ASHIDA Hisashi

    Department of Science and Technology on Food Safety Professor
Last Updated :2024/05/29

Researcher Information

Degree

  • Ph.D(Kyoto University)

Research funding number

  • 40379087

J-Global ID

Research Interests

  • ビフィズス菌   乳酸菌   腸内細菌   応用微生物学   糖鎖生物学   グリコシダーゼ   食品機能学   食品工学   免疫学   細胞生物学   オートファジー   昆虫学   分類学   生物多様性   Applied Microbiology   Food Science and Technology   Glycobiology   Cell Biology   Biodiversity   

Research Areas

  • Life sciences / Applied microbiology
  • Life sciences / Food sciences
  • Life sciences / Applied biochemistry
  • Life sciences / Applied molecular and cellular biology
  • Environmental science/Agricultural science / Entomology

Academic & Professional Experience

  • 2012/04 - Today  Faculty of Biology-Oriented Science and Technology, Kinki UniversityProfessor
  • 2008/06 - 2012/03  Kyoto UniversityGraduate School of Biostudies准教授
  • 2006/01 - 2008/05  Kyoto UniversityGraduate School of Biostudies助手/助教
  • 2004/04 - 2005/12  Osaka UniversityResearch Institute for Microbial Diseases助手
  • 2001/09 - 2004/03  Osaka UniversityResearch Institute for Microbial Diseases博士研究員
  • 2000/07 - 2001/08  Tulane University,Post doctoral fellow
  • 1997/04 - 2000/07  Kyoto UniversityGraduate School of Agriculture
  • 1997/04 - 2000/03  同志社国際中・高等学校 非常勤講師
  • 1993/07 - 1997/03  株式会社京都第一科学
  • 1988/04 - 1993/06  ヒガシマル醤油株式会社

Education

  • 2000/09 - 2000/09  京都大学博士(農学) 農論2331号
  • 1983/04 - 1988/03  Kyoto University  Faculty of Agriculture  食品工学科

Association Memberships

  • JAPAN SOCIETY FOR LACTIC ACID BACTERIA   日本甲虫学会   日本洞窟学会   日本進化学会   日本応用糖質科学会   日本糖質学会   日本農芸化学会   日本生化学会   

Published Papers

  • Toshihiko Katoh; Hisashi Ashida
    Methods in Molecular Biology Springer US 337 - 344 1064-3745 2024/02
  • Kusumia kukai (Coleoptera, Carabidae, Trechinae), a New Species Found near Kôyasan in the Northern Part of the Kii Peninsula, Central Japan
    Hisashi Ashida
    Elytra, New Series 13 (2) 175 - 180 2023/11 [Refereed]
  • Toshihiko Katoh; Chihaya Yamada; Michael D Wallace; Ayako Yoshida; Aina Gotoh; Moe Arai; Takako Maeshibu; Toma Kashima; Arno Hagenbeek; Miriam N Ojima; Hiromi Takada; Mikiyasu Sakanaka; Hidenori Shimizu; Keita Nishiyama; Hisashi Ashida; Junko Hirose; Maria Suarez-Diez; Makoto Nishiyama; Ikuo Kimura; Keith A Stubbs; Shinya Fushinobu; Takane Katayama
    Nature Chemical Biology 19 778 - 789 2023/03 [Refereed]
     
    Mucinolytic bacteria modulate host-microbiota symbiosis and dysbiosis through their ability to degrade mucin O-glycans. However, how and to what extent bacterial enzymes are involved in the breakdown process remains poorly understood. Here we focus on a glycoside hydrolase family 20 sulfoglycosidase (BbhII) from Bifidobacterium bifidum, which releases N-acetylglucosamine-6-sulfate from sulfated mucins. Glycomic analysis showed that, in addition to sulfatases, sulfoglycosidases are involved in mucin O-glycan breakdown in vivo and that the released N-acetylglucosamine-6-sulfate potentially affects gut microbial metabolism, both of which were also supported by a metagenomic data mining analysis. Enzymatic and structural analysis of BbhII reveals the architecture underlying its specificity and the presence of a GlcNAc-6S-specific carbohydrate-binding module (CBM) 32 with a distinct sugar recognition mode that B. bifidum takes advantage of to degrade mucin O-glycans. Comparative analysis of the genomes of prominent mucinolytic bacteria also highlights a CBM-dependent O-glycan breakdown strategy used by B. bifidum.
  • A New Species of the Genus Jujiroa (Coleoptera, Carabidae, Platyninae) from the Kii Peninsula, Central Japan
    Nobuya Wada; Hisashi Ashida
    Elytra, Tokyo, New Series 12 (2) 265 - 270 2022/12 [Refereed]
  • Rika Hirano; Aiko Kume; Chisato Nishiyama; Ryosuke Honda; Hideto Shirasawa; Yiwei Ling; Yuta Sugiyama; Misaki Nara; Hiromi Shimokawa; Hiroki Kawada; Takashi Koyanagi; Hisashi Ashida; Shujiro Okuda; Mitsuharu Matsumoto; Hiroki Takagi; Shin Kurihara
    Microorganisms MDPI AG 10 (4) 697 - 697 2022/03 [Refereed]
     
    Polyamines are aliphatic hydrocarbons with terminal amino groups and are essential for biological activities. It has been reported that polyamines have health-promoting effects in animals, such as the extension of lifespan by polyamine intake. The identification of a high polyamine-producing bacterium from foods could lead to the development of a novel probiotic candidate. We aimed to identify high polyamine-producing bacteria from food, and isolated and collected bacteria from vegetables and fermented foods produced in Japan. We successfully acquired Latilactobacillus curvatus KP 3-4 isolated from Kabura-zushi as a putrescine producing lactic acid bacteria. Comparing the polyamine synthesis capability of L. curvatus KP 3-4 with that of typical probiotic lactic acid bacteria and L. curvatus strains available from the Japan Collection of Microorganisms, it was found that only L. curvatus KP 3-4 was capable of exporting high levels of putrescine into the culture supernatant. The enhancement of putrescine production by the addition of ornithine, and whole-genome analysis of L. curvatus KP 3-4, suggest that putrescine is synthesized via ornithine decarboxylase. The administration of L. curvatus KP 3-4 to germ-free mice increased the concentration of putrescine in the feces.
  • Masahiro Komeno; Yuki Yoshihara; Junya Kawasaki; Wataru Nabeshima; Koshi Maeda; Yuki Sasaki; Kiyotaka Fujita; Hisashi Ashida
    Applied Microbiology and Biotechnology Springer Science and Business Media LLC 106 1957 - 1965 0175-7598 2022/03 [Refereed]
     
    Arabinoxylan (AX) and arabinoxylooligosaccharides (AXOs) are carbohydrate sources utilized by Bifidobacterium longum subsp. longum. However, their degradation pathways are poorly understood. In this study, we characterized two genes, BLLJ_1850 and BLLJ_1851, in the hemicellulose-degrading gene cluster (BLLJ_1836-BLLJ_1859) of B. longum subsp. longum JCM 1217. Both recombinant enzymes expressed in Escherichia coli exhibited exo-α-L-arabinofuranosidase activity toward p-nitrophenyl-α-L-arabinofuranoside. BlArafE (encoded by BLLJ_1850) contains the glycoside hydrolase family 43 (GH43), subfamily 22 (GH43_22), and GH43_34 domains. The BlArafE GH43_22 domain was demonstrated to release α1,3-linked Araf from AX, but the function of BlArafE GH43_34 could not be clearly identified in this study. BlArafD (encoded by BLLJ_1851) contains GH43 unclassified subfamily (GH43_UC) and GH43_26 domains. The BlArafD GH43_UC domain showed specificity for α1,2-linked Araf in α1,2- and α1,3-Araf double-substituted structures in AXOs, while BlArafD GH43_26 was shown to hydrolyze α1,5-linked Araf in the arabinan backbone. Co-incubation of BlArafD and BlArafE revealed that these two enzymes sequentially removed α1,2-Araf and α1,3-Araf from double-substituted AXOs in this order. B. longum strain lacking BLLJ_1850-BLLJ_1853 did not grow in the medium containing α1,2/3-Araf double-substituted AXOs, suggesting that BlArafE and BlArafD are important for the assimilation of AX. KEY POINTS: • BlArafD GH43 unclassified subfamily domain is a novel α1,2-L-arabinofuranosidase. • BlArafE GH43 subfamily 22 domain is an α1,3-L-arabinofuranosidase. • BlArafD and BlArafE cooperatively degrade α1,2/3-Araf double-substituted arabinoxylan.
  • Yuki Sasaki; Masahiro Komeno; Akihiro Ishiwata; Ayako Horigome; Toshitaka Odamaki; Jin-Zhong Xiao; Katsunori Tanaka; Yukishige Ito; Kanefumi Kitahara; Hisashi Ashida; Kiyotaka Fujita
    Applied and Environmental Microbiology American Society for Microbiology 88 (6) e02187-21  0099-2240 2022/02 [Refereed]
     
    Gum arabic is an arabinogalactan protein (AGP) that is effective as a prebiotic for the growth of bifidobacteria in the human intestine. We recently identified a key enzyme in the glycoside hydrolase (GH) family 39, 3- O -α- d -galactosyl-α- l -arabinofuranosidase (GAfase), for the assimilation of gum arabic AGP in Bifidobacterium longum subsp. longum . The enzyme released α- d -Gal p -(1→3)- l -Ara and β- l -Ara p -(1→3)- l -Ara from gum arabic AGP and facilitated the action of other enzymes for degrading the AGP backbone and modified sugar. In this study, we identified an α- l -arabinofuranosidase (BlArafE; encoded by BLLJ_1850), a multidomain enzyme with both GH43_22 and GH43_34 catalytic domains, as a critical enzyme for the degradation of modified α- l -arabinofuranosides in gum arabic AGP. Site-directed mutagenesis approaches revealed that the α1,3/α1,4-Ara f double-substituted gum arabic AGP side chain was initially degraded by the GH43_22 domain and subsequently cleaved by the GH43_34 domain to release α1,3-Ara f and α1,4-Ara f residues, respectively. Furthermore, we revealed that a tetrasaccharide, α- l -Rha p -(1→4)-β- d -Glc p A-(1→6)-β- d -Gal p -(1→6)- d -Gal, was a limited degradative oligosaccharide in the gum arabic AGP fermentation of Bi. longum JCM7052. The oligosaccharide was produced from gum arabic AGP by the cooperative action of the three cell surface-anchoring enzymes, GAfase, exo-β1,3-galactanase (Bl1,3Gal), and BlArafE, on Bi. longum JCM7052. Furthermore, the tetrasaccharide was utilized by the commensal bacteria. IMPORTANCE Terminal galactose residues of the side chain of gum arabic arabinogalactan protein (AGP) are mainly substituted by α1,3/α1,4-linked Ara f and β1,6-linked α- l -Rha p -(1→4)-β- d -Glc p A residues. This study found a multidomain α- l -arabinofuranosidase (BlArafE) with GH43_22 and GH43_34 catalytic domains showing cooperative action for degrading α1,3/α1,4-linked Ara f of the side chain of gum arabic AGP. In particular, the GH43_34 domain of BlArafE was a novel α- l -arabinofuranosidase for cleaving the α1,4-Ara f linkage of terminal galactose. α- l -Rha p -(1→4)-β- d -Glc p A-(1→6)-β- d -Gal p -(1→6)- d -Gal tetrasaccharide was released from gum arabic AGP by the cooperative action of GAfase, GH43_24 exo-β-1,3-galactanase (Bl1,3Gal), and BlArafE and remained after Bi. longum JCM7052 culture. Furthermore, in vitro assimilation test of the remaining oligosaccharide using Bacteroides species revealed that cross-feeding may occur from bifidobacteria to other taxonomic groups in the gut.
  • Hideya Shintani; Hisashi Ashida; Tomoya Shintani
    Human Nutrition & Metabolism Elsevier BV 26 200134 - 200134 2666-1497 2021/12 [Refereed]
  • A New Species of the Trechiama notoi Complex (Coleoptera, Carabidae, Trechinae) from the Northwestern Area of Kyoto Prefecture, Central Japan: Dedicated to the Late Dr. Shun-Ichi Uéno
    Nobuya Wada; Hisashi Ashida
    Elytra, Tokyo, New Series 11 (Supplement) 51 - 56 2021/10 [Refereed]
  • Takayuki Ito; Shogo Nishio; Maho Hirabayashi; Seiji Miyatani; Hisashi Ashida
    Memoirs of The Faculty of B.O.S.T. of Kindai University 45 1 - 9 2021/03 [Refereed]
  • Toshihiko Katoh; Miriam N. Ojima; Mikiyasu Sakanaka; Hisashi Ashida; Aina Gotoh; Takane Katayama
    Microorganisms MDPI AG 8 (4) 481  2020/03 [Refereed]
     
    Certain species of the genus Bifidobacterium represent human symbionts. Many studies have shown that the establishment of symbiosis with such bifidobacterial species confers various beneficial effects on human health. Among the more than ten (sub)species of human gut-associated Bifidobacterium that have significantly varied genetic characteristics at the species level, Bifidobacterium bifidum is unique in that it is found in the intestines of a wide age group, ranging from infants to adults. This species is likely to have adapted to efficiently degrade host-derived carbohydrate chains, such as human milk oligosaccharides (HMOs) and mucin O-glycans, which enabled the longitudinal colonization of intestines. The ability of this species to assimilate various host glycans can be attributed to the possession of an adequate set of extracellular glycoside hydrolases (GHs). Importantly, the polypeptides of those glycosidases frequently contain carbohydrate-binding modules (CBMs) with deduced affinities to the target glycans, which is also a distinct characteristic of this species among members of human gut-associated bifidobacteria. This review firstly describes the prevalence and distribution of B. bifidum in the human gut and then explains the enzymatic machinery that B. bifidum has developed for host glycan degradation by referring to the functions of GHs and CBMs. Finally, we show the data of co-culture experiments using host-derived glycans as carbon sources, which underpin the interesting altruistic behavior of this species as a cross-feeder.
  • Ashida Hisashi; Fujimoto Taku; Kurihara Shin; Nakamura Masayuki; Komeno Masahiro; Huang Yibo; Katayama Takane; Kinoshita Takashi; Takegawa Kaoru
    Journal of Applied Glycoscience The Japanese Society of Applied Glycoscience 67 (1) 23 - 29 1344-7882 2020/02 [Refereed]
     

    Bifidobacterium longum subsp. infantis ATCC 15697 possesses five α-L-fucosidases, which have been previously characterized toward fucosylated human milk oligosaccharides containing α1,2/3/4-linked fucose [Sela et al.: Appl. Environ. Microbiol., 78, 795-803 (2012)]. In this study, two glycoside hydrolase family 29 α-L-fucosidases out of five (Blon_0426 and Blon_0248) were found to be 1,6-α-L-fucosidases acting on core α1,6-fucose on the N-glycan of glycoproteins. These enzymes readily hydrolyzed p-nitrophenyl-α-L-fucoside and Fucα1-6GlcNAc, but hardly hydrolyzed Fucα1-6(GlcNAcβ1-4)GlcNAc, suggesting that they de-fucosylate Fucα1-6GlcNAcβ1-Asn-peptides/proteins generated by the action of endo-β-N-acetylglucosaminidase. We demonstrated that Blon_0426 can de-fucosylate Fucα1-6GlcNAc-IgG prepared from Rituximab using Endo-CoM from Cordyceps militaris. To generate homogenous non-fucosylated N-glycan-containing IgG with high antibody-dependent cellular cytotoxicity (ADCC) activity, the resulting GlcNAc-IgG has a potential to be a good acceptor substrate for the glycosynthase mutant of Endo-M from Mucor hiemalis. Collectively, our results strongly suggest that Blon_0426 and Blon_0248 are useful for glycoprotein glycan remodeling.

  • Komeno M; Hayamizu H; Fujita K; Ashida H
    Applied and Environmental Microbiology 85 (6) e02582-18  0099-2240 2019/01 [Refereed]
     
    Arabinose-containing poly- or oligosaccharides are suitable carbohydrate sources for Bifidobacterium longum subsp. longum. However, their degradation pathways are poorly understood. In this study, we cloned and characterized the previously uncharacterized glycoside hydrolase family 43 (GH43) enzymes B. longum subsp. longum ArafC (BlArafC; encoded by BLLJ_1852) and B. longum subsp. longum ArafB (BlArafB; encoded by BLLJ_1853) from B. longum subsp. longum JCM 1217. Both enzymes exhibited α-L-arabinofuranosidase activity toward p-nitrophenyl-α- L-arabinofuranoside but no activity toward p-nitrophenyl-β-D-xylopyranoside. The specificities of the two enzymes for L-arabinofuranosyl linkages were different. BlArafC catalyzed the hydrolysis of α1,2- and α1,3-L-arabinofuranosyl linkages found on the side chains of both arabinan and arabinoxylan. It released L-arabinose 100 times faster from arabinan than from arabinoxylan but did not act on arabinogalactan. On the other hand, BlArafB catalyzed the hydrolysis of the α1,5-Larabinofuranosyl linkage found on the arabinan backbone. It released L-arabinose from arabinan but not from arabinoxylan or arabinogalactan. Coincubation of BlArafC and BlArafB revealed that these two enzymes are able to degrade arabinan in a synergistic manner. Both enzyme activities were suppressed with EDTA treatment, suggesting that they require divalent metal ions. The GH43 domains of BlArafC and BlArafB are classified into GH43 subfamilies 27 and 22, respectively, but show very low similarity (less than 15% identity) with other biochemically characterized members in the corresponding subfamilies. The B. longum subsp. longum strain lacking the GH43 gene cluster that includes BLLJ_1850 to BLLJ_1853 did not grow in arabinan medium, suggesting that BlArafC and BlArafB are important for assimilation of arabinan.
  • Ashida H; Tanigawa K; Kiyohara M; Katoh T; Katayama T; Yamamoto K
    Bioscience, Biotechnology, and Biochemistry Oxford University Press (OUP) 82 (11) 2030 - 2039 0916-8451 2018/11 [Refereed]
     
    ABSTRACT Sialidases catalyze the removal of terminal sialic acid from various complex carbohydrates. In the gastrointestinal tract, sialic acid is commonly found in the sugar chain of mucin, and many enteric commensals use mucin as a nutrient source. We previously identified two different sialidase genes in Bifidobacterium bifidum, and one was cloned and expressed as an extracellular protein designated as exo-α-sialidase SiaBb2. The other exo-α-sialidase gene (siabb1) from the same bifidobacterium encodes an extracellular protein (SiaBb1) consisting of 1795 amino acids with a molecular mass of 189 kDa. SiaBb1 possesses a catalytic domain that classifies this enzyme as a glycoside hydrolase family 33 member. SiaBb1 preferentially hydrolyzes α2,3-linked sialic acid over α2,6-linked sialic acid from sialoglycan, which is the same as SiaBb2. However, SiaBb1 has an SGNH hydrolase domain with sialate-O-acetylesterase activity and an N-terminal signal sequence and C-terminal transmembrane region. SiaBb1 is the first bifunctional sialidase identified with esterase activity. Abbreviations: GalNAc: N-acetyl-D-galactosamine; Fuc: L-fucose; Gal: D-galactose
  • Masahiro Komeno; Azusa Ito; Natsumi Ohigashi; Yuki Yoshihara; Toshio Suzuki; Kouhei Nagai; Hisashi Ashida
    Mem Faculty B.O.S.T., Kindai University (42) 1 - 12 2018/10 [Refereed]
  • Shintani T; Kosuge Y; Ashida H
    Journal of Applied Glycoscience 65 (3) 37 - 43 1880-7291 2018/08 [Refereed]
  • Takayuki Higashiyama; Midori Umekawa; Masaya Nagao; Toshihiko Katoh; Hisashi Ashida; Kenji Yamamoto
    Carbohydrate Research Elsevier Ltd 455 92 - 96 1873-426X 2018/01 [Refereed]
     
    The chemo-enzymatic synthesis of an artificially N-glycosylated derivative of glucagon, a peptide hormone that regulates the blood sugar level, is described. We synthesized the glycosylated glucagon by chemical synthesis of an N-acetylglucosaminyl peptide and enzymatic transfer of an oligosaccharide using the transglycosylation activity of the glycosynthase-like mutant of Mucor hiemalis endo-β-N-acetylglucosaminidase (Endo-M) and sialo-oligosaccharide oxazoline as a donor substrate. The sialo-oligosaccharide-attached glucagon synthesized showed high resistance against protease degradation and stimulated the release of glucose from mouse hepatocytes when added to cells. The synthetic glucagon showed slightly higher activity than native glucagon and has potential as a therapeutic agent for treating diabetic patients.
  • Toshihiko Katoh; Takako Maeshibu; Kei-ichi Kikkawa; Aina Gotoh; Yusuke Tomabechi; Motoharu Nakamura; Wei-Hsiang Liao; Masanori Yamaguchi; Hisashi Ashida; Kenji Yamamoto; Takane Katayama
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY TAYLOR & FRANCIS LTD 81 (10) 2018 - 2027 0916-8451 2017/10 [Refereed]
     
    Human gut symbiont bifidobacteria possess carbohydrate-degrading enzymes that act on the O-linked glycans of intestinal mucins to utilize those carbohydrates as carbon sources. However, our knowledge about mucin type O-glycan degradation by bifidobacteria remains fragmentary, especially regarding how they decompose sulfated glycans, which are abundantly found in mucin sugar-chains. Here, we examined the abilities of several Bifidobacterium strains to degrade a sulfated glycan substrate and identified a 6-sulfo--d-N-acetylglucosaminidase, also termed sulfoglycosidase, encoded by bbhII from Bifidobacterium bifidum JCM 7004. A recombinant BbhII protein showed a substrate preference toward 6-sulfated and 3,4-disulfated N-acetylglucosamines over non-sulfated and 3-sulfated N-acetylglucosamines. The purified BbhII directly released 6-sulfated N-acetylglucosamine from porcine gastric mucin and the expression of bbhII was moderately induced in the presence of mucin. This de-capping activity may promote utilization of sulfated glycans of mucin by other bacteria including bifidobacteria, thereby establishing the symbiotic relationship between human and gut microbes.
  • Mayo Sato; Dorothee Liebschner; Yusuke Yamada; Naohiro Matsugaki; Takatoshi Arakawa; Siobhan S. Wills; Mitchell Hattie; Keith A. Stubbs; Tasuku Ito; Toshiya Senda; Hisashi Ashida; Shinya Fushinobu
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 292 (29) 12126 - 12138 0021-9258 2017/07 [Refereed]
     
    The alpha-N-acetylgalactosaminidase from the probiotic bacterium Bifidobacterium bifidum (NagBb) belongs to the glycoside hydrolase family 129 and hydrolyzes the glycosidic bond of Tnantigen (GalNAc alpha 1-Ser/Thr). NagBb is involved in assimilation of O-glycans on mucin glycoproteins by B. bifidum in the human gastrointestinal tract, but its catalytic mechanism has remained elusive because of a lack of sequence homology around putative catalytic residues and of other structural information. Here we report the X-ray crystal structure of NagBb, representing the first GH129 family structure, solved by the single-wavelength anomalous dispersion method based on sulfur atoms of the native protein. We determined ligand-free, GalNAc, and inhibitor complex forms of NagBb and found that Asp-435 and Glu-478 are located in the catalytic domain at appropriate positions for direct nucleophilic attack at the anomeric carbon and proton donation for the glycosidic bond oxygen, respectively. A highly conserved Asp-330 forms a hydrogen bond with the O4 hydroxyl of GalNAc in the -1 subsite, and Trp-398 provides a stacking platform for the GalNAc pyranose ring. Interestingly, a metal ion, presumably Ca2+, is involved in the recognition of the GalNAc N-acetyl group. Mutations at Asp-435, Glu-478, Asp-330, and Trp-398 and residues involved in metal coordination (including an all-Ala quadruple mutant) significantly reduced the activity, indicating that these residues and the metal ion play important roles in substrate recognition and catalysis. Interestingly, NagBb exhibited some structural similarities to the GH101 endo-alpha-N-acetylgalactosaminidases, but several critical differences in substrate recognition and reaction mechanism account for the different activities of these two enzymes.
  • Yuta Sugiyama; Toshihiko Katoh; Yuji Honda; Aina Gotoh; Hisashi Ashida; Shin Kurihara; Kenji Yamamoto; Takane Katayama
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY TAYLOR & FRANCIS LTD 81 (2) 283 - 291 0916-8451 2017/02 [Refereed]
     
    We have recently generated a highly efficient 1,2-alpha-L-fucosynthase (BbAfcA N423H mutant) by protein engineering of 1,2-alpha-L-fucosidase from Bifidobacterium bifidum JCM 1254. This synthase could specifically introduce H-antigens (Fuc alpha 1-2Gal) into the non-reducing ends of oligosaccharides and in O-linked glycans in mucin glycoprotein. In the present study, we show an extended application of the engineered 1,2-alpha-L-fucosynthase by demonstrating its ability to insert Fuc residues into N- and O-glycans in fetuin glycoproteins, GM1 ganglioside, and a plant-derived xyloglucan nonasaccharide. This application study broadens the feasibility of this novel H-antigen synthesis technique in functional glycomics.
  • Yuich Abe; Hiroshi Inoue; Hisashi Ashida; Yusuke Maeda; Taroh Kinoshita; Sakae Kitada
    JOURNAL OF INVERTEBRATE PATHOLOGY ACADEMIC PRESS INC ELSEVIER SCIENCE 142 71 - 81 0022-2011 2017/01 [Refereed]
     
