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

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.

SPINDLY inhibits class I TCP proteolysis to promote sensitivity to cytokinin

“…The OGT activity of SEC has been demonstrated (Kim et al, 2011), and a recent study by Zentella et al (2016) showed that SEC interacts with and O-GlcNAcylates DELLAs, reduces their activity, and promotes GA responses. SPY, similar to SEC, contains multiple tetratricopeptide repeats at the N terminus, which bind substrate proteins, and a putative OGT catalytic domain at the C terminus (Silverstone et al, 2007).…”

Identification and Origin of N-Linked β-d-N-Acetylglucosamine Monosaccharide Modifications on Arabidopsis Proteins

“…Proteins from the fucta/fuctb/xylt triple mutant were treated with PNGase F and digested with endo-Lys-C or trypsin. Peptides bearing modifications with terminal GlcNAc were then enriched by lectin affinity chromatography (Kim et al, 2011). In this procedure, terminal GlcNAc is enzymatically capped with Gal using bovine b-1,4-galactosyltransferase, creating the disaccharide N-acetyl-D-lactosamine (LacNAc).…”

“…O-GlcNAc-modified CT-5 was prepared by coexpressing it with SECRET AGENT as described previously (Kim et al, 2011). Detection of GlcNAcylated proteins on the blots was performed as described (Kim et al, 2011).…”

“…Detection of GlcNAcylated proteins on the blots was performed as described (Kim et al, 2011). b-Elimination of O-GlcNAc on the protein blots was performed as described previously (Duk et al, 1997).…”

“…Details of RCA I affinity chromatography procedures are described (Kim et al, 2011). Protein extraction for the purification of TGG1 and TGG2 by Con A affinity chromatography was performed as described previously (Pessina et al, 1990) and according to the Con A resin manufacturer's protocol (Pharmacia Biotech).…”