2021年03月, 国立研究開発法人新エネルギー・産業技術総合開発機構（NEDO）, TCP 最優秀賞　　　　　　　　　　　　　　　
光診断薬Picklesで患者さんの未来を明るく照らす
Horizon Illumination Lab Optics

2015年04月, WILEY, The FEBS Journal Top-Cited Paper Award　　　　　　　　　　　　　　　
天野 麻穂

2013年, 資生堂女性研究者サイエンスグラント受賞　　　　　　　　　　　　　　　
グライコミクスによる昆虫の休眠機構の体系的解明
天野 麻穂

2011年, 鳥類の大規模糖鎖解析による感染症予防データベースの構築　　　　　　　　　　　　　　　
ノーステック財団「研究開発助成事業」スタートアップ研究補助金
天野 麻穂

2008年, （財）秋山記念生命科学振興財団 研究奨励賞受賞　　　　　　　　　　　　　　　
大規模グライコミクスに向けた植物由来全糖タンパク抽出系の構築
天野 麻穂

2006年, ヒト腸管由来レクチンが有する乳酸菌コロニー形成促進機構の解析, （財）糧食研究会 一般公募委託研究採択　　　　　　　　　　　　　　　
天野 麻穂

2006年, ダノン健康・栄養普及協会 学術研究助成金受賞　　　　　　　　　　　　　　　
乳酸菌を用いた自己免疫疾患に対するワクチンの作製
天野 麻穂

2006年, 公益信託林女性自然科学者研究助成基金 研究助成受賞　　　　　　　　　　　　　　　
galectin-1が眼内免疫機構に果たす役割についての研究
天野 麻穂

Interaction between PI3K and the VDAC2 channel tethers Ras-PI3K-positive endosomes to mitochondria and promotes endosome maturation.
Aya O Satoh, Yoichiro Fujioka, Sayaka Kashiwagi, Aiko Yoshida, Mari Fujioka, Hitoshi Sasajima, Asuka Nanbo, Maho Amano, Yusuke Ohba
Cell reports, 42, 3, 112229, 112229, 2023年03月28日, ［国際誌］
英語, 研究論文（学術雑誌）, Intracellular organelles of mammalian cells communicate with one another during various cellular processes. The functions and molecular mechanisms of such interorganelle association remain largely unclear, however. We here identify voltage-dependent anion channel 2 (VDAC2), a mitochondrial outer membrane protein, as a binding partner of phosphoinositide 3-kinase (PI3K), a regulator of clathrin-independent endocytosis downstream of the small GTPase Ras. VDAC2 tethers endosomes positive for the Ras-PI3K complex to mitochondria in response to cell stimulation with epidermal growth factor and promotes clathrin-independent endocytosis, as well as endosome maturation at membrane association sites. With an optogenetics system to induce mitochondrion-endosome association, we find that, in addition to its structural role in such association, VDAC2 is functionally implicated in the promotion of endosome maturation. The mitochondrion-endosome association thus plays a role in the regulation of clathrin-independent endocytosis and endosome maturation.

A method for the generation of pseudovirus particles bearing SARS coronavirus spike protein in high yields.
Yoichiro Fujioka, Sayaka Kashiwagi, Aiko Yoshida, Aya O Satoh, Mari Fujioka, Maho Amano, Yohei Yamauchi, Yusuke Ohba
Cell structure and function, 47, 1, 43, 53, 2022年04月28日, ［国内誌］
英語, 研究論文（学術雑誌）, The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 spike protein in high yield. We found that a large proportion of pseudovirions produced with the conventional transient expression system lacked coronavirus spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus spike protein allowed the efficient production of progeny pseudoviruses decorated with spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.Keywords: severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, pseudovirus, vesicular stomatitis virus (VSV), spike protein.

Direct visualization of glucagon-like peptide-1 secretion by fluorescent fusion proteins.
Atsushi Tsuzuki, Yoichiro Fujioka, Aiko Yoshida, Sayaka Kashiwagi, Maho Amano, Tohru Hira, Akinobu Nakamura, Hideaki Miyoshi, Tatsuya Atsumi, Yusuke Ohba
Journal of diabetes investigation, 13, 7, 1134, 1139, 2022年04月04日, ［国内誌］
英語, 研究論文（学術雑誌）, Live-cell imaging with fluorescent proteins (FPs) is a powerful tool for investigating the exocytosis processes of hormones. However, the secretion process of glucagon-like peptide-1 (GLP-1) has not been visualized by FPs, which might be because tagging FPs inhibits GLP-1 synthesis through the post-translational processing from proglucagon. Here, we have developed FP-tagged GLP-1 by inserting FPs into the middle of GLP-1 and adding the proglucagon signal peptide. Confocal imaging confirmed that GLP-1 fused to FPs with high folding efficiency showed granular structure, in which secretory vesicle markers colocalized. The fluorescence intensity of FP in the culture supernatant from cells treated with KCl or forskolin was significantly increased compared with those from untreated cells. Furthermore, FP-tagged GLP-1 enables direct visualization of stimulation-dependent exocytosis of GLP-1 at a single granule resolution with total internal reflection fluorescence microscopy. FP-tagged GLP-1 might facilitate the screening of GLP-1 secretagogues and the discovery of new antidiabetic drugs.

学際研究プロジェクトにおける異分野研究者間コミュニケーション ―インタビュー調査によるプロジェクト維持要因の仮説作成―　　　　　　　　　　　　　　　
天野麻穂, 片岡良美, 川本思心
年報『科学・技術・社会』, 29, 51, 68, 2020年, ［査読有り］, ［筆頭著者］
日本語, 研究論文（学術雑誌）

Localization of BCR-ABL to Stress Granules Contributes to Its Oncogenic Function.
Sayaka Kashiwagi, Yoichiro Fujioka, Takeshi Kondo, Aya O Satoh, Aiko Yoshida, Mari Fujioka, Hitoshi Sasajima, Maho Amano, Takanori Teshima, Yusuke Ohba
Cell structure and function, 44, 2, 195, 204, 2019年12月26日, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, The oncogenic tyrosine kinase BCR-ABL activates a variety of signaling pathways and plays a causative role in the pathogenesis of chronic myelogenous leukemia (CML); however, the subcellular distribution of this chimeric protein remains controversial. Here, we report that BCR-ABL is localized to stress granules and that its granular localization contributes to BCR-ABL-dependent leukemogenesis. BCR-ABL-positive granules were not colocalized with any markers for membrane-bound organelles but were colocalized with HSP90a, a component of RNA granules. The number of such granules increased with thapsigargin treatment, confirming that the granules were stress granules. Given that treatment with the ABL kinase inhibitor imatinib and elimination of the N-terminal region of BCR-ABL abolished granule formation, kinase activity and the coiled-coil domain are required for granule formation. Whereas wild-type BCR-ABL rescued the growth defect in IL-3-depleted Ba/F3 cells, mutant BCR-ABL lacking the N-terminal region failed to do so. Moreover, forced tetramerization of the N-terminus-deleted mutant could not restore the growth defect, indicating that granule formation, but not tetramerization, through its N-terminus is critical for BCR-ABL-dependent oncogenicity. Our findings together provide new insights into the pathogenesis of CML by BCR-ABL and open a window for developing novel therapeutic strategies for this disease.Key words: BCR-ABL, subcellular localization, stress granule.

Glycosphingolipid GM2 Induces Invasiveness in Irradiation-tolerant Lung Cancer Cells　　　　　　　　　　　　　　　
Ishihara S, Aoki K, Mizutani T, Amano M, Nishimura SI, Haga H
Cell Struct Funct, 43, 2, 177, 185, 2018年, ［査読有り］
英語, 研究論文（学術雑誌）

Visualising the dynamics of live pancreatic microtumours self-organised through cell-in-cell invasion.
Miyatake Y, Kuribayashi-Shigetomi K, Ohta Y, Ikeshita S, Subagyo A, Sueoka K, Kakugo A, Amano M, Takahashi T, Okajima T, Kasahara M
Sci Rep, 8, 1, 14054, Springer Science and Business Media LLC, 2018年, ［査読有り］
英語, 研究論文（学術雑誌）

N-glycomic and microscopic subcellular localization analyses of NPP1, 2 and 6 strongly indicate that trans-golgi compartments participate in the golgi to plastid traffic of nucleotide pyrophosphatase/phosphodiesterases in rice
Kentaro Kaneko, Takeshi Takamatsu, Takuya Inomata, Kazusato Oikawa, Kimiko Itoh, Kazuko Hirose, Maho Amano, Shin-Ichiro Nishimura, Kiminori Toyooka, Ken Matsuoka, Javier Pozueta-Romero, Toshiaki Mitsui
Plant and Cell Physiology, 57, 8, 1610, 1628, Oxford University Press, 2016年08月01日
英語, 研究論文（学術雑誌）, Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1-NPP6. Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)-Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2-GFP and NPP6-GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER-Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/ mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs.

N-Glycomic and Microscopic Subcellular Localization Analyses of NPP1, 2 and 6 Strongly Indicate that trans-Golgi Compartments Participate in the Golgi to Plastid Traffic of Nucleotide Pyrophosphatase/Phosphodiesterases in Rice
Kentaro Kaneko, Takeshi Takamatsu, Takuya Inomata, Kazusato Oikawa, Kimiko Itoh, Kazuko Hirose, Maho Amano, Shin-Ichiro Nishimura, Kiminori Toyooka, Ken Matsuoka, Javier Pozueta-Romero, Toshiaki Mitsui
PLANT AND CELL PHYSIOLOGY, 57, 8, 1610, 1628, OXFORD UNIV PRESS, 2016年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1-NPP6. Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)-Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2-GFP and NPP6-GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER-Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs.

Alteration of serum N-glycan profile in patients with autoimmune pancreatitis
Takeshi Tomoda, Kazuhiro Nouso, Hironari Kato, Koji Miyahara, Chihiro Dohi, Yuki Morimoto, Hideaki Kinugasa, Yutaka Akimoto, Kazuyuki Matsumoto, Naoki Yamamoto, Yasuhiro Noma, Shigeru Horiguchi, Koichiro Tsutsumi, Maho Amano, Shin-Ichiro Nishimura, Kazuhide Yamamoto
PANCREATOLOGY, 16, 1, 44, 51, KARGER, 2016年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Objectives: The aims of this study were to determine the change in whole-serum N-glycan profile in autoimmune pancreatitis (AIP) patients and to investigate its clinical utility.
Methods: We collected serum from 21 AIP patients before any treatment, and from 60 healthy volunteers (HLTs). Serum glycan profile was measured by comprehensive and quantitative high-throughput glycome analysis.
Results: Of the 53 glycans detected, 14 were differentially expressed in AIP patients. Pathway analysis demonstrated that agalactosyl and monogalactosyl bi-antennary glycans were elevated in AIP patients. Among the 14 glycans, #3410, #3510, and #4510 showed high area under receiver operating characteristic (AUROC) values (0.955, 0.964, and 0.968 respectively) for the diagnosis of AIP. These three glycans were mainly bound to immunoglobulin G; however, their serum levels were significantly higher, even in AIP patients who showed lower serum IgG4 levels, than in HLTs.
Conclusions: We demonstrated, for the first time, whole-serum glycan profiles of AIP patients and showed that the levels of glycans #3410, #3510, and #4510 were increased in AIP patients. These glycans might be valuable biomarkers of AIR Copyright (C) 2015, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved.

Short Communication: Increase of Sialylated N-Glycansin Eyes with Neovascular Glaucoma Secondary to Proliferative Diabetic Retinopathy
Saori Inafuku, Kousuke Noda, Maho Amano, Shin-Ichiro Nishimura, Susumu Ishida
CURRENT EYE RESEARCH, 41, 5, 721, 724, TAYLOR & FRANCIS INC, 2016年, ［査読有り］
英語, 研究論文（学術雑誌）, Purpose: To investigate the alteration of N-glycans in the vitreous fluid of patients with neovascular glaucoma (NVG) secondary to proliferative diabetic retinopathy (PDR).
Methods: Vitreous samples were collected from 18 patients with PDR (including 7 with NVG and 11 without NVG), and 17 patients without diabetes. Profiles of N-glycans were analyzed by glycoblotting-based high throughput protocol, which we recently developed. Protein levels of vascular endothelial growth factor (VEGF)-A were measured by ELISA.
Results: The concentration of total N-glycans and the concentration of N-glycans with sialic acids were significantly higher in NVG group compared with those in non-NVG group or control group, whereas there was no significant difference in concentrations of high-mannose N-glycans among three groups. There was a moderate correlation between the concentrations of sialylated N-glycans and VEGF-A.
Conclusions: Our data demonstrate the distinct changes of N-glycan profile and the increase of sialylated N-glycans in eyes with NVG secondary to PDR.

Large-Scale Glycomics of Livestock: Discovery of Highly Sensitive Serum Biomarkers Indicating an Environmental Stress Affecting Immune Responses and Productivity of Holstein Dairy Cows
Ibrahim F. Rehan, Koichiro Ueda, Tomohiro Mitani, Maho Amano, Hiroshi Hinou, Tetsu Ohashi, Seiji Kondo, Shin-Ichiro Nishimura
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 63, 48, 10578, 10590, AMER CHEMICAL SOC, 2015年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Because various stresses strongly influence the food productivity of livestock, biomarkers to indicate unmeasurable environmental stress in domestic animals are of increasing importance. Thermal comfort is one of the basic principles of dairy cow welfare that enhances productivity. To discover sensitive biomarkers that monitor such environmental stresses in dairy cows, we herein performed, for the first time, large-scale glycomics on 336 lactating Holstein cow serum samples over 9 months between February and October. Glycoblotting combined with MALDI-TOF/MS and DMB/HPLC allowed for comprehensive glycomics of whole serum glycoproteins. The results obtained revealed seasonal alterations in serum N-glycan levels and their structural characteristics, such as an increase in high-mannose type N-glycans in spring, the occurrence of di/triantennary complex type N-glycans terminating with two or three Neu5Gc residues in summer and autumn, and N-glycans in winter dominantly displaying Neu5Ac. A multivariate analysis revealed a correlation between the serum expression levels of these season-specific glycoforms and productivity.

Impaired ATP6V0A2 expression contributes to Golgi dispersion and glycosylation changes in senescent cells
Miyako Udono, Kaoru Fujii, Gakuro Harada, Yumi Tsuzuki, Keishi Kadooka, Pingbo Zhang, Hiroshi Fujii, Maho Amano, Shin-Ichiro Nishimura, Kosuke Tashiro, Satoru Kuhara, Yoshinori Katakura
SCIENTIFIC REPORTS, 5, 17342, NATURE PUBLISHING GROUP, 2015年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Many genes and signaling pathways have been found to be involved in cellular senescence program. In the present study, we have identified 16 senescence-associated genes by differential proteomic analysis of the normal human diploid fibroblast cell line, TIG-1, and focused on ATP6V0A2. The aim of this study is to clarify the role of ATP6V0A2, the causal gene for ARCL2, a syndrome of abnormal glycosylation and impaired Golgi trafficking, in cellular senescence program. Here we showed that ATP6V0A2 is critical for cellular senescence; impaired expression of ATP6V0A2 disperses the Golgi structure and triggers senescence, suggesting that ATP6V0A2 mediates these processes. FITC-lectin staining and glycoblotting revealed significantly different glycosylation structures in presenescent (young) and senescent (old) TIG-1 cells; reducing ATP6V0A2 expression in young TIG-1 cells yielded structures similar to those in old TIG-1 cells. Our results suggest that senescence-associated impaired expression of ATP6V0A2 triggers changes in Golgi structure and glycosylation in old TIG-1 cells, which demonstrates a role of ATP6V0A2 in cellular senescence program.

