7 research outputs found
The Chemical Neurobiology of Carbohydrates
The cell surface displays a complex array of oligosaccharides, glycoproteins, and glycolipids. This diverse mixture of glycans contains a wealth of information, modulating a wide range of processes such as cell migration, proliferation, transcriptional regulation, and differentiation. Glycosylation is one of the most ubiquitous forms of post-translational modification, with more than 50% of the human proteome estimated to be glycosylated. Glycosylation adds another dimension to the complexity of cellular signaling and expands the ability of a cell to modulate protein function. The structural complexity of glycan modifications ranges from the addition of a single monosaccharide unit to polysaccharides containing hundreds of sugars in branched or linear arrays. This chemical diversity enables glycans to impart a vast array of functions, from structural stability and proteolytic protection to protein recognition and modulation of cell signaling networks.
Emerging evidence suggests a pivotal role for glycans in regulating nervous system development and function. For instance, glycosylation influences various neuronal processes, such as neurite outgrowth and morphology, and may contribute to the molecular events that underlie learning and memory. Glycosylation is an efficient modulator of cell signaling and has been implicated in memory consolidation pathways. Genetic ablation of glycosylation enzymes often leads to developmental defects and can influence various organismal behaviors such as stress and cognition. Thus, the complexity of glycan functions help to orchestrate proper neuronal development during embryogenesis, as well as influence behaviors in the adult organism.
The importance of glycosylation is further highlighted by defects in glycan structures that often lead to human disease, as exhibited by congenital disorders of glycosylation (CDG).25–29 These are usually inherited disorders resulting from defects in glycan biosynthesis, which are accompanied by severe developmental abnormalities, mental retardation, and difficulties with motor coordination. Such disorders highlight the importance of glycan biosynthesis in human health and development. Because therapeutic treatments are currently limited, investigations into the structure–activity relationships of glycans, as well as disease-associated alterations to glycan structure, are crucial for developing strategies to combat these diseases.
Understanding the structure–function relationships of glycans has been hampered by a lack of tools and methods to facilitate their analysis. In contrast to nucleic acids and proteins, oligosaccharides often have branched structures, and their biosynthesis is not template-encoded. As such, the composition and sequence of oligosaccharides cannot be easily predicted, and genetic manipulations are considerably less straightforward. Analytical techniques for investigating oligosaccharide composition, sequence, and tertiary structure are still undergoing development and are far from routine, unlike methods for DNA and protein analysis. Lastly, glycan structures are not under direct genetic control and, thus, are often heterogeneous. This heterogeneity complicates structure–function analyses by traditional biochemical approaches that rely on the isolation and purification of glycans from natural sources.
The problems associated with oligosaccharide analysis have hindered efforts to understand the biology of oligosaccharides yet have given chemists a unique opportunity to develop new methods to overcome these challenges. The development of chemical tools for the analysis of glycan structure and function is essential to advance our understanding of the roles of glycoconjugates in regulating diverse biological processes. In this review, we will highlight the emerging area of glyconeurobiology with an emphasis on current chemical approaches for elucidating the biological functions of glycans in the nervous system
Identification of the Plasticity-Relevant Fucose-α(1−2)-Galactose Proteome from the Mouse Olfactory Bulb
Fucose-α(1−2)-galactose [Fucα(1−2)Gal] sugars have been implicated in the molecular mechanisms that underlie neuronal development, learning, and memory. However, an understanding of their precise roles has been hampered by a lack of information regarding Fucα(1−2)Gal glycoproteins. Here, we report the first proteomic studies of this plasticity-relevant epitope. We identify five classes of putative Fucα(1−2)Gal glycoproteins: cell adhesion molecules, ion channels and solute carriers/transporters, ATP-binding proteins, synaptic vesicle-associated proteins, and mitochondrial proteins. In addition, we show that Fucα(1−2)Gal glycoproteins are enriched in the developing mouse olfactory bulb (OB) and exhibit a distinct spatiotemporal expression that is consistent with the presence of a “glycocode” to help direct olfactory sensory neuron (OSN) axonal pathfinding. We find that expression of Fucα(1−2)Gal sugars in the OB is regulated by the α(1−2)fucosyltransferase FUT1. FUT1-deficient mice exhibit developmental defects, including fewer and smaller glomeruli and a thinner olfactory nerve layer, suggesting that fucosylation contributes to OB development. Our findings significantly expand the number of Fucα(1−2)Gal glycoproteins and provide new insights into the molecular mechanisms by which fucosyl sugars contribute to neuronal processes