    Parasporin-2 (PS2), alternatively named Cry46Aa1, an anticancer protein derived from Bacillus thuringiensis strain A1547, causes specific cell damage via PS2 oligomerization in the cell membrane. Although PS2 requires glycosylphosphatidylinositol (GPI)-anchored proteins for its cytocidal action, their precise role is unknown. Here, we report that the glycan of GPI induces PS2 oligomerization, which causes cell death. Cytotoxicity, cell-binding and oligomerization of the toxin were not observed in GPI-anchored protein deficient Chinese hamster ovary cells. Expression and protease-treatment analyses showed that the actions of the toxin were dependent on the glycan core, not the polypeptide moiety, of GPI-anchored proteins. However, surface expression of some GPI-anchored proteins is observed in PS2-insensitive cells. These data suggest that GPI-anchored proteins do not determine the target specificity, but instead function as a kind of coreceptor, in the cytocidal action of PS2. (C) 2016 Elsevier Inc. All rights reserved.
  • Yuta Sugiyama; Aina Gotoh; Toshihiko Katoh; Yuji Honda; Erina Yoshida; Shin Kurihara; Hisashi Ashida; Hidehiko Kumagai; Kenji Yamamoto; Motomitsu Kitaoka; Takane Katayama
    GLYCOBIOLOGY OXFORD UNIV PRESS INC 26 (11) 1235 - 1247 0959-6658 2016/11 [Refereed]
     
    Fuc alpha 1-2 Gal linkages, or H-antigens, constitute histo-blood group antigens and are involved in various physiological processes. In addition, recent studies have shown that the H-antigen-containing glycans play an important role, not only in establishing harmonious relationship between gut microbes and the host, but also in preventing gut dysbiosis-related diseases. Therefore, development of an efficient method for introducing Fuc residue at Gal residue at the nonreducing end of glycans via alpha-(1 -> 2) linkage is desired for research as well as medicinal purposes. In this study, we succeeded in derivatizing inverting 1,2-alpha-L-fucosidase (AfcA) into a highly efficient 1,2-alpha-L-fucosynthase. The synthase specifically synthesized H type 1-, type 2-, type 3- and type 4-chaincontaining oligosaccharides with yields of 57-75% based on acceptor depletion. The synthase was also able to specifically introduce Fuc residues into Lewis a/x antigens to produce Lewis b/y antigens, with yields of 43% and 62%, respectively. In addition, the enzyme efficiently introduced Hantigens into sugar chains of porcine gastric mucins, as revealed by lectin blotting and mass spectroscopy analysis of the sugars. Detailed acceptor specificity analysis using various monosaccharides and oligosaccharides unraveled unique substrate recognition feature of this synthase at the subsite (+1), which can be explained by our previous X-ray crystallographic study of AfcA. These results show that the synthase developed in this study could serve as an alternative to other H-antigen synthesis methods involving alpha-1,2-fucosyltransferases and retaining alpha-fucosidase.
  • Shimada Y; Ashida H
    Mem. Faculty. B.O.S.T. Kindai University (38) 1 - 10 1342-7202 2016/10 [Refereed]
  • Yoshimi Shimada; Takamasa Kagawa; Takafumi Fuke; Asako Horinishi; Yoshihiko Ozaki; Hisashi Ashida
    Mem. Faculty. B.O.S.T. Kindai University (37) 1 - 10 2016/05 [Refereed]
  • Aina Gotoh; Toshihiko Katoh; Yuta Sugiyama; Shin Kurihara; Yuji Honda; Haruko Sakurama; Taiho Kambe; Hisashi Ashida; Motomitsu Kitaoka; Kenji Yamamoto; Takane Katayama
    CARBOHYDRATE RESEARCH ELSEVIER SCI LTD 408 18 - 24 0008-6215 2015/05 [Refereed]
     
    We describe the novel substrate specificities of two independently evolved lacto-N-biosidases (LnbX and LnbB) towards the sugar chains of globo-and ganglio-series glycosphingolipids. LnbX, a non-classified member of the glycoside hydrolase family, isolated from Bifidobacterium longum subsp. longum, was shown to liberate galacto-N-biose (GNB: Gal beta 1-3GalNAc) and 2'-fucosyl GNB (a type-4 trisaccharide) from Gb5 pentasaccharide and globo H hexasaccharide, respectively. LnbB, a member of the glycoside hydrolase family 20 isolated from Bifidobacterium bifidum, was shown to release GNB from Gb5 and GA1 oligosaccharides. This is the first report describing enzymatic release of beta-linked GNB from natural substrates. These unique activities may play a role in modulating the microbial composition in the gut ecosystem, and may serve as new tools for elucidating the functions of sugar chains of glycosphingolipids. (C) 2015 Elsevier Ltd. All rights reserved.
  • Yoshimi Shimada; Yuka Watanabe; Takura Wakinaka; Yoshihisa Funeno; Masayuki Kubota; Thida Chaiwangsri; Shin Kurihara; Kenji Yamamoto; Takane Katayama; Hisashi Ashida
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY SPRINGER 99 (9) 3941 - 3948 0175-7598 2015/05 [Refereed]
     
    alpha-Linked N-acetylglucosamine is one of the major glyco-epitopes in O-glycan of gastroduodenal mucin. Here, we identified glycoside hydrolase (GH) family 89 alpha-N-acetylglucosaminidase, termed AgnB, from Bifidobacterium bifidum JCM 1254, which is essentially specific to GlcNAc alpha 1-4Gal structure. AgnB is a membrane-anchored extracellular enzyme consisting of a GH89 domain and four carbohydrate-binding module (CBM) 32 domains. Among four CBM32 domains, three tandem ones at C-terminus showed to bind porcine gastric mucin, suggesting that these domains enhance the enzyme activity by increasing affinity for multivalent substrates. AgnB might be important for assimilation of gastroduodenal mucin by B. bifidum and also applicable to production of prebiotic oligosaccharides from porcine gastric mucin.
  • Yasushi Tani; Yasunori Amaishi; Tori Funatsu; Masahiro Ito; Saki Itonori; Yoji Hata; Hisashi Ashida; Kenji Yamamoto
    BIOTECHNOLOGY LETTERS SPRINGER 36 (12) 2507 - 2513 0141-5492 2014/12 [Refereed]
     
    Glucosylceramide and galactosylceramide were detected in three Aspergillus species: Aspergillus oryzae, Aspergillus sojae and Aspergillus. awamori, using borate-coated TLC. The cerebrosides from A. oryzae were further purified by ion exchange and iatrobeads column chromatographies with or without borate, and determined the composition of sugar, fatty acid and sphingoid base by GC/MS, MALDI-TOF/MS and H-1-NMR. We identified them as beta-glucosylceramide and beta-galactosylceramide. The ceramide moiety of both cerebrosides consisted mainly of 2-hydroxystearic acid and either 9-methyl-octadeca-4, 8-sphingadienine or octadeca-4, 8-sphingadienine. To our knowledge, this is the first study to provide evidence for the presence of beta-galactosylceramide in A. oryzae.
  • Haruko Sakurama; Shigenobu Kishino; Yoshie Uchibori; Yasunori Yonejima; Hisashi Ashida; Keiko Kita; Satomi Takahashi; Jun Ogawa
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY SPRINGER 98 (9) 4021 - 4032 0175-7598 2014/05 [Refereed]
     
    Baicalin (baicalein 7-O-beta-d-glucuronide) is one of the major flavonoid glucuronides found in traditional herbal medicines. Because its aglycone, baicalein, is absorbed more quickly and shows more effective properties than baicalin, the conversion of baicalin into baicalein by beta-glucuronidase (GUS) has drawn the attention of researchers. Recently, we have found that Lactobacillus brevis subsp. coagulans can convert baicalin to baicalein. Therefore, we aimed to identify and characterize the converting enzyme from L. brevis subsp. coagulans. First, we purified this enzyme from the cell-free extracts of L. brevis subsp. coagulans and cloned its gene. Surprisingly, this enzyme was found to be a GUS belonging to glycoside hydrolase (GH) family 30 (designated as LcGUS30), and its amino acid sequence has little similarity with any GUS belonging to GH families 1, 2, and 79 that have been reported so far. We then established a high-level expression and simple purification system of the recombinant LcGUS30 in Escherichia coli. The detailed analysis of the substrate specificity revealed that LcGUS30 has strict specificity toward glycon but not toward aglycones. Interestingly, LcGUS30 prefers baicalin rather than estrone 3-(beta-d-glucuronide), one of the human endogenous steroid hormones. These results indicated that L. brevis subsp. coagulans and LcGUS30 should serve as powerful tools for the construction of a safe bioconversion system for baicalin. In addition, we propose that this novel type of GUS forms a new group in subfamily 3 of GH family 30.
  • Toshihiko Katoh; Juri Takase; Yasushi Tani; Ryuta Amamoto; Naofumi Aoshima; Michael Tiemeyer; Kenji Yamamoto; Hisashi Ashida
    GLYCOBIOLOGY OXFORD UNIV PRESS INC 23 (10) 1142 - 1151 0959-6658 2013/10 [Refereed]
     
    Endoplasmic reticulum (ER) alpha-glucosidase I is an enzyme that trims the distal alpha 1,2-linked glucose (Glc) residue from the Glc(3)Man(9)GlcNAc(2) oligosaccharide following its addition to nascent glycoproteins in the initial step of processing. This reaction is critical to the subsequent processing of N-glycans and thus defects in alpha-glucosidase I gene in human cause congenital disorder of glycosylation (CDG) type IIb. We identified the Caenorhabditis elegans alpha-glucosidase I gene (F13H10.4, designated agl-1) that encodes a polypeptide with 36% identity to human alpha-glucosidase I. The agl-1 cDNA restored the expression of complex-type N-glycans on the cell surface of alpha-glucosidase I-defective Chinese hamster ovary Lec23 cells. RNAi knockdown of agl-1 [agl-1(RNAi)] produced worms that were visibly similar to wild-type, but life-span was reduced to about half of the control. Analyses of N-glycosylation in agl-1(RNAi) animals by western blotting and mass spectrometry showed reduction of paucimannose and complex-type glycans and dramatic increase of glucosylated oligomannose glycans. In addition, a significant amount of unusual terminally fucosylated N-glycans were found in agl-1(RNAi) animals. ER stress response was also provoked, leading to the accumulation of large amounts of triglucosylated free oligosaccharides (FOSs) (Glc(3)Man(4-5)GlcNAc(1-2)) in agl-1(RNAi) animals. Acceleration of ER-associated degradation in response to the accumulation of unfolded glycoproteins and insufficient interaction with calnexin/calreticulin in the ER lumen likely accounts for the increase of FOSs. Taken together, these studies in C. elegans demonstrate that decreased ER alpha-glucosidase I affects the entire N-glycan profile and induces chronic ER stress, which may contribute to the pathophysiology of CDG-IIb in humans.
  • Haruko Sakurama; Masashi Kiyohara; Jun Wada; Yuji Honda; Masanori Yamaguchi; Satoru Fukiya; Atsushi Yokota; Hisashi Ashida; Hidehiko Kumagai; Motomitsu Kitaoka; Kenji Yamamoto; Takane Katayama
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 288 (35) 25194 - 25206 0021-9258 2013/08 [Refereed]
     
    Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N- tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Gal beta 1-3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase(+) strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAc beta 1-3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fuc alpha 1-2Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc), and sialyllacto-N-tetraose a (Neu5Ac alpha 2-3Gal beta 1-3GlcNAc beta 1-3-Gal beta 1-4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-beta-lacto-N-bioside I (Gal beta 1-3-GlcNAc beta-pNP) and GalNAc beta 1-3GlcNAc beta-pNP.GalNAc beta 1-3-GlcNAc beta linkage has been found in O-mannosyl glycans of alpha-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca2+ and Mg2+) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.
  • Satoshi Murakami; Yuki Takaoka; Hisashi Ashida; Kenji Yamamoto; Hisashi Narimatsu; Yasunori Chiba
    Glycobiology 23 (6) 736 - 44 0959-6658 2013/06 [Refereed]
     
    In four yeast strains, Ogataea minuta, Candida parapolymorpha, Pichia anomala and Zygosaccharomyces rouxii, we identified endo-β-N-acetylglucosaminidase (ENGase) homologous sequences by database searches; in each of the four species, a corresponding enzyme activity was also confirmed in crude cell extract obtained from each strain. The O. minuta ENGase (Endo-Om)-encoding gene was directly amplified from O. minuta genomic DNA and sequenced. The Endo-Om-encoding gene contained a 2319-bp open-reading frame; the deduced amino acid sequence indicated that the putative protein belonged to glycoside hydrolase family 85. The gene was introduced into O. minuta, and the recombinant Endo-Om was overexpressed and purified. When the enzyme assay was performed using an agalacto-biantennary oligosaccharide as a substrate, Endo-Om exhibited both hydrolysis and transglycosylation activities. Endo-Om exhibited hydrolytic activity for high-mannose, hybrid, biantennary and (2,6)-branched triantennary N-linked oligosaccharides, but not for tetraantennary, (2,4)-branched triantennary, bisecting N-acetylglucosamine structure and core-fucosylated biantennary N-linked oligosaccharides. Endo-Om also was able to hydrolyze N-glycans attached to RNase B and human transferrin under both denaturing and nondenaturing conditions. Thus, the present study reports the detection and characterization of a novel yeast ENGase.
  • Tasuku Ito; Takane Katayama; Mitchell Hattie; Haruko Sakurama; Jun Wada; Ryuichiro Suzuki; Hisashi Ashida; Takayoshi Wakagi; Kenji Yamamoto; Keith A. Stubbs; Shinya Fushinobu
    Journal of Biological Chemistry 288 (17) 11795 - 11806 0021-9258 2013/04 [Refereed]
     
    Human milk oligosaccharides contain a large variety of oligosaccharides, of which lacto-N-biose I (Gal-β 1, 3-GlcNAc LNB) predominates as a major core structure. A unique metabolic pathway specific for LNB has recently been identified in the human commensal bifidobacteria. Several strains of infant gut-associated bifidobacteria possess lacto-N-biosidase, a membrane-anchored extracellular enzyme, that liberates LNB from the nonreducing end of human milk oligosaccharides and plays a key role in the metabolic pathway of these compounds. Lacto-N-biosidase belongs to the glycoside hydrolase family 20, and its reaction proceeds via a substrate-assisted catalytic mechanism. Several crystal structures of GH20 β-N-acetylhexosaminidases, which release monosaccharide GlcNAc from its substrate, have been determined, but to date, a structure of lacto-N-biosidase is unknown. Here, we have determined the first three-dimensional structures of lacto-N-biosidase from Bifidobacterium bifidum JCM1254 in complex with LNB and LNB-thiazoline (Gal-β1, 3-GlcNAc-thiazoline) at 1.8-Å resolution. Lacto-N-biosidase consists of three domains, and the C-terminal domain has a unique β-trefoil-like fold. Compared with other β-N-acetyl-hexosaminidases, lacto-N-biosidase has a wide substrate-binding pocket with a - 2 subsite specific for β-1, 3-linked Gal, and the residues responsible for Gal recognition were identified. The bound ligands are recognized by extensive hydrogen bonds at all of their hydroxyls consistent with the enzyme's strict substrate specificity for the LNB moiety. The GlcNAc sugar ring of LNB is in a distorted conformation near 4E, whereas that of LNB-thiazoline is in a 4C1 conformation. A possible conformational pathway for the lacto-N-biosidase reaction is discussed. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
  • Takura Wakinaka; Masashi Kiyohara; Shin Kurihara; Akiko Hirata; Thida Chaiwangsri; Takayuki Ohnuma; Tamo Fukamizo; Takane Katayama; Hisashi Ashida; Kenji Yamamoto
    GLYCOBIOLOGY OXFORD UNIV PRESS INC 23 (2) 232 - 240 0959-6658 2013/02 [Refereed]
     
    Bifidobacterium bifidum is one of the most frequently found bifidobacteria in the intestines of newborn infants. We previously reported that B. bifidum possesses unique metabolic pathways for O-linked glycans on gastrointestinal mucin (Yoshida E, Sakurama H, Kiyohara M, Nakajima M, Kitaoka M, Ashida H, Hirose J, Katayama T, Yamamoto K, Kumagai H. 2012. Bifidobacterium longum subsp. infantis uses two different beta-galactosidases for selectively degrading type-1 and type-2 human milk oligosaccharides. Glycobiology. 22: 361-368). The nonreducing termini of O-linked glycans on mucin are frequently covered with histo-blood group antigens. Here, we identified a gene agabb from B. bifidum JCM 1254, which encodes glycoside hydrolase (GH) family 110 alpha-galactosidase. AgaBb is a 1289-amino acid polypeptide containing an N-terminal signal sequence, a GH110 domain, a carbohydrate-binding module (CBM) 51 domain, a bacterial Ig-like (Big) 2 domain and a C-terminal transmembrane region, in this order. The recombinant enzyme expressed in Escherichia coli hydrolyzed alpha 1,3-linked Gal in branched blood group B antigen [Gal alpha 1-3(Fuca1-2)Gal beta 1-R], but not in a linear xenotransplantation antigen (Gal alpha 1-3Gal beta 1-R). The enzyme also acted on group B human salivary mucin and erythrocytes. We also revealed that CBM51 specifically bound blood group B antigen using both isothermal titration calorimetry and a solid-phase binding assay, and it enhanced the affinity of the enzyme toward substrates with multivalent B antigens. We suggest that this enzyme plays an important role in degrading B antigens to acquire nutrients from mucin oligosaccharides in the gastrointestinal tracts.
  • Haruko Sakurama; Erika Tsutsumi; Hisashi Ashida; Takane Katayama; Kenji Yamamoto; Hidehiko Kumagai
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY TAYLOR & FRANCIS LTD 76 (5) 1022 - 1024 0916-8451 2012/05 [Refereed]
     
    Recent studies suggest that alpha-L-fucosidases of glycoside hydrolase family 29 can be divided into two subfamilies based on substrate specificity and phylogenetic clustering. To explore the validity of this classification, we enzymatically characterized two structure-solved alpha-L-fucosidases representing the respective subfamilies. Differences in substrate specificities are discussed in relation to differences in active-site structures between the two enzymes.
  • Haruko Sakurama; Shinya Fushinobu; Masafumi Hidaka; Erina Yoshida; Yuji Honda; Hisashi Ashida; Motomitsu Kitaoka; Hidehiko Kumagai; Kenji Yamamoto; Takane Katayama
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 287 (20) 16709 - 16719 0021-9258 2012/05 [Refereed]
     
    alpha-L-Fucosyl residues attached at the non-reducing ends of glycoconjugates constitute histo-blood group antigens Lewis (Le) and ABO and play fundamental roles in various biological processes. Therefore, establishing a method for synthesizing the antigens is important for functional glycomics studies. However, regiospecific synthesis of glycosyl linkages, especially alpha-L-fucosyl linkages, is quite difficult to control both by chemists and enzymologists. Here, we generated an alpha-L-fucosynthase that specifically introduces Le(a) and Le(x) antigens into the type-1 and type-2 chains, respectively; i.e. the enzyme specifically accepts the disaccharide structures (Gal beta 1-3/4GlcNAc) at the non-reducing ends and attaches a Fuc residue via an alpha-(1,4/3)-linkage to the GlcNAc. X-ray crystallographic studies revealed the structural basis of this strict regio- and acceptor specificity, which includes the induced fit movement of the catalytically important residues, and the difference between the active site structures of 1,3-1,4-alpha-L-fucosidase (EC 3.2.1.111) and alpha-L-fucosidase (EC 3.2.1.51) in glycoside hydrolase family 29. The glycosynthase developed in this study should serve as a potentially powerful tool to specifically introduce the Le(a/x) epitopes onto labile glycoconjugates including glycoproteins. Mining glycosidases with strict specificity may represent the most efficient route to the specific synthesis of glycosidic bonds.
  • Erina Yoshida; Haruko Sakurama; Masashi Kiyohara; Masahiro Nakajima; Motomitsu Kitaoka; Hisashi Ashida; Junko Hirose; Takane Katayama; Kenji Yamamoto; Hidehiko Kumagai
    GLYCOBIOLOGY OXFORD UNIV PRESS INC 22 (3) 361 - 368 0959-6658 2012/03 [Refereed]
     
    The breast-fed infant intestine is often colonized by particular bifidobacteria, and human milk oligosaccharides (HMOs) are considered to be bifidogenic. Recent studies showed that Bifidobacterium longum subsp. infantis can grow on HMOs as the sole carbon source. This ability has been ascribed to the presence of a gene cluster (HMO cluster-1) contained in its genome. However, the metabolism of HMOs by the organism remains unresolved because no enzymatic studies have been completed. In the present study, we characterized beta-galactosidases of this subspecies to understand how the organism degrades type-1 (Gal beta 1-3GlcNAc) and type-2 (Gal beta 1-4GlcNAc) isomers of HMOs. The results revealed that the locus tag Blon_2016 gene, which is distantly located from the HMO cluster-1, encodes a novel beta-galactosidase (Bga42A) with a significantly higher specificity for lacto-N-tetraose (LNT; Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc) than for lacto-N-biose I (Gal beta 1-3GlcNAc), lactose (Lac) and type-2 HMOs. The proposed name of Bga42A is LNT beta-1,3-galactosidase. The Blon_2334 gene (Bga2A) located within the HMO cluster-1 encodes a beta-galactosidase specific for Lac and type-2 HMOs. Real-time quantitative reverse transcription-polymerase chain reaction analysis revealed the physiological significance of Bga42A and Bga2A in HMO metabolism. The organism therefore uses two different beta-galactosidases to selectively degrade type-1 and type-2 HMOs. Despite the quite rare occurrence in nature of beta-galactosidases acting on type-1 chains, the close homologs of Bga42A were present in the genomes of infant-gut associated bifidobacteria that are known to consume LNT. The predominance of type-1 chains in HMOs and the conservation of Bga42A homologs suggest the coevolution of these bifidobacteria with humans.
  • Wattana Sriphannam; Saisamorn Lumyong; Piyanuch Niumsap; Hisashi Ashida; Kenji Yamamoto; Chartchai Khanongnuch
    JOURNAL OF MICROBIOLOGY MICROBIOLOGICAL SOCIETY KOREA 50 (1) 119 - 126 1225-8873 2012/02 [Refereed]
     
    Lactic acid bacteria from healthy breast-fed infants were isolated and screened for beta-galactosidase production in MRS broth. Among 49 isolates that exhibited the yellow clear zone on MRS agar supplemented with bromocresol blue, the isolate CM33 was selected as being the highest beta-galactosidase producer and was identified as Lactobacillus fermentum based on its morphological characteristics and 16S rDNA nucleotide sequence. L. fermentum CM33 exhibited a good survival rate under the simulated stomach passage model, comparable to known probiotic strains L. gallinarum JCM2011 and L. agilis JCM1187. L. fermentum CM33 was antagonistic to pathogenic bacteria Listeria monocytogenes, Escherichia coli 0157:H7, Salmonella typhi, and Salmonella enteriditis, using the well diffusion method. In addition, the selected lactobacilli exhibited a high growth rate when cultivated in modified MRS containing commercial galactooligosaccharide (GOS) as a sole carbon source, as well as in glucose. A preliminary study on the enzymatic synthesis of oligosaccharide using crude beta-galactosidase revealed the capability for oligosaccharide synthesis by the transgalactosylation activity.
  • Masashi Kiyohara; Takashi Nakatomi; Shin Kurihara; Shinya Fushinobu; Hideyuki Suzuki; Tomonari Tanaka; Shin-ichiro Shoda; Motomitsu Kitaoka; Takane Katayama; Kenji Yamamoto; Hisashi Ashida
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 287 (1) 693 - 700 0021-9258 2012/01 [Refereed]
     
    Bifidobacteria inhabit the lower intestine of mammals including humans where the mucin gel layer forms a space for commensal bacteria. We previously identified that infant-associated bifidobacteria possess an extracellular membrane-bound endo-alpha-N-acetylgalactosaminidase (EngBF) that may be involved in degradation and assimilation of mucin-type oligosaccharides. However, EngBF is highly specific for core-1-type O-glycan (Gal beta 1-3GalNAc alpha 1-Ser/Thr), also called T antigen, which is mainly attached onto gastroduodenal mucins. By contrast, core-3-type O-glycans (GlcNAc beta 1-3GalNAc alpha 1-Ser/Thr) are predominantly found on the mucins in the intestines. Here, we identified a novel alpha-N-acetylgalactosaminidase (NagBb) from Bifidobacterium bifidum JCM 1254 that hydrolyzes the Tn antigen (GalNAc alpha 1-Ser/Thr). Sialyl and galactosyl core-3 (Gal beta 1-3/4GlcNAc beta 1-3(Neu5Ac alpha 2-6)GalNAc alpha 1-Ser/Thr), a major tetrasaccharide structure on MUC2 mucin primarily secreted from goblet cells in human sigmoid colon, can be serially hydrolyzed into Tn antigen by previously identified bifidobacterial extracellular glycosidases such as alpha-sialidase (SiaBb2), lacto-N-biosidase (LnbB), beta-galactosidase (BbgIII), and beta-N-acetylhexosaminidases (BbhI and BbhII). Because NagBb is an intracellular enzyme without an N-terminal secretion signal sequence, it is likely involved in intracellular degradation and assimilation of Tn antigen-containing polypeptides, which might be incorporated through unknown transporters. Thus, bifidobacteria possess two distinct pathways for assimilation of O-glycans on gastroduodenal and intestinal mucins. NagBb homologs are conserved in infant-associated bifidobacteria, suggesting a significant role for their adaptation within the infant gut, and they were found to form a new glycoside hydrolase family 129.
  • Medium component improvement for β-galactosidase production by a probiotic strain
    Sriphannam W; Unban K; Ashida H; Yamamoto K; Khanongnuch C
    African Journal of Biotechnology 11 (51) 11242 - 11251 2012/01 [Refereed]
  • Fumiki Nakatani; Yasu S. Morita; Hisashi Ashida; Kisaburo Nagamune; Yusuke Maeda; Taroh Kinoshita
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 415 (2) 421 - 425 0006-291X 2011/11 [Refereed]
     
