O-GlcNAcylation of the Plum pox virus capsid protein catalyzed by SECRET AGENT: Characterization of O-GlcNAc sites by electron transfer dissociation mass spectrometry

Young Cheon Kim, Namrata D. Udeshi, Jeremy L. Balsbaugh, Jeffrey Shabanowitz, Donald F. Hunt, Neil E. Olszewski

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21 Scopus citations

Abstract

The capsid protein of Plum pox virus (PPV-CP) is modified with O-linked β-N-acetylglucosamine (O-GlcNAc). In Arabidopsis thaliana this modification is made by an O-GlcNAc transferase named SECRET AGENT (SEC). Modification of PPV-CP by SEC is hypothesized to have a direct role in the infection process, because virus titer and rate of spread are reduced in SEC mutants. Previous studies used deletion mapping and site-directed mutagenesis to identify four O-GlcNAc sites on the capsid protein that are modified by Escherichia coli-expressed SEC. The infection process was not affected when two of these sites were mutated suggesting that O-GlcNAcylation of these sites does not have a significant role in the infection process or that a subset of the modifications is sufficient. Since it is possible that the mutational mapping approach missed or incorrectly identified O-GlcNAc sites, the modifications produced by E. coli-expressed SEC were characterized using mass spectrometry. O-GlcNAcylated peptides were enzymatically tagged with galactose, the products were enriched on immobilized Ricinus communis agglutinin I and sequenced by electron transfer dissociation (ETD) mass spectrometry. Five O-GlcNAc sites on PPV-CP were identified. Two of these sites were not identified in by the previous mutational mapping. In addition, one site previously predicted by mutation mapping was not detected, but modification of this site was not supported when the mutation mapping was repeated. This study suggests that mapping modification sites by ETD mass spectrometry is more comprehensive and accurate than mutational mapping.

Original languageEnglish (US)
Pages (from-to)869-876
Number of pages8
JournalAmino Acids
Volume40
Issue number3
DOIs
StatePublished - Mar 2011

Bibliographical note

Funding Information:
We thank Neal Jahren and Katie Saathoff for making valuable suggestions for improving the manuscript. This work was supported by National Science Foundation grant MCB-0820666 to NEO and National Institutes of Health grant GM37537 to DFH.

Keywords

  • Arabidopsis thaliana
  • Capsid protein
  • Electron transfer dissociation tandem mass spectrometry
  • O-GlcNAc
  • O-GlcNAc transferase
  • Plum pox virus
  • SECRET AGENT

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