Oxford University Press (OUP) Acta Medica Okayama 1347-6947 89 6 2025 PNGase activity and free N-glycans in phloem fluid prepared from Nerium oleander (oleander tree) 872 875 EN Fuki Otaguro Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yoshinobu Kimura Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Megumi Maeda Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Free N-glycans (FNGs) occur ubiquitously in growing plants. Recently, it was reported that these FNGs interact with auxin. In this study, we investigated whether PNGase activity responsible for producing the FNGs occurs in the extracellular fluid, where auxin is present during its polar transfer. Here, we report the occurrences of PNGase activity and FNGs in the phloem fluid. No potential conflict of interest relevant to this article was reported.

Frontiers Media SA Acta Medica Okayama 1664-462X 12 2021 Purification, Characterization, and Gene Expression of Rice Endo-beta-N-Acetylglucosaminidase, Endo-Os 647684 EN Megumi Maeda Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University Naoko Okamoto Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University Node Araki Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University Yoshinobu Kimura Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University 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-beta-N-acetylglucosaminidase (endo-beta-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-beta-GlcNAc-ase is required. In our previous work, we described the purification, characterization, and subcellular distribution of some plant endo-beta-GlcNAc-ases and preliminarily reported the gene information of rice endo-beta-GlcNAc-ase (Endo-Os). Furthermore, we analyzed the changes in gene expression of endo-beta-GlcNAc-ase during tomato fruit maturation and constructed a mutant line of Arabidopsis thaliana, in which the two endo-beta-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 Man alpha 1-2Man alpha 1-3Man beta 1 unit; this substrate specificity was almost the same as that of other plant endo-beta-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. No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 1664-462X 11 2021 Cytosolic Free N-Glycans Are Retro-Transported Into the Endoplasmic Reticulum in Plant Cells 610124 EN Makoto Katsube Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University Natsuki Ebara Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University Megumi Maeda Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University Yoshinobu Kimura Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University During endoplasmic reticulum (ER)-associated degradation, free N-glycans (FNGs) are produced from misfolded nascent glycoproteins via the combination of the cytosolic peptide N-glycanase (cPNGase) and endo-beta-N-acetylglucosaminidase (ENGase) in the plant cytosol. The resulting high-mannose type (HMT)-FNGs, which carry one GlcNAc residue at the reducing end (GN1-FNGs), are ubiquitously found in developing plant cells. In a previous study, we found that HMT-FNGs assisted in protein folding and inhibited beta-amyloid fibril formation, suggesting a possible biofunction of FNGs involved in the protein folding system. However, whether these HMT-FNGs occur in the ER, an organelle involved in protein folding, remained unclear. On the contrary, we also reported the presence of plant complex type (PCT)-GN1-FNGs, which carry the Lewis(a) epitope at the non-reducing end, indicating that these FNGs had been fully processed in the Golgi apparatus. Since plant ENGase was active toward HMT-N-glycans but not PCT-N-glycans that carry beta 1-2xylosyl and/or alpha 1-3 fucosyl residue(s), these PCT-GN1-FNGs did not appear to be produced from fully processed glycoproteins that harbored PCT-N-glycans via ENGase activity. Interestingly, PCT-GN1-FNGs were found in the extracellular space, suggesting that HMT-GN1-FNGs formed in the cytosol might be transported back to the ER and processed in the Golgi apparatus through the protein secretion pathway. As the first step in elucidating the production mechanism of PCT-GN1-FNGs, we analyzed the structures of free oligosaccharides in plant microsomes and proved that HMT-FNGs (Man(9-7)GlcNAc(1) and Man(9-8)GlcNAc(2)) could be found in microsomes, which almost consist of the ER compartments. No potential conflict of interest relevant to this article was reported.

TAYLOR & FRANCIS Acta Medica Okayama 0916-8451 83 7 2019 Plant complex type free N-glycans occur in tomato xylem sap 1310 1314 EN Yuta Tsujimori Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science , Okayama University Mikako Ogura Faculty of Agriculture, Division of Agricultural Science , Okayama University Md. Ziaur Rahman Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science , Okayama University Megumi Maeda Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science , Okayama University Yoshinobu Kimura Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science , Okayama University Free N-glycans (FNGs) are ubiquitous in growing plants. Further, acidic peptide:N-glycanase is believed to be involved in the production of plant complex-type FNGs (PCT-FNGs) during the degradation of dysfunctional glycoproteins. However, the distribution of PCT-FNGs in growing plants has not been analyzed. Here, we report the occurrence of PCT-FNGs in the xylem sap of the stem of the tomato plant. No potential conflict of interest relevant to this article was reported.

岡山大学農学部 Acta Medica Okayama 2186-7755 108 2019 花粉アレルゲンの糖鎖構造特性と糖鎖ポリマーの合成 15 18 EN Megumi Maeda Graduate School of Environmental and Life Science, Okayama University No potential conflict of interest relevant to this article was reported.

Taylor & Francis Acta Medica Okayama 09168451 82 7 2018 Novel assay system for acidic Peptide:N-glycanase (aPNGase) activity in crude plant extract 1172 1175 EN Ryota Uemura Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science , Okayama University Mikako Ogura Faculty of Agriculture , Okayama University Chihiro Matsumaru Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science , Okayama University Tsuyoshi Akiyama Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science , Okayama University Megumi Maeda Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science , Okayama University Yoshinobu Kimura Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science , Okayama University Acidic peptide:N-glycanase (aPNGase) plays a pivotal role in plant glycoprotein turnover. For the construction of aPNGase-knockout or -overexpressing plants, a new method to detect the activity in crude plant extracts is required because endogenous peptidases present in the extract hamper enzyme assays using fluorescence-labeled N-glycopeptides as a substrate. In this study, we developed a new method for measuring aPNGase activity in crude extracts from plant materials No potential conflict of interest relevant to this article was reported.

Spandidos Acta Medica Okayama 1019-6439 50 1 2016 Accelerated cell cycle progression of human regulatory T cell-like cell line caused by continuous exposure to asbestos fibers 66 74 EN Suni Lee Department of Hygiene, Kawasaki Medical School Hidenori Matsuzaki Department of Hygiene, Kawasaki Medical School Megumi Maeda Department of Biofunctional Chemistry, Division of Bioscience, Okayama University Graduate School of Natural Science and Technology Shoko Yamamoto Department of Hygiene, Kawasaki Medical School Naoko Kumagai-Takei Tamayo Hatayama Department of Hygiene, Kawasaki Medical School Miho Ikeda Department of Hygiene, Kawasaki Medical School Kei Yoshitome Department of Hygiene, Kawasaki Medical School Yasumitsu Nishimura Department of Hygiene, Kawasaki Medical School Takemi Otsuki Department of Hygiene, Kawasaki Medical School Asbestos exposure causes malignant tumors such as lung cancer and malignant mesothelioma. Based on our hypothesis in which continuous exposure to asbestos of immune cells cause reduction of antitumor immunity, the decrease of natural killer cell killing activity with reduction of NKp46 activating receptor expression, inhibition of cytotoxic T cell clonal expansion, reduced CXCR3 chemokine receptor expression and production of interferon-γ production in CD4+ T cells were reported using cell line models, freshly isolated peripheral blood immune cells from health donors as well as asbestos exposed patients such as pleural plaque and mesothelioma. In addition to these findings, regulatory T cells (Treg) showed enhanced function through cell-cell contact and increased secretion of typical soluble factors, interleukin (IL)-10 and transforming growth factor (TGF)-β, in a cell line model using the MT-2 human polyclonal T cells and its sublines exposed continuously to asbestos fibers. Since these sublines showed a remarkable reduction of FoxO1 transcription factor, which regulates various cell cycle regulators in asbestos-exposed sublines, the cell cycle progression in these sublines was examined and compared with that of the original MT-2 cells. Results showed that cyclin D1 expression was markedly enhanced, and various cyclin-dependent kinase-inhibitors were reduced with increased S phases in the sublines. Furthermore, the increase of cyclin D1 expression was regulated by FoxO1. The overall findings indicate that antitumor immunity in asbestos-exposed individuals may be reduced in Treg through changes in the function and volume of Treg. No potential conflict of interest relevant to this article was reported.