Glycome characterization of immunoglobulin G from buffalo (Bubalus bubalis) colostrum
L. S. Mamatha Bhanu, M. Amano, S. -I. Nishimura, H. S. Aparna
GLYCOCONJUGATE JOURNAL, 32, 8, 625, 634, SPRINGER, 2015年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Immunoglobulin G (IgG) is a major glycoprotein in ruminant colostrum. First day buffalo colostrum protein was purified on Sephadex G-100 and its mass was determined by MALDI-TOF as 147.848 KDa. The PMF data of protein subunits revealed its homology to IgG, which was supported by the identification of peptide sequences LLIYGATSR and VYNEYLPAPIVR corresponding to light and heavy chains of IgG by CID MS/MS analysis. The N-glycan microheterogeneity was established based on chemoselective glycoblotting technique with the identification of high mannose, neutral complex/hybrid and sialylated complex/hybrid glycans. A complete structural assignment of 54 N-linked oligosaccharides were identified and the ratio of sialyl oligosaccharides was found to be higher compared to neutral saccharides. The fucosylation observed in more than 20 oligosaccharides, high mannose and trisialyl oligosaccharides were present in diminutive amount. The high non-fucosyl and sialyl oligosaccharides in buffalo colostrum IgG provide ample scope for its utilization in targeted therapies to elicit effective ADCC and anti-inflammatory responses.

Rapid Endolysosomal Escape and Controlled Intracellular Trafficking of Cell Surface Mimetic Quantum-Dots-Anchored Peptides and Glycopeptides
Roger S. Tan, Kentaro Naruchi, Maho Amano, Hiroshi Hinou, Shin-Ichiro Nishimura
ACS CHEMICAL BIOLOGY, 10, 9, 2073, 2086, AMER CHEMICAL SOC, 2015年09月, ［査読有り］
英語, 研究論文（学術雑誌）, A novel strategy for the development of a high performance nanoparticules platform was established by means of cell surface mimetic quantum-dots (QDs)-anchored peptides/glycopeptides, which was developed as a model system for nanopartide-based drug delivery (NDD) vehicles with defined functions helping the specific intracellular trafficking after initial endocytosis. In this paper, we proposed a standardized protocol for the preparation of multifunctional QDs that allows for efficient cellular uptake and rapid escaping from the endolysosomal system and subsequent cytoplasmic molecular delivery to the target cellular compartment. Chemoselective ligation of the ketone-functionalized hexahistidine derivative facilitated both efficient endocytic entry and rapid endolysosomal escape of the aminooxy/phosphorylcholine self-assembled monolayer-coated QDs (AO/PCSAM-QDs) to the cytosol in various cell lines such as human normal and cancer cells, while modifications of these QDs with cell-penetrating arginine-rich peptides showed poor cellular uptake and induced self-aggregation of AO/PCSAM-QDs. Combined use of hexahistidylated AO/PCSAM-QDs with serglycine-like glycopeptides, namely synthetic proteoglycan initiators (PGIs), elicited the entry and controlled intracellular trafficking, Golgi localization, and also excretion of these nanoparticles, which suggested that the present approach would provide an ideal platform for the design of high performance NDD systems.

A comprehensive glycome profiling of Huntington's disease transgenic mice
Solomon T. Gizaw, Toshiaki Koda, Maho Amano, Keiko Kamimura, Tetsu Ohashi, Hiroshi Hinou, Shin-Ichiro Nishimura
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1850, 9, 1704, 1718, ELSEVIER SCIENCE BV, 2015年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Background: Huntington's disease (HD) is an autosomal, dominantly inherited and progressive neurodegenerative disease, nosologically classified as the presence of intranuclear inclusion bodies and the loss of GABA-containing neurons in the neostriatum and subsequently in the cerebellar cortex. Abnormal processing of neuronal proteins can result in the misfolding of proteins and altered post-translational modification of newly synthesized proteins. Total glycomics, namely, N-glycomics, O-glycomics, and glycosphingolipidomics (GSL-omics) of HD transgenic mice would be a hallmark for central nervous system disorders in order to discover disease specific biomarkers.
Methods: Glycoblotting method, a high throughput glycomic protocol, and matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) were used to study the total glycome expression levels in the brain tissue (3 mice of each sex) and sera (5 mice of each sex) of HD transgenic and control mice. All experiments were performed twice and differences in the expression levels of major glycoforms were compared between HD transgenic and control mice.
Results: We estimated the structure and expression levels of 87 and 58 N-glycans in brain tissue and sera, respectively, of HD transgenic and control mice. The present results clearly indicated that the brain glycome and their expression levels are significantly gender specific when compared with those of other tissues and serum. Core-fucosylated and bisecting-GlcNAc types of N-glycans were found in increased levels in the brain tissue HD transgenic mice. Accordingly, core-fucosylated and sialic acid (particularly N-glycolylneuraminic acid, NeuGc) for biantennary type glycans were found in increased amounts in the sera of HD transgenic mice compared to that of control mice. Core 3 type O-glycans were found in increased levels in male and in decreased levels in both the striatum and cortexes of female HD transgenic mice. Furthermore, serum levels of core 1 type O-glycans decreased and were undetected for core 2 type O-glycans for HD transgenic mice. In glycosphingolipids, GD1a in brain tissue and GM2-NeuGc serum levels were found to have increased and decreased, respectively, in HD transgenic mice compared to those of the control group mice.
Conclusion: Total glycome expression levels are significantly different between HD transgenic and control group mice.
General significance: Glycoblotting combined with MALDI-TOF/MS total glycomics warrants a comprehensive, effective, novel and versatile technique for qualitative and quantitative analysis of total glycome expression levels. Furthermore, glycome-focused studies of both environmentally and genetically rooted neurodegenerative diseases are promising candidates for the discovery of potential disease glyco-biomarkers in the post-genome era. (C) 2015 Elsevier B.V. All rights reserved.

Alteration of N-glycan profiles in patients with chronic hepatitis and hepatocellular carcinoma
Koji Miyahara, Kazuhiro Nouso, Chihiro Dohi, Yuki Morimoto, Hideaki Kinugasa, Nozomu Wada, Yasuto Takeuchi, Kenji Kuwaki, Hideki Onishi, Fusao Ikeda, Yasuhiro Miyake, Shinichiro Nakamura, Hidenori Shiraha, Akinobu Takaki, Maho Amano, Shin-Ichiro Nishimura, Kazuhide Yamamoto
HEPATOLOGY RESEARCH, 45, 9, 986, 993, WILEY-BLACKWELL, 2015年09月, ［査読有り］
英語, 研究論文（学術雑誌）, AimMost of the modification of N-glycosylation reported in cancers including hepatocellular carcinoma (HCC) were based on the examinations of a small number of patients or particular proteins. The aim of this study is to reveal changes in whole serum N-glycan profiles systematically during the process of hepatocarcinogenesis and to elucidate their clinical application.
MethodsWe analyzed sera from 105 patients with chronic hepatitis/liver cirrhosis (CH/LC) and age-/sex-matched healthy volunteers (HLT), as well as from 114 patients with HCC. Serum N-glycan profiles were measured comprehensively by a new, quantitative, high-throughput method and compared with clinical parameters.
ResultsThe total amount of N-glycan expression was significantly higher in patients with CH/LC than in HLT; however, no differences were observed between CH/LC and HCC patients. In HCC patients, multi-antennary glycans with fucose residues, particularly m/z 3195, were increased compared with CH/LC patients. The expression of m/z 3195 was high, especially in patients with a high number of intrahepatic lesions (>3), large tumor size (>3cm), macroscopic vascular invasion or metastasis. The ratio of pairs of glycans on the same path of the biosynthesis pathway (m/z 3195/1914) showed a higher area under the receiver-operator curve of 0.810 than any other single glycan to distinguish HCC from CH/LC.
ConclusionWe demonstrate the full spectrum of the alterations of serum N-glycans comprehensively in patients with liver disease, and elucidate the possible use of glycans as novel biomarkers of liver disease progression.

Alteration of N-Glycan Profiles in Diabetic Retinopathy
Saori Inafuku, Kousuke Noda, Maho Amano, Tetsu Ohashi, Chikako Yoshizawa, Wataru Saito, Miyuki Murata, Atsuhiro Kanda, Shin-Ichiro Nishimura, Susumu Ishida
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, 56, 9, 5316, 5322, ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2015年08月, ［査読有り］
英語, 研究論文（学術雑誌）, PURPOSE. To investigate the alteration of vitreal N-glycans in patients with proliferative diabetic retinopathy (PDR).
METHODS. Plasma and vitreous samples were collected from 17 patients (10 females and 7 males) with PDR (PDR group) and 17 nondiabetic patients (8 females and 9 males) with epiretinal membrane (ERM) and idiopathic macular hole (MH) (non-diabetes mellitus [DM] group). Profiles of N-glycans were analyzed by a glycoblotting-based high-throughput protocol that we recently developed. Human retinal microvascular endothelial cells (HRMECs) were cultivated with culture media containing either low glucose (5 mM) or high glucose (25 mM), and expression levels of sialyltransferases were analyzed by real-time PCR and ELISA.
RESULTS. Amount of N-glycans in the vitreous fluid of the PDR group was significantly higher than that of the non-DM group (495.5 +/- 37.4 vs. 142.7 +/- 30.8 pmol/100 mu g protein, P < 0.005), whereas there was no significant difference in the plasma samples between the PDR and the non-DM group. In addition, profile analysis showed that N-glycans with sialic acids increased in the vitreous of the PDR group (328.4 +/- 25.8 pmol/100 mu g protein) compared to the non-DM group (92.1 +/- 21.2 pmol/100 mu g protein, P < 0.0005). Expression levels of sialyltransferases ST3GAL1 and ST3GAL4 were upregulated in the HRMECs after high-glucose stimulation. Consistent with the real-time PCR data, high-glucose stimulation elevated the protein levels of ST3GAL1 (117.4 +/- 14.9 pg/mg, P < 0.01) and ST3GAL4 (6.1 +/- 0.9 pg/mg, P < 0.05) in the HRMECs compared with the cells cultured with low-glucose culture media (ST3GAL1, 64.4 +/- 5.8 pg/mg; ST3GAL4, 3.8 +/- 0.3 pg/mg).
CONCLUSIONS. Our data demonstrate distinct changes in the N-glycan profile and an increase in sialylated N-glycans in eyes with PDR.

Serum N-glycan profiles in patients with intraductal papillary mucinous neoplasms of the pancreas
Yutaka Akimoto, Kazuhiro Nouso, Hironari Kato, Koji Miyahara, Chihiro Dohi, Yuki Morimoto, Hideaki Kinugasa, Takeshi Tomoda, Naoki Yamamoto, Koichiro Tsutsumi, Kenji Kuwaki, Hideki Onishi, Fusao Ikeda, Shinichiro Nakamura, Hidenori Shiraha, Akinobu Takaki, Hiroyuki Okada, Maho Amano, Shin-Ichiro Nishimura, Kazuhide Yamamoto
PANCREATOLOGY, 15, 4, 432, 438, KARGER, 2015年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Background/objectives: Diagnosing the invasiveness of intraductal papillary mucinous neoplasms (IPMNs) is difficult, especially by blood test. Alterations in serum glycan profiles have been reported for several cancers, but changes in serum glycan profiles have not been investigated in patients with IPMNs. The objectives of this study were to determine the serum N-glycan profile and to investigate its clinical utility in patients with IPMNs.
Methods: We measured serum N-glycan profiles in 79 patients with IPMNs, including 13 invasive IPMNs, by performing comprehensive glycome analysis and assessed the relationship between N-glycan changes and clinical parameters.
Results: Seventy glycans were identified and their expression profiles were significantly different depending on the cyst size, the presence of an enhancing solid component, and the histological grade of the IPMN. Nine glycans were highly expressed in patients with invasive IPMNs. The glycan m/z 3195, which is a fucosylated tri-antennary glycan, had the highest diagnostic value for distinguishing invasive IPMNs from non-invasive IPMNs (area under the receiver operating characteristic curve = 0.803). Multivariate analyses revealed high levels of m/z 3195 [odds ratio (OR), 20.5; 95% confidence interval (CI) 2.60-486.4] and the presence of enhancing solid components (OR, 35.8; 95% Cl, 5.39-409.6) were significant risk factors for invasive IPMNs.
Conclusions: We performed a comprehensive evaluation of the changes in serum N-glycan profiles in patients with IPMNs for the first time. We determined that increased expression of fucosylated complex-type glycans, especially m/z 3195, is a potential marker for invasive IPMNs. Copyright (C) 2015, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved.

Prognostic Value of Altered N-Glycosylation of Circulating Glycoproteins in Patients With Unresectable Pancreatic Cancer Treated With Gemcitabine
Koji Miyahara, Kazuhiro Nouso, Yuki Morimoto, Hideaki Kinugasa, Hironari Kato, Naoki Yamamoto, Koichiro Tsutsumi, Kenji Kuwaki, Hideki Onishi, Fusao Ikeda, Shinichiro Nakamura, Hidenori Shiraha, Akinobu Takaki, Taku Nakahara, Yoshiaki Miura, Hidehisa Asada, Maho Amano, Shin-Ichiro Nishimura, Kazuhide Yamamoto
PANCREAS, 44, 4, 551, 556, LIPPINCOTT WILLIAMS & WILKINS, 2015年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Objectives: The objectives of this study were to examine the whole serum N-glycan profile of patients with unresectable pancreatic cancer and to evaluate the ability of glycans to predict gemcitabine treatment efficacy and patient survival.
Methods: We collected serum from 52 patients with advanced pancreatic cancer before they began gemcitabine monotherapy. The serum glycan profile was measured through comprehensive quantitative high-throughput glycome analysis and compared with the treatment efficacy and patient survival.
Results: Of the 61 glycans detected, the serum levels of glycan 4310 (molecular weight [m/z] 1549.566), 6301 (m/z 2032.724), and 9200 (m/z 2010.692) were high in patients with a short time to tumor progression (TTP). Multivariate analysis revealed that a high glycan 9200 concentration was an independent risk factor for shorter TTP (hazard ratio, 2.11; 95% confidence interval, 1.07-4.17) and poor overall survival (hazard ratio, 2.56; 95% confidence interval, 1.08-6.19). The median TTP of patients with up-regulation of 9200 after gemcitabine treatment was shorter than for the remaining patients (91 vs 301 days; P = 0.0005). A similar relationship was observed for overall survival (median, 181 vs 561 days; P = 0.001).
Conclusions: Glycan 9200 is a possible biomarker predicting gemcitabine efficacy survival in patients with unresectable pancreatic cancer.

Use of non-invasive serum glycan markers to distinguish non-alcoholic steatohepatitis from simple steatosis
Yasushi Yamasaki, Kazuhiro Nouso, Koji Miyahara, Nozomu Wada, Chihiro Dohi, Yuki Morimoto, Hideaki Kinugasa, Yasuto Takeuchi, Tetsuya Yasunaka, Kenji Kuwaki, Hideki Onishi, Fusao Ikeda, Yasuhiro Miyake, Shinichiro Nakamura, Hidenori Shiraha, Akinobu Takaki, Yoshiaki Iwasaki, Maho Amano, Shin-Ichiro Nishimura, Kazuhide Yamamoto
JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, 30, 3, 528, 534, WILEY-BLACKWELL, 2015年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Background and AimsSerum glycans have been reported to be promising diagnostic markers for many inflammatory diseases and cancers. The aims of this study were to investigate whole glycan expression in patients with non-alcoholic fatty liver diseases and to evaluate the potential use of glycan profiles as new clinical biomarkers to distinguish non-alcoholic steatohepatitis (NASH) from simple steatosis (SS).
MethodsWe collected sera from 42 histologically proven NASH and 15 SS patients prior to treatment. Serum glycan profiles were measured by comprehensive, quantitative, high-throughput glycome analysis, and diagnostic values of serum glycans for NASH prediction were examined.
ResultsAmong the 41 serum glycans examined, the expression levels of 8 glycans in NASH were significantly higher than those of SS. Out of these eight glycans, three glycans (m/z 1955, 2032, and 2584) showed high areas under the receiver operating characteristic curve (0.833, 0.863, and 0.866, respectively) for distinguishing NASH from SS. In multivariate analyses with clinical parameters and serum glycans, these three glycans were significant predictive factors for distinguishing NASH from SS. The odds ratio of m/z 1955, 2032, and 2584 were 48.5, 6.46, and 11.8, respectively. These glycans also correlated significantly with lobular inflammation, ballooning, and fibrosis, but not with steatosis.
ConclusionWe clearly demonstrated whole-serum glycan profiles in NASH patients, and the feasibility of serum glycans (m/z 1955, 2032, and 2584) as new noninvasive biomarkers for distinguishing NASH from SS.

