title: Identification and origin of N-linked β-D-N-acetylglucosamine monosaccharide modifications on Arabidopsis proteins.
abstract: Many plant proteins are modified with N-linked oligosaccharides at asparagine-X-serine/threonine sites during transit through the endoplasmic reticulum and the Golgi. We have identified a number of Arabidopsis (Arabidopsis thaliana) proteins with modifications consisting of an N-linked N-acetyl-D-glucosamine monosaccharide (N-GlcNAc). Electron transfer dissociation mass spectrometry analysis of peptides bearing this modification mapped the modification to asparagine-X-serine/threonine sites on proteins that are predicted to transit through the endoplasmic reticulum and Golgi. A mass labeling method was developed and used to study N-GlcNAc modification of two thioglucoside glucohydrolases (myrosinases), TGG1 and TGG2 (for thioglucoside glucohydrolase). These myrosinases are also modified with high-mannose (Man)-type glycans. We found that N-GlcNAc and high-Man-type glycans can occur at the same site. It has been hypothesized that N-GlcNAc modifications are generated when endo-β-N-acetylglucosaminidase (ENGase) cleaves N-linked glycans. We examined the effects of mutations affecting the two known Arabidopsis ENGases on N-GlcNAc modification of myrosinase and found that modification of TGG2 was greatly reduced in one of the single mutants and absent in the double mutant. Surprisingly, N-GlcNAc modification of TGG1 was not affected in any of the mutants. These data support the hypothesis that ENGases hydrolyze high-Man glycans to produce some of the N-GlcNAc modifications but also suggest that some N-GlcNAc modifications are generated by another mechanism. Since N-GlcNAc modification was detected at only one site on each myrosinase, the production of the N-GlcNAc modification may be regulated.
authors.affiliation: Department of Plant Biology, Microbial and Plant Genomics Institute, St. Paul, Minnesota 55108, USA.
authors.forename: Young-Cheon
authors.initials: YC
authors.lastname: Kim
authors.forename: Neal
authors.initials: N
authors.lastname: Jahren
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Stone
authors.forename: Namrata D
authors.initials: ND
authors.lastname: Udeshi
authors.forename: Todd W
authors.initials: TW
authors.lastname: Markowski
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Witthuhn
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Shabanowitz
authors.forename: Donald F
authors.initials: DF
authors.lastname: Hunt
authors.forename: Neil E
authors.initials: NE
authors.lastname: Olszewski
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001216:Asparagine / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004789:Enzyme Activation; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D008968:Molecular Conformation; D011134:Polysaccharides / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D001216:Asparagine; D006026:Glycoside Hydrolases; C511862:TGG1 protein, Arabidopsis; C511863:TGG2 protein, Arabidopsis; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D000117:Acetylglucosamine