Bioorthogonal chemical reporter methodology for visualization, isolation and analysis of glycoconjugates

Boons, Geert‐Jan

doi:10.1039/9781849730891-00152

Cited by 17 publications

(5 citation statements)

References 109 publications

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“…Chemical reporter strategies have been applied to visualize glycans in cells and living organisms with success (18)(19)(20)(21)(22)(23)(24)(25), however, insights into the functional relevance of glycans in normal and disease biology have yet to emerge from these applications. Our findings pave the way for the use of chemical reporter strategies to study the impact of altered glycosylation on the pathophysiology of human diseases.…”

Section: Discussionmentioning

confidence: 99%

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Abnormal accumulation and recycling of glycoproteins visualized in Niemann–Pick type C cells using the chemical reporter strategy

Mbua

Flanagan-Steet

Johnson

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Niemann-Pick type C (NPC) disease is characterized by impaired cholesterol efflux from late endosomes and lysosomes and secondary accumulation of lipids. Although impaired trafficking of individual glycoproteins and glycolipids has been noted in NPC cells and other storage disorders, there is currently no effective way to monitor their localization and movement en masse. Using a chemical reporter strategy in combination with pharmacologic treatments, we demonstrate a disease-specific and previously unrecognized accumulation of a diverse set of glycoconjugates in NPC1-null and NPC2-deficient fibroblasts within endocytic compartments. These labeled vesicles do not colocalize with the cholesterol-laden compartments of NPC cells. Experiments using the endocytic uptake marker dextran show that the endosomal accumulation of sialylated molecules can be largely attributed to impaired recycling as opposed to altered fusion of vesicles. Treatment of either NPC1-null or NPC2-deficient cells with cyclodextrin was effective in reducing cholesterol storage as well as the endocytic accumulation of sialoglycoproteins, demonstrating a direct link between cholesterol storage and abnormal recycling. Our data further demonstrate that this accumulation is largely glycoproteins, given that inhibitors of O-glycan initiation or N-glycan processing led to a significant reduction in staining intensity. Taken together, our results provide a unique perspective on the trafficking defects in NPC cells, and highlight the utility of this methodology in analyzing cells with altered recycling and turnover of glycoproteins.

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Section: Discussionmentioning

confidence: 99%

“…The chemical reporter strategy, pioneered by Bertozzi and coworkers (16)(17)(18)(19), is emerging as a versatile approach to visualizing and capturing glycoconjugates of living systems. In this approach, a unique chemical functionality (reporter) is incorporated into glycoconjugates by feeding a modified biosynthetic precursor.…”

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confidence: 99%

Abnormal accumulation and recycling of glycoproteins visualized in Niemann–Pick type C cells using the chemical reporter strategy

Mbua

Flanagan-Steet

Johnson

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…The ability to label glycans using chemical glycobiology methods has ushered in new opportunities to investigate the functions of these molecules in living systems (1)(2)(3). These studies are beginning to yield insight into how glycan profiles changes during development and how the content, localization, and trafficking of glycans is altered in the context of many human diseases (4 -9).…”

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confidence: 99%

Selective Exo-Enzymatic Labeling Detects Increased Cell Surface Sialoglycoprotein Expression upon Megakaryocytic Differentiation

Yu¹,

Zhao²,

Sun

et al. 2016

Journal of Biological Chemistry

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Selective exo-enzymatic labeling (or SEEL) uses recombinant glycosyltransferases and nucleotide-sugar analogues to allow efficient labeling of cell surface glycans. SEEL can circumvent many of the possible issues associated with metabolic labeling, including low incorporation of sugar precursors, and allows for sugars to be added selectively to different types of glycans by virtue of the inherent specificity of the glycosyltransferases. Here we compare the labeling of sialoglycoproteins in undifferentiated and differentiated human erythroleukemia cells (HEL) using SEEL using the sialyltransferases ST6Gal1 and ST3Gal1, which label N-and O-glycans, respectively. Our results show that the profile of glycoproteins detected varies between undifferentiated HEL cells and those differentiated to megakaryocytes, with a shift to more N-linked sialoglycoproteins in the differentiated cells. The efficiency of SEEL for both sialyltransferases in HEL cells was greatly increased with prior neuraminidase treatment highlighting the necessity for the presence of available acceptors with this labeling method. Following metabolic labeling or SEEL, tagged glycoproteins were enriched by immunoprecipitation and identified using mass spectrometry. The proteomic findings demonstrated that the detection of many glycoproteins is markedly improved by SEEL labeling, and that unique glycoproteins can be identified using either ST6Gal1 or ST3Gal1. Furthermore, this analysis enabled the identification of increased surface expression of several sialylated cell adhesion molecules, including the known megakaryocytic markers integrin␤3 and CD44, upon differentiation of HEL cells to adherent megakaryocytes.The ability to label glycans using chemical glycobiology methods has ushered in new opportunities to investigate the functions of these molecules in living systems (1-3). These studies are beginning to yield insight into how glycan profiles changes during development and how the content, localization, and trafficking of glycans is altered in the context of many human diseases (4 -9). Most methodologies utilize metabolic labeling as a way to incorporate functionalized sugars into glycans during biosynthesis. The extent of labeling in some cell types may be limited by the presence of endogenous non-labeled sugar precursors that can dilute out the azide-sugars available for incorporation. Variable distribution of azide-sugars into different glycan classes (e.g. glycoproteins versus glycolipids) following metabolic labeling can also skew or limit the types of glycans that can be analyzed. As an alternative approach, labeling methods have been developed that instead rely on the use of recombinant or purified glycosyltransferases to install functionalized sugars directly onto existing glycans (10 -12). This method, recently termed SEEL (selective exoenzymatic labeling), 4 leverages the inherent selectivity of the recombinant glycosyltransferases to label certain types (e.g. N-linked or O-linked) of glycans. Prior work using the sialyltransferase ST6Gal...

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“…The development of bioorthogonal chemistry empowered wide applications of metabolic cell surface SA engineering, such as detection, imaging, and isolation of sialyl glycoconjugates of living cells, tissues, and organisms. − Various reactive functional groups such as ketones, azides, alkynes, thiol, alkenes, and diazirines can be incorporated into SAs on the cell surface and functionalized under physiological conditions and in a chemically selective way for imaging, isolation, and derivatization applications . Among them, the azido functional group is most often incorporated into cell surface SAs by using N -azidoacetylmannosamine (ManNAz).…”

Section: Metabolic Labeling Approaches To Profiling Sas On the Cell S...mentioning

confidence: 99%

Recent Chemical Biology Approaches for Profiling Cell Surface Sialylation Status

et al. 2018

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Sialic acids (SAs) often exist as the terminal sugars of glycans of either glycoproteins or glycolipids on the cell surface and thus are directly involved in biological processes, such as cell-cell, cell-ligand, and cell-pathogen interactions. Cell surface SA expression levels and their linkages are collectively termed cell surface sialylation status, which represent varying cellular states and contribute to the overall functionality of a cell. Accordingly, systemic and specific profiling of the cell surface sialyation status is critical in deciphering the structures and functions of cell surface glycoconjugates and the molecular mechanisms of their underlying biological processes. In recent decades, several advanced chemical biology approaches have been developed to profile the cell surface sialyation status of both in vitro and in vivo samples, including metabolic labeling, direct chemical modification, and boronic acid coupling approaches. Various investigative technologies have also been explored for their unique competence, including fluorescent imaging, flow cytometry, Raman imaging, magnetic resonance imaging (MRI), and matrix-assisted laser desorption ionization imaging mass spectrometry. In particular, the sialylation status of a specific glycoprotein on the cell surface has been investigated. This review highlights the recent advancements in chemical biology approaches for profiling cell surface sialyation status. It is expected that this review will provide researchers different choices for both biological and biomedical research and applications.

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Bioorthogonal chemical reporter methodology for visualization, isolation and analysis of glycoconjugates

Cited by 17 publications

References 109 publications

Abnormal accumulation and recycling of glycoproteins visualized in Niemann–Pick type C cells using the chemical reporter strategy

Abnormal accumulation and recycling of glycoproteins visualized in Niemann–Pick type C cells using the chemical reporter strategy

Selective Exo-Enzymatic Labeling Detects Increased Cell Surface Sialoglycoprotein Expression upon Megakaryocytic Differentiation

Recent Chemical Biology Approaches for Profiling Cell Surface Sialylation Status

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