Spandidos Acta Medica Okayama 1019-6439 50 6 2017 Induction of IL-17 production from human peripheral blood CD4+ cells by asbestos exposure 2024 2032 EN Megumi Maeda Department of Biofunctional Chemistry, Division of Bioscience, Okayama University Graduate School of Natural Science and Technology Ying Chen Department of Hygiene, Kawasaki Medical School Suni Lee Department of Hygiene, Kawasaki Medical School Naoko Kumagai-Takei Department of Hygiene, Kawasaki Medical School Kei Yoshitome Department of Hygiene, Kawasaki Medical School Hidenori Matsuzaki Department of Hygiene, Kawasaki Medical School Shoko Yamamoto Department of Hygiene, Kawasaki Medical School Tamayo Hatayama Department of Hygiene, Kawasaki Medical School Miho Ikeda Department of Hygiene, Kawasaki Medical School Yasumitsu Nishimura Department of Hygiene, Kawasaki Medical School Takemi Otsuki Department of Hygiene, Kawasaki Medical School We have previously reported that chronic, recurrent and low-dose exposure to asbestos fibers causes a reduction in antitumor immunity. Investigation of natural killer (NK) cells using an in vitro cell line model and comprising in vitro activation using freshly isolated NK cells co-cultured with chrysotile fibers, as well as NK cells derived from asbestos-exposed patients with pleural plaque (PP) or malignant mesothelioma (MM), revealed decreased expression of NK cell activating receptors such as NKG2D, 2B4 and NKp46. An in vitro differentiation and clonal expansion model for CD8+ cytotoxic T lymphocytes (CTLs) showed reduced cytotoxicity with decreased levels of cytotoxic molecules such as granzyme B and perforin, as well as suppressed proliferation of CTLs. Additionally, analysis of T helper cells showed that surface CXCR3, chemokine receptor, and the productive potential of interferon (IFN)γ were reduced following asbestos exposure in an in vitro cell line model and in peripheral CD4+ cells of asbestos-exposed patients. Moreover, experiments revealed that asbestos exposure enhanced regulatory T cell (Treg) function. This study also focused on CXCR3 expression and the Th-17 cell fraction. Following activation with T-cell receptor and co-culture with various concentrations of chrysotile fibers using freshly isolated CD4+ surface CXCR3 positive and negative fractions, the intracellular expression of CXCR3, IFNγ and IL-17 remained unchanged when co-cultured with chrysotile. However, subsequent re-stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin resulted in enhanced IL-17 production and expression, particularly in CD4+ surface CXCR3 positive cells. These results indicated that the balance and polarization between Treg and Th-17 fractions play an important role with respect to the immunological effects of asbestos and the associated reduction in antitumor immunity. No potential conflict of interest relevant to this article was reported.

岡山大学農学部 Acta Medica Okayama 2186-7755 106 2017 Ginkgo biloba α-fucosidase with activity towards plant complex type N-glycans containing the Lewis a epitope: Purification and characterization 5 12 EN Satsuki Itano Graduate School of Environmental and life Science, Okayama University Megumi Maeda Graduate School of Environmental and life Science, Okayama University Md. Ziaur Rahman Institute of Food and Radiation Biology, Atomic Energy Research Establishment, Bangladesh Atomic Energy Commission Yoshinobu Kimura Graduate School of Environmental and life Science, Okayama University 　We have identified, and purified to homogeneity, a high molecular weight Ginkgo biloba α-fucosidase (α-fucosidase Gb, 120 kDa estimated by SDS‒PAGE) with activity against α-fucosylated oligosaccharides. When a Lewis a epitope-containing N-glycan was used as a substrate, α-fucosidase Gb showed optimum activity at approximately pH 5.5, suggesting that it functions in acidic environments such as the vacuole. It remains uncertain, however, whether this Ginkgo α-fucosidase belongs to the GH29 family, since its N-terminal sequence could not be determined, probably due to a chemical modification. α-Fucosidase Gb showed substantial activity towards the α1,3-fucosyl linkage in Lacto-N-fucopentaose III and an α1,4-fucosyl linkage in the Lewis a epitope found in plant complex type N-glycans, indicating an involvement in the degradation process of α-fucosylated oligosaccharides or N-glycoproteins. No potential conflict of interest relevant to this article was reported.

岡山大学農学部 Acta Medica Okayama 2186-7755 103 2014 Changes in Glycinin‒Digesting Protease Activity During Soybean Germination. 1 4 EN Md. Akhtaruzzaman Megumi Maeda Keiko Kitagawa Shigeaki Takagi Yoshinobu Kimura 　Changes in glycinin-digesting protease activity during soybean germination have been investigated. The glycinin-digesting protease activities of imbibed or germinated soybean seed were assayed by RP‒HPLC using a tryptic peptide from CM‒glycinin or by SDS‒PAGE using CM‒glycinin as the endogenous substrate. Proteolytic activities of the germinated soybean seeds were found through the whole period of germination, the activities were maintained significantly unchanged during germination for 4 days, and then those specific activities declined slowly. AE‒HPLC analysis of the glycinin-digesting protease in the imbibed or germinated soybean seeds showed unchanged peaks corresponding to glycinin- digesting activity, suggesting that the glycinin-digesting protease was not induced during germination but had already been synthesized during seed maturation. No potential conflict of interest relevant to this article was reported.