TY - JOUR
T1 - Purification, Characterization, and Gene Expression of Rice Endo-β-N-Acetylglucosaminidase, Endo-Os
AU - Maeda, Megumi
AU - Okamoto, Naoko
AU - Araki, Norie
AU - Kimura, Yoshinobu
N1 - Funding Information:
This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (JSPS KAKENHI Grant Numbers 15KK0282 and 18K05559 to MM, 17K08197 and 20K05959 to YK).
Funding Information:
We are grateful to the Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University for the ESI-MS analysis. Funding. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (JSPS KAKENHI Grant Numbers 15KK0282 and 18K05559 to MM, 17K08197 and 20K05959 to YK).
Publisher Copyright:
© Copyright © 2021 Maeda, Okamoto, Araki and Kimura.
PY - 2021/8/10
Y1 - 2021/8/10
N2 - 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.
AB - 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.
KW - endo-β-N-acetylglucosaminidase
KW - ER associated degradation
KW - free N-glycans
KW - Oryza sativa
KW - peptide:N-glycanase
UR - http://www.scopus.com/inward/record.url?scp=85113397554&partnerID=8YFLogxK
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U2 - 10.3389/fpls.2021.647684
DO - 10.3389/fpls.2021.647684
M3 - Article
AN - SCOPUS:85113397554
SN - 1664-462X
VL - 12
JO - Frontiers in Plant Science
JF - Frontiers in Plant Science
M1 - 647684
ER -