Determination of Nucleotides and Sugar Nucleotides Involved in Protein Glycosylation by High-Performance Anion-Exchange Chromatography: Sugar Nucleotide Contents in Cultured Insect Cells and Mammalian Cells

Tomiya, Noboru; Ailor, Eric; Lawrence, Shawn M.; Betenbaugh, Michael J.; Lee, Yuan C.

doi:10.1006/abio.2001.5091

Cited by 188 publications

(155 citation statements)

References 70 publications

(40 reference statements)

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“…Small molecule metabolites were collected via filtration through a spin concentrator with a 10-kDa molecular weight cutoff (Amicon). HPAEC analysis was performed essentially as described (43).…”

Section: Methodsmentioning

confidence: 99%

Metabolic cross-talk allows labeling of O-linked β- N -acetylglucosamine-modified proteins via the N -acetylgalactosamine salvage pathway

Boyce

Carrico

Ganguli

et al. 2011

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

296

325

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Hundreds of mammalian nuclear and cytoplasmic proteins are reversibly glycosylated by O-linked β-N-acetylglucosamine (O-GlcNAc) to regulate their function, localization, and stability. Despite its broad functional significance, the dynamic and posttranslational nature of O-GlcNAc signaling makes it challenging to study using traditional molecular and cell biological techniques alone. Here, we report that metabolic cross-talk between the N-acetylgalactosamine salvage and O-GlcNAcylation pathways can be exploited for the tagging and identification of O-GlcNAcylated proteins. We found that N-azidoacetylgalactosamine (GalNAz) is converted by endogenous mammalian biosynthetic enzymes to UDP-GalNAz and then epimerized to UDP-N-azidoacetylglucosamine (GlcNAz). O-GlcNAc transferase accepts UDP-GlcNAz as a nucleotide-sugar donor, appending an azidosugar onto its native substrates, which can then be detected by covalent labeling using azide-reactive chemical probes. In a proof-of-principle proteomics experiment, we used metabolic GalNAz labeling of human cells and a bioorthogonal chemical probe to affinity-purify and identify numerous O-GlcNAcylated proteins. Our work provides a blueprint for a wide variety of future chemical approaches to identify, visualize, and characterize dynamic O-GlcNAc signaling.

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Section: Methodsmentioning

confidence: 99%

Metabolic cross-talk allows labeling of O-linked β- N -acetylglucosamine-modified proteins via the N -acetylgalactosamine salvage pathway

Boyce

Carrico

Ganguli

et al. 2011

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

296

325

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“…UDP-Sugars-When different cell lines were compared, cells were seeded on 10-cm plates and cultured until 90% confluence, counted from one plate, whereas cells from parallel plates were used to isolate and measure UDP-sugars as described earlier (26), using the sodium hydroxide/sodium acetate gradient as originally described by Tomiya et al (27) with some modifications. Because with this method the isomers UDP-GalNAc and UDP-GlcNAc were incompletely separated in a part of the samples, the results are presented as UDP-HexNAc, which includes both.…”

Section: Methodsmentioning

confidence: 99%

Hyaluronan Synthase 1 (HAS1) Requires Higher Cellular UDP-GlcNAc Concentration than HAS2 and HAS3*

Rilla

Oikari

Jokela

et al. 2013

Journal of Biological Chemistry

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“…Nucleotide diphosphate sugars were extracted from cells according to established procedures (33,34). EMEG Ϫ/Ϫ cells were plated onto 10-cm plates and grown to 50% confluence and then treated with either 5 mM GlcNAc or 5 mM GlcNGc or PBS as a control.…”

Section: Nucleotide Diphosphate Extractionmentioning

confidence: 99%

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups

Macauley

Chan

Zandberg

et al. 2012

Journal of Biological Chemistry

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Determination of Nucleotides and Sugar Nucleotides Involved in Protein Glycosylation by High-Performance Anion-Exchange Chromatography: Sugar Nucleotide Contents in Cultured Insect Cells and Mammalian Cells

Cited by 188 publications

References 70 publications

Metabolic cross-talk allows labeling of O-linked β- N -acetylglucosamine-modified proteins via the N -acetylgalactosamine salvage pathway

Metabolic cross-talk allows labeling of O-linked β- N -acetylglucosamine-modified proteins via the N -acetylgalactosamine salvage pathway

Hyaluronan Synthase 1 (HAS1) Requires Higher Cellular UDP-GlcNAc Concentration than HAS2 and HAS3*

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups

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