The loss of luteal progesterone production in women is associated with a galectin switch via α2,6-sialylation of glycoconjugates.
Nio-Kobayashi J, Boswell L, Amano M, Iwanaga T, Duncan WC
The Journal of clinical endocrinology and metabolism, 99, 12, 4616, 4624, 12, 2014年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Context: Luteal progesterone is fundamental for reproduction, but the molecular regulation of the corpus luteum (CL) in women remains unclear. Galectin-1 and galectin-3 bind to the sugar chains on cells to control key biological processes including cell function and fate.
Methods: The expression and localization of LGALS1 and LGALS3 were analyzed by quantitative PCR and histochemical analysis, with special reference to alpha 2,6-sialylation of glycoconjugates in carefully dated human CL collected across the menstrual cycle and after exposure to human chorionic gonadotrophin (hCG) in vivo. The effects of hCG and prostaglandin E-2 on the expression of galectins and an alpha 2,6-sialyltransferase 1 (ST6GAL1) in granulosa lutein cells were analyzed in vitro.
Results: Galectin-1 was predominantly localized to healthy granulosa lutein cells and galectin-3 was localized to macrophages and regressing granulosa lutein cells. Acute exposure to luteotrophic hormones (hCG and prostaglandin E2) up-regulated LGALS1 expression (P < .001). ST6GAL1, which catalyzes alpha 2,6-sialylation to block galectin-1 binding, increased during luteolysis (P < .05) as did LGALS3 (P < .05). Luteotrophic hormones reduced ST6GAL1 and LGALS3 in vivo (P < .05) and in vitro (P < .001). There was an inverse correlation between the expression of ST6GAL1 and HSD3B1 (P < .01) and a distinct cellular relationship among alpha 2,6-sialylation, 3 beta-hydroxysteroid dehydrogenase, and galectin expression.
Conclusions: Galectin-1 is a luteotrophic factor whose binding is inhibited by alpha 2,6-sialylation in the human CL during luteolysis. ST6GAL1 and galectin-3 expression is increased during luteolysis and associated with a loss of progesterone synthesis. Luteotrophic hormones differentially regulate galectin-1 and galectin-3/alpha 2,6-sialylation in granulosa lutein cells, suggesting a novel galectin switch regulated by luteotrophic stimuli during luteolysis and luteal rescue.

Quantitative glycomics monitoring of induced pluripotent- and embryonic stem cells during neuronal differentiation
Michiyo Terashima, Maho Amano, Tomohiro Onodera, Shin-Ichiro Nishimura, Norimasa Iwasaki
STEM CELL RESEARCH, 13, 3, 454, 464, ELSEVIER SCIENCE BV, 2014年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Alterations in the structure of cell surface glycoforms occurring during the stages of stem cell differentiation remain unclear. We describe a rapid glycoblotting-based cellular glycomics method for quantitatively evaluating changes in glycoform expression and structure during neuronal differentiation of murine induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). Our results show that changes in the expression of cellular N-glycans are comparable during the differentiation of iPSCs and ESCs. The expression of bisect-type N-glycans was significantly up-regulated in neurons that differentiated from both iPSCs and ESCs. From a glycobiological standpoint, iPSCs are an alternative neural cell source in addition to ESCs. (C) 2014 The Authors. Published by Elsevier B.V.

A comparison of N-glycan profiles in human plasma and vitreous fluid
Saori Inafuku, Kousuke Noda, Maho Amano, Tetsu Ohashi, Chikako Yoshizawa, Wataru Saito, Atsuhiro Kanda, Shin-Ichiro Nishimura, Susumu Ishida
GRAEFES ARCHIVE FOR CLINICAL AND EXPERIMENTAL OPHTHALMOLOGY, 252, 8, 1235, 1243, SPRINGER, 2014年08月, ［査読有り］
英語, 研究論文（学術雑誌）, To investigate the concentration and composition of N-glycans in plasma and vitreous samples obtained from patients with non-proliferative vitreoretinal diseases.
Plasma and vitreous samples were collected from 11 patients with idiopathic macular hole (MH) and 9 patients with epiretinal membrane (ERM). The samples were pretreated for enzymatic cleaving, and subsequently glycans released from proteins were captured on BlotGlyco H beads. Sialic acids were methyl-esterified. Processed glycans were tagged with aminooxy-functionalized peptide reagent (aoWR) and released from the beads, followed by detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The concentration and composition of N-glycans in the samples were assessed.
Concentration of N-glycans in vitreous samples (132 +/- 29 pmol/100 mu g protein) was significantly lower compared with those in plasma samples (714 +/- 29 pmol/100 mu g protein, p < 0.001). Predominant N-glycan in both plasma (39.7 +/- 1.1 %) and vitreous fluid (37.2 +/- 3.1 %) was identical, and the composition was presumed as [ (Hex)2(HexNAc)2(NeuAc)2+ (Man)3(GlcNAc)2]. By contrast, the second-ranked N-glycan in vitreous samples (15.6 +/- 1.5 %) was the seventh in plasma (2.3 +/- 0.2 %).
The current data provide useful information on N-glycan profile in the vitreous fluid, which is distinct from that in the plasma.

A straightforward protocol for the preparation of high performance microarray displaying synthetic MUC1 glycopeptides
Takahiko Matsushita, Wataru Takada, Kota Igarashi, Kentaro Naruchi, Risho Miyoshi, Fayna Garcia-Martin, Maho Amano, Hiroshi Hinou, Shin-Ichiro Nishimura
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1840, 3, 1105, 1116, ELSEVIER SCIENCE BV, 2014年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Background: Human serum MUC1 peptide fragments bearing aberrant O-glycans are secreted from columnar epithelial cell surfaces and known as clinically important serum biomarkers for the epithelial carcinoma when a specific monoclonal antibody can probe disease-relevant epitopes. Despite the growing importance of MUC1 glycopeptides as biomarkers, the precise epitopes of most anti-MUC1 monoclonal antibodies remains unclear.
Methods: A novel protocol for the fabrication of versatile microarray displaying peptide/glycopeptide library was investigated for the construction of highly sensitive and accurate epitope mapping assay of various anti-MUC1 antibodies.
Results: Selective imine-coupling between aminooxy-functionalized methaciylic copolymer with phosphorylcholine unit and synthetic MUC1 glycopeptides-capped by a ketone linker at N-terminus provided a facile and seamless protocol for the preparation of glycopeptides microarray platform. It was demonstrated that anti-KL-6 monoclonal antibody shows an extremely specific and strong binding affinity toward MUC1 fragments carrying sialyl T antigen (Neu5Ac alpha 2,3Gal beta 1,3GalNAc alpha 1 ->) at Pro-Asp-Thr-Arg motif when compared with other seven anti-MUD monoclonal antibodies such as VU-3D, VU-12E1, VU-11E2, Ma552, VU-3C6, SM3, and DF3. The present microarray also uncovered the occurrence of IgG autoantibodies in healthy human sera that bind specifically with sialyl T antigen attached at five potential O-glycosylation sites of MUD tandem repeats.
Conclusion: We established a straightforward strategy toward the standardized microarray platform allowing highly sensitive and accurate epitope mapping analysis by reducing the background noise due to nonspecific protein adsorption.
General significance: The present approach would greatly accelerate the discovery research of new class autoantibodies as well as the development of therapeutic mAbs reacting specifically with disease-relevant epitopes. (C) 2013 Elsevier B.V. All rights reserved.

Serum N-Glycan Alteration Associated with Renal Cell Carcinoma Detected by High Throughput Glycan Analysis
Shingo Hatakeyama, Maho Amano, Yuki Tobisawa, Tohru Yoneyama, Norihiko Tsuchiya, Tomonori Habuchi, Shin-Ichiro Nishimura, Chikara Ohyama
JOURNAL OF UROLOGY, 191, 3, 805, 813, ELSEVIER SCIENCE INC, 2014年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Purpose: Biomarkers for the early detection and prediction of survival in patients with renal cell carcinoma have not been established. We developed what is to our knowledge a novel glycoblotting method that allows high throughput, comprehensive, quantitative analysis of glycans in human serum. In this study we identified alterations in serum N-glycans associated with renal cell carcinoma.
Materials and Methods: We performed a comprehensive N-glycan structural analysis of serum from 64 patients with renal cell carcinoma and 34 age matched, healthy volunteers using glycoblotting methods and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The peak intensity of N-glycan was analyzed using logistic regression analysis and ROCs were used to select candidate N-glycans. Candidate N-glycans with a statistically significant relationship to renal cell carcinoma or overall survival were independently evaluated using a Cox regression model to determine superiority compared to other conventional renal cell carcinoma biomarkers.
Results: We identified 56 types of N-glycans in serum from healthy volunteers and patients with renal cell carcinoma. Peaks 40 and 43 were significantly more intense in patients than in volunteers. Peak 19 intensity was significantly higher and peak 49 intensity was significantly lower in patients with renal cell carcinoma who survived for a longer period. Multivariate analysis revealed that peaks 19 and 49 were independent predictors of overall survival.
Conclusions: Serum N-glycan analysis is a promising approach to discovering new biomarkers for renal cell carcinoma. Further study is warranted to validate our results.

A straightforward protocol for the preparation of high performance microarray displaying synthetic MUC1 glycopeptides.
Matsushita T, Takada W, Igarashi K, Naruchi K, Miyoshi R, Garcia-Martin F, Amano M, Hinou H, Nishimura S
Biochimica et biophysica acta, 1840, 3, 1105, 1116, 3, 2014年03月, ［査読有り］
研究論文（学術雑誌）

Discovery of novel differentiation markers in the early stage of chondrogenesis by glycoform-focused reverse proteomics and genomics
Takeshi Ishihara, Kiyoshi Kakiya, Koji Takahashi, Hiroto Miwa, Masatomo Rokushima, Tomoyo Yoshinaga, Yoshikazu Tanaka, Takaomi Ito, Hiroko Togame, Hiroshi Takemoto, Maho Amano, Norimasa Iwasaki, Akio Minami, Shin-Ichiro Nishimura
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1840, 1, 645, 655, ELSEVIER SCIENCE BV, 2014年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Background: Osteoarthritis (OA) is one of the most common chronic diseases among adults, especially the elderly, which is characterized by destruction of the articular cartilage. Despite affecting more than 100 million individuals all over the world, therapy is currently limited to treating pain, which is a principal symptom of OA New approaches to the treatment of OA that induce regeneration and repair of cartilage are strongly needed.
Methods: To discover potent markers for chondrogenic differentiation, glycoform-focused reverse proteomics and genomics were performed on the basis of glycoblotting-based comprehensive approach.
Results: Expression levels of high-mannose type N-glycans were up-regulated significantly at the late stage of differentiation of the mouse chondroprogenitor cells. Among 246 glycoproteins carrying this glycotype identified by ConA affinity chromatography and LC/MS, it was demonstrated that 52% are classified as cell surface glycoproteins. Gene expression levels indicated that mRNAs for 15 glycoproteins increased distinctly in the earlier stages during differentiation compared with Type II collagen. The feasibility of mouse chondrocyte markers in human chondrogenesis model was demonstrated by testing gene expression levels of these 15 glycoproteins during differentiation in human mesenchymal stem cells.
Conclusion: The results showed clearly an evidence of up-regulation of 5 genes, ectonucleotide pyrophosphatase/ phosphodiesterase family member 1, collagen alpha-1(III) chain, collagen alpha-1(XI) chain, aquaporin-1, and netrin receptor UNC5B, in the early stages of differentiation.
General significance: These cell surface 5 glycoproteins become highly sensitive differentiation markers of human chondrocytes that contribute to regenerative therapies, and development of novel therapeutic reagents. (C) 2013 Published by Elsevier B.V.

Serum Glycan as a Prognostic Marker in Patients With Advanced Hepatocellular Carcinoma Treated With Sorafenib
Koji Miyahara, Kazuhiro Nouso, Yasuhiro Miyake, Shinichiro Nakamura, Shuntaro Obi, Maho Amano, Kazuko Hirose, Shin-ichiro Nishimura, Kazuhide Yamamoto
HEPATOLOGY, 59, 1, 355, 356, WILEY-BLACKWELL, 2014年01月, ［査読有り］
英語

Discovery of novel differentiation markers in the early stage of chondrogenesis by glycoform-focused reverse proteomics and genomics.
Ishihara T, Kakiya K, Takahashi K, Miwa H, Rokushima M, Yoshinaga T, Tanaka Y, Ito T, Togame H, Takemoto H, Amano M, Iwasaki N, Minami A, Nishimura S
Biochimica et biophysica acta, 1840, 1, 645, 655, 1, 2014年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Osteoarthritis (OA) is one of the most common chronic diseases among adults, especially the elderly, which is characterized by destruction of the articular cartilage. Despite affecting more than 100 million individuals all over the world, therapy is currently limited to treating pain, which is a principal symptom of OA. New approaches to the treatment of OA that induce regeneration and repair of cartilage are strongly needed. METHODS: To discover potent markers for chondrogenic differentiation, glycoform-focused reverse proteomics and genomics were performed on the basis of glycoblotting-based comprehensive approach. RESULTS: Expression levels of high-mannose type N-glycans were up-regulated significantly at the late stage of differentiation of the mouse chondroprogenitor cells. Among 246 glycoproteins carrying this glycotype identified by ConA affinity chromatography and LC/MS, it was demonstrated that 52% are classified as cell surface glycoproteins. Gene expression levels indicated that mRNAs for 15 glycoproteins increased distinctly in the earlier stages during differentiation compared with Type II collagen. The feasibility of mouse chondrocyte markers in human chondrogenesis model was demonstrated by testing gene expression levels of these 15 glycoproteins during differentiation in human mesenchymal stem cells. CONCLUSION: The results showed clearly an evidence of up-regulation of 5 genes, ectonucleotide pyrophosphatase/phosphodiesterase family member 1, collagen alpha-1(III) chain, collagen alpha-1(XI) chain, aquaporin-1, and netrin receptor UNC5B, in the early stages of differentiation. GENERAL SIGNIFICANCE: These cell surface 5 glycoproteins become highly sensitive differentiation markers of human chondrocytes that contribute to regenerative therapies, and development of novel therapeutic reagents.

Potential usage for in vivo lectin screening in live animals utilizing cell surface mimetic glyco-nanoparticles, phosphorylcholine-coated quantum dots (PC-QDs)
Maho Amano, Hiroshi Hinou, Risho Miyoshi, Shin-Ichiro Nishimura
Methods in Molecular Biology, 1200, 361, 369, Humana Press Inc., 2014年, ［査読有り］
英語, 研究論文（学術雑誌）, Utilizing glycosylated derivatives as a tag, we are able to explore novel counter-receptor of endogenous lectins or lectin-like molecules in vivo. We have established the standardized methodology including preparation of glycosylated derivatives and construction of a platform for tracing the molecules in vivo at first. Combined use of an aminooxy-terminated thiol derivative and a phosphorylcholine (PC) derivative provides quantum dots (QDs) with novel functions for the chemical ligation of ketone-functionalized compounds and the prevention of nonspecific protein adsorption concurrently. In order to track the derivatives in vivo, near-infrared (NIR) fluorescence imaging of QDs displaying various simple sugars (glyco-PC-QDs) after administration into the tail vein of the mouse can be performed. It has revealed that distinct long-term delocalization over 2 h can be observed depending on the species of glycans ligated to PC-QDs at least in the liver. Until today we have performed live animal imaging utilizing various kinds of sialyl glyco-PC-QDs. They are still retained stably in whole body after 2 h while they showed significantly different in vivo dynamics in the tissue distribution, suggesting that structure/sequence of the neighboring sugar residues in the individual sialyl oligosaccharides might influence the final organ-specific distribution, which should be equivalent to the distribution of sialic acid-recognizing lectins. Here we describe a standardized protocol using ligand-displayed PC-QDs for live cell/animal imaging by versatile NIR fluorescence photometry without influence of size-dependent accumulation/excretion pathway for nanoparticles (e.g., viruses)∈>
∈10 nm in hydrodynamic diameter by the liver. © 2014 Springer Science+Business Media New York.

