Synapsin-1 and tau reciprocal O-GlcNAcylation and phosphorylation sites in mouse brain synaptosomes.
- Author:
Min Jueng KANG
1
;
Chaeyoung KIM
;
Hyobin JEONG
;
Byoung Kyu CHO
;
Ae Lan RYOU
;
Daehee HWANG
;
Inhee MOOK-JUNG
;
Eugene C YI
Author Information
1. WCU Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology and College of Medicine or College of Pharmacy, Seoul National University, Seoul, Korea. euyi@snu.ac.kr
- Publication Type:Original Article ; Research Support, Non-U.S. Gov't
- Keywords:
BEMAD;
O-GlcNAcylation;
phosphorylation;
synapsin-1;
synaptosome;
tau
- MeSH:
Acetylglucosamine/*metabolism;
Amino Acid Sequence;
Animals;
Brain/*metabolism;
Chromatography, Affinity;
Glycosylation;
Mice;
Molecular Sequence Data;
Peptides/isolation & purification;
Phosphorylation;
Synapsins/chemistry/*metabolism;
Synaptosomes/*metabolism;
Tandem Mass Spectrometry;
tau Proteins/chemistry/*metabolism
- From:Experimental & Molecular Medicine
2013;45(6):e29-
- CountryRepublic of Korea
- Language:English
-
Abstract:
O-linked N-acetylglucosamine (O-GlcNAc) represents a key regulatory post-translational modification (PTM) that is reversible and often reciprocal with phosphorylation of serine and threonine at the same or nearby residues. Although recent technical advances in O-GlcNAc site-mapping methods combined with mass spectrometry (MS) techniques have facilitated study of the fundamental roles of O-GlcNAcylation in cellular processes, an efficient technique for examining the dynamic, reciprocal relationships between O-GlcNAcylation and phosphorylation is needed to provide greater insights into the regulatory functions of O-GlcNAcylation. Here, we describe a strategy for selectively identifying both O-GlcNAc- and phospho-modified sites. This strategy involves metal affinity separation of O-GlcNAcylated and phosphorylated peptides, beta-elimination of O-GlcNAcyl or phosphoryl functional groups from the separated peptides followed by dithiothreitol (DTT) conjugation (BEMAD), affinity purification of DTT-conjugated peptides using thiol affinity chromatography, and identification of formerly O-GlcNAcylated or phosphorylated peptides by MS. The combined metal affinity separation and BEMAD approach allows selective enrichment of O-GlcNAcylated peptides over phosphorylated counterparts. Using this approach with mouse brain synaptosomes, we identified the serine residue at 605 of the synapsin-1 peptide, 603QASQAGPGPR612, and the serine residue at 692 of the tau peptide, 688SPVVSGDTSPR698, which were found to be potential reciprocal O-GlcNAcylation and phosphorylation sites. These results demonstrate that our strategy enables mapping of the reciprocal site occupancy of O-GlcNAcylation and phosphorylation of proteins, which permits the assessment of cross-talk between these two PTMs and their regulatory roles.
