Document Detail

Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners.
MedLine Citation:
PMID:  22411826     Owner:  NLM     Status:  MEDLINE    
O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic proteins in higher eukaryotes. Despite its ubiquity and essentiality in mammals, functional roles for the O-GlcNAc modification remain poorly defined. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. We used this method to identify interaction partners for the O-GlcNAc-modified FG-repeat nucleoporins. We observed crosslinking between FG-repeat nucleoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Further, we propose that the method reported here could find widespread use in investigating the functional consequences of O-GlcNAcylation.
Seok-Ho Yu; Michael Boyce; Amberlyn M Wands; Michelle R Bond; Carolyn R Bertozzi; Jennifer J Kohler
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2012-03-12
Journal Detail:
Title:  Proceedings of the National Academy of Sciences of the United States of America     Volume:  109     ISSN:  1091-6490     ISO Abbreviation:  Proc. Natl. Acad. Sci. U.S.A.     Publication Date:  2012 Mar 
Date Detail:
Created Date:  2012-03-28     Completed Date:  2012-05-21     Revised Date:  2014-09-16    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  United States    
Other Details:
Languages:  eng     Pagination:  4834-9     Citation Subset:  IM    
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MeSH Terms
Acetylglucosamine / chemistry,  metabolism*
Active Transport, Cell Nucleus / radiation effects
Cell Nucleus / metabolism,  radiation effects
Cross-Linking Reagents / metabolism*
Diazomethane / chemistry,  metabolism
HeLa Cells
Models, Biological
Mutagenesis / radiation effects
Nuclear Pore Complex Proteins / chemistry,  metabolism*
Peptides / chemistry,  metabolism
Protein Binding / radiation effects
Protein Processing, Post-Translational / radiation effects*
Repetitive Sequences, Amino Acid
Staining and Labeling / methods*
Uridine Diphosphate / metabolism
Grant Support
GM66047/GM/NIGMS NIH HHS; R01 GM066047/GM/NIGMS NIH HHS; //Howard Hughes Medical Institute
Reg. No./Substance:
0/Cross-Linking Reagents; 0/Nuclear Pore Complex Proteins; 0/Peptides; 58-98-0/Uridine Diphosphate; 60A625P70P/Diazomethane; V956696549/Acetylglucosamine

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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