Dr. Neil E. Olszewski

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Browsing Dr. Neil E. Olszewski by Author "Huntand, Donald F."

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    Identification and Origin of N-Linked β-D-N-Acetylglucosamine Monosaccharide Modifications on Arabidopsis Proteins
    (American Society of Plant Biologists, 2012-11-08) Olszewski, Neil E.; Kim, Young-Cheon; Jahren, Neal; Stone, Mathew D.; Udeshi, Namrata D.; Markowski, Todd W.; Witthuhn, Bruce A.; Shabanowitz, Jeffrey; Huntand, Donald F.
    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.