    The procyclic stage of Trypanosoma brucei is covered by glycosylphosphatidylinositol (GPI)-anchored surface proteins called procyclins. The procyclin GPI anchor contains a side chain of N-acetyllactosamine repeats terminated by sialic acids. Sialic acid modification is mediated by trans-sialidases expressed on the parasite's cell surface. Previous studies suggested the presence of more than one active trans-sialidases, but only one has so far been reported. Here we cloned and examined enzyme activities of four additional trans-sialidase homologs, and show that one of them, Tb927.8.7350, encodes another active transsialidase, designated as TbSA C2. In an in vitro assay, TbSA C2 utilized alpha 2-3 sialyllactose as a donor, and produced an alpha 2-3-sialylated product, suggesting that it is an alpha 2-3 trans-sialidase. We suggest that TbSA C2 plays a role in the sialic acid modification of the trypanosome cell surface. (C) 2011 Elsevier Inc. All rights reserved.
  • Sadaki Asakuma; Emi Hatakeyama; Tadasu Urashima; Erina Yoshida; Takane Katayama; Kenji Yamamoto; Hidehiko Kumagai; Hisashi Ashida; Junko Hirose; Motomitsu Kitaoka
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 286 (40) 34583 - 34592 0021-9258 2011/10 [Refereed]
     
    The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono-and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.
  • Mitsuharu Matsumoto; Shin Kurihara; Ryoko Kibe; Hisashi Ashida; Yoshimi Benno
    PLOS ONE PUBLIC LIBRARY SCIENCE 6 (8) e23652  1932-6203 2011/08 [Refereed]
     
    Background: Chronic low-grade inflammation is recognized as an important factor contributing to senescence and age-related diseases. In mammals, levels of polyamines (PAs) decrease during the ageing process; PAs are known to decrease systemic inflammation by inhibiting inflammatory cytokine synthesis in macrophages. Reductions in intestinal luminal PAs levels have been associated with intestinal barrier dysfunction. The probiotic strain Bifidobacterium animalis subsp. lactis LKM512 is known to increase intestinal luminal PA concentrations. Methodology/Principal Findings: We supplemented the diet of 10-month-old Crj:CD-1 female mice with LKM512 for 11 months, while the controls received no supplementation. Survival rates were compared using Kaplan-Meier survival curves. LKM512-treated mice survived significantly longer than controls (P<0.001); moreover, skin ulcers and tumors were more common in the control mice. We then analyzed inflammatory and intestinal conditions by measuring several markers using HPLC, ELISA, reverse transcription-quantitative PCR, and histological slices. LKM512 mice showed altered 16S rRNA gene expression of several predominant intestinal bacterial groups. The fecal concentrations of PAs, but not of short-chain fatty acids, were significantly higher in LKM512-treated mice (P<0.05). Colonic mucosal function was also better in LKM512 mice, with increased mucus secretion and better maintenance of tight junctions. Changes in gene expression levels were evaluated using the NimbleGen mouse DNA microarray. LKM512 administration also downregulated the expression of ageing-associated and inflammation-associated genes and gene expression levels in 21-month-old LKM512-treated mice resembled those in 10-month-old untreated (younger) mice. Conclusion/Significance: Our study demonstrated increased longevity in mice following probiotic treatment with LKM512, possibly due to the suppression of chronic low-grade inflammation in the colon induced by higher PA levels. This indicates that ingestion of specific probiotics may be an easy approach for improving intestinal health and increasing lifespan. Further studies are required to clarify its effectiveness in humans.
  • Masashi Kiyohara; Kana Tanigawa; Thida Chaiwangsri; Takane Katayama; Hisashi Ashida; Kenji Yamamoto
    GLYCOBIOLOGY OXFORD UNIV PRESS INC 21 (4) 437 - 447 0959-6658 2011/04 [Refereed]
     
    Bifidobacteria are health-promoting enteric commensals that are assumed to proliferate predominantly in the intestines of breast-fed infants by assimilating human milk oligosaccharides (HMOs) that are frequently fucosylated and/or sialylated. We previously identified two different alpha-l-fucosidases in Bifidobacterium bifidum and showed that the strain furnishes an extracellular degradation pathway for fucosylated HMOs. However, the catabolism of sialylated HMOs by bifidobacteria has remained unresolved. Here we describe the identification and characterization of an exo-alpha-sialidase in bifidobacteria. By expression cloning, we isolated a novel exo-alpha-sialidase gene (siabb2) from B. bifidum JCM1254, which encodes a protein (SiaBb2) consisting of 835-amino-acid residues with a predicted molecular mass of 87 kDa. SiaBb2 possesses an N-terminal signal sequence, a sialidase catalytic domain classified into the glycoside hydrolase family 33 (GH33) and a C-terminal transmembrane region, indicating that the mature SiaBb2 is an extracellular membrane-anchored enzyme. The recombinant enzyme expressed in Escherichia coli showed the highest activity in an acidic pH range from 4.0 to 5.0, and at 50 degrees C. Notably, 80% activity remained after 30 min incubation at 80 degrees C, indicating that the enzyme is highly thermostable. SiaBb2 liberated sialic acids from sialyloligosaccharides, gangliosides, glycoproteins and colominic acid; however, the linkage preference of the enzyme was remarkably biased toward the alpha 2,3-linkage rather than alpha 2,6- and alpha 2,8-linkages. Expression of siabb2 in B. longum 105-A, which has no endogenous exo-alpha-sialidase, enabled this strain to degrade sialyloligosaccharides present in human milk. Our results suggest that SiaBb2 plays a crucial role in bifidobacterial catabolism of sialylated HMOs.
  • Masaya Fujita; Akiko Tsuchida; Akiko Hirata; Natsumi Kobayashi; Kohtaro Goto; Kenji Osumi; Yuriko Hirose; Jun Nakayama; Takashi Yamanoi; Hisashi Ashida; Mamoru Mizuno
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 286 (8) 6479 - 6489 0021-9258 2011/02 [Refereed]
     
    In mammals, alpha-linked GlcNAc is primarily found in heparan sulfate/heparin and gastric gland mucous cell type mucin. alpha-N-Acetylglucosaminidases (alpha GNases) belonging to glycoside hydrolase family 89 are widely distributed from bacteria to higher eukaryotes. Human lysosomal alpha GNase is well known to degrade heparin and heparan sulfate. Here, we reveal the substrate specificity of alpha GNase (AgnC) from Clostridium perfringens strain 13, a bacterial homolog of human alpha GNase, by chemically synthesizing a series of disaccharide substrates containing alpha-linked GlcNAc. AgnC was found to release GlcNAc from GlcNAc alpha 1,4Gal beta 1pMP and GlcNAc alpha 1pNP substrates (where pMP and pNP represent p-methoxyphenyl and p-nitrophenyl, respectively). AgnC also released GlcNAc from porcine gastric mucin and cell surface mucin. Because AgnC showed no activity against any of the GlcNAc alpha 1,2Gal beta 1pMP, GlcNAc alpha 1,3Gal beta 1pMP, GlcNAc alpha 1,6Gal beta 1pMP, and GlcNAc alpha 1,4GlcA beta 1pMP substrates, this enzyme may represent a specific glycosidase required for degrading alpha-GlcNAc-capped O-glycans of the class III mucin secreted from the stomach and duodenum. Deletion of the C-terminal region containing several carbohydrate-binding module 32 (CBM32) domains significantly reduced the activity for porcine gastric mucin; however, activity against GlcNAc alpha 1,4Gal beta 1pMP was markedly enhanced. Dot blot and ELISA analyses revealed that the deletion construct containing the C-terminal CBM-C2 to CBM-C6 domains binds strongly to porcine gastric mucin. Consequently, tandem CBM32 domains located near the C terminus of AgnC should function by increasing the affinity for branched or clustered alpha-GlcNAc-containing glycans. The agnC gene-disrupted strain showed significantly reduced growth on the class III mucin-containing medium compared with the wild type strain, suggesting that AgnC might have an important role in dominant growth in intestines.
  • Mika Miwa; Tomohiro Horimoto; Masashi Kiyohara; Takane Katayama; Motomitsu Kitaoka; Hisashi Ashida; Kenji Yamamoto
    Glycobiology OXFORD UNIV PRESS INC 20 (11) 1402 - 1409 0959-6658 2010/11 [Refereed]
     
    Bifidobacteria are predominant in the intestines of breast-fed infants and offer health benefits to the host. Human milk oligosaccharides (HMOs) are considered to be one of the most important growth factors for intestinal bifidobacteria. HMOs contain two major structures of core tetrasaccharide: lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc; type 1 chain) and lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc; type 2 chain). We previously identified the unique metabolic pathway for lacto-N-tetraose in Bifidobacterium bifidum. Here, we clarified the degradation pathway for lacto-N-neotetraose in the same bifidobacteria. We cloned one beta-galactosidase (BbgIII) and two beta-N-acetylhexosaminidases (BbhI and BbhII), all of which are extracellular membrane-bound enzymes. The recombinant BbgIII hydrolyzed lacto-N-neotetraose into Gal and lacto-N-triose II, and furthermore the recombinant BbhI, but not BbhII, catalyzed the hydrolysis of lacto-N-triose II to GlcNAc and lactose. Since BbgIII and BbhI were highly specific for lacto-N-neotetraose and lacto- N-triose II, respectively, they may play essential roles in degrading the type 2 oligosaccharides in HMOs.
  • Umekawa M; Higashiyama T; Koga Y; Tanaka T; Noguchi M; Kobayashi A; Shoda S; Huang W; Wang LX; Ashida H; Yamamoto K
    Biochim. Biophys. Acta General Subjects 11 1800 1203 - 1209 0006-3002 2010/11 [Refereed]
  • Ryuichiro Suzuki; Takane Katayama; Byung-Jun Kim; Takayoshi Wakagi; Hirofumi Shoun; Hisashi Ashida; Kenji Yamamoto; Shinya Fushinobu
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 285 (44) 34279 - 34287 0021-9258 2010/10 [Refereed]
     
    Thiamine diphosphate (ThDP)-dependent enzymes are ubiquitously present in all organisms and catalyze essential reactions in various metabolic pathways. ThDP-dependent phosphoketolase plays key roles in the central metabolism of heterofermentative bacteria and in the pentose catabolism of various microbes. In particular, bifidobacteria, representatives of beneficial commensal bacteria, have an effective glycolytic pathway called bifid shunt in which 2.5 mol of ATP are produced per glucose. Phosphoketolase catalyzes two steps in the bifid shunt because of its dual-substrate specificity; they are phosphorolytic cleavage of fructose 6-phosphate or xylulose 5-phosphate to produce aldose phosphate, acetyl phosphate, and H(2)O. The phosphoketolase reaction is different from other well studied ThDP-dependent enzymes because it involves a dehydration step. Although phosphoketolase was discovered more than 50 years ago, its three-dimensional structure remains unclear. In this study we report the crystal structures of xylulose 5-phosphate/ fructose 6-phosphate phosphoketolase from Bifidobacterium breve. The structures of the two intermediates before and after dehydration (alpha,beta-dihydroxyethyl ThDP and 2-acetylThDP) and complex with inorganic phosphate give an insight into the mechanism of each step of the enzymatic reaction.
  • Ryuichiro Suzuki; Byung-Jun Kim; Tsuyoshi Shibata; Yuki Iwamoto; Takane Katayama; Hisashi Ashida; Takayoshi Wakagi; Hirofumi Shoun; Shinya Fushinobu; Kenji Yamamoto
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS WILEY-BLACKWELL PUBLISHING, INC 66 941 - 943 1744-3091 2010/08 [Refereed]
     
    The xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene from Bifidobacterium breve was cloned and overexpressed in Escherichia coli. The enzyme was purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method. Crystals were obtained at 293 K using 0.05 mM thiamine diphosphate, 0.25 mM MgCl(2), 24%(w/v) PEG 6000 and 0.1 M Bicine pH 9.0. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 174.8, c = 163.8 A, and diffracted to beyond 1.7 A resolution.
  • Myco Umemura; Yutaka Makimura; Masae Itoh; Takeshi Yamamoto; Toshiki Mine; Seiji Mitani; Ichiro Simizu; Hisashi Ashida; Kenji Yamamoto
    CARBOHYDRATE POLYMERS ELSEVIER SCI LTD 81 (2) 330 - 334 0144-8617 2010/06 
    We have succeeded in one-step synthesis of an efficient binding-inhibitor for influenza virus, which is composed of only sugar chains. This binding-inhibitor utilizes the carbohydrate recognition of influenza virus, thus it can prevent the virus from infection. We modified chitosan with multiple sialyl saccharides, alpha 2,6-sialyllactose or free sialyl glycan, using reductive amination reaction. The resulting inhibitors showed sufficient inhibitory activity against influenza virus infection in MDCK cells compared to that of alpha 2,6-sialyllactose or free sialyl glycan. Unlike the other binding-inhibitors of influenza virus, this virus inhibitor of sugar chains requires only one step in its synthesis. Therefore this inhibitor is suitable for use in products such as filters and masks. (C) 2010 Elsevier Ltd. All rights reserved.
  • Yasushi Tani; Tori Funatsu; Hisashi Ashida; Masahiro Ito; Saki Itonori; Mutsumi Sugita; Kenji Yamamoto
    GLYCOBIOLOGY OXFORD UNIV PRESS INC 20 (4) 433 - 441 0959-6658 2010/04 [Refereed]
     
    Hirsutella rhossiliensis, a nematophagous fungus belonging to the Ascomycota, is resistant to aureobasidin A (AbA). In this fungus, the biosynthetic pathway leading to mannosylinositolphosphoceramides, which is inhibited by AbA, was not detected. Instead, this fungus contains neutral complex glycosphingolipids (GSLs) and monoglycosylceramides. Except for monoglycosylceramides, neutral GSLs share a neogala-series core structure, Gal beta 1-6Gal beta 1-Cer. Among the GSLs of H. rhossiliensis, three novel GSLs with terminal Man and Glc residues on the sugar chain were elucidated. We analyzed GSL structure using compositional sugar, fatty acid, and sphingoid analyses, methylation analysis, matrix-assisted laser desorption ionization time-of-flight/mass spectrometry (MALDI-TOF MS), and (1)H nuclear magnetic resonance spectroscopy (NMR). The following structures were determined: Man alpha 1-3Gal beta 1-6Gal beta 1-6Gal beta 1-Cer; Glc alpha 1-2Gal beta 1-6Gal beta 1-6Gal beta 1-Cer; and Man alpha 1-3Gal beta 1-6(Glc alpha 1-4)Gal beta 1-6Gal beta 1-Cer. In the ceramides, the fatty acids were predominantly saturated h24:0-acids and the sphingoids were predominately t18:0- or t18:1-sphingoids. In contrast, the ceramides of Glc beta 1-Cer contained d18:2- and d19:2-sphingoids. These findings indicate the presence of a novel biosynthetic pathway of neogala-series GSLs in fungi.
  • Fujimoto Taku; Kiyohara Masashi; Ashida Hisashi; Yamamoto Kenji
    Journal of Applied Glycoscience Supplement The Japanese Society of Applied Glycoscience 2010 68 - 68 2010
  • Nakatomi Takashi; Kiyohara Masashi; Ashida Hisashi; Yamamoto Kenji
    Journal of Applied Glycoscience Supplement The Japanese Society of Applied Glycoscience 2010 69 - 69 2010
  • Hisashi Ashida; Hayato Ozawa; Kiyotaka Fujita; Shun'ichi Suzuki; Kenji Yamamoto
    GLYCOCONJUGATE JOURNAL SPRINGER 27 (1) 125 - 132 0282-0080 2010/01 [Refereed]
     
    Endo-alpha-N-acetylgalactosaminidase catalyzes the release of Gal beta 1-3GalNAc from the core 1-type O-glycan (Gal beta 1-3GalNAc alpha 1-Ser/Thr) of mucin glycoproteins and synthetic p-nitrophenyl (pNP) alpha-linked substrates. Here, we report the enzymatic syntheses of core 1 disaccharide-containing glycopeptides using the transglycosylation activity of endo-alpha-N-acetylgalactosaminidase (EngBF) from Bifidobacterium longum. The enzyme directly transferred Gal beta 1-3GalNAc to serine or threonine residues of bioactive peptides such as PAMP-12, bradykinin, peptide-T and MUC1a when Gal beta 1-3GalNAc alpha 1-pNP was used as a donor substrate. The enzyme was also found to catalyze the reverse-hydrolysis reaction. EngBF synthesized the core 1 disaccharide-containing oligosaccharides when the enzyme was incubated with either glucose or lactose and Gal beta 1-3GalNAc prepared from porcine gastric mucin using bifidobacterial cells expressing endo-alpha-N-acetylgalactosaminidase. Synthesized oligosaccharides are promising prebiotics for bifidobacteria.
  • Midori Umekawa; Cishan Li; Takayuki Higashiyama; Wei Huang; Hisashi Ashida; Kenji Yamamoto; Lai-Xi Wang
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 285 (1) 511 - 521 0021-9258 2010/01 [Refereed]
     
    Endo-M, an endo-beta-N-acetylglucosaminidase from Mucor hiemalis, is a family 85 glycoside hydrolase. This enzyme is unique in that it can transfer en bloc the oligosaccharide of various types of N-glycans onto different acceptors, and thereby it enzymatically generates diverse glycoconjugates. In this study, we performed mutational and kinetic studies focusing on a key catalytic asparagine 175 of Endo-M. We have shown that most of the Asn-175 mutants had significantly diminished hydrolysis activity but acted as glycosynthases capable of using synthetic sugar oxazoline for transglycosylation. Our results confirm the critical role of this asparagine residue in promoting the formation of an oxazolinium ion intermediate in the first step of the substrate-assisted catalysis. Interestingly, the N175Q mutant was found to possess dramatically enhanced glycosynthase-like activity with sugar oxazoline in comparison with N175A and a transglycosidase-like activity with "natural" N-glycan as well. These results also implicated the significance of amide side chain in the asparagine 175 of Endo-M for promoting oxazoline transglycosylation in the second step of the catalysis. The highly efficient syntheses of glycopeptides/glycoproteins by N175Q combined with synthetic sugar oxazolines or natural N-glycan substrates were exemplified. In addition, we also identified several previously unknown residues that seem to play a role in the catalysis of Endo-M.
  • Tomoya Shintani; Fumiyoshi Yamazaki; Toshihiko Katoh; Midori Umekawa; Yoshiharu Matahira; Seiji Hori; Akira Kakizuka; Kazuhide Totani; Kenji Yamamoto; Hisashi Ashida
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 391 (4) 1775 - 1779 0006-291X 2010/01 [Refereed]
     
    Autophagy is a cellular process that nonspecifically degrades cytosolic components and is involved in many cellular responses. We found that amino sugars with a free amino group such as glucosamine, galactosamine and mannosamine induced autophagy via an rnTOR-independent pathway. Glucosamine-induced autophagy at concentrations of at least 500 mu M to over 40 mM. In the presence of 40 mM glucosamine, autophagy induction was initiated at 6 h and reached a plateau at 36 h. Glucosamine-induced autophagy could remove accumulated ubiquitin-conjugated proteins as well as 79-glutamine repeats. Therefore, Orally administered glucosamine could contribute to the prevention of neurodegenerative diseases and promotion of antiaging effects. (C) 2010 Elsevier Inc. All rights reserved.
  • Jun Wada; Takuro Ando; Masashi Kiyohara; Hisashi Ashida; Motomitsu Kitaoka; Masanori Yamaguchi; Hidehiko Kumagai; Takane Katayama; Kenji Yamamoto
    Applied and Environmental Microbiology 75 (19) 6414  0099-2240 2009/10 [Refereed]
  • Suzuki R; Katayama T; Kitaoka M; Kumagai H; Wakagi T; Shoun H; Ashida H; Yamamoto K; Fushinobu S
    J. Biochem. (Tokyo) 3 146 (3) 389 - 398 0021-924X 2009/09 [Refereed]
  • Hisashi Ashida; Akiko Miyake; Masashi Kiyohara; Jun Wada; Erina Yoshida; Hidehiko Kumagai; Takane Katayama; Kenji Yamamoto
    GLYCOBIOLOGY OXFORD UNIV PRESS INC 19 (9) 1010 - 1017 0959-6658 2009/09 [Refereed]
     
    Bifidobacteria are predominant bacteria present in the intestines of breast-fed infants and offer important health benefits for the host. Human milk oligosaccharides are one of the most important growth factors for bifidobacteria and are frequently fucosylated at their non-reducing termini. Previously, we identified 1,2-alpha-l-fucosidase (AfcA) belonging to the novel glycoside hydrolase (GH) family 95, from Bifidobacterium bifidum JCM1254 (Katayama T, Sakuma A, Kimura T, Makimura Y, Hiratake J, Sakata K, Yamanoi T, Kumagai H, Yamamoto K. 2004. Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-l-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95). J Bacteriol. 186:4885-4893). Here, we identified a gene encoding a novel 1,3-1,4-alpha-l-fucosidase from the same strain and termed it afcB. The afcB gene encodes a 1493-amino acid polypeptide containing an N-terminal signal sequence, a GH29 alpha-l-fucosidase domain, a carbohydrate binding module (CBM) 32 domain, a found-in-various-architectures (FIVAR) domain and a C-terminal transmembrane region, in this order. The recombinant enzyme was expressed in Escherichia coli and was characterized. The enzyme specifically released alpha 1,3- and alpha 1,4-linked fucosyl residues from 3-fucosyllactose, various Lewis blood group substances (a, b, x, and y types), and lacto-N-fucopentaose II and III. However, the enzyme did not act on glycoconjugates containing alpha 1,2-fucosyl residue or on synthetic alpha-fucoside (p-nitrophenyl-alpha-l-fucoside). The afcA and afcB genes were introduced into the B. longum 105-A strain, which has no intrinsic alpha-l-fucosidase. The transformant carrying afcA could utilize 2'-fucosyllactose as the sole carbon source, whereas that carrying afcB was able to utilize 3-fucosyllactose and lacto-N-fucopentaose II. We suggest that AfcA and AfcB play essential roles in degrading alpha 1,2- and alpha 1,3/4-fucosylated milk oligosaccharides, respectively, and also glycoconjugates, in the gastrointestinal tracts.
  • Dirk J. Lefeber; Johannes Schoenberger; Eva Morava; Mailys Guillard; Karin M. Huyben; Kiek Verriip; Olga Grafakou; Athanasios Evangelioi; Frank W. Preijers; Panagiota Manta; Jef Yildiz; Stephanie Gruenewald; Martha Spilioti; Christa van den Elzen; Dominique Klein; Daniel Hess; Hisashi Ashida; Jan Hofsteenge; Yusuke Maeda; Lambert van den Heuvel; Martin Lammens; Ludwig Lehle; Ron A. Wevers
    AMERICAN JOURNAL OF HUMAN GENETICS CELL PRESS 85 (1) 76 - 86 0002-9297 2009/07 [Refereed]
     
    Alpha-dystroglycanopathies such as Walker Warburg syndrome represent an important subgroup of the muscular dystrophies that have been related to defective O-mannosylation of alpha-dystroglycan. In many patients, the underlying genetic etiology remains unsolved. Isolated muscular dystrophy has not been described in the congenital disorders of glycosylation (CDG) caused by N-linked protein glycosylation defects. Here, we present a genetic N-glycosylation disorder with muscular dystrophy in the group of CDG type I. Extensive biochemical investigations revealed a strongly reduced dolichol-phosphate-mannose (Dol-P-Man) synthase activity. Sequencing of the three DPM subunits and complementation of DPM3-deficient CHO2.38 cells showed a pathogenic p.L85S missense mutation in the strongly conserved coiled-coil domain of DPM3 that tethers catalytic DPM1 to the ER membrane. Cotransfection experiments in CHO cells showed a reduced binding capacity of DPM3(L85S) for DPM1. Investigation of the four Dol-P-Man-dependent glycosylation pathways in the ER revealed strongly reduced O-mannosylation of alpha-dystroglycan in a muscle biopsy, thereby explaining the clinical phenotype of muscular dystrophy. This mild Dol-P-Man biosynthesis defect due to DPM3 mutations is a cause for alpha-dystroglycanopathy, thereby bridging the congenital disorders of glycosylation with the dystroglycanopathies.
  • Masashi Kiyohara; Asaki Tachizawa; Mamoru Nishimoto; Motomitsu Kitaoka; Hisashi Ashida; Kenji Yamamoto
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY TAYLOR & FRANCIS LTD 73 (5) 1175 - 1179 0916-8451 2009/05 [Refereed]
     
    We demonstrated the prebiotic effect of lacto-N-biose I (Gal beta 1-3GlcNAc) on bifidobacteria in vitro. Lacto-N-biose I, a building unit of the type-I milk oligosaccharides, enhanced the growth of many bifidobacteria, especially Bifidobacterium bifidum, B. breve, and B. longum, which are predominant in the intestines of breast-fed infants. It might be a substantial, natural prebiotic in human colostrums.
  • Fujita Masaya; Tsuchida Akiko; Goto Kohtaro; Osumi Kenji; Mizuno Mamoru; Ashida Hisashi; Nakayama Jun
    Journal of Applied Glycoscience Supplement The Japanese Society of Applied Glycoscience 2009 70 - 70 2009
  • Suzuki Ryuichiro; Katayama Takane; Ashida Hisashi; Yamamoto Kenji; Kitaoka Motomitsu; Kumagai Hidehiko; Wakagi Takayoshi; Shoun Hirofumi; Fushinobu Shinya
    Journal of Applied Glycoscience Supplement The Japanese Society of Applied Glycoscience 2009 71 - 71 2009
  • Miwa Mika; Horimoto Tomohito; Kiyohara Masashi; Ashida Hisashi; Yamamoto Kenji
    Journal of Applied Glycoscience Supplement The Japanese Society of Applied Glycoscience 2009 73 - 73 2009
  • Ito Tasuku; Suzuki Ryuichiro; Katayama Takane; Wakagi Takayoshi; Shoun Hirofumi; Ashida Hisashi; Yamamoto Kenji; Kitaoka Motomitsu; Fushinobu Shinya
    Journal of Applied Glycoscience Supplement The Japanese Society of Applied Glycoscience 2009 72 - 72 2009
  • Nihira Takanori; Katayama Takane; Ashida Hisashi; Yamamoto Kenji; Yaoi Katsuro; Mitsuishi Yasushi; Kitaoka Motomitsu
    Journal of Applied Glycoscience Supplement The Japanese Society of Applied Glycoscience 2009 84 - 84 2009
  • Trechiama ion (Coleoptera, Trechinae), a remarkable new species from Chûgoku District, West Japan
    Ashida H; Souma A
    Elytra, Tokyo 36 381 - 386 2008/12
  • Hisashi Ashida; Riichi Maki; Hayato Ozawa; Yasushi Tani; Masashi Kiyohara; Masaya Fujita; Akihiro Imamura; Hideharu Ishida; Makoto Kiso; Kenji Yamamoto
    GLYCOBIOLOGY OXFORD UNIV PRESS INC 18 (9) 727 - 734 0959-6658 2008/09 [Refereed]
     
