Bibliographic Details
| Title: |
Purification, Characterization, and Gene Expression of Rice Endo-β-N-Acetylglucosaminidase, Endo-Os |
| Authors: |
Megumi Maeda, Naoko Okamoto, Norie Araki, Yoshinobu Kimura |
| Source: |
Frontiers in Plant Science, Vol 12 (2021) |
| Publisher Information: |
Frontiers Media S.A., 2021. |
| Publication Year: |
2021 |
| Collection: |
LCC:Plant culture |
| Subject Terms: |
endo-β-N-acetylglucosaminidase, free N-glycans, Oryza sativa, ER associated degradation, peptide:N-glycanase, Plant culture, SB1-1110 |
| More Details: |
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. |
| Document Type: |
article |
| File Description: |
electronic resource |
| Language: |
English |
| ISSN: |
1664-462X |
| Relation: |
https://www.frontiersin.org/articles/10.3389/fpls.2021.647684/full; https://doaj.org/toc/1664-462X |
| DOI: |
10.3389/fpls.2021.647684 |
| Access URL: |
https://doaj.org/article/2bf6eb38929144498ce3437baa782ac6 |
| Accession Number: |
edsdoj.2bf6eb38929144498ce3437baa782ac6 |
| Database: |
Directory of Open Access Journals |
