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.

Figure 1.

PNGase and ENGase reactions. PNGase and ENGase cleave the common core of N-linked glycans, which is composed of GlcNAc (closed squares) and Man (closed circles), at the sites indicated. In plants, the common core can be modified with Fuc (open triangle), which inhibits PNGase F, and Xyl (inverted triangle). When a glycan is removed by PNGase F, the modified Asn (N) is converted to an Asp (D).

Figure 2.

PNGase F-resistant glycans with terminal GlcNAc on fucta/fuctb/xylt triple mutant proteins are resistant to β-elimination chemistry. Glycans with terminal GlcNAc on a blot containing PNGase F-treated fucta/fuctb/xylt triple mutant proteins were detected by labeling with [³H]Gal. The lane on the left shows the blot prior to β-elimination, and the right lane (β-elm.) shows the same blot after subjecting it to β-elimination chemistry, which removes O-linked glycans.

Figure 3.

TGG1 and TGG2 are N-GlcNAc modified. Proteins from the fucta/fuctb/xylt triple mutant were digested with Lys-C (or trypsin) and treated with PNGase F. Glycans with terminal GlcNAc were capped with Gal, and GlcNAc-modified peptides were enriched by RCA I lectin affinity chromatography (Supplemental Fig. S2). The enriched peptides were then analyzed by mass spectrometry. A, ETD MS/MS spectrum recorded on [M+3H]⁺³ ions (m/z 515.2663) from the LacNAc (365.1322)-modified TGG2 peptide FgNSTEARLLK. B, [M+3H]⁺³ ions (m/z 830.0481) corresponding to the LacNAc-modified TGG1 peptide gNATGHAPGPPFNAASYYYPK. Predicted c′- and z′·-type ions are listed above and below the peptide sequence, respectively. Singly and doubly charged fragment ions are listed as monoisotopic masses. Ions observed and labeled in the spectrum are underlined. Ions corresponding to charge-reduced species and those resulting from neutral losses are bracketed. Modified residues are preceded by “g” to signify modification by a single LacNAc moiety.

Figure 4.

TGG1 and TGG2 from wild-type plants are N-GlcNAc modified. A, Immunoblot detection of TGG1 and TGG2 from wild-type (WT) and triple mutant plants following treatment with or without PNGase F. PNGase F treatment removes high-Man glycans, which increases the rate of TGG1 and TGG2 migration during electrophoresis (Ueda et al., 2006). B, Immunoblots showing the effect of PEGylating wild-type and triple mutant proteins after treatment with or without PNGase F. Asterisks indicate the PEGylated proteins.

Figure 5.

High-Man-type glycans occur at multiple sites on TGG1, including N379, the site of N-GlcNAc modification. A, TGG1 was enriched by Con A-Sepharose affinity chromatography, treated with PNGase F, and further purified by 2D PAGE. TGG1 was cut out from a 2D gel, digested with trypsin, and analyzed by MS/MS. The CID MS/MS spectrum was recorded on [M+2H]⁺² ions (m/z 786.8574) corresponding to the peptide dNATGHAPGPPFNAASYYYPK deamidated at Asn (N379). Predicted b′- and y′-type ions are listed above and below the peptide sequence, respectively. Singly and doubly charged fragment ions are listed as monoisotopic masses. Ions observed and labeled in the spectrum are underlined. The modified residue is preceded by “d” to signify deamidated Asn. B, An immunoblot showing that extensive PEGylation of TGG1 occurs after treatment with Endo H. Asterisks indicate the PEGylated proteins.

Figure 6.

ENGase is required for N-GlcNAc modification of TGG2. Immunoblots show PEGylation of TGG1 and TGG2 from wild-type (WT), AtENGase85B^Salk_031210, AtENGase85A^Sail_714_D09, and AtENGase85B^Salk_031210 AtENGase85A^Sail_714_D09 double mutant plants. Asterisks indicate PEGylated TGG1 and TGG2.