Dataset Information

Azide- and Alkyne-Bearing Metabolic Chemical Reporters of Glycosylation Show Structure-Dependent Feedback Inhibition of the Hexosamine Biosynthetic Pathway.

ABSTRACT: Metabolic chemical reporters (MCRs) of protein glycosylation are analogues of natural monosaccharides that bear reactive groups, like azides and alkynes. When they are added to living cells and organisms, these small molecules are biosynthetically transformed into nucleotide donor sugars and then used by glycosyltransferases to modify proteins. Subsequent installation of tags by bioorthogonal chemistries can then enable the visualization and enrichment of these glycoproteins. Although this two-step procedure is powerful, the use of MCRs has the potential to change the endogenous production of the natural repertoire of donor sugars. A major route for the generation of these glycosyltransferase substrates is the hexosamine biosynthetic pathway (HBP), which results in uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Interestingly, the rate-determining enzyme of the HBP, glutamine fructose-6-phosphate amidotransferase (GFAT), is feedback inhibited by UDP-GlcNAc. This raises the possibility that a build-up of UDP-MCRs would block the biosynthesis of UDP-GlcNAc, resulting in off target effects. Here, we directly test this possibility with recombinant human GFAT and a small panel of synthetic UDP-MCRs. We find that MCRs with larger substitutions at the N-acetyl position do not inhibit GFAT, whereas those with modifications of the 2- or 6-hydroxy group do. These results further illuminate the considerations that should be applied to the use of MCRs.

SUBMITTER: Walter LA

PROVIDER: S-EPMC6261355 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Azide- and Alkyne-Bearing Metabolic Chemical Reporters of Glycosylation Show Structure-Dependent Feedback Inhibition of the Hexosamine Biosynthetic Pathway.

Walter Lisa A LA Batt Anna R AR Darabedian Narek N Zaro Balyn W BW Pratt Matthew R MR

Chembiochem : a European journal of chemical biology 20180810 18

Metabolic chemical reporters (MCRs) of protein glycosylation are analogues of natural monosaccharides that bear reactive groups, like azides and alkynes. When they are added to living cells and organisms, these small molecules are biosynthetically transformed into nucleotide donor sugars and then used by glycosyltransferases to modify proteins. Subsequent installation of tags by bioorthogonal chemistries can then enable the visualization and enrichment of these glycoproteins. Although this two-s ...[more]

PMID: 29979493

Similar Datasets

Project description:O-linked β-N-acetylglucosamine (O-GlcNAc) is emerging as an essential protein post-translational modification in a range of organisms. It is involved in various cellular processes such as nutrient sensing, protein degradation, gene expression, and is associated with many human diseases. Despite its importance, identifying O-GlcNAcylated proteins is a major challenge in proteomics. Here, using peracetylated N-azidoacetylglucosamine (Ac4 GlcNAz) as a bioorthogonal chemical handle, we described a gel-based mass spectrometry method for the identification of proteins with O-GlcNAc modification in A549 cells. In addition, we made a labeling efficiency comparison between two modes of azide-alkyne bioorthogonal reactions in click chemistry: copper-catalyzed azide-alkyne cycloaddition (CuAAC) with Biotin-Diazo-Alkyne and stain-promoted azide-alkyne cycloaddition (SPAAC) with Biotin-DIBO-Alkyne. After conjugation with click chemistry in vitro and enrichment via streptavidin resin, proteins with O-GlcNAc modification were separated by SDS-PAGE and identified with mass spectrometry. Proteomics data analysis revealed that 229 putative O-GlcNAc modified proteins were identified with Biotin-Diazo-Alkyne conjugated sample and 188 proteins with Biotin-DIBO-Alkyne conjugated sample, among which 114 proteins were overlapping. Interestingly, 74 proteins identified from Biotin-Diazo-Alkyne conjugates and 46 verified proteins from Biotin-DIBO-Alkyne conjugates could be found in the O-GlcNAc modified proteins database dbOGAP (http://cbsb.lombardi.georgetown.edu/hulab/OGAP.html). These results suggested that CuAAC with Biotin-Diazo-Alkyne represented a more powerful method in proteomics with higher protein identification and better accuracy compared to SPAAC. The proteomics credibility was also confirmed by the molecular function and cell component gene ontology (GO). Together, the method we reported here combining metabolic labeling, click chemistry, affinity-based enrichment, SDS-PAGE separation, and mass spectrometry, would be adaptable for other post-translationally modified proteins in proteomics.

Dataset Information

Azide- and Alkyne-Bearing Metabolic Chemical Reporters of Glycosylation Show Structure-Dependent Feedback Inhibition of the Hexosamine Biosynthetic Pathway.

Publications

Azide- and Alkyne-Bearing Metabolic Chemical Reporters of Glycosylation Show Structure-Dependent Feedback Inhibition of the Hexosamine Biosynthetic Pathway.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets