Abstract
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.
Highlights
Free N-glycans (FNGs) occur ubiquitously in developing or proliferating plants, such as seedlings, developing seeds or fruits, and cells in culture
An endo-β-GlcNAc-ase was purified from rice-cultured cells by a combination of ion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration
To identify the endo-β-GlcNAc-ase gene, O. sativa Endo-Os was completely purified from the cultured rice cells
Summary
Free N-glycans (FNGs) occur ubiquitously in developing or proliferating plants, such as seedlings, developing seeds or fruits, and cells in culture. Among the plant FNGs, the GN1 high-mannose type free N-glycans (GN1-HMT-FNGs) are believed to be produced from misfolded glycoproteins by the sequential action of two enzymes, cytosolic peptide:N-glycanase (cPNGase) and endoβ-N-acetylglucosaminidase (endo-β-GlcNAc-ase; Diepold et al, 2007; Masahara-Negishi et al, 2012). GN1-HMTFNGs completely disappeared and all the HMT-FNGs produced were GN2-type FNGs, suggesting that endo-β-GlcNAc-ase acts on the products (GN2-HMT-FNGs) of cPNGase but not on the misfolded glycoproteins directly. In 2002, Suzuki et al (2002) purified endo-β-GlcNAc-ase from hen oviducts and identified a human ortholog gene based on the purified enzyme. They reported that there are two orthologs of the animal
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