Identification of the Plasticity-Relevant Fucose-α(1−2)-Galactose Proteome from the Mouse Olfactory Bulb
Fucose-α(1−2)-galactose [Fucα(1−2)Gal] sugars have been implicated in the molecular mechanisms that underlie neuronal development, learning, and memory. However, an understanding of their precise roles has been hampered by a lack of information regarding Fucα(1−2)Gal glycoproteins. Here, we report the first proteomic studies of this plasticity-relevant epitope. We identify five classes of putative Fucα(1−2)Gal glycoproteins: cell adhesion molecules, ion channels and solute carriers/transporters, ATP-binding proteins, synaptic vesicle-associated proteins, and mitochondrial proteins. In addition, we show that Fucα(1−2)Gal glycoproteins are enriched in the developing mouse olfactory bulb (OB) and exhibit a distinct spatiotemporal expression that is consistent with the presence of a “glycocode” to help direct olfactory sensory neuron (OSN) axonal pathfinding. We find that expression of Fucα(1−2)Gal sugars in the OB is regulated by the α(1−2)fucosyltransferase FUT1. FUT1-deficient mice exhibit developmental defects, including fewer and smaller glomeruli and a thinner olfactory nerve layer, suggesting that fucosylation contributes to OB development. Our findings significantly expand the number of Fucα(1−2)Gal glycoproteins and provide new insights into the molecular mechanisms by which fucosyl sugars contribute to neuronal processes
Development of Sulfonamide Photoaffinity Inhibitors for Probing Cellular γ‑Secretase
γ-Secretase
is a multiprotein complex that catalyzes intramembrane proteolysis
associated with Alzheimer’s disease and cancer. Here, we have
developed potent sulfonamide clickable photoaffinity probes that target
γ-secretase <i>in vitro</i> and in cells by incorporating
various photoreactive groups and walking the clickable alkyne handle
to different positions around the molecule. We found that benzophenone
is preferred over diazirine as a photoreactive group within the sulfonamide
scaffold for labeling γ-secretase. Intriguingly, the placement
of the alkyne at different positions has little effect on probe potency
but has a significant impact on the efficiency of labeling of γ-secretase.
Moreover, the optimized clickable photoprobe, 163-BP3, was utilized
as a cellular probe to effectively assess the target engagement of
inhibitors with γ-secretase in primary neuronal cells. In addition,
biotinylated 163-BP3 probes were developed and used to capture the
native γ-secretase complex in the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate
(CHAPSO) solubilized state. Taken together, these next generation
clickable and biotinylated sulfonamide probes offer new tools to study
γ-secretase in biochemical and cellular systems. Finally, the
data provide insights into structural features of the sulfonamide
inhibitor binding site in relation to the active site and into the
design of clickable photoaffinity probes
Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging
Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging
Bioorthogonal
reactions, including the strain-promoted azide–alkyne
cycloaddition (SPAAC) and inverse electron demand Diels–Alder
(iEDDA) reactions, have become increasingly popular for live-cell
imaging applications. However, the stability and reactivity of reagents
has never been systematically explored in the context of a living
cell. Here we report a universal, organelle-targetable system based
on HaloTag protein technology for directly comparing bioorthogonal
reagent reactivity, specificity, and stability using clickable HaloTag
ligands in various subcellular compartments. This system enabled a
detailed comparison of the bioorthogonal reactions in live cells and
informed the selection of optimal reagents and conditions for live-cell
imaging studies. We found that the reaction of sTCO with monosubstituted
tetrazines is the fastest reaction in cells; however, both reagents
have stability issues. To address this, we introduced a new variant
of sTCO, Ag-sTCO, which has much improved stability and can be used
directly in cells for rapid bioorthogonal reactions with tetrazines.
Utilization of Ag complexes of conformationally strained <i>trans</i>-cyclooctenes should greatly expand their usefulness especially when
paired with less reactive, more stable tetrazines