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Cytosolic N-GlcNAc proteins are formed by the action of endo-β-N-acetylglucosaminidase.

ABSTRACT: NGLY1 is a widely conserved eukaryotic cytosolic deglycosylating enzyme involved in the endoplasmic reticulum-associated degradation (ERAD) process, which eliminates misfolded proteins through retrograde translocation and proteasomal degradation. A human genetic disorder called NGLY1-deficiency has been reported, indicating the functional importance of NGLY1 in humans. Evidence suggests that Ngly1-KO is embryonic lethal in mice, while additional deletion of the Engase gene, encoding another cytosolic deglycosylating enzyme (endo-β-N-acetylglucosaminidase; ENGase), partially rescued lethality. Upon compromised Ngly1 activity, ENGase-mediated deglycosylation of misfolded glycoproteins may cause excess formation of N-GlcNAc proteins in the cytosol, leading to detrimental effects in the mice. Whether endogenous N-GlcNAc proteins are really formed in Ngly1-KO cells/animals or not remains unclarified. Here, comprehensive identification of O- and N-GlcNAc proteins was carried out using purified cytosol from wild type, Ngly1-KO, Engase-KO, and Ngly1/Engase double KO mouse embryonic fibroblasts. It was revealed that while there is no dramatic change in the level of O-GlcNAc proteins among cells examined, there was a vast increase of N-GlcNAc proteins in Ngly1-KO cells upon proteasome inhibition. Importantly, few N-GlcNAc proteins were observed in Engase-KO or Ngly1/Engase double-KO cells, clearly indicating that the cytosolic ENGase is responsible for the formation of N-GlcNAc proteins. The excess formation of N-GlcNAc proteins may at least in part account for the pathogenesis of NGLY1-deficiency.

SUBMITTER: Maynard JC

PROVIDER: S-EPMC7508226 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In the endoplasmic reticulum-associated degradation system of plant and animal cells, high-mannose type free N-glycans (HMT-FNGs) are produced from misfolded glycoproteins prior to proteasomal degradation, and two enzymes, cytosolic peptide:N-glycanase (cPNGase) and endo-β-N-acetylglucosaminidase (endo-β-GlcNAc-ase), are involved in the deglycosylation. Although the physiological functions of these FNGs in plant growth and development remain to be elucidated, detailed characterization of cPNGase and endo-β-GlcNAc-ase is required. In our previous work, we described the purification, characterization, and subcellular distribution of some plant endo-β-GlcNAc-ases and preliminarily reported the gene information of rice endo-β-GlcNAc-ase (Endo-Os). Furthermore, we analyzed the changes in gene expression of endo-β-GlcNAc-ase during tomato fruit maturation and constructed a mutant line of Arabidopsis thaliana, in which the two endo-β-GlcNAc-ase genes were knocked-out based on the Endo-Os gene. In this report, we describe the purification, characterization, amino acid sequence, and gene cloning of Endo-Os in detail. Purified Endo-Os, with an optimal pH of 6.5, showed high activity for high-mannose type N-glycans bearing the Manα1-2Manα1-3Manβ1 unit; this substrate specificity was almost the same as that of other plant endo-β-GlcNAc-ases, suggesting that Endo-Os plays a critical role in the production of HTM-FNGs in the cytosol. Electrospray ionization-mass spectrometry analysis of the tryptic peptides revealed 17 internal amino acid sequences, including the C terminus; the N-terminal sequence could not be identified due to chemical modification. These internal amino acid sequences were consistent with the amino acid sequence (UniProt ID: Q5W6R1) deduced from the Oryza sativa cDNA clone AK112067 (gene ID: Os05g0346500). Recombinant Endo-Os expressed in Escherichia coli using cDNA showed the same enzymatic properties as those of native Endo-Os.

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Cytosolic N-GlcNAc proteins are formed by the action of endo-β-N-acetylglucosaminidase.

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