Metabolic cross-talk allows labeling of O-linked beta-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway.
Proc Natl Acad Sci U S A
; 108(8): 3141-6, 2011 Feb 22.
Article
em En
| MEDLINE
| ID: mdl-21300897
ABSTRACT
Hundreds of mammalian nuclear and cytoplasmic proteins are reversibly glycosylated by O-linked ß-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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Acetilgalactosamina
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Acetilglucosamina
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Marcadores de Afinidade
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Receptor Cross-Talk
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Redes e Vias Metabólicas
Limite:
Humans
Idioma:
En
Ano de publicação:
2011
Tipo de documento:
Article