<Emphasis Type="Italic">O</Emphasis>-GlcNAcylation of the <Emphasis Type="Italic">Plum pox virus</Emphasis> capsid protein catalyzed by SECRET AGENT: characterization of <Emphasis Type="Italic">O</Emphasis>-GlcNAc sites by electron transfer dissociation mass spectrometry |
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Authors: | Young-Cheon Kim Namrata D Udeshi Jeremy L Balsbaugh Jeffrey Shabanowitz Donald F Hunt Neil E Olszewski |
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Institution: | (1) Department of Plant Biology, 250 Biological Sciences Center, Microbial and Plant Genomics Institute, 1445 Gortner Ave., St. Paul, MN 55108, USA;(2) Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA;(3) Department of Pathology, University of Virginia, Charlottesville, VA 22904, USA; |
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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. |
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