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.
Highlights
Many plant proteins are modified with N-linked oligosaccharides at asparagine-X-serine/threonine sites during transit through the endoplasmic reticulum and the Golgi
Plant complex and hybrid glycans are resistant to removal by PNGase F because the protein-proximal GlcNAc is modified with Fuc
Using an assay that detects terminal GlcNAc by capping it with [3H]Gal, we found that many proteins still had glycans terminating with GlcNAc after PNGase F treatment (Fig. 2). b-Elimination chemistry was used to determine if these glycans were N or O linked; O-linked glycans are removed by this chemistry
Summary
Many plant proteins are modified with N-linked oligosaccharides at asparagine-X-serine/threonine sites during transit through the endoplasmic reticulum and the 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 modified with high-mannose (Man)-type glycans. Many plant proteins are modified with N-linked (Glc) (Man)9(GlcNAc) glycan(s) at the Asn of the Asn-X-Ser/ Thr sequon, where X is any amino acid except Pro, as they enter the endoplasmic reticulum (ER; Pattison and Amtmann, 2009). These N-glycans are modified by the removal and addition of sugars as the protein transits through the secretory system. Matrix-assisted laser-desorption ionization time-of-flight analysis of trypsin fragments derived from antibodies expressed in transgenic maize (Zea mays) and tobacco (Nicotiana tabacum) detects fragments with masses consistent with N-GlcNAc modification (Bakker et al, 2006; Rademacher et al, 2008)
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