Serum Glycan Markers for Evaluation of Disease Activity and Prediction of Clinical Course in Patients with Ulcerative Colitis
Koji Miyahara, Kazuhiro Nouso, Shunsuke Saito, Sakiko Hiraoka, Keita Harada, Sakuma Takahashi, Yuki Morimoto, Sayo Kobayashi, Fusao Ikeda, Yasuhiro Miyake, Hidenori Shiraha, Akinobu Takaki, Hiroyuki Okada, Maho Amano, Kazuko Hirose, Shin-Ichiro Nishimura, Kazuhide Yamamoto
PLoS ONE, 8, 10, e74861, 10, 2013年10月07日, ［査読有り］
英語, 研究論文（学術雑誌）, Background:The aims of this study were to determine the change of whole-serum N-glycan profile in ulcerative colitis (UC) patients and to investigate its clinical utility.Methods:We collected serum from 75 UC patients at the time of admission and the same number of age/sex-matched healthy volunteers. Serum glycan profile was measured by comprehensive quantitative high-throughput glycome analysis and was compared with disease activity and prognosis.Results:Out of 61 glycans detected, 24 were differentially expressed in UC patients. Pathway analysis demonstrated that highly sialylated multi-branched glycans and agalactosyl bi-antennary glycans were elevated in UC patients
in addition, the glycan ratio m/z 2378/1914, which also increased in UC, showed the highest Area under Receiver Operating Characteristic curve (0.923) for the diagnosis of UC. Highly sialylated multi-branched glycans and the glycan ratio m/z 2378/1914 were higher in the patients with total colitis, Clinical Activity Index >
10, Mayo endoscopic score 3, or a steroid-refractory status. In particular, the glycan ratio m/z 2378/1914 (above median) was an independent prognostic factor for the need for an operation (hazard ratio, 2.67
95% confidence interval, 1.04-7.84).Conclusions:Whole-serum glycan profiles revealed that the glycan ratio m/z 2378/1914 and highly sialylated multi-branched glycans increase in UC patients, and are correlated with disease activity. The glycan ratio m/z 2378/1914 was an independent predictive factor of the prognosis of UC. © 2013 Miyahara et al.

Clinical utility of high-throughput glycome analysis in patients with pancreatic cancer
Kazuhiro Nouso, Maho Amano, Yoichi M. Ito, Koji Miyahara, Yuki Morimoto, Hironari Kato, Koichiro Tsutsumi, Takeshi Tomoda, Naoki Yamamoto, Shinichiro Nakamura, Sayo Kobayashi, Kenji Kuwaki, Hiroaki Hagihara, Hideki Onishi, Yasuhiro Miyake, Fusao Ikeda, Hidenori Shiraha, Akinobu Takaki, Taku Nakahara, Shin-Ichiro Nishimura, Kazuhide Yamamoto
JOURNAL OF GASTROENTEROLOGY, 48, 10, 1171, 1179, SPRINGER JAPAN KK, 2013年10月, ［査読有り］
英語, 研究論文（学術雑誌）, Most of the glycan changes reported in cancers were based on the examinations of a small number of patients or particular proteins. The aim of this study was to determine the changes of the serum N-glycan profile comprehensively in a large number of pancreatic cancer patients and investigate its clinical utility.
Glycan levels in the serum of 92 pancreatic cancer patients and 243 healthy volunteers (HLT) were examined by comprehensive quantitative high-throughput glycome analysis and were compared with clinical parameters.
Out of 66 glycans detected, 15 were differentially expressed in pancreatic cancer, and 10 out of the 15 glycans were significantly up-regulated in cases with distant metastasis. There was a clear increase in overall expression of serum glycans, especially highly-branched glycans with fucose moieties, in pancreatic cancer. Among these 15 glycans, a tri-antennary complex type glycan (m/z 3195) showed the highest area under the receiver operating characteristic curve (AUROC = 0.799) for the diagnosis of pancreatic cancer. The ratio of pairs of glycans on the same path of the biosynthesis pathway (m/z 3195/1914) was found to be significantly higher in pancreatic cancer than in HLT (median = 1.11 and 0.41, respectively; p < 0.0001, AUROC = 0.831). For this pair ratio, the hazard ratio for survival (2.60, 95 % CI = 1.44-4.79) was higher than that of any single glycan and 1-year survival of patients with a high and low ratio was 36.9 and 69.2 %, respectively, (p = 0.001).
Comprehensive glycome analysis can be used to know the presence of pancreatic cancer, distant metastasis, and patient prognosis, simultaneously.

潰瘍性大腸炎の活動性・予後予測における血清糖鎖マーカーの有用性　　　　　　　　　　　　　　　
宮原 孝治, 能祖 一裕, 平岡 佐規子, 森元 裕貴, 高橋 索真, 小林 沙代, 斎藤 俊介, 原田 馨太, 山本 和秀, 天野 麻穂, 西村 紳一郎
日本臨床分子医学会学術総会プログラム・抄録集, 50回, 76, 76, 日本臨床分子医学会, 2013年04月
日本語

腎細胞癌における血清免疫グロブリンの糖鎖変異についての検討　　　　　　　　　　　　　　　
畠山 真吾, 米山 徹, 飛澤 悠葵, 盛 和行, 米山 高弘, 橋本 安弘, 古家 琢也, 天野 麻穂, 西村 紳一郎, 大山 力
日本泌尿器科学会雑誌, 104, 2, 216, 216, (一社)日本泌尿器科学会, 2013年03月, ［査読有り］
日本語

Glycoblotting-based high throughput protocol for the structural characterization of hyaluronan degradation products during enzymatic fragmentation
Takayuki Furukawa, Misaki Arai, Fayna Garcia-Martin, Maho Amano, Hiroshi Hinou, Shin-Ichiro Nishimura
GLYCOCONJUGATE JOURNAL, 30, 2, 171, 182, SPRINGER, 2013年02月, ［査読有り］
英語, 研究論文（学術雑誌）, Increasing interests have been focused on the functional roles of hyaluronan degradation products, namely hyaluronan oligosaccharides, as signal molecules regulating cell growth, differentiation, malignancy, and inflammatory responses. It is clear that molecular size of hyaluronan oligosaccharides might be crucial for defining possible and dynamic roles in supporting and suppressing homeostatic cellular processes. The present paper communicates a facile and efficient approach based on glycoblotting method for the characterization of hyaluronan fragments liberated from three different sources of hyaluronan (rooster comb, bovine vitreous humor, and Streptococcus) by in vitro degradation using two typical hyaluronidases of bovine testicular (EC 3.2.1.35) and Streptomyces hyalurolyticus (EC 4.2.2.1). It was demonstrated that glycoblotting method allows for high throughput and quantitative analysis of hyaluronan fragments within a wide dynamic range (1 similar to 1,000 pmole) when 5 mu g of hyaluronan digests were applied for this enrichment protocol. Molecular size and distribution of hyaluronan fragments were proved to be influenced strongly by conditions and hyaluronidases employed while source of hyaluronan did not affect the degradation profiles. Strikingly, the present method uncovered the existence of the smallest and unusual hyaluronan degradation fragments such as a disaccharide GlcA beta 1-3GlcNAc during the digestion by bovine hyaluronidase and a trisaccharide GlcA beta 1-3GlcNAc beta 1-4GlcA derivative by Streptomyces hyaluronidase. Bovine testis hyaluronidases afforded hyaluronan tetra- and hexasaccharides as major products. On the other hand, it was demonstrated that Streptomyces hyaluronidase can produce odd number fragments from three to nine sugar residues while even number fragments from four to fourteen sugar residues were major products.

Serum N-Glycan Profiling Predicts Prognosis in Patients Undergoing Hemodialysis
Shingo Hatakeyama, Maho Amano, Yuki Tobisawa, Tohru Yoneyama, Megumi Tsushima, Kazuko Hirose, Takahiro Yoneyama, Yasuhiro Hashimoto, Takuya Koie, Hisao Saitoh, Kanemitsu Yamaya, Tomihisa Funyu, Shin-Ichiro Nishimura, Chikara Ohyama
SCIENTIFIC WORLD JOURNAL, HINDAWI PUBLISHING CORPORATION, 2013年, ［査読有り］
英語, 研究論文（学術雑誌）, Background. The aim of this study is to evaluate the usefulness of serum N-glycan profiling for prognosis in hemodialysis patients. Methods. Serum N-glycan analysis was performed in 100 hemodialysis patients in June 2008 using the glycoblotting method, which allows high-throughput, comprehensive, and quantitative N-glycan analysis. All patients were longitudinally followed up for 5 years. To evaluate the independent predictors for prognosis, patients' background, blood biochemistry, and N-glycans intensity were analyzed using Cox regression multivariate analysis. Selected N-glycans and independent factors were evaluated using the log-rank test with the Kaplan-Meier method to identify the predictive indicators for prognosis. Each patient was categorized according to the number of risk factors to evaluate the predictive potential of the risk criteria for prognosis. Results. In total, 56 N-glycan types were identified in the hemodialysis patients. Cox regression multivariate analysis showed cardiovascular events, body mass index, maximum intima media thickness, and the serum N-glycan intensity of peak number 49 were predictive indicators for overall survival. Risk classification according to the number of independent risk factors revealed significantly poor survival by increasing the number of risk factors. Conclusions. Serum N-glycan profiling may have a potential to predict prognosis in patients undergoing hemodialysis.

N- and O-glycome analysis of serum and urine from bladder cancer patients using a high-throughput glycoblotting method　　　　　　　　　　　　　　　
Takeuchi M, Amano M, Kitamura H, Tsukamoto T, Masumori N, Hirose K, Ohashi T, Nishimura SI
3, 108, 2013年, ［査読有り］
英語, 研究論文（学術雑誌）

Effect of Ganglioside GM3 Synthase Gene Knockout on the Glycoprotein N-Glycan Profile of Mouse Embryonic Fibroblast
Noriko Nagahori, Tadashi Yamashita, Maho Amano, Shin-Ichiro Nishimura
CHEMBIOCHEM, 14, 1, 73, 82, WILEY-V C H VERLAG GMBH, 2013年01月, ［査読有り］
英語, 研究論文（学術雑誌）, The structural and clinical significance of cellular glycoproteins and glycosphingolipids (GSLs) are often separately discussed. Considering the biosynthetic pathway of glycoconjugates, glycans of cell-surface glycoproteins and GSLs might partially share functions in maintaining cellular homeostatis. The purpose of this study is to establish a general and comprehensive glycomics protocol for cellular GSLs and N-glycans of glycoproteins. To test the feasibility of a glycoblotting-based protocol, whole glycans released both from GSLs and glycoproteins were profiled concurrently by using GM3 synthase-deficient mouse embryonic fibroblast GM3(-/-). GM3(-/-) cells did not synthesize GM3 or any downstream product of GM3 synthase. Instead, expression levels of o-series gangliosides involving GM1-b and GD1-alpha increased dramatically, whereas a-/b-series gangliosides were predominantly detected in wild-type (WT) cells. We also discovered that glycoprotein N-glycan profiles of GM3(-/-) cells are significantly altered as compared to WT cells, although GM3 synthase is responsible only for GSLs synthesis and is not associated with glycoprotein N-glycan biosynthesis. The present approach allows for high-throughput profiling of cellular glycomes enriched by different classes of glycoconjugates, and our results demonstrated that gene knockout of the enzymes responsible for GSL biosynthesis significantly influences the N-glycans of glycoproteins.

Serum N-glycan profiling predicts prognosis in patients undergoing hemodialysis.
Shingo Hatakeyama, Maho Amano, Yuki Tobisawa, Tohru Yoneyama, Megumi Tsushima, Kazuko Hirose, Takahiro Yoneyama, Yasuhiro Hashimoto, Takuya Koie, Hisao Saitoh, Kanemitsu Yamaya, Tomihisa Funyu, Shin-Ichiro Nishimura, Chikara Ohyama
TheScientificWorldJournal, 2013, 268407, 268407, 2013年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: The aim of this study is to evaluate the usefulness of serum N-glycan profiling for prognosis in hemodialysis patients. METHODS: Serum N-glycan analysis was performed in 100 hemodialysis patients in June 2008 using the glycoblotting method, which allows high-throughput, comprehensive, and quantitative N-glycan analysis. All patients were longitudinally followed up for 5 years. To evaluate the independent predictors for prognosis, patients' background, blood biochemistry, and N-glycans intensity were analyzed using Cox regression multivariate analysis. Selected N-glycans and independent factors were evaluated using the log-rank test with the Kaplan-Meier method to identify the predictive indicators for prognosis. Each patient was categorized according to the number of risk factors to evaluate the predictive potential of the risk criteria for prognosis. RESULTS: In total, 56 N-glycan types were identified in the hemodialysis patients. Cox regression multivariate analysis showed cardiovascular events, body mass index, maximum intima media thickness, and the serum N-glycan intensity of peak number 49 were predictive indicators for overall survival. Risk classification according to the number of independent risk factors revealed significantly poor survival by increasing the number of risk factors. CONCLUSIONS: Serum N-glycan profiling may have a potential to predict prognosis in patients undergoing hemodialysis.

Tumour suppressor p16INK4a-anoikis-favouring decrease in N/O-glycan/cell surface sialylation by down-regulation of enzymes in sialic acid biosynthesis in tandem in a pancreatic carcinoma model
Maho Amano, Hanna Eriksson, Joachim C. Manning, Katharina M. Detjen, Sabine Andre, Shin-Ichiro Nishimura, Janne Lehtio, Hans-Joachim Gabius
FEBS JOURNAL, 279, 21, 4062, 4080, WILEY-BLACKWELL, 2012年11月, ［査読有り］
英語, Tumour suppressor p16INK4a is known to exert cell-cycle control via cyclin-dependent kinases. An emerging aspect of its functionality is the orchestrated modulation of N/O-glycosylation and galectin expression to induce anoikis in human Capan-1 pancreatic carcinoma cells. Using chemoselective N/O-glycan enrichment technology (glycoblotting) and product characterization, we first verified a substantial decrease in sialylation. Tests combining genetic (i.e. transfection with a2,6-sialyltransferase-specific cDNA) or metabolic (i.e. medium supplementation with N-acetylmannosamine to track down a bottleneck in sialic acid biosynthesis) engineering with cytofluorometric analysis of lectin binding indicated a role of limited substrate availability, especially for a2,6-sialylation, which switches off reactivity for anoikis-triggering homodimeric galectin-1. Quantitative MS analysis of protein level changes confirmed an enhanced galectin-1 presence along with an influence on glycosyltransferases (beta 1,4-galactosyltransferase-IV, a2,3-sialyltransferase-I) and detected p16INK4a-dependent down-regulation of two enzymes in the biosynthesis pathway for sialic acid [i.e. the bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) and N-acetylneuraminic acid 9-phosphate synthase] (P < 0.001). By contrast, quantitative assessment for the presence of nuclear CMP-N-acetylneuraminic acid synthase (which is responsible for providing the donor for enzymatic sialylation that also acts as feedback inhibitor of the epimerase activity of GNE) revealed a trend for an increase. Partial restoration of sialylation in GNE-transfected cells supports the implied role of sialic acid availability for the glycophenotype. Fittingly, the extent of anoikis was reduced in double-transfected (p16INK4a/GNE) cells. Thus, a second means of modulating cell reactivity to the growth effector galectin-1 is established in addition to the common route of altering a2,6-sialyltransferase expression: regulating enzymes of the pathway for sialic acid biosynthesis.