    Endo-alpha-N-acetylgalactosaminidase (endo-alpha-GalNAc-ase) catalyzes the hydrolysis of the O-glycosidic bond between alpha-GalNAc at the reducing end of mucin-type sugar chains and serine/threonine of proteins to release oligosaccharides. Previously, we identified the gene en-gBF encoding endo-alpha-GalNAc-ase from Bifidobacterium longum, which specifically released the disaccharide Gal beta 1-3GalNAc (Fujita K, Oura F, Nagamine N, Katayama T, Hiratake J, Sakata K, Kumagai H, Yamamoto K. 2005. Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-alpha-N-acetylgalactosaminidase from Bifidobacterium longum. J Biol Chem. 280:37415-37422). Here we cloned a similar gene named engCP from Clostridium perfringens, a pathogenic enterobacterium, and characterized the gene product EngCP. Detailed analyses on substrate specificities of EngCP and EngBF using a series of p-nitrophenyl-alpha-glycosides chemically synthesized by the di-tert-butylsilyiene-directed method revealed that both enzymes released Hex/HexNAc beta 1-3GalNAc (Hex = Gal or Glc). EngCP could also release the core 2 trisaccharide Gal beta 1-3(GlcNAc beta 1-6)GalNAc, core 8 disaccharide Gal alpha 1-3GalNAc, and monosaccharide GalNAc. Our results suggest that EngCP possesses broader substrate specificity than EngBF. Actions of the two enzymes on native glycoproteins and cell surface glycoproteins were also investigated.
  • Myco Umemura; Masae Itoh; Yutaka Makimura; Kohji Yamazaki; Midori Umekawa; Ayano Masui; Yoshiharu Matahira; Mari Shibata; Hisashi Ashida; Kenji Yamamoto
    JOURNAL OF MEDICINAL CHEMISTRY AMER CHEMICAL SOC 51 (15) 4496 - 4503 0022-2623 2008/08 [Refereed]
     
    We verified here the inhibitory activity of a sialylglycopolymer prepared from natural products, chitosan and hen egg yolk, against influenza virus infection and estimated the requirements of the molecule for efficient inhibition. The inhibitory activity clearly depended on two factors, the length (the degree of polymerization: DP) of the chitosan backbone and the amount (the degree of substitution: DS) of conjugated sialyloligosaccharide side chain. The inhibitory efficiency increased in accordance with the DP value, with the highest inhibitory activity obtained when the DP was 1430. The inhibition of virus infection reached more than 90% as the DS value increased up to 15.6% when the neighboring sialyloligosaccharide side chains came as close as 4 nm, which was nearly the distance between two receptor-binding pockets in a hemagglutinin trimer. These results demonstrate that the sialylglycopolymer could be an excellent candidate of the safe and efficient anti-influenza drug.
  • Jun Wada; Takuro Ando; Masashi Kiyohara; Hisashi Ashida; Motomitsu Kitaoka; Masanori Yamaguchi; Hidehiko Kumagai; Takane Katayama; Kenji Yamamoto
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY AMER SOC MICROBIOLOGY 74 (13) 3996 - 4004 0099-2240 2008/07 [Refereed]
     
    Breast-fed infants often have intestinal microbiota dominated by bifidobacteria in contrast to formula-fed infants. We found that several bifidobacterial strains produce a lacto-N-biosidase that liberates lacto-N-biose I (Gal beta 1,3GlcNAc; type 1 chain) from lacto-N-tetraose (Gal beta 1,3GlcNAc beta 1,3Gal beta 1,4G1c), which is a major component of human milk oligosaccharides, and subsequently isolated the gene from Bifidobacterium bifidum JCM1254. The gene, designated lnbB, was predicted to encode a protein of 1,112 amino acid residues containing a signal peptide and a membrane anchor at the N and C termini, respectively, and to possess the domain of glycoside hydrolase family 20, carbohydrate binding module 32, and bacterial immunoglobulin-like domain 2, in that order, from the N terminus. The recombinant enzyme showed substrate preference for the unmodified P-linked lacto-N-biose I structure. Lacto-N-biosidase activity was found in several bifidobacterial strains, but not in the other enteric bacteria, such as clostridia, bacteroides, and lactobacilli, under the tested conditions. These results, together with our recent finding of a novel metabolic pathway specific for lacto-N-biose I in bifidobacterial cells, suggest that some of the bifidobacterial strains are highly adapted for utilizing human milk oligosaccharides with a type I chain.
  • Ryuichiro Suzuki; Jun Wada; Takane Katayama; Shinya Fushinobu; Takayoshi Wakagi; Hirofumi Shoun; Hayuki Sugimoto; Akiyoshi Tanaka; Hidehiko Kumagai; Hisashi Ashida; Motomitsu Kitaoka; Kenji Yamamoto
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 283 (19) 13165 - 13173 0021-9258 2008/05 [Refereed]
     
    Recently, a gene cluster involving a phosphorylase specific for lacto-N-biose I (LNB; Gal beta 1-3GlcNAc) and galacto-N-biose (GNB; Gal beta 1-3GalNAc) has been found in Bifidobacterium longum. We showed that the solute-binding protein of a putative ATP-binding cassette-type transporter encoded in the cluster crystallizes only in the presence of LNB or GNB, and therefore we named it GNB/LNB-binding protein (GL-BP). Isothermal titration calorimetry measurements revealed that GL-BP specifically binds LNB and GNB with K-d values of 0.087 and 0.010 mu M, respectively, and the binding process is enthalpy-driven. The crystal structures of GL-BP complexed with LNB, GNB, and lacto-N-tetraose ( Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc) were determined. The interactions between GL-BP and the disaccharide ligands mainly occurred through water-mediated hydrogen bonds. In comparison with the LNB complex, one additional hydrogen bond was found in the GNB complex. These structural characteristics of ligand binding are in agreement with the thermodynamic properties. The overall structure of GL-BP was similar to that of maltose-binding protein; however, the mode of ligand binding and the thermodynamic properties of these proteins were significantly different.
  • Midori Umekawa; Wei Huang; Bing Li; Kiyotaka Fujita; Hisashi Ashida; Lai-Xi Wang; Kenji Yamamoto
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 283 (8) 4469 - 4479 0021-9258 2008/02 [Refereed]
     
    Endo-beta-N-acetylglucosaminidase from Mucor hiemalis(Endo-M), a family 85 glycoside hydrolase, acts on the beta 1,4 linkage of N,N'-diacetylchitobiose moiety in the N-linked glycans of glycoproteins and catalyzes not only the hydrolysis reaction but also the transglycosylation reaction that transfers the releasing sugar chain to an acceptor other than water to form a new glycosidic linkage. The transglycosylation activity of Endo-M holds a great promise for the chemo-enzymatic synthesis and glyco-engineering of glycoproteins, but the inherent hydrolytic activity for product hydrolysis and low transglycosylation have hampered its broad applications. This paper describes the site-directed mutagenesis on residues in the putative catalytic region of Endo-M to generate mutants with superior transglycosylation activity. Two interesting mutants were discovered. The Y217F mutant was found to possess much enhanced transglycosylation activity and yet much diminished hydrolytic activity in comparison with the wild-type Endo-M. Kinetic analyses revealed that the K(m) value of Y217F for an acceptor substrate 4-methylumbel-liferyl-beta-D-N-acetylglucosaminide was only one-tenth of that of the wild-type, implicating a much higher affinity of Y217F for the acceptor substrate than the wild-type. The other mutant, N175A, acts like a glycosynthase. It was found that mutation at Asn175"knocked out" the hydrolytic activity, but the mutant was able to take the highly active sugar oxazolines ( the transition state mimics) as donor substrates for transglycosylation. This is the first glycosynthase derived from endo-beta-N-acetylglucosaminidases that proceed via a substrate-assisted mechanism. Our findings provide further insights on the substrate-assisted mechanism of GH85. The usefulness of the novel glycosynthase was exemplified by the efficient synthesis of a human immunodeficiency deficiency virus, type 1 (HIV-1) glycopeptide with potent anti-HIV activity.
  • Miyake Akiko; Kiyohara Masashi; Katayama Takane; Ashida Hisashi; Yamamoto kenji
    Journal of Applied Glycoscience Supplement The Japanese Society of Applied Glycoscience 2008 138 - 138 2008
  • Fujita Masaya; Tsuchida Akiko; Kobayashi Natsumi; Goto Kohtaro; Osumi Kenji; Mizuno Mamoru; Yamanoi Takashi; Ashida Hisashi; Haneda Katsuji; Nakayama jun
    Journal of Applied Glycoscience Supplement The Japanese Society of Applied Glycoscience 2008 6 - 6 2008
  • Suzuki Ryuichiro; Fushinobu Shinya; Katayama Takane; Ashida Hisashi; Kumagai Hidehiko; Yamamoto Kenji; Kitaoka Motomitsu; Wakagi Takayoshi; Shoun Hirofumi
    Journal of Applied Glycoscience Supplement The Japanese Society of Applied Glycoscience 2008 147 - 147 2008
  • Ashida Hisashi; Kato Toshihiko; Kawahara Akihito; Tanaka Yuki; Yamamoto Kenji
    Journal of Applied Glycoscience Supplement The Japanese Society of Applied Glycoscience 2008 151 - 151 2008
  • Jun Wada; Ryuichiro Suzuki; Shinya Fushinobu; Motomitsu Kitaoka; Takayoshi Wakagi; Hirofumi Shoun; Hisashi Ashida; Hidehiko Kumagai; Takane Katayama; Kenji Yamamoto
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS INT UNION CRYSTALLOGRAPHY 63 751 - 753 2053-230X 2007/09 [Refereed]
     
    A recombinant galacto-N-biose-/ lacto-N-biose I-binding protein ( GL-BP) from Bifidobacterium longum JCM1217 has been prepared and crystallized by the hanging-drop vapour-diffusion method using 10 mg ml(-1) purified enzyme, 0.01 M zinc sulfate, 0.1 M MES buffer pH 5.9 -6.4 and 20 -22%( v/ v) PEG MME 550 in the presence of 5 mM disaccharide ligands. Suitable crystals grew after 10 d incubation at 293 K. The crystals belong to space group C222(1), with unit-cell parameters a = 106.3, b = 143.6, c = 114.6 angstrom for the lacto-N-biose I complex and a = 106.4, b = 143.4, c = 115.5 angstrom for the galacto-N-biose complex, and diffracted to 1.85 and 1.99 angstrom resolution, respectively.
  • Toshihiko Kato; Akihito Kawahara; Hisashi Ashida; Kenji Yamamoto
    JOURNAL OF BIOCHEMISTRY OXFORD UNIV PRESS 142 (2) 175 - 181 0021-924X 2007/08 [Refereed]
     
    Peptide:N-glycanase (PNGase) is the enzyme responsible for de-N-glycosylation of misfolded glycoproteins in the cytosol. Here, we report the molecular identification and characterization of PNGase (png-1, F56G4.5) from Caenorhabditis elegans. This enzyme released both high mannose- and complex-type N-glycans from glycopeptides and denatured glycoproteins. Deglycosylation activity was inhibited by Zn2+ and z-VAD-fmk, but not by EDTA. PNG-1 has a thioredoxin-like domain in addition to a transglutaminase domain, the core domain of PNGases, and exhibited protein disulphide reductase activity in vitro. Our biochemical studies revealed that PNG-1 is a unique bifunctional protein possessing two enzyme activities.
  • Youn Uck Kim; Hisashi Ashida; Kenichiro Mori; Yusuke Maeda; Yeongjin Hong; Taroh Kinoshita
    JOURNAL OF BIOCHEMISTRY OXFORD UNIV PRESS 142 (1) 123 - 129 0021-924X 2007/07 [Refereed]
     
