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Exploring the O-GlcNAc proteome: Direct identification of O-GlcNAc-modified proteins from the brain

Khidekel, Nelly and Ficarro, Scott B. and Peters, Eric C. and Hsieh-Wilson, Linda C. (2004) Exploring the O-GlcNAc proteome: Direct identification of O-GlcNAc-modified proteins from the brain. Proceedings of the National Academy of Sciences of the United States of America, 101 (36). pp. 13132-13137. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:KHIpnas04

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Abstract

The covalent modification of intracellular proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) is emerging as a crucial regulatory posttranslational modification akin to phosphorylation. Numerous studies point to the significance of O-GlcNAc in cellular processes such as nutrient sensing, protein degradation, and gene expression. Despite its importance, the breadth and functional roles of O-GlcNAc are only beginning to be elucidated. Advances in our understanding will require the development of new strategies for the detection and study of O-GlcNAc-modified proteins in vivo. Herein we report the direct, high-throughput analysis of O-GlcNAc-glycosylated proteins from the mammalian brain. The proteins were identified by using a chemoenzymatic approach that exploits an engineered galactosyltransferase enzyme to selectively label O-GlcNAc proteins with a ketone-biotin tag. The tag permits enrichment of low-abundance O-GlcNAc species from complex mixtures and localization of the modification to short amino acid sequences. Using this approach, we discovered 25 O-GlcNAc-glycosylated proteins from the brain, including regulatory proteins associated with gene expression, neuronal signaling, and synaptic plasticity. The functional diversity represented by this set of proteins suggests an expanded role for O-GlcNAc in regulating neuronal function. Moreover, the chemoenzymatic strategy described here should prove valuable for identifying O-GlcNAc-modified proteins in various tissues and facilitate studies of the physiological significance of O-GlcNAc across the proteome.


Item Type:Article
Additional Information:Copyright © 2004 by the National Academy of Sciences. Edited by Peter G. Schultz, The Scripps Research Institute, La Jolla, CA, and approved July 27, 2004 (received for review May 15, 2004). Published online before print August 30, 2004, 10.1073/pnas.0403471101. This paper was submitted directly (Track II) to the PNAS office. We thank Drs. Pradman Qasba and Boopathy Ramakrishnan for generously providing the mutant GalT enzyme, Hwan-Ching Tai and Scott Brittain for helpful discussions, and Dr. Andrew Su for assistance with Celera database searches. This research was supported by National Institutes of Health Training Grant T32GM07616, a Parson’s Foundation Fellowship (to N.K.), National Science Foundation CAREER Award CHE-0239861, and an Alfred P. Sloan Fellowship.
Subject Keywords:linked N-acetylglucosamine, activating transcription factor-2, RNA polymerase-II, mass-spectrometry, posttranslational modifications, gene-expression, glycosylation, phosphorylation, phosphoproteome
Record Number:CaltechAUTHORS:KHIpnas04
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:KHIpnas04
Alternative URL:http://dx.doi.org/10.1073/pnas.0403471101
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:1005
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:29 Nov 2005
Last Modified:14 Nov 2014 19:18

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