Molecular shuttle between extracellular and cytoplasmic space allows for monitoring of GAG biosynthesis in human articular chondrocytes
Hiroko Hoshi, Ken Shimawaki, Yasuhiro Takegawa, Tatsuya Ohyanagi, Maho Amano, Hiroshi Hinou, Shin-Ichiro Nishimura
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1820, 9, 1391, 1398, ELSEVIER SCIENCE BV, 2012年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Background: Cell surface proteoglycans play vital functional roles in various biological processes such as cell proliferation, differentiation, adhesion, inflammation, immune response, sustentation of cartilage tissue and intensity of tissues. We show here that serglycin-like synthetic glycopeptides function efficiently as a molecular shuttle to hijack glycosaminoglycan (GAG) biosynthetic pathway within cells across the plasma membrane.
Methods: Fluorescence (FITC)-labeled tetrapeptide (H-Ser(1)-Gly(2)-Ser(3)-Gly(4)-OH) carrying Gal beta(1 -> 4)Xyl beta 1 -> defined as proteoglycan initiator (PGI) monomer and its tandem repeating PGI polymer was employed for direct imaging of cellular uptake and intracellular traffic by confocal laser-scanning microscopy. Novel method for enrichment analysis of GAG-primed PGIs by combined use of anti-FITC antibody and LC/mass spectrometry was established.
Results: PGI monomer was incorporated promptly into human articular chondrocytes and distributed in whole cytoplasm including ER/Golgi while PGI polymer localized specifically in nucleus. It was demonstrated that PGIs become good substrates for GAG biosynthesis within the cells and high molecular weight GAGs primed by PGIs is chondroitin sulfate involving N-acetyl-D-galactosamine residues substituted by 4-O-sulfate or 6-O-sulfate group as major components. PGIs activated chondrocytes proliferation and induced up-regulation of the expression level of type II collagen, suggesting that PGIs can function as new class cytokine-like molecules to stimulate cell growth.
Conclusion: Synthetic serglycin-type PGIs allow for live cell imaging during proteoglycan biosynthesis and structural characterization of GAG-primed PGIs by an antibody-based enrichment protocol.
General significance: Novel glycomics designated for investigating proteoglycan biosynthesis, namely real-time GAGomics using synthetic glycopeptides as PG's, should facilitate greatly dynamic profiling of GAGs in the living cells. This article is part of a Special Issue entitled Glycoproteomics. (C) 2012 Elsevier B.V. All rights reserved.

Molecular shuttle between extracellular and cytoplasmic space allows for monitoring of GAG biosynthesis in human articular chondrocytes.
Hoshi H, Shimawaki K, Takegawa Y, Ohyanagi T, Amano M, Hinou H, Nishimura S
Biochimica et biophysica acta, 1820, 1391, 1398, 9, 2012年09月, ［査読有り］

Effects of Single Genetic Damage in Carbohydrate-Recognizing Proteins in Mouse Serum N-Glycan Profile Revealed by Simple Glycotyping Analysis
Maho Amano, Ryo Hashimoto, Shin-Ichiro Nishimura
CHEMBIOCHEM, 13, 3, 451, 464, WILEY-BLACKWELL, 2012年02月, ［査読有り］
英語, 研究論文（学術雑誌）, Gene knock-out of C-type lectin receptors expressed in dendritic cells induced significant alteration of serum N-glycans compared with that of gender-matched controls. Glycotyping analysis suggested that putative-core fucosylation is strongly influenced by differences in the dominant mechanisms after carbohydrate recognition by pattern-recognition receptors, endocytosis of ligands, or induction of cytokines/chemokines. However, the loss of galectin-9, a ligand for T-helper type 1-specific cell-surface molecule, did not affect most N-glycan profiles. Interestingly, lack of the Chst3 gene (chondroitin 6-sulfotransferase) appeared to influence markedly the expression of most N-glycans, especially highly modified glycoforms bearing multiple Neu5Gc, Fuc, and LacNAc units. In contrast, genetic mutations in B4galnt1 and B4galnt2 (GalNAc transferase, responsible for the synthesis of many gangliosides) induced no discernable alteration. These results indicate that the biosynthesis of N-glycans of serum glycoproteins can be affected not only by direct genetic mutations in the glycosyltransferases but also by changes in metabolite availability in sugar nucleotide synthesis and Golgi N-glycosylation pathways caused concertedly in whole cells, tissues, and organs by milder deficiencies in immune cell-surface lectins. Many common chronic conditions, such as autoimmunity, metabolic syndrome, and aging/dementia result.

Glycomics for Drug Discovery: Metabolic Perturbation in Androgen-Independent Prostate Cancer Cells Induced by Unnatural Hexosamine Mimics
Shin-Ichiro Nishimura, Megumi Hato, Satoshi Hyugaji, Fei Feng, Maho Amano
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 51, 14, 3386, 3390, WILEY-V C H VERLAG GMBH, 2012年, ［査読有り］
英語, 研究論文（学術雑誌）

Alterations of High-Mannose Type N-Glycosylation in Human and Mouse Osteoarthritis Cartilage
Atsushi Urita, Tomoya Matsuhashi, Tomohiro Onodera, Hiroaki Nakagawa, Megumi Hato, Maho Amano, Naoki Seito, Akio Minami, Shin-Ichiro Nishimura, Norimasa Iwasaki
ARTHRITIS AND RHEUMATISM, 63, 11, 3428, 3438, WILEY-BLACKWELL, 2011年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Objective. The process of N-glycosylation is involved in the pathogenesis of various diseases. However, little is known about the contribution of changes in N-glycans in osteoarthritis (OA). The aim of this study was to identify the alterations in N-glycans in human OA cartilage, to characterize the messenger RNA (mRNA) expression of N-glycan biosynthesis enzyme genes (N-glycogenes) in mouse articular chondrocytes during cartilage degradation, and to analyze the relationship between altered N-glycan patterns and mechanisms of cartilage degradation.
Methods. Alterations in N-glycans were analyzed in human OA cartilage and degraded mouse cartilage by high-performance liquid chromatography and mass spectrometry. N-glycogene mRNA expression in mouse chondrocytes was measured using reverse transcription-polymerase chain reaction. To assess the relationship between the altered N-glycans and degradation of mouse cartilage, experiments involving either knockdown or overexpression of N-glycogenes were performed in mouse articular chondrocytes.
Results. Alterations in high-mannose type N-glycans were observed in both human OA cartilage and degraded mouse cartilage. The expression of beta 1,2N-acetylglucosaminyltransferase I (GlcNAc-TI) mRNA, which converts high-mannose type N-glycans, was significantly increased in degraded mouse cartilage. Mouse chondrocytes with suppressed GlcNAc-TI expression had reduced levels of matrix metalloproteinase 13 (MMP-13) and ADAMTS-5 (aggrecanase 2) mRNA following stimulation with interleukin-1 alpha (IL-1 alpha). In contrast, mouse chondrocytes overexpressing GlcNAc-TI had increased levels of MMP-13 and ADAMTS-5 mRNA following stimulation with IL-1 alpha.
Conclusion. These findings indicate that alterations in high-mannose type N-glycans and N-glycogenes in chondrocytes correlate with the release of MMP-13 and ADAMTS-5 during cartilage degradation. These findings suggest that N-glycans play a crucial role in the initiation and progression of OA.

Alteration of the N-glycome of bovine milk glycoproteins during early lactation
Shota Takimori, Hideyuki Shimaoka, Jun-Ichi Furukawa, Tadashi Yamashita, Maho Amano, Naoki Fujitani, Yasuhiro Takegawa, Lennart Hammarstrom, Imre Kacskovics, Yasuro Shinohara, Shin-Ichiro Nishimura
FEBS JOURNAL, 278, 19, 3769, 3781, WILEY-BLACKWELL, 2011年10月, ［査読有り］
英語, 研究論文（学術雑誌）, Milk provides nutritional, immunological and developmental components for newborns. Whereas identification of such components has been performed by targeting proteins and free oligosaccharides, structural and functional analyses of the N-glycome of milk glycoproteins are scarce. In this study, we investigated, for the first time, the alterations of the bovine milk N-glycome during early lactation (1 day, 1, 2, 3 and 4 weeks postpartum), characterizing more than 80 N-glycans. The glycomic profile of colostrum on day 1 after calving differed substantially from that in other periods during early lactation. The proteins in colostrum obtained 1 day postpartum were more highly sialylated than milk samples obtained at other time points, and the N-glycolylneuraminic acid (Neu5Gc)/N-acetylneuraminic acid (Neu5Ac) ratio was significantly higher on day 1, showing a gradual decline with time. In order to dissect the N-glycome of colostrum, alterations of the N-glycosylation profile of major bovine milk proteins during the early lactation stage were elucidated, revealing that the alteration is largely attributable to qualitative and quantitative N-glycosylation changes of IgG, the major glycoprotein in colostrum. Furthermore, by preparing and analyzing IgGs in which the N-glycan structure and subtypes were well characterized, we found that the interaction between IgG and FcRn was not affected by the structure of the N-glycans attached to IgG. We also found that bovine FcRn binds IgG(2) better than IgG(1), strongly suggesting that the role of FcRn in the bovine mammary gland is to recycle IgG(2) from the udder to blood, rather than to secrete IgG(1) into colostrum.

Insight into Glycan Diversity and Evolutionary Lineage Based on Comparative Avio-N-glycomics and Sialic Acid Analysis of 88 Egg Whites of Galloanserae
Kazuko Hirose, Maho Amano, Ryo Hashimoto, Yuan Chuan Lee, Shin-Ichiro Nishimura
BIOCHEMISTRY, 50, 21, 4757, 4774, AMER CHEMICAL SOC, 2011年05月, ［査読有り］
英語, 研究論文（学術雑誌）, A large set of glycome information was obtained from egg white proteins of 88 samples from Galloanserae (63 Anseriformes and 25 Galliformes). The data were obtained on whole N-glycan structures and types of sialic acids of these egg whites by glycoblotting-based high-throughput and quantitative glycomics. The results revealed clear trends and complexity patterns as well as diversity among taxonomic groups. It is well-known that chicken, a representative domesticated poultry involved in Galliformes, can become an influenza host. However, our data demonstrate that duck, wild goose, and swan of Anseriformes are representative migratory birds that are known as natural hosts of the influenza virus. Hierarchical clustering analysis of the expression pattern of N-glycome (total of 61 N-glycan peaks) revealed that the members of Galloanserae can be classified into two major groups and five submajor clusters (clusters 1-5) on the basis of simple m/z values obtained by MALDI-TOF MS. It is dear that expression patterns of N-glycomes in the five dusters are influenced significantly by the features such as the body size of the birds, rather than by the difference of the family. On the other hand, quantitative analysis showed that the total amounts of sialic acids in egg whites of Galliformes were distinctly larger than those of Anseriformes. However, it was also revealed in Anseriformes that Neu5Gc and KDN, in addition to common Neu5Ac, were expressed significantly in both N- and O-glycans of glycoproteins and glycosphingolipids, suggesting the influence of their lifestyles and diet. This is the first report that KDN exists in egg white. These results and the environmental factors are discussed preliminarily with respect to their evolutionary lineage.

血液透析患者血清中の糖鎖性動脈硬化因子の探索
山本 勇人, 對馬 惠, 山谷 金光, 畠山 真吾, 岡本 亜希子, 齋藤 久夫, 舟生 富壽, 天野 麻穂, 西村 紳一郎, 鈴木 裕一朗, 杉山 尚樹, 米山 徹, 大山 力
泌尿器外科, 24, 臨増, 522, 522, 医学図書出版(株), 2011年04月, ［査読有り］
日本語

腎細胞癌における血清糖タンパク糖鎖の網羅的解析
畠山 真吾, 山本 勇人, 對馬 惠, 岡本 亜希子, 齋藤 久夫, 舟生 富壽, 鈴木 裕一朗, 杉山 尚樹, 米山 徹, 盛 和行, 天野 麻穂, 西村 紳一郎, 大山 力
泌尿器外科, 24, 臨増, 531, 531, 医学図書出版(株), 2011年04月, ［査読有り］
日本語

グライコブロッティング法が明らかにする翻訳後修飾の意義と大規模糖鎖解析への展開
天野麻穂, 三浦嘉晃, 西村紳一郎
生化学, 83, 1, 5, 12, Japanese Biochemical Society, 2011年, ［査読有り］, ［招待有り］
日本語

Glycoblotting-Assisted O-Glycomics: Ammonium Carbamate Allows for Highly Efficient O-Glycan Release from Glycoproteins
Yoshiaki Miura, Kentaro Kato, Yasuhiro Takegawa, Masaki Kurogochi, Jun-ichi Furukawa, Yasuro Shinohara, Noriko Nagahori, Maho Amano, Hiroshi Hinou, Shin-Ichiro Nishimura
ANALYTICAL CHEMISTRY, 82, 24, 10021, 10029, AMER CHEMICAL SOC, 2010年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Glycoblotting, high throughput method for N-glycan enrichment analysis based on the specific chemical ligation between aminooxy/hyclrazide-polymers/solids and reducing N-glycans released from whole serum and cellular glycoproteins, was proved to be feasible for selective enrichment analysis of O-glycans of common (mucin) glycoproteins. We established a standard protocol of glycoblotting-based O-glycomics in combination with nonenzymatic chemical treatment to release reducing O-glycans predominantly from various glycoprotein samples. It was demonstrated that the nonreductive condition employing a simple ammonium salt, ammonium carbamate, made glycoblotting-based enrichment analysis of O-glycans possible without significant loss or unfavorable side reactions. A general workflow of glycoblotting using a hydrazide bead (BlotGlyco H), on-bead chemical manipulations, and subsequent mass spectrometry allowed for rapid O-glycomics of human milk osteopontin (OPN) and urinary MUC1 glycoproteins purified from healthy donors in a quantitative manner. It was revealed that structures of O-glycans in human milk OPN were varied with habitual fucosylation and N-acetyllactosamine units. It was also suggested that purified human urinary MUC1 was modified preferentially by sialylated O-glycans (94% of total) with 7:3 ratio of core 1 to core 2 type O-glycans. Versatility of the present strategy is evident because this method was proved to be suited for the enrichment analysis of general biological and clinical samples such as human serum and urine, cultured human cancer cells, and formalin-fixed paraffin-embedded tissue sections. It is our belief that the present protocols would greatly accelerate discovery of disease-relevant O-glycans as potential biomarkers.

Sialic Acid-focused Quantitative Mouse Serum Glycoproteomics by Multiple Reaction Monitoring Assay
Masaki Kurogochi, Takahiko Matsushista, Maho Amano, Jun-ichi Furukawa, Yasuro Shinohara, Masato Aoshima, Shin-Ichiro Nishimura
MOLECULAR & CELLULAR PROTEOMICS, 9, 11, 2354, 2368, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Despite increasing importance of protein glycosylation, most of the large-scale glycoproteomics have been limited to profiling the sites of N-glycosylation. However, in-depth knowledge of protein glycosylation to uncover functions and their clinical applications requires quantitative glycoproteomics eliciting both peptide and glycan sequences concurrently. Here we describe a novel strategy for the multiplexed quantitative mouse serum glycoproteomics based on a specific chemical ligation, namely, reverse glycoblotting technique, focusing sialic acids and multiple reaction monitoring (MRM). LC-MS/MS analysis of de-glycosylated peptides identified 270 mouse serum peptides (95 glycoproteins) as sialylated glycopeptides, of which 67 glycopeptides were fully characterized by MS/MS analyses in a straightforward manner. We revealed the importance of a fragment ion containing innermost N-acetylglucosamine (GlcNAc) residue as MRM transitions regardless the sequence of the peptides. Versatility of the reverse glycoblotting-assisted MRM assays was demonstrated by quantitative comparison of 25 targeted glycopeptides from 16 proteins between mice with homo and hetero types of diabetes disease model. Molecular & Cellular Proteomics 9:2354-2368, 2010.

