficiency of the ligand-accelerated CuAAC 20−38-fold in living systems with no apparent toxicity. With a combination of this azide and BTTPS, a tris(triazolylmethyl)amine-based ligand for Cu(I), we were able to detect newly synthesized cell-surface glycans by flow cytometry using as low as 1 nM of a metabolic precursor. This supersensitive chemistry enabled us to monitor the dynamic glycan biosynthesis in mammalian cells and in early zebrafish embryogenesis. In live mammalian cells, we discovered that it takes approximately 30−45 min for a monosaccharide building block to be metabolized and incorporated into cell-surface glycoconjugates. In zebrafish embryos, the labeled glycans could be detected as early as the two-cell stage. To our knowledge, this was the first time that newly synthesized glycans were detected at the cleavage period (0.75−2 hpf) in an animal model using bioorthogonal chemistry.