Double-Knockout of Putative Endo-β-N-acetylglucosaminidase (ENGase) Genes inArabidopsis thaliana: Loss of ENGase Activity Induced Accumulation of High-Mannose Type FreeN-Glycans BearingN,N′-Acetylchitobiosyl Unit

Kimura, Yoshinobu; Yuki, Takeoka; Inoue, Masami; Maeda, Megumi; Fujiyama, Kazuhito

doi:10.1271/bbb.110148

Cited by 22 publications

(17 citation statements)

References 7 publications

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“…Interestingly, neither ENGase seems to have a role in producing N-GlcNAc modifications on TGG1. Since Arabidopsis is believed to have just two ENGases (Fischl et al, 2011;Kimura et al, 2011), these results suggest that multiple mechanisms exist for the production of N-GlcNAc modifications. Bacterial N-linked glycosyltransferases that glycosylated Asn-X-Ser/Thr sites using either UDP-Glc or UDP-Gal have recently been identified (Choi et al, 2010;Grass et al, 2010), but we were unable to identify a gene encoding a similar enzyme in the Arabidopsis genome.…”

Section: Discussionmentioning

confidence: 97%

“…Free oligosaccharides with one GlcNAc at the reducing end are found in animals and plants (Suzuki and Funakoshi, 2006;Chantret and Moore, 2008;Funakoshi and Suzuki, 2009), but these could arise from ENGase acting on glycoproteins or the oligosaccharides after they have been released from the protein. Arabidopsis mutants lacking ENGase activity have a large increase in free oligosaccharides with two GlcNAc at the reducing end (Fischl et al, 2011;Kimura et al, 2011), suggesting that ENGase acts primarily on free oligosaccharides. Our finding that TGG2 from the AtENGase85B AtENGase85A double mutant is not N-GlcNAc modified (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The Arabidopsis genome also contains a predicted ENGase pseudogene, At3g61010, which is unlikely to encode a functional ENGase (Fischl et al, 2011). It is also possible that one or both alleles used in the double mutant are not null alleles, but this is unlikely because the AtENGase85B allele used here was also used previously to create an ENGase double mutant that had no detectable ENGase activity (Fischl et al, 2011;Kimura et al, 2011). Although we employed a different AtENGase85A allele than was used in other studies and the T-DNA is inserted in an intron, TGG2 modification was almost undetectable in single mutants with this allele, suggesting that the allele encodes little or no functional ENGase.…”

Section: Discussionmentioning

confidence: 99%

“…Arabidopsis has two functional ENGase genes, At3g11040 (AtENGase85B) and At5g05460 (AtENGase85A), and a pseudogene. Previous studies have found that free oligosaccharides with one GlcNAc at the reducing end are not detectable in AtENGase85A AtENGase85B double mutants, suggesting that the double mutant lacks all ENGase activity (Fischl et al, 2011;Kimura et al, 2011). We tested the hypothesis that Arabidopsis ENGase is responsible for the production of N-GlcNAc by determining if the abundance of this modification on TGG1 and TGG2 is affected in ENGase single and double mutants.…”

Section: Engase Activity Is Required For Glcnac Modification Of Tgg2mentioning

confidence: 95%

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

et al. 2012

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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-b-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.

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Engase Activity Is Required For Glcnac Modification Of Tgg2mentioning

confidence: 95%

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

et al. 2012

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“…An Arabidopsis thaliana double-knockout mutant was constructed in which the expression of two putative ENGase genes was suppressed [124,125] and no ENGase activity was detected. In the mutant the high mannose FNGs carried the proximal GlcNAc1 at reducing end, revealing the probable action of endogenous PNGase.…”

Section: Plant N-glycan-engasesmentioning

confidence: 99%

Endo-N-Acetyl-β-D-Glucosaminidases and Peptide-N4-(N-acetyl-β-D-Glucosaminyl) Asparagine Amidases: More Than Just Tools

Karamanos¹

2013

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Abstract:Since the discovery of endo-N-acetyl-β-D-glucosaminidases (ENGase) and peptide-N4-(N-acetyl-β-D-glucosaminyl) asparagine amidases (PNGase) most of the published work described their use for structural studies. Less attention was given to the potential roles of those enzymes in the physiology of the cells/organisms they produced them. The scope of this review is firstly to analyse the data on the occurrence and characteristics of murein-, chitin-, and N-glycan-ENGases acting on GlcNAc-containing polymers in three structural families, namely murein, chitin, and N-glycosylproteins, and of PNGases, only acting on N-glycosylproteins, and secondly to discuss the biological roles of the enzymes in the producing cells. The analysis demonstrates the remarkable diversity of the enzymes, and simultaneously the interest of studying their substrate specificity and their structural features. Many examples illustrate the importance of the structure/function relationships studies. Diverse biological roles were anticipated, e.g. they are useful for feeding purposes, are implicated in pathogenesis processes, modulate the activity of macromolecules, and help in the destruction of misfolded proteins. Their effect can be direct or indirect, through the reaction products. Current knowledge only partially explains the biological roles of ENGases and PNGases, thus further studies are expected for determining novel possibilities and elucidating other cell pathways.

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Generation and degradation of free asparagine-linked glycans

Harada

Hirayama

Suzuki

2015

Cell. Mol. Life Sci.

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Asparagine (N)-linked protein glycosylation, which takes place in the eukaryotic endoplasmic reticulum (ER), is important for protein folding, quality control and the intracellular trafficking of secretory and membrane proteins. It is known that, during N-glycosylation, considerable amounts of lipid-linked oligosaccharides (LLOs), the glycan donor substrates for N-glycosylation, are hydrolyzed to form free N-glycans (FNGs) by unidentified mechanisms. FNGs are also generated in the cytosol by the enzymatic deglycosylation of misfolded glycoproteins during ER-associated degradation. FNGs derived from LLOs and misfolded glycoproteins are eventually merged into one pool in the cytosol and the various glycan structures are processed to a near homogenous glycoform. This article summarizes the current state of our knowledge concerning the formation and catabolism of FNGs.

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Double-Knockout of Putative Endo-β-N-acetylglucosaminidase (ENGase) Genes inArabidopsis thaliana: Loss of ENGase Activity Induced Accumulation of High-Mannose Type FreeN-Glycans BearingN,N′-Acetylchitobiosyl Unit

Cited by 22 publications

References 7 publications

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

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

Endo-N-Acetyl-β-D-Glucosaminidases and Peptide-N4-(N-acetyl-β-D-Glucosaminyl) Asparagine Amidases: More Than Just Tools

Generation and degradation of free asparagine-linked glycans

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