Threshold in Stage-specific Embryonic Glycotypes Uncovered by a Full Portrait of Dynamic N-Glycan Expression during Cell Differentiation
Maho Amano, Misa Yamaguchi, Yasuhiro Takegawa, Tadashi Yamashita, Michiyo Terashima, Jun-ichi Furukawa, Yoshiaki Miura, Yasuro Shinohara, Norimasa Iwasaki, Akio Minami, Shin-ichiro Nishimura
MOLECULAR & CELLULAR PROTEOMICS, 9, 3, 523, 537, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Although various glycoforms appear to participate independently in multiple molecular interactions in cellular adhesion that contribute to embryogenesis and organogenesis, a full portrait of the glycome diversity and the effect of the structural variations of cellular glycoforms on individual cell stages in proliferation and differentiation remain unclear. Here we describe a novel concept for the characterization of dynamic glycoform alteration during cell differentiation by means of "glycoblotting-based cellular glycomics," the only method allowing for rapid and quantitative glycan analysis. We demonstrated that processes of dynamic cellular differentiation of mouse embryonic carcinoma cells, P19CL6 and P19C6, and mouse embryonic stem cells into cardiomyocytes or neural cells can be monitored and characterized quantitatively by profiling entire N-glycan structures of total cell glycoproteins. Whole N-glycans enriched and identified by the glycoblotting method (67 glycans for P19CL6, 75 glycans for P19C6, and 72 glycans for embryonic stem cells) were profiled and bar-coded quantitatively with respect to the ratio of subgroups composed of characteristic glycoforms, namely glycotypes. Molecular & Cellular Proteomics 9: 523-537, 2010.

Carbohydrate Moieties Contribute Significantly to the Physicochemical Properties of French Bean 7S Globulin Phaseolin
Aiko Kimura, Mary Rose G. Tandang-Silvas, Takako Fukuda, Cerrone Cabanos, Yasuhiro Takegawa, Maho Amano, Shin-Ichiro Nishimura, Yasuki Matsumura, Shigeru Utsumi, Nobuyuki Maruyama
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 58, 5, 2923, 2930, AMER CHEMICAL SOC, 2010年03月, ［査読有り］
英語, 研究論文（学術雑誌）, We have previously reported that the solubility of French bean 7S globulin (phaseolin) at low ionic strength and its emulsifying stability are remarkably high compared with those of 7S globulins prepared from other plant species, including soybean (Kimura et al. J. Agric. Food Chem. 2008, 56, 10273-10279). In this study, we examined the role of carbohydrate moieties in the properties of phaseolin. Three preparations of phaseolin were analyzed: (i) N7S, prepared from defatted seed meal and having intact carbohydrate moieties: (ii) R7S, expressed in E coli and lacking N-linked glycans; and (iii) EN7S, having partial N-linked glycans after treatment with Endo H. The solubilities of N7S and EN7S were much higher than that of R7S at a low ionic strength (mu = 0.08). N7S exhibited good emulsifying ability under the conditions examined, but R7S did not. In terms of emulsion stability, an emulsion of R7S separated into two phases after 1 h at mu = 0.01, 0.08, and 0.5, whereas the emulsion of N7S was stable for 5 days at mu = 0.01 and for at least 10 days at mu = 0.08 and 0.5. The emulsion stability of EN7S was comparable to that of N7S under most conditions examined. These results indicate the carbohydrate modifications are necessary for the good solubility, emulsifying ability, and emulsion stability of phaseolin. Further, a structural analysis of the carbohydrate moieties indicates that truncated carbohydrate moieties are sufficient for conferring these physicochemical properties to phaseolin.

LARGE-SCALE GLYCOMICS FOR DISCOVERING CANCER-ASSOCIATED N-GLYCANS BY INTEGRATING GLYCOBLOTTING AND MASS SPECTROMETRY
Maho Amano, Shin-Ichiro Nishimura
METHODS IN ENZYMOLOGY, VOL 478: GLYCOMICS, 478, 109, 125, ELSEVIER ACADEMIC PRESS INC, 2010年, ［査読有り］
英語, 論文集(書籍)内論文, It has known that the glycosylation plays an important role in the biological states, such as development, aging, and diseases. Although genomic and proteomic approaches have been intensively studied for diagnosis and disease treatment, glycomics have been laggard compared to them due to the hardness of the purification procedure from crude biological materials.
Recently, we have developed "glycoblotting" method, a high-throughput and quantitative technique for comprehensive glycomics, which enables to enrich and quantify glycans from crude biological materials, such as serum, tissue biopsy, and cell lysate [Niikura, K., Kamitani, R., Kurogochi, M., Uematsu, R., Shinohara, Y., Nakagawa, H., Deguchi, K., Monde, K., Kondo, H., and Nishimuram S.-I. (2005). Versatile glycoblotting nanoparticles for high-throughput protein glycomics. Chem. Eur. J. 11, 3825-3834; Nishimuara, S.-I., Niikura, K., Kurogochi, M., Matsushita, T., Fumoto, M., Hinou, H., Kamitani, R., Nakagawa, H., Deguchi, K., Miura, N., Monde, K., and Kondo, H. (2005). High-throughput protein glycomics: Combined use of chemoselective glycoblotting and MALDI-TOF/TOF mass spectrometry. Angew. Chem. Int. Ed. 44, 91-96]. The automated machine for glycoblotting, "SweetBlot," fixed to use optimized protocol allows us to obtain quantitative profile of 40-50 kinds of major glycoforms from 5 mu l of human serum within 11 h. Based on the method, we have detected potential differences of N-glycome between sera from hepatocellular carcinoma (HCC) and healthy donor [Miura, Y., Hato, M., Shinohara, Y., Kuramoto, H., Furukawa, J.-i, Kurogochi, M., Shimaoka, H., Tada, M., Nakanishi, K., Ozaki, M., Todo, S., and Nishimura, S.-I. (2008). BlotGlycoABC (TM), an integrated glycoblotting technique for rapid and large scale clinical glycomics. Mol. Cell. Proteomics 7, 370-377]. The method also permitted cellular quantitative N-glycomics to monitor the process of dynamic cellular differentiation of mouse embryonic stem cells into neural cells [Amano, M., Yamaguchi, M., Takegawa, Y., Yamashita, T., Terashima, M., Furukawa, Miura, Y., Shinohara, Y., Iwasaki, N., Minami, A., and Nishimura, S.-I. (2010). Threshold in stage-specific embryonic glycotypes uncovered by a full portrait of dynamic N-glycan expression during cell differentiation. Mol. Cell. Proteomics 9, 523-537]. In this chapter, we will discuss glycoblotting method including the potentials not only for exploration of glycan-related cancer biomarker but also for detection of cellular differentiation.

高速化が進む糖鎖解析-糖鎖構造研究を加速する新しい解析法：グライコブロッティング法-
天野麻穂, 西村紳一郎
現代化学, 435, 435, 55, 61, 東京化学同人, 2007年, ［査読有り］
日本語

Reverse glycoblotting allows rapid-enrichment glycoproteomics of biopharmaceuticals and disease-related biomarkers
Masaki Kurogochi, Maho Amano, Masataka Fumoto, Akio Takimoto, Hirosato Kondo, Shin-ichiro Nishimura
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 46, 46, 8808, 8813, WILEY-V C H VERLAG GMBH, 2007年, ［査読有り］
英語, 研究論文（学術雑誌）

Haploinsufficiency of C2GnT-I glycosyltransferase renders T lymphoma cells resistant to cell death
Paula V. Cabrera, Maho Amano, Junya Mitoma, Jessica Chan, Jonathan Said, Minoru Fukuda, Linda G. Baum
BLOOD, 108, 7, 2399, 2406, AMER SOC HEMATOLOGY, 2006年10月, ［査読有り］
英語, 研究論文（学術雑誌）, Neoplastic T cells in mycosis fungoides (MF) are resistant to apoptotic agents, including galectin-1 that is abundant in skin. Although MF cells are typically CB7(-), and thus galectin-1 resistant, CD7(+) HH cells, derived from a patient with MF, were also resistant to galectin-1. HH cells demonstrate altered cell surface glycosylation, with loss of core 2 01 glycan ligands for galectin-1 created by core 2 beta 1,6-N-acetylglucosaminyltransferase (C2GnT-I). Loss of core 2 O-glycans on tumor cells was also seen in primary CD7+ MF lesions. Surprisingly, HH cells are heterozygous for a C2GnT-I point mutation, yet this mutation resulted in a dramatic reduction in cellular glycosyltransferase activity. Expression of wild-type C2GnT-I in human HH cells, or murine lymphoma cells that lack C2GnT-I, restored core 2 O-glycan expression and susceptibility to galectin-1, whereas mutant enzyme lacked activity and did not restore core 2 O-glycan expression or susceptibility to galectin-1. Mutant enzyme did not have a dominant negative effect by affecting dimerization or activity of wild-type enzyme; rather, C2GnT-I haploinsufficiency is sufficient for loss of core 2 O-glycan expression and galectin-1 resistance. Thus, glycosyltransferase haploinsufficiency results in altered cellular glycosylation and resistance to cell death, identifying a new survival mechanism for T-lymphoma cells.

Control of T cell fate by galectins and their ligand-through regulation of the expression of glycosyltransferases
M Amano
SEIKAGAKU, 76, 9, 1203, 1206, JAPANESE BIOCHEMICAL SOC, 2004年09月, ［査読有り］
日本語

Effect of fermented milk containing Lactobacillus johnsonii La1 (LC1) on Defecation in healthy Japanese adults –A double blind placebo controlled study-
天野 麻穂
Bioscience and Microflora, 23, 4, 139, 147, JAPAN BIFIDUS FOUNDATION, 2004年, ［査読有り］
英語, 研究論文（学術雑誌）, To elucidate the effect of fermented milk containing Lactobacillus johnsonii La1 (LC1_®) on defecation, a double blind placebo controlled study was conducted. Healthy Japanese adults (24-67 years of age, n=57, male: 31, female: 26) were randomly divided into two groups, and the subjects in the LC1 group (n=30) consumed LC1 fermented milk containing L. johnsonii La1 at 1×10⁹ cfu/90 g (90 g per day) for 21 days and the subjects in the control group (n=27) consumed placebo fermented milk without the La1 strain in the same manner. In the subjects of the LC1 group with mild constipation (n=9), less than one defecation per day in the 3 weeks observation period, defecation frequency both in times per week and days per week significantly increased during consumption of LC1 compared to before consumption (p<0.05), and the frequency recovered to normal status with at least one defecation per day in 4 out of 9 of subjects (44%) after 3 weeks LC1 consumption. Subjects with mild constipation in the control group (n=7) showed no significant changes in defecation frequency. The frequency in the subjects with normal defecation did not change in either group. L. johnsonii La1 was found in all feces from 10 subjects in the LC1 group participating in fecal collection, suggesting that La1 strain efficiently reached the gastrointestinal tract as a viable form. Excess amount (360 g per day) of LC1 intake for 2 weeks in the other healthy subjects (n=10) caused an increase in defecation frequency, which was kept at the normal level of less than twice per day. These results suggest that LC1_®, a probiotic fermented milk containing L. johnsonii La1, is beneficial for improving mild constipation without any safety concerns.

Galectin-1-mediated apoptosis in mycosis fungoides: The roles of CD7 and cell surface glycosylation
AA Roberts, M Amano, C Felten, M Galvan, G Sulur, L Pinter-Brown, U Dobbeling, G Burg, J Said, LG Baum
MODERN PATHOLOGY, 16, 6, 543, 551, LIPPINCOTT WILLIAMS & WILKINS, 2003年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Sezary cells, the malignant T cells in mycosis fungoides/Sezary syndrome, resist a variety of apoptosis-inducing agents, a feature that contributes to the poor response to therapy in mycosis fungoides. Galectin-1 is a mammalian lectin that triggers T cell apoptosis. For T cells to be susceptible to galectin-l-induced apoptosis, the T cells must express specific glycoprotein receptors, such as CD7, that bear the specific oligosaccharides recognized by galectin-1. Because Sezary cells are characteristically CD7-, lack of CD7 expression has been proposed to render Sezary cells resistant to galectin-1-induced death. However, the role played by aberrant cell surface glycosylation in resistance of Sezary cells to galectin-1 has not been examined. In this study, we demonstrated abundant galectin-1 in mycosis fungoides skin lesions, indicating that Sezary cells are exposed to galectin-1 in vivo. To determine specific characteristics of Sezary cells that contribute to galectin-1 resistance, we assessed CD7 expression and cell surface glycosylation. of Sezary cells in mycosis fungoides lesions and of four Sezary T cell lines. Sezary cells in primary lesions and Sezary T cell lines demonstrated a characteristic "glycotype" with sialylated core 1 O-glycans that promote galectin-1 resistance. Expression of CD7 was necessary but not sufficient for galectin-l-induced death of Sezary cell lines. In addition, CD7- Sezary cell lines, and Sezary cells within mycosis fungoides; lesions, expressed galectin-1, whereas CD7-positive Sezary cell lines did not express galectin-1. We propose that both loss of CD7 expression and altered cellular glycosylation contribute to apoptosis resistance of malignant T cells in mycosis fungoides.

The ST6Gal I sialyltransferase selectively modifies N-glycans on CD45 to negatively regulate galectin-1-induced CD45 clustering, phosphatase modulation, and T cell death
M Amano, M Galvan, JL He, LG Baum
JOURNAL OF BIOLOGICAL CHEMISTRY, 278, 9, 7469, 7475, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2003年02月, ［査読有り］
英語, 研究論文（学術雑誌）, The addition of sialic acid to T cell surface glycoproteins influences essential T cell functions such as selection in the thymus and homing in the peripheral circulation. Sialylation of glycoproteins can be regulated by expression of specific sialyltransferases that transfer sialic acid in a specific linkage to defined saccharide acceptor substrates and by expression of particular glycoproteins bearing saccharide acceptors preferentially recognized by different sialyltransferases. Addition of alpha2,6-linked sialic acid to the Galbeta1,4GlcNAc sequence, the preferred ligand for galectin-1, inhibits recognition of this saccharide ligand by galectin-1. SAalpha2,6Gal sequences, created by the ST6Gal I enzyme, are present on medullary thymocytes resistant to galectin-l-induced death but not on galectin-l-susceptible cortical thymocytes. To determine whether addition of alpha2,6-linked sialic acid to lactosamine sequences on T cell glycoproteins inhibits galectin-1 death, we expressed the ST6Gal I enzyme in a galectin-l-sensitive murine T cell line. ST6Gal I expression reduced galectin-1 binding to the cells and reduced susceptibility of the cells to galectin-1-induced cell death. Because the ST6Gal I preferentially utilizes N-glycans as acceptor substrates, we determined that N-glycans are essential for galectin-1-induced T cell death. Expression of the ST6Gal I specifically resulted in increased sialylation of N-glycans on CD45, a receptor tyrosine phosphatase that is a T cell receptor for galectin-1. ST6Gal I expression abrogated the reduction in CD45 tyrosine phosphatase activity that results from galectin-1 binding. Sialylation of CD45 by the ST6Gal I also prevented galectin-1-induced clustering of CD45 on the T cell surface, an initial step in galectin-1 cell death. Thus, regulation of glycoprotein sialylation may control susceptibility to cell death at specific points during T cell development and peripheral activation.

Characterization of Carbohydrate Epitope of Pollen and Wheat Allergens
小川 温子, 天野 麻穂, 土方 亜子, 加藤 真利, 上平 知子, 末次 勧, 石塚 稲夫
Journal of Applied Glycoscience, 50, 2, 327, 331, The Japanese Society of Applied Glycoscience, 2003年
日本語, Plant N -linked oligosaccharides of complex type generally possess a characteristic core structure with xylose β-2 linked to β-mannose and fucose a 1-3 linked to N-acetylglucosamine at the reducing end, which rarely found in animals. Such glycans of plant glycoproteins have been found to induce immunogenic responses in animals. This brief report introduces the current knowledge on the structure, antigenicity and allergenicity of plant carbohydrate epitopes (plant glycotopes) obtained from our studies on the allergens of Japanese cedar pollinosis and baker's asthma. It also presents relationship between CCD (cross-reactive carbohydrate determinant) of allergens from common vegetables or fruits and that of the Japanese cedar pollen in this study.