    GPI mannosyltransferase I (GPI-MT-I) transfers the first mannose to a GPI-anchor precursor, glucosamine-(acyl)phosphatidylinositol [GIcN-(acyl)PI]. Mammalian GPIMT-I consists of two components, PIG-M and PIG-X, which are homologous to Gpil4p and Pbn1p in Saccharomyces cerevisiae, respectively. In the present study, we disrupted yeast GPI14 and analysed the phenotype of gpi14 yeast. The gpi14 haploid cells were inviable and accumulated GIcN-(acyl)PI. We cloned PIG-M homologues from human, Plasmodium falciparum (PfPIG-M) and Trypanosoma brucei (TbGPI14), and tested whether they could complement gpi14-disrupted yeast. None of them restored GPI-MT-I activity and cell growth in gpi14-disrupted yeast. However, gpi14disrupted yeast cells with human PIG-M, but not with PfPIG-M or TbGPI14, grew slowly but significantly when they were supplemented with rat PIG-X. This suggests that the association of PIG-X and PIG-M for GPI-MT-I activity is not interchangeable between mammals and the other lower eukaryotes.
  • Toshihiko Kato; Kumiko Kitamura; Megumi Maeda; Yoshinobu Kimura; Takane Katayama; Hisashi Ashida; Kenji Yamamoto
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 282 (30) 22080 - 22088 0021-9258 2007/07 [Refereed]
     
    Free oligosaccharides (FOSs) in the cytosol of eukaryotic cells are mainly generated during endoplasmic reticulum (ER)-associated degradation (ERAD) of misfolded glycoproteins. We analyzed FOS of the nematode Caenorhabditis elegans to elucidate its detailed degradation pathway. The major FOSs were high mannose-type ones bearing 3-9 Man residues. About 94% of the total FOSs had one GlcNAc at their reducing end (FOS-GN1), and the remaining 6% had two GlcNAc (FOS-GN2). A cytosolic endo-beta-N-acetylglucosaminidase mutant (tm1208) accumulated FOS-GN2, indicating involvement of the enzyme in conversion of FOS-GN2 into FOS-GN1. The most abundant FOS in the wild type was Man(5)GlcNAc(1), the M5A' isomer (Man alpha 1- 3(Man alpha 1-6)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc), which is different from the corresponding M5B' (Man alpha 1-2Man alpha 1- 2Man alpha 1-3(Man alpha 1-6)Man beta 1-4GlcNAc) in mammals. Analyses of FOS in worms treated with Golgi alpha-mannosidase I inhibitors revealed decreases in Man(5)GlcNAc(1) and increases in Man(7)GlcNAc(1). These results suggested that Golgi alpha-mannosidase I-like enzyme is involved in the production of Man(5-6)- GlcNAc(1), which is unlike in mammals, in which cytosolic alpha-mannosidase is involved. Thus, we assumed that major FOSs in C. elegans were generated through Golgi trafficking. Analysis of FOSs from a Golgi alpha-mannosidase II mutant (tm1078) supported this idea, because GlcNAc(1)Man(5)GlcNAc(1), which is formed by the Golgi-resident GlcNAc-transferase I, was found as a FOS in the mutant. We concluded that significant amounts of misfolded glycoproteins in C. elegans are trafficked to the Golgi and are directly or indirectly retro-translocated into the cytosol to be degraded.
  • Y Hong; K Nagamune; YS Morita; F Nakatani; H Ashida; Y Maeda; T Kinoshita
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 281 (17) 11595 - 11602 0021-9258 2006/04 [Refereed]
     
    The protozoan parasite Trypanosoma brucei is coated by glycosylphosphatidylinositol ( GPI)-anchored proteins. During GPI biosynthesis, inositol in phosphatidylinositol becomes acylated. Inositol is deacylated prior to attachment to variant surface glycoproteins in the bloodstream form, whereas it remains acylated in procyclins in the procyclic form. We have cloned a T. brucei GPI inositol deacylase ( GPIdeAc2). In accordance with the acylation/deacylation profile, the level of GPIdeAc2 mRNA was 6-fold higher in the bloodstream form than in the procyclic form. Knockdown of GPIdeAc2 in the bloodstream form caused accumulation of an inositol-acylated GPI, a decreased VSG expression on the cell surface and slower growth, indicating that inositol-deacylation is essential for the growth of the bloodstream form. Overexpression of GPIdeAc2 in the procyclic form caused an accumulation of GPI biosynthetic intermediates lacking inositol-linked acyl chain and decreased cell surface procyclins because of release into the culture medium, indicating that overexpression of GPIdeAc2 is deleterious to the surface coat of the procyclic form. Therefore, the GPI inositol deacylase activity must be tightly regulated in trypanosome life cycle.
  • Y Tashima; R Taguchi; C Murata; H Ashida; T Kinoshita; Y Maeda
    MOLECULAR BIOLOGY OF THE CELL AMER SOC CELL BIOLOGY 17 (3) 1410 - 1420 1059-1524 2006/03 [Refereed]
     
    Biosynthesis of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the ER has been extensively studied, whereas the molecular events during the transport of GPI-APs from the ER to the cell surface are poorly understood. Here, we established new mutant cell lines whose surface expressions of GPI-APs were greatly decreased despite normal biosynthesis of GPI-APs in the ER. We identified a gene responsible for this defect, designated PGAP2 (for Post-GPI-Attachment to Proteins 2), which encoded a Golgi/ER-resident membrane protein. The low surface expression of GPI-APs was due to their secretion into the culture medium. GPI-APs were modified/cleaved by two reaction steps in the mutant cells. First, the GPI anchor was converted to lyso-GPI before exiting the trans-Golgi network. Second, lyso-GPI-APs were cleaved by a phospholipase D after transport to the plasma membrane. Therefore, PGAP2 deficiency caused transport to the cell surface of lyso-GPI-APs that were sensitive to a phospholipase D. These results demonstrate that PGAP2 is involved in the processing of GPI-APs required for their stable expression at the cell surface.
  • H Ashida; Y Maeda; T Kinoshita
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 281 (2) 896 - 904 0021-9258 2006/01 [Refereed]
     
    Dolichol-phosphate mannose (DPM) synthase is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor, N-glycan precursor, protein O-mannose, and C-mannose. We previously identified DPM3, the third component of this enzyme, which was co-purified with DPM1 and DPM2. Here, we have established mutant Chinese hamster ovary (CHO) 2.38 cells that were defective in DPM3. CHO2.38 cells were negative for GPI-anchored proteins, and microsomes from these cells showed no detectable DPM synthase activity, indicating that DPM3 is an essential component of this enzyme. A coiled-coil domain near the C terminus of DPM3 was important for tethering DPM1, the catalytic subunit of the enzyme, to the endoplasmic reticulum membrane and, therefore, was critical for enzyme activity. On the other hand, two transmembrane regions in the N-terminal portion of DPM3 showed no specific functions. DPM1 was rapidly degraded by the proteasome in the absence of DPM3. Free DPM1 was strongly associated with the C terminus of Hsc70-interacting protein (CHIP), a chaperone-dependent E3 ubiquitin ligase, suggesting that DPM1 is ubiquitinated, at least in part, by CHIP.
  • YC Hong; K Nagamune; K Ohishi; YS Morita; H Ashida; Y Maeda; T Kinoshita
    FEBS LETTERS ELSEVIER SCIENCE BV 580 (2) 603 - 606 0014-5793 2006/01 [Refereed]
     
    Glycosylphosphatidylinositol (GPI) is widely used by eukaryotic cell surface proteins for membrane attachment. De novo synthesized GPI precursors are attached to proteins posttranstationally by the enzyme complex, GPI transamidase. TbGPI16, a component of the trypanosome transamidase, shares similarity with human PIG-T. Here, we show that TbGPI16 is the orthologue of PIG-T and an essential component of GPI transamidase by creating a TbGPI16 knockout. TbGPI16 forms a disulfide-linked complex with TbGP18. A cysteine to serine mutant of TbGPI16 was unable to fully restore the surface expression of GPI-anchored proteins upon transfection into the knockout cells, indicating that its disulfide linkage with TbGP18 is important for the full transamidase activity. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • W Tempel; ZJ Liu; PS Horanyi; L Deng; D Lee; GN Newton; JP Rose; H Ashida; SC Li; YT Li; BC Wang
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS WILEY-LISS 59 (1) 141 - 144 0887-3585 2005/04 [Refereed]
  • KM Anderson; H Ashida; K Maskos; A Dell; SC Li; YT Li
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 280 (9) 7720 - 7728 0021-9258 2005/03 [Refereed]
     
    We have isolated an endo-beta-galactosidase designated E-ABase from Clostridium perfringens ATCC 10543 capable of liberating both the A trisaccharide (A-Tri; GalNalpha1-->3(Fucalpha1-->2)Gal) and B trisaccharide (B-Tri; Galalpha1-->3( Fucalpha1-->2)Gal) from glycoconjugates containing blood group A and B glycotopes, respectively. We have subsequently cloned the gene (eabC) that encodes E-ABase from this organism. This gene was found to be identical to the CPE0329 gene of C. perfringens strain 13, whose product was labeled as a hypothetical protein (Shimizu, T., Ohtani, K., Hirakawa, H., Ohshima, K., Yamashita, A., Shiba, T., Ogasawara, N., Hattori, M., Kuhara, S., and Hayashi, H. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 996 - 1001). Since the amino acid sequence of E-ABase does not bear detectable similarity to any of the 97 existing families of glycoside hydrolases, we have proposed to assign this unusual enzyme to a new family, GH98. We also expressed eabC in Escherichia coli BL21(DE3) and obtained 27 mg of fully active recombinant E-ABase from 1 liter of culture. Recombinant E-ABase not only destroyed the blood group A and B antigenicity of human type A and B erythrocytes, but also released A-Tri and B-Tri from blood group A (+)- and B+-containing glycoconjugates. The structures of A-Tri and B-Tri liberated from A(+) porcine gastric mucin and B+ human ovarian cyst glycoprotein were established by NMR spectroscopy. The unique specificity of E-ABase should make it useful for studying the structure and function of blood group A- and B-containing glycoconjugates as well as for identifying other glycosidases belonging to the new GH98 family.
  • JY Kang; YJ Hong; H Ashida; N Shishioh; Y Murakami; YS Morita; Y Maeda; T Kinoshita
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 280 (10) 9489 - 9497 0021-9258 2005/03 [Refereed]
     
    Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors many proteins to the eukaryotic cell surface. The biosynthetic pathway of GPI is mediated by sequential additions of sugars and other components to phosphatidylinositol. Four mannoses in the GPI are transferred from dolichol-phosphate-mannose (Dol-PMan) and are linked through different glycosidic linkages. Therefore, four Dol-P-Man-dependent mannosyl-transferases, GPI-MT-I, -MT-II, -MT-III, and -MT-IV for the first, second, third, and fourth mannoses, respectively, are required for generation of GPI. GPI-MT-I (PIG-M), GPI-MT-III (PIG-B), and GPI-MT-IV (SMP3) were previously reported, but GPI-MT-II remains to be identified. Here we report the cloning of PIG-V involved in transferring the second mannose in the GPI anchor. Human PIG-V encodes a 493-amino acid, endoplasmic reticulum (ER) resident protein with eight putative transmembrane regions. Saccharomyces cerevisiae protein encoded in open reading frame YBR004c, which we termed GPI18, has 25% amino acid identity to human PIG-V. Viability of the yeast gpi18 deletion mutant was restored by human PIG-V cDNA. PIG-V has two functionally important conserved regions facing the ER lumen. Taken together, we suggest that PIG-V is the second mannosyltransferase in GPI anchor biosynthesis.
  • N Shishioh; YJ Hong; K Ohishi; H Ashida; Y Maeda; T Kinoshita
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 280 (10) 9728 - 9734 0021-9258 2005/03 [Refereed]
     
    Many eukaryotic cell surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). GPI is synthesized from phosphatidylinositol by stepwise reactions and attached en bloc to nascent proteins. In mammalian cells, the major GPI species transferred to proteins is termed H7. By attachment of an additional ethanolamine phosphate (EtNP) to the second mannose, H7 can be converted to H8, which acts as a minor type of protein-linked GPI and also exists as a free GPI on the cell surface. Yeast GPI7 is involved in the transfer of EtNP to the second mannose, but the corresponding mammalian enzyme has not yet been clarified. Here, we report that the human homolog of Gpi7p (hGPI7) forms a protein complex with PIG-F and is involved in the H7-to-H8 conversion. We knocked down hGPI7 by RNA interference and found that H7 accumulated with little production of H8. Immunoprecipitation experiments revealed that hGPI7 was associated with and stabilized by PIG-F, which is known to bind to and stabilize PIG-O, a protein homologous to hGPI7. PIG-O is a transferase that adds EtNP to the third mannose, rendering GPI capable of attaching to proteins. We further found that the overexpression of hGPI7 decreased the level of PIG-O and, therefore, decreased the level of EtNP transferred to the third mannose. Finally, we propose a mechanism for the regulation of GPI biosynthesis through competition between the two independent enzymes, PIG-O and hGPI7, for the common stabilizer, PIG-F.
  • H Ashida; YJ Hong; Y Murakami; N Shishioh; N Sugimoto; YU Kim; Y Maeda; T Kinoshita
    MOLECULAR BIOLOGY OF THE CELL AMER SOC CELL BIOLOGY 16 (3) 1439 - 1448 1059-1524 2005/03 [Refereed]
     
    Within the endoplasmic reticulum (ER), mannoses and glucoses, donated from dolichol-phosphate-mannose and -glucose, are transferred to N-glycan and GPI-anchor precursors, and serine/threonine residues in many proteins. Glycosyltransferases that mediate these reactions are ER-resident multitransmembrane proteