Metabolic labeling of sialic acids in living animals with alkynyl sugars.
Ontology highlight
ABSTRACT:
SUBMITTER: Chang PV
PROVIDER: S-EPMC2868584 | biostudies-literature | 2009
REPOSITORIES: biostudies-literature
Publications
Metabolic labeling of sialic acids in living animals with alkynyl sugars.
Chang Pamela V PV Chen Xing X Smyrniotis Chris C Xenakis Alexander A Hu Tianshun T Bertozzi Carolyn R CR Wu Peng P
Angewandte Chemie (International ed. in English) 20090101 22
Sialome sweet sialome: As sialic acids are involved in many host-pathogen recognition events and are markers of embryonic and malignant tissues, there is great interest in methods for the enrichment and identification of sialylated glycoproteins from complex tissues. Now N-(4-pentynoyl)mannosamine can be used to metabolically label sialylated glycoproteins in living animals, enabling future identification of new biomarkers. ...[more]
Similar Datasets
Project description:The posttranslational modification of neural cell-adhesion molecule (NCAM) with polysialic acid (PSA) and the spatiotemporal distribution of PSA-NCAM play an important role in the neuronal development. In this work, we developed a tissue-based strategy for metabolically incorporating an unnatural monosaccharide, peracetylated N-azidoacetyl-D-mannosamine, in the sialic acid biochemical pathway to present N-azidoacetyl sialic acid to PSA-NCAM. Although significant neurotoxicity was observed in the conventional metabolic labeling that used the dissociated neuron cells, neurotoxicity disappeared in this modified strategy, allowing for investigation of the temporal and spatial distributions of PSA in the primary hippocampal neurons. PSA-NCAM was synthesized and recycled continuously during neuronal development, and the two-color labeling showed that newly synthesized PSA-NCAMs were transported and inserted mainly to the growing neurites and not significantly to the cell body. This report suggests a reliable and cytocompatible method for in vitro analysis of glycans complementary to the conventional cell-based metabolic labeling for chemical glycobiology.
| S-EPMC4311863 | biostudies-other
Project description:Sialic acids are used as a receptor by several viruses and variations in the linkage type or C-5 modifications affect the binding properties. A species barrier for multiple viruses is present due to α2,3- or α2,6-linked sialic acids. The C-5 position of the sialic acid can be modified to form N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), which acts as a determinant for host susceptibility for pathogens such as influenza A virus, rotavirus, and transmissible gastroenteritis coronavirus. Neu5Gc is present in most mammals such as pigs and horses but is absent in humans, ferrets, and dogs. However, little is known about C-5 content in wildlife species or how many C-5 modified sialic acids are present on N-linked glycans or glycolipids. Using our previously developed tissue microarray system, we investigated how 2 different lectins specific for Neu5Gc can result in varying detection levels of Neu5Gc glycans. We used these lectins to map Neu5Gc content in wild Suidae, Cervidae, tigers, and European hedgehogs. We show that Neu5Gc content is highly variable among different species. Furthermore, the removal of N-linked glycans reduces the binding of both Neu5Gc lectins while retention of glycolipids by omitting methanol treatment of tissues increases lectin binding. These findings highlight the importance of using multiple Neu5Gc lectins as the rich variety in which Neu5Gc is displayed can hardly be detected by a single lectin.
| S-EPMC9387512 | biostudies-literature
Project description:Metabolic labeling of glycans with synthetic sugar analogs has emerged as an attractive means for introducing nonnatural chemical functionality into glycoproteins. However, the complexities of glycan biosynthesis prevent the installation of nonnatural moieties at defined, predictable locations within glycoproteins at high levels of incorporation. Here, we demonstrate that the conserved N-acetyglucosamine (GlcNAc) residues within chitobiose cores of N-glycans in the model organism Saccharomyces cerevisiae can be specifically targeted for metabolic replacement by unnatural sugars. We introduced an exogenous GlcNAc salvage pathway into yeast, allowing cells to metabolize GlcNAc provided as a supplement to the culture medium. We then rendered the yeast auxotrophic for production of the donor nucleotide-sugar uridine-diphosphate-GlcNAc (UDP-GlcNAc) by deletion of the essential gene GNA1. We demonstrate that gna1Delta strains require a GlcNAc supplement and that expression plasmids containing both exogenous components of the salvage pathway, GlcNAc transporter NGT1 from Candida albicans and GlcNAc kinase NAGK from Homo sapiens, are required for rescue in this context. Further, we show that cells successfully incorporate synthetic GlcNAc analogs N-azidoacetyglucosamine (GlcNAz) and N-(4-pentynoyl)-glucosamine (GlcNAl) into cell-surface glycans and secreted glycoproteins. To verify incorporation of the nonnatural sugars at N-glycan core positions, endoglycosidase H (endoH)-digested peptides from a purified secretory glycoprotein, Ygp1, were analyzed by mass spectrometry. Multiple Ygp1 N-glycosylation sites bearing GlcNAc, isotopically labeled GlcNAc, or GlcNAz were identified; these modifications were dependent on the supplement added to the culture medium. This system enables the production of glycoproteins that are functionalized for specific chemical modifications at their glycosylation sites.
| S-EPMC2840165 | biostudies-literature
Project description:The lipooligosaccharides (LOS) of Haemophilus ducreyi are highly sialylated, a modification that has been implicated in resistance to host defense and in virulence. In previous work, we demonstrated that H. ducreyi scavenges sialic acid from the extracellular milieu and incorporates those residues into LOS. Here we report that H. ducreyi can use unnatural sialic acids bearing elongated N-acyl groups from three to seven carbon atoms in length, resulting in outer membrane presentation of unnatural sialyl-LOS. The unnatural variant comprises approximately 90% of cell surface sialosides when exogenous substrates are added to the media at micromolar concentrations, despite the availability of natural sialic acid in the growth media. Although they represent the majority of cell surface sialosides, analogs with longer N-acyl groups diminish the overall level of LOS sialylation, culminating in complete inhibition of LOS sialylation by N-octanoyl sialic acid. Thus, sialylation of H. ducreyi LOS can be modulated with respect to the structure of the terminal sialic acid residue and the extent to which the LOS acceptor is modified by supplying the bacteria with various sialic acid analogs.
| S-EPMC152251 | biostudies-literature
Project description:The analysis of sialylated glycans is critical for understanding the role of sialic acid in normal biological processes as well as in disease. However, the labile nature of sialic acid typically renders routine analysis of this monosaccharide by mass spectrometric methods difficult. To overcome this difficulty we pursued derivatization methodologies, extending established acetohydrazide approaches to aniline-based methods, and finally to optimized p-toluidine derivatization. This new quantitative glycoform profiling method with use of MALDI-TOF in positive ion mode was validated by first comparing N-glycans isolated from fetuin and serum and was then exploited to analyze the effects of increased metabolic flux through the sialic acid pathway in SW1990 pancreatic cancer cells by using a colabeling strategy with light and heavy toluidine. The latter results established that metabolic flux, in a complementary manner to the more well-known impact of sialyltransferase expression, can critically modulate the sialylation of specific glycans while leaving others virtually unchanged.
| S-EPMC3681956 | biostudies-literature
Project description:Saffron is one of the most expensive spices of the world. Since this spice is triploid and meiosis is unusual, it cannot reproduce sexually like other plants; rather, it is propagated vegetatively via an underground corm, which can withstand a long dry dormant period before sprouting. Thus, corms are indispensable to saffron propagation. To identify and analyse signature metabolites associated with the 'dormancy-sprouting' process, non-targeted GC-MS was performed at different stages of corm development. Comparative metabolite profiling reflected dissimilar profiles among the stages as portrayed by differential cluster patterns of metabolites in the PCA and PLS-DA analysis. Correlation analysis revealed the interdependencies of individual metabolites and metabolic pathway. At the onset of stage 2, characterized by the initiation and differentiation of leaf primordia, a shift from dormancy to active metabolism occurred as derived from the increased abundance of sugars and other metabolites involved in the tricarboxylic acid cycle, glycolytic, amino acid and fatty acid pathways. These changes contribute to sprouting and vegetative growth of the corm. The present study provides new insights into saffron corm composition and metabolite changes associated with various stages of corm development and may pave the way for achieving agronomical improvements in this economically important spice.