Identification of the major allergens in wheat flour responsible for baker’s asthma.
Amano M, Ogawa H, Kojima K, Kamidaira T, Suetsugu S, Yoshihama M, Satoh T, Samejima T, Matsumoto I
Biochemical Journal, 330, 1229, 1234, PORTLAND PRESS, 1998年03月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Baker's asthma, a typical occupational allergic disease, is a serious problem in the food industries. In this study, purification and identification of major allergens recognized by IgEs in sera of allergic patients were performed. Major immunoreactive proteins were purified from the albumin fraction by gel filtration on a Toyopearl HW-50 column followed by reverse-phase HPLC. The N-terminal amino acid sequences and molecular masses measured by MS indicated that the major immunoreactive proteins are members of the alpha-amylase inhibitor family, 0.19 and 0.28. Significant leukotriene release by each purified protein was observed in cell-associated stimulation tests, suggesting in vivo activity of these antigens. Carbohydrate analyses of major allergens indicated that they are monoglycosylated but not N-glycosylated in spite of the presence of a potential N-glycosylation site. Recombinant 0.19 expressed in Escherichia coli showed the same reactivity with IgE as native wheat 0.19 in Western blotting and ELISA using methyl vinyl ether maleic anhydride co-polymer as an immobilizing reagent, suggesting that the allergenic epitopes are located in the peptide portions.

Structures and contribution to the antigenicity of oligosaccharides of Japanese cedar (Cryptomeria japonica) pollen allergen Cry j I: Relationship between the structures and antigenic epitopes of plant N-linked complex-type glycans
H Ogawa, A Hijikata, M Amano, K Kojima, H Fukushima, Ishizuka, I, Y Kurihara, Matsumoto, I
GLYCOCONJUGATE JOURNAL, 13, 4, 555, 566, CHAPMAN HALL LTD, 1996年08月, ［査読有り］
英語, 研究論文（学術雑誌）, The oligosaccharide structures of Cry j I, a major allergenic glycoprotein of Cryptomeria japonica (Japanese cedar, sugi), were analysed by 400 MHz H-1-NMR and two-dimensional sugar mapping analyses. The four major fractions comprised a series of biantennary complex type N-linked oligosaccharides that share a fucose/xylose-containing core and glucosamine branches including a novel structure with a nongalactosylated fucosylglucosamine branch.
Rabbit polyclonal anti-Cry j I IgG antibodies cross-reacted with three different plant glycoproteins having the same or shorter N-linked oligosaccharides as Cry j I. ELISA and ELISA inhibition studies with intact glycoproteins, glycopeptides and peptides indicated that both anti-Cry j I IgGs and anti-Sophora japonica bark lectin II (B-SJA-II) IgGs included oligosaccharide-specific antibodies with different specificities, and that the epitopic structures against anti-Cry j I IgGs include a branch containing alpha 1-6 linked fucose and a core containing fucose/xylose, while those against anti-B-SJA-II IgGs include nonreducing terminal mannose residues. The crossreactivities of human allergic sera to miraculin and Clerodendron Trichotomum lectin (CTA) were low and inhibition studies suggested that the oligosaccharides on Cry j I contribute little or only conformationally to the reactivity of specific IgE antibodies.

弱い相互作用のインターフェースと細胞応答の同時可視化を実現するイメージング技術
吉田 藍子, 藤岡 容一朗, 天野 麻穂, 大場 雄介, Drug Delivery System, 37, 2, 102, 111, 2022年03月25日, ［査読有り］, ［招待有り］
Japan Society of Drug Delivery System, 日本語, 記事・総説・解説・論説等（学術雑誌）

ウイルスを創る,ウイルスを視る ウイルスの細胞侵入を視る
藤岡容一朗, 天野麻穂, 大場雄介, 医学のあゆみ, 280, 9, 2022年

病原体と宿主のイベント 9.宿主における病原体のダイナミクス
藤岡容一朗, 天野麻穂, 大場雄介, 実験医学, 39, 2, 2021年

眼科のトランスレーショナルリサーチ 多機能蛋白質に着目した糖尿病網膜症に対する創薬研究
野田 航介, 村田 美幸, 稲福 沙織, 松田 剛, 吉田 志帆, 董 陽子, 木下 哲志, 安藤 亮, 藤谷 顕雄, 齋藤 理幸, 董 震宇, 森 祥平, 加瀬 諭, 吉澤 史子, 齋藤 航, 神田 敦宏, 石田 晋, 眞島 行彦, 笹瀬 智彦, 天野 麻穂, 大橋 哲, 西村 伸一郎, 今川 貴仁, 白仁田 明生, 日本眼科学会雑誌, 122, 3, 223, 248, 2018年03月
近年の基礎研究は、血管内皮増殖因子(vascular endothelial growth factor:VEGF)が糖尿病網膜症の病態形成に主要な役割を演じることを明らかとした。そして、同分子群に対する阻害薬の臨床応用は糖尿病網膜症の治療予後を劇的に改善し、現在我々はanti-VEGF eraと呼ばれるこの時代において同疾患の治療体系が刻々と変貌するのを目の当たりにしている。かつては光凝固と硝子体手術のみが進行した糖尿病網膜症に対する治療手段であったことを考えると隔世の感がある。しかしながらその一方で、情報システムの革新と研究技術の進歩を背景に蓄積される基礎および臨床研究の成果は、網膜症病態の複雑さ、VEGF単独阻害による治療の限界、そしてその弊害の可能性なども浮き彫りにした。そして、その必然としてVEGF以外の病態責任分子を標的とした糖尿病網膜症に対する創薬研究が全世界で現在行われ、複数の分子標的製剤が糖尿病網膜症の治療オプションとなるpost anti-VEGF eraが目前に迫ってきている。糖尿病網膜症の発症および進展には、慢性炎症、そして酸化ストレスの関与が知られている。本研究においては、糖鎖など新規標的分子の探索的研究を行うとともに、この二つの病態に関わる分子としてvascular adhesion protein-1(VAP-1)/semicarbazide sensitive amine oxidase(SSAO)の糖尿病網膜症病態における役割についての検討を主に行った。VAP-1/SSAOは血管内皮細胞に発現する白血球接着分子だが、その一方で酵素活性も持つ多機能蛋白質"moonlighting protein"であり、慢性炎症と酸化ストレスの双方に関わる重要な分子の一つである。本研究ではVAP-1/SSAOが糖尿病網膜症の病態形成に白血球接着分子として関与する一方、遊離型蛋白質としてその眼内に蓄積すること、そしてその機序にVEGFや蛋白質分解酵素matrix metalloproteinasesが関与することを明らかにした。また、VAP-1/SSAOは酵素として過酸化水素および不飽和アルデヒドの一種アクロレインを産生し、血管内皮細胞における酸化ストレス亢進に寄与することとその機序を見出した。以上の検討結果に基づいて、本稿では糖尿病網膜症におけるVAP-1/SSAO阻害剤による治療可能性について述べたい。(著者抄録), (公財)日本眼科学会, 日本語

文理融合の障壁となる諸要因の探索 : 研究者へのインタビュー調査とゲーム式調査法の開発　　　　　　　　　　　　　　　
天野麻穂, 川本思心, CoSTEP研修科年次報告書, 2, 1, 1, 5, 2018年, ［招待有り］
日本語, 速報，短報，研究ノート等（大学，研究機関紀要）

北海道胆振東部地震からの教訓　　　　　　　　　　　　　　　
天野麻穂, 産学官連携ジャーナル, 14, 12, 23, 25, 2018年, ［招待有り］
日本語, 記事・総説・解説・論説等（その他）

文理融合の障壁となる諸要因の探索　　　　　　　　　　　　　　　
天野麻穂, 川本思心, CoSTEP研修科年次報告書, 1, 1, 1, 4, 2017年
日本語, 速報，短報，研究ノート等（学術雑誌）

糖尿病網膜症の硝子体および血漿におけるN型糖鎖プロファイル　　　　　　　　　　　　　　　
稲福 沙織, 野田 航介, 天野 麻穂, 大橋 哲, 齋藤 航, 村田 美幸, 神田 敦宏, 西村 紳一郎, 石田 晋, 日本糖尿病眼学会誌, 20, 114, 114, 2016年03月
日本糖尿病眼学会, 日本語

Alteration of N-glycan Profiles in Diabetic Retinopathy
Saori Inafuku, Kousuke Noda, Maho Amano, Miyuki Murata, Wataru Saito, Tetsu Ohashi, Atsuhiro Kanda, Shin-Ichiro Nishimura, Susumu Ishida, INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, 56, 7, 2015年06月
ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 英語, 研究発表ペーパー・要旨（国際会議）

Serum N-Glycan Profiles in Patients With Intraductal Papillary Mucinous Neoplasms of Pancreas
Yutaka Akimoto, Kazuhiro Nouso, Hironari Kato, Koji Miyahara, Chihiro Dohi, Yuki Morimoto, Hideaki Kinugasa, Takeshi Tomoda, Naoki Yamamoto, Koichiro Tsutsumi, Shinichiro Nakamura, Hidenori Shiraha, Akinobu Takaki, Hiroyuki Okada, Maho Amano, Shin-ichiro Nishimura, Kazuhide Yamamoto, GASTROENTEROLOGY, 148, 4, S518, S518, 2015年04月
W B SAUNDERS CO-ELSEVIER INC, 英語, 研究発表ペーパー・要旨（国際会議）

Alteration of Serum N-Glycan Profile in Patients With Hepatocellular Carcinoma and Their Clinical Application
Kazuhiro Nouso, Koji Miyahara, Chihiro Dohi, Yuki Morimoto, Hideaki Kinugasa, Nozomu Wada, Yasuto Takeuchi, Kenji Kuwaki, Hideki Onishi, Fusao Ikeda, Shinichiro Nakamura, Hidenori Shiraha, Akinobu Takaki, Maho Amano, Shin-Ichiro Nishimura, Kazuhide Yamamoto, GASTROENTEROLOGY, 148, 4, S1022, S1022, 2015年04月
W B SAUNDERS CO-ELSEVIER INC, 英語, 研究発表ペーパー・要旨（国際会議）

Intracellular traffic of cell surface mimetic quantum dots-anchored glycopeptides
Shin-Ichiro Nishimura, Roger Tan, Kentaro Naruchi, Maho Amano, Hiroshi Hinou, ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 249, 2015年03月
AMER CHEMICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

糖尿病網膜症の硝子体および血漿におけるN型糖鎖の網羅的解析　　　　　　　　　　　　　　　
稲福 沙織, 野田 航介, 天野 麻穂, 大橋 哲, 吉澤 史子, 齋藤 航, 村田 美幸, 神田 敦宏, 西村 紳一郎, 石田 晋, 日本眼科学会雑誌, 119, 臨増, 160, 160, 2015年03月
(公財)日本眼科学会, 日本語

糖負荷刺激によるシアル酸転移酵素の誘導　　　　　　　　　　　　　　　
稲福 沙織, 野田 航介, 天野 麻穂, 大橋 哲, 齋藤 航, 村田 美幸, 神田 敦宏, 西村 紳一郎, 石田 晋, 日本眼科学会雑誌, 119, 臨増, 199, 199, 2015年03月
(公財)日本眼科学会, 日本語

糖尿病モデルマウスの比較糖鎖解析
坂西裕介, 比能洋, 天野麻穂, 幸田敏明, 上村佳子, 西村紳一郎, 高分子学会北海道支部研究発表会講演要旨集, 49th, 2015年

肝疾患の病態と糖鎖抗原の意義 網羅的糖鎖解析による肝細胞癌バイオマーカーの検討　　　　　　　　　　　　　　　
宮原 孝治, 能祖 一裕, 森元 裕貴, 衣笠 秀明, 和田 望, 竹内 康人, 安中 哲也, 桑木 健志, 大西 秀樹, 池田 房雄, 三宅 康弘, 中村 進一郎, 白羽 英則, 高木 章乃夫, 天野 麻穂, 西村 紳一郎, 山本 和秀, 肝臓, 55, Suppl.1, A154, A154, 2014年04月
(一社)日本肝臓学会, 日本語

Prediction of Survival in Patients with Hepatocellular Carcinoma Treated with Sorafenib by Comprehensive Serum Glycan Analysis
Kazuhiro Nouso, Koji Miyahara, Yuuki Morimoto, Yasuto Takeuchi, Hiroaki Hagihara, Kenji Kuwaki, Hideki Onishi, Fusao Ikeda, Yasuhiro Miyake, Shinichiro Nakamura, Hidenori Shiraha, Akinobu Takaki, Koichi Takaguchi, Takahisa Sato, Shinpei Sato, Shuntaro Obi, Kazuko Hirose, Maho Amano, Shin-Ichiro Nishimura, Kazuhide Yamamoto, HEPATOLOGY, 58, 1242A, 1242A, 2013年10月
WILEY-BLACKWELL, 英語, 研究発表ペーパー・要旨（国際会議）

血清糖鎖マーカーによる進行肝細胞癌の予後予測　　　　　　　　　　　　　　　
宮原 孝治, 能祖 一裕, 森元 裕貴, 和田 望, 竹内 康人, 萩原 宏明, 桑木 健志, 大西 秀樹, 中村 進一郎, 白羽 英則, 天野 麻穂, 西村 紳一郎, 山本 和秀, 肝臓, 54, Suppl.2, A638, A638, 2013年09月
(一社)日本肝臓学会, 日本語

ABERRANT GLYCOSYLATION OF N-GLYCAN ON IMMUNOGLOBULIN CLOSELY RELATES SURVIVAL OF THE PATIENTS WITH IN RENAL CELL CARCINOMA
Shingo Hatakeyama, Yuki Tobisawa, Tohru Yoneyama, Yuichiro Suzuki, Hayato Yamamoto, Kazuyuki Mori, Takahiro Yoneyama, Yasuhiro Hashimoto, Takuya Koie, Norihiko Tsuchiya, Tomonori Habuchi, Maho Amano, Shin-Ichiro Nishimura, Chikara Ohyama, JOURNAL OF UROLOGY, 189, 4, E186, E187, 2013年04月
ELSEVIER SCIENCE INC, 英語, 研究発表ペーパー・要旨（国際会議）

ぶどう膜炎における網羅的糖鎖解析　　　　　　　　　　　　　　　
南場 研一, 北市 伸義, 安藤 亮, 竹本 裕子, 水内 一臣, 堀江 幸弘, 大野 重昭, 天野 麻穂, 西村 紳一郎, 石田 晋, 日本眼科学会雑誌, 117, 臨増, 318, 318, 2013年03月
(公財)日本眼科学会, 日本語

Autoantibodies to cancer-associated MUC1 fragments in healthy human sera discovered by high performance glycopeptide microarray platform
Nishimura S. I, Matsushita T, Takada W, Igarashi K, Naruchi K, Garcia-Martin F, Amano M, Hinou H, Abstracts of Papers of the American Chemical Society, 246, 2013年, ［査読有り］

Serum N-glycan profiling has potential to predict cardiovascular disease (CVD) event and prognosis in hemodialysis (HD) patients
Shingo Hatakeyama, Yuki Tobisawa, Tohru Yoneyama, Maho Amano, Shin-Ichiro Nishimura, Chikara Ohyama, GLYCOBIOLOGY, 22, 11, 1558, 1559, 2012年11月
OXFORD UNIV PRESS INC, 英語, 研究発表ペーパー・要旨（国際会議）

グライコブロッティング法を用いた異なる種類のヒアルロン酸分解酵素生成ヒアルロン酸断片の同定
荒井みさき, 古川貴之, ガルシア ファイナ, 比能洋, 天野麻穂, 西村紳一郎, 日本薬学会年会要旨集, 132nd, 3, 185, 2012年03月05日
日本語

Glycoblotting 法による健常成人血清中の糖鎖プロファイル解析
木村 聡, 天野 麻穂, 西村 紳一郎, 臨床病理, 59, 6, 585, 585, 2011年06月25日
日本語

HIGH THROUGH PUT, COMPREHENSIVE AND QUANTITATIVE GLYCOMICS OF WHOLE SERUM GLYCOPROTEINS FOR DIAGNOSIS OF RENAL CELL CARCINOMA
Shingo Hatakeyama, Maho Amano, Megumi Tsushima, Hatato Yamamoto, Tohru Yoneyama, Yusuke Ishibashi, Hiromi Murasawa, Kengo Imanishi, Teppei Okamoto, Noriko Tokui, Yuichiro Suzuki, Naoki Sugiyama, Sigemasa Kudo, Takahiro Yoneyama, Takuya Koie, Norihiko Tsuchiya, Tomonori Habuchi, Shin-Ichiro Nishimura, Chikara Ohyama, JOURNAL OF UROLOGY, 185, 4, E385, E385, 2011年04月
ELSEVIER SCIENCE INC, 英語, 研究発表ペーパー・要旨（国際会議）

APP-013 腎細胞癌における血清N-結合型糖鎖の網羅的解析による新規バイオマーカーの検索(総会賞応募ポスター,第99回日本泌尿器科学会総会)
畠山 真吾, 舟生 富壽, 天野 麻穂, 西村 紳一郎, 大山 力, 山本 隼人, 米山 徹, 盛 和行, 工藤 茂将, 米山 高弘, 古家 琢也, 神村 典孝, 對馬 惠, 日泌尿会誌, 102, 2, 299, 299, 2011年
一般社団法人 日本泌尿器科学会, 日本語

プラスチド局在糖タンパク質NPPのグライコミクス解析
金古 堅太郎, 甲州 努, 梅澤 幸歩, 石本 卓也, 天野 麻穂, 西村 紳一郎, 三ツ井 敏明, 日本プロテオーム学会大会要旨集, 2011, 0, 139, 139, 2011年
日本プロテオーム学会（日本ヒトプロテオーム機構）, 日本語

イネプラスチド局在型ヌクレオチドピロホスファターゼ／ホスホジエステラーゼのN-グライコーム解析：機能糖タンパク質がゴルジ装置の後期区画からプラスチドに輸送される
三ツ井 敏明, ポズエタ ロメロハビア, 金古 堅太郎, 甲州 努, 梅澤 幸歩, 古賀 彩, 白矢 武士, 広瀬 和子, 天野 麻穂, 西村 紳一郎, 日本プロテオーム学会大会要旨集, 2011, 0, 65, 65, 2011年
日本プロテオーム学会（日本ヒトプロテオーム機構）, 日本語

Serum N-Glycan Profile in Renal Cell Carcinoma
Shingo Hatakeyama, Maho Amano, Naoki Fujita, Noriko Tokui, Toru Yoneyama, Kengo Imanishi, Hayato Yamamoto, Shigemasa Kudo, Takahiro Yoneyama, Yasuhiro Hashimoto, Takuya Koie, Noritaka Kamimura, Shin-Ichiro Nishimura, Chikara Ohyama, GLYCOBIOLOGY, 20, 11, 1501, 1501, 2010年11月
OXFORD UNIV PRESS INC, 英語, 研究発表ペーパー・要旨（国際会議）

イネにおけるプラスチド局在糖タンパク質ヌクレオチドピロホスファターゼの糖鎖構造解析
金古堅太郎, 甲州努, 梅澤幸歩, 北嶋(古賀)彩, 天野麻穂, 西村紳一郎, 豊岡公徳, 伊藤紀美子, 三ツ井敏明, 生化学, 82, 9, 2010年

本当は，どこを見ているの？～癌特異的糖ペプチド抗原の探索～
天野 麻穂, 臨床化学, 39, 1, 81, 81, 2010年, ［査読有り］, ［招待有り］
日本語, 書評論文,書評,文献紹介等

Large-Scale Serum Glycomics of CFG KO Mice Based on the Glycoblotting Method
Maho Amano, Shin-Ichiro Nishimura, GLYCOBIOLOGY, 19, 11, 1303, 1303, 2009年11月
OXFORD UNIV PRESS INC, 英語, 研究発表ペーパー・要旨（国際会議）

Glycoform-Focused Reverse Genomics (GFRG) in Mouse Ovulation
Maho Amano, Mari Yokoyama, Masaki Kurogochi, Yasuhiro Takegawa, Noriko Nagahori, Tadashi Yamashita, Shin-Ichiro Nishimura, GLYCOBIOLOGY, 19, 11, 1340, 1340, 2009年11月
OXFORD UNIV PRESS INC, 英語, 研究発表ペーパー・要旨（国際会議）

グライコミミックを用いた糖鎖生合成機構の制御
日向寺慧, 羽藤愛美, 天野麻穂, FENG Fei, 三浦嘉晃, 篠原康郎, 比能洋, 西村紳一郎, 日本化学会講演予稿集, 89th, 2, 2009年

大規模高速糖鎖構造解析法の開発と応用
天野麻穂, 比能洋, 古川潤一, 篠原康郎, 西村紳一郎, 日本化学会講演予稿集, 88th, 2, 2008年

エタノールによるメチル基代謝作用の攪乱　　　　　　　　　　　　　　　
天野 麻穂, 栄養学レビュー, 14, 387, 391, 2006年, ［査読有り］, ［招待有り］
日本語, 記事・総説・解説・論説等（学術雑誌）

最新栄養学（第９版）　　　　　　　　　　　　　　　
天野 麻穂, 免疫機能と感染症に対する栄養学的な調節
2007年, ［共訳］

「食品とからだ―免疫・アレルギーのしくみ」（共著）　　　　　　　　　　　　　　　
朝倉書店, 2003年

Animal Cell Technology; Challenges for the 21st century　　　　　　　　　　　　　　　
Amano, M, Totsuka, M, Fujine, K, Yamada, S, Kaminogawa, S, Expression of a soluble alpha-beta heterodimeric T cell receptor in animal cells
Springer, 1999年, ［共著］

マテリアル・シンバイオシスのための生命物理化学
科学研究費助成事業
2020年11月19日 - 2025年03月31日
山吉 麻子, 前仲 勝実, 荏原 充宏, 望月 慎一, 長谷 耕二, 大場 雄介, 宇都 甲一郎, 白石 貢一, 植畑 拓也, 森 健, 天野 麻穂, 山本 剛史
本研究課題の目的は、学術変革領域「マテリアル・シンバイオシスのための生命物理化学」の研究推進と領域運営である。
【領域会議等の開催】2021年度9月より公募研究班の領域参画が始まった。領域目標を達成するためには、個々の研究の活性化に加え、領域全体が一体となって相互補完的な融合研究を実施することが肝要である。そこで、領域全メンバー参加型の第１回領域会議（2021/11/4-11/5）をハイブリッド開催し、最新の研究成果の共有と共同研究推進の場を提供した。また別途、A01-A03各班での班会議を開催し情報共有を行った。総括班員による班会議も定期開催し、領域体制の最適化に勤めた。
【領域共催のシンポジウムの開催】日本薬剤学会第36年会において、ラウンドテーブル「シンバイオティック・マテリアルの実現と新しい創薬モダリティを考える」を企画・開催した（2021/1/13）。また、日本DDS学会第37年会において、若手ワークショップ「腸内細菌叢の共生原理に学ぶDDS」を企画・開催した（2019/6/29）。領域メンバーはもとより、国内外研究協力者が領域研究に関する研究発表を行い、領域研究を外部に向けて発信した。さらに、男女共同参画推進を目的としたシンポジウムを共催した（「北九州サイエンスガールプロジェクト（北九州市立大学公開講座）」2021/10/2, 10/9, 10/16; 「令和3年度 長崎大学リケジョ育成プログラム 志セミナー」, 2021/12/4）。
【イメージングブートキャンプ】領域班員、若手研究者に、物質共生学研究で重要となる細胞イメージング技術を修得する場を提供することを目的に、イメージングブートキャンプ2021 （2021/9/7-9）を共催した。
【共同研究推進】分野横断的かつ積極的な共同研究を推進するために、総括班よりマテリアル合成支援や超高速AFM解析支援等を提供した。
日本学術振興会, 学術変革領域研究(A), 長崎大学, 20H05871

網羅的糖鎖解析による新規癌マーカーの探索と診断技術の開発
科学研究費助成事業
2013年05月31日 - 2018年03月31日
西村 紳一郎, 能祖 一裕, 比能 洋, 大山 力, 神山 俊哉, 天野 麻穂
世界初の「疾患糖鎖構造データベースの構築」を目標として、医師を含む臨床チームとの強力な連携により３５００件を超える患者検体を用いた大規模網羅的糖鎖解析による新規バイオマーカーの探索を進めた。特に消化器癌と泌尿器癌に焦点を絞りバイオマーカーとして有望な糖鎖構造情報を獲得するため、独創的な「全自動糖鎖解析装置」による大規模糖鎖解析からの疾患糖鎖データベースの構築に挑戦した。具体的には膵臓癌、膵炎、肝細胞癌、肝炎、潰瘍性大腸炎、大腸癌、自己免疫性膵炎等の消化器疾患領域、および腎細胞癌、腎炎、前立腺癌等の泌尿器疾患領域を中心に疾患糖鎖情報を収集して世界初の疾患関連糖鎖データベースの構築が実現した。
日本学術振興会, 基盤研究(S), 北海道大学, 25220206

網羅的糖鎖解析グライコミクスによるぶどう膜炎炎症増悪マーカー探索
科学研究費助成事業
2013年04月01日 - 2016年03月31日
南塲 研一, 北市 伸義, 西村 紳一郎, 天野 麻穂
今回我々はぶどう膜炎の発症時に特異的に上昇する糖鎖について検討をおこなった。インフリキシマブ導入後もぶどう膜炎を生じる患者、特に抗インフリキシマブ抗体が陽性患者、およびコントロールとしてインフリキシマブ導入後経過良好な患者の血清について、新しい糖鎖構造解析グライコブロッティング法をもちいて網羅的糖鎖解析をおこなった。2群間比較でm/z1680, m/z2337, m/z2379, m/z3195の４つの糖鎖において比較的明確な差異が認められ、いずれも無効例に多くみられるという結果であった。
日本学術振興会, 基盤研究(C), 北海道大学, 25462745

グライコブロッティング法を用いた糖尿病網膜症関連糖鎖の探索
科学研究費助成事業
2012年04月01日 - 2014年03月31日
石田 晋, 西村 伸一郎, 天野 麻穂
核酸と蛋白質に続く第三の鎖状生命分子として、糖鎖分子が近年注目されている。蛋白質のが受ける糖鎖修飾は「翻訳後修飾」の中でも重要なプロセスであり、糖鎖構造の変化は蛋白機能にも影響を与える。本研究において、我々は硝子体中のN型糖鎖を網羅的に解析する手法を検討した。非糖尿病網膜症（黄斑上膜、黄斑円孔）患者２０例における血漿中と硝子体中のN型糖鎖を比較検討したところ、糖鎖量は硝子体中では血漿中と比較して統計学的有意に低かった。また、硝子体中と血漿中の双方で最も多く含まれていたN型糖鎖は共通であり、その構造は(Hex)2 (HexNAc)2 (NeuAc)2 + (Man)3(GlcNAc)2であった。
日本学術振興会, 挑戦的萌芽研究, 北海道大学, 24659754

新たな網羅的糖鎖解析グライコブロッティング法によるぶどう膜炎の病態解明
科学研究費助成事業
2010年 - 2012年
南場 研一, 西村 紳一郎, 北市 伸義, 天野 麻穂
新しい網羅的糖鎖構造解析グライコブロッティング法を用いてぶどう膜炎の患者での糖鎖解析をおこなった。ぶどう膜炎患者では血清中の糖鎖量が増えていること、また、活動期に変動する糖鎖が3種類見られた。ベーチェット病ではHLA-B51との関連、眼外症状との関連性を示す糖鎖がみられたが、眼炎症発作との関連はみられなかった。マウス実験的自己免疫性ぶどう膜網膜炎モデルでの糖鎖変動を調べたところ、免疫により変動する糖鎖はみられたが、ぶどう膜炎特異的に変動していると思われる糖鎖はみられなかった。
日本学術振興会, 基盤研究(C), 北海道大学, 22591927

グライコミクスによる変形性関節症の早期診断に有効な血清糖鎖マーカー探索
科学研究費助成事業
2011年 - 2011年
西田 欽也, 岩崎 倫政, 西村 伸一郎, 天野 麻穂
変形性関節症の早期診断のための血清マーカーの探索を目的に研究を進めた。変形性膝関節症(OA)患者および健常者の血清5-20μ1を用いた。血清中からグライコブロッティング法を用いて高速(5時間程度)かつ高効率に、糖蛋白質からN-結合型糖鎖の遊離精製を行った。得られたN-結合型糖鎖をMALDI-TOF/MSに供することで、各糖鎖構造を推定し、内部標準物質のピーク面積からの比例計算により定量化を行った。解析は現在進行中である。患者検体はX線所見にて膝OAと診断されたKellgren-Lawrence分類grade 2-4の患者より採取している。糖鎖解析は、本申請者が中心となり西村、天野らの協力のもと行っている。独自に開発したグライコブロッティング法は従来の解析法に比べ飛躍的に解析時間の高速化と高効率性を実現し、これまで困難とされていた大規模糖鎖解析を可能とし、本研究で予定している検体数の解析を早急に進めているところである。統計学的解析によりAUC(Area Under the Curve)が0.85以上の糖鎖およびその組み合わせを抽出する。先行研究の結果より、5から10程度の候補糖鎖および組み合わせが予想される。さらに、糖鎖関連分子のOAの病因における機能的役割を解明するためにスフィンゴ糖脂質を軟骨組織特異的にノックアウトしたマウス(UgcgKOマウス)を用いてOAモデルを作成し、その機能解析を行った。その結果、GSLsは軟骨の発生や分化には必須ではないが、正常な軟骨代謝を維持し、OA進行を抑制する機能を持つことが示唆された。
日本学術振興会, 挑戦的萌芽研究, 北海道大学, 23659702

網膜ミクログリアによるT細胞アポトーシスの機序解明とぶどう膜炎治療
科学研究費助成事業
2006年 - 2006年
木本 高志, 天野 麻穂
網膜可溶性抗原(S抗原)のペプチド合成を行った。ルイス系ラット(体重150〜1759)の雌の足蹠に、ペプチドを接種した。免疫接種後10日目から前眼部炎症による両眼の角膜毛様充血をみた。眼球摘出後、網膜の凍結切片を作成した。接種後11日目から網膜内に炎症細胞の浸潤をみるようになった。接種後15日目以降になると、視細胞層は消失して、網膜構造が崩壊して後部ぶどう膜炎の発症が確認できた。正常網膜の凍結切片を作成して、galectin-9抗体による免疫染色では、網膜に陽性細胞をみなかったが、実験的ぶどう膜炎の網膜では、galectin-9抗体陽性が網膜色素上皮細胞(RPE)、内網状層にみた。内網状層の陽性細胞は、局在部位から網膜ミクログリアと思われた。さらにRPEの細胞ラインであるARPE-19 and hTERTを培養させた。それらの細胞は、galectin-1抗体に陽性であった。RPEを活性化したT細胞と共存させて培養すると、T細胞にアポトーシスを生じた。
また、ルイス系ラット(生後2〜3日)の前眼部組織、水晶体、硝子体を切除して、RPEを除いて網膜を剥離した。網膜細胞間を分離させて、網膜細胞をDMEM-FBS培養液に95%air-5%CO_2下で入れ、浮遊した細胞を除去して,接着した細胞のみを1ng/ml murine granulocytemacrophage(GM)-CSF(Sigma)を含有したDMEM-FBS培養液中で培養した。培養後10日後から浮遊してきた細胞を分離して、CD11b抗体(OX42)による免疫染色を行うと陽性であったことから、網膜ミクロクリアと同定し、培養系実験を確立させた。
今回の研究結果から、実験的ぶどう膜炎の発症経過に網膜ミクログリアとRPEがガレクチン糖鎖ファミリーを介してT細胞に働きかけていることが示唆された。
日本学術振興会, 萌芽研究, 関西医科大学, 18659518