| S-EPMC5605653 | biostudies-literature
Project description:Fucosylated glycans critically regulate the physiological functions of proteins and cells. Alterations in levels of fucosylated glycans are associated with various diseases. For detection and functional modulation of fucosylated glycans, chemical biology approaches using fucose (Fuc) analogs are useful. However, little is known about how efficiently each unnatural Fuc analog is utilized by enzymes in the biosynthetic pathway of fucosylated glycans. We show here that three clickable Fuc analogs with similar but distinct structures labeled cellular glycans with different efficiency and protein specificity. For instance, 6-alkynyl (Alk)-Fuc modified O-Fuc glycans much more efficiently than 7-Alk-Fuc. The level of GDP-6-Alk-Fuc produced in cells was also higher than that of GDP-7-Alk-Fuc. Comprehensive in vitro fucosyltransferase assays revealed that 7-Alk-Fuc is commonly tolerated by most fucosyltransferases. Surprisingly, both protein O-fucosyltransferases (POFUTs) could transfer all Fuc analogs in vitro, likely because POFUT structures have a larger space around their Fuc binding sites. These findings demonstrate that labeling and detection of fucosylated glycans with Fuc analogs depend on multiple cellular steps, including conversion to GDP form, transport into the ER or Golgi, and utilization by each fucosyltransferase, providing insights into design of novel sugar analogs for specific detection of target glycans or inhibition of their functions.
| S-EPMC7503990 | biostudies-literature
Project description:Sialic acid sugars on mammalian cells regulate numerous biological processes, while aberrant expression of sialic acid is associated with diseases such as cancer and pathogenic infection. Inhibition of the sialic acid biosynthesis may therefore hold considerable therapeutic potential. To effectively decrease the sialic acid expression, we synthesized C-5-modified 3-fluoro sialic acid sialyltransferase inhibitors. We found that C-5 carbamates significantly enhanced and prolonged the inhibitory activity in multiple mouse and human cell lines. As an underlying mechanism, we have identified that carbamate-modified 3-fluoro sialic acid inhibitors are more efficiently metabolized to their active cytidine monophosphate analogues, reaching higher effective inhibitor concentrations inside cells.
| S-EPMC6348442 | biostudies-literature
Project description:Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality present in the cell. The tetrazine ligation is very suitable as a bioorthogonal reaction because of its selectivity and high reaction rates with several alkenes and alkynes. Recently, we described vinylboronic acids (VBAs) as novel hydrophilic bioorthogonal moieties that react efficiently with dipyridyl- s-tetrazines and used them for protein modification in cell lysate. It is not clear, however, whether VBAs are suitable for labeling experiments in living cells because of the possible coordination with, for example, vicinal carbohydrate diols. Here, we evaluated VBAs as bioorthogonal reactants for labeling of proteins in living cells using an irreversible inhibitor of the proteasome and compared the reactivity to that of an inhibitor containing norbornene, a widely used reactant for the tetrazine ligation. No large differences were observed between the VBA and norbornene probes in a two-step labeling approach with a cell-penetrable fluorescent tetrazine, indicating that the VBA gives little or no side reactions with diols and can be used efficiently for protein labeling in living cells.
| S-EPMC5942871 | biostudies-literature
Project description:Dynamic turnover of cell-surface glycans is involved in a myriad of biological events, making this process an attractive target for in?vivo molecular imaging. Metabolic glycan labeling coupled with bioorthogonal chemistry has paved the way for visualizing glycans in living organisms. However, a two-step labeling sequence is required, which suffers from the tissue-penetration difficulties of the imaging probes. Here, by exploring the substrate promiscuity of endogenous glycosyltransferases, we developed a single-step fluorescent glycan labeling strategy by using fluorophore-tagged analogues of the nucleotide sugars. Injecting fluorophore-tagged sialic acid and fucose into the yolk of zebrafish embryos at the one-cell stage enables systematic imaging of sialylation and fucosylation in live zebrafish embryos at distinct developmental stages. From these studies, we obtained insights into the role of sialylated and fucosylated glycans in zebrafish hematopoiesis.
| S-EPMC6820142 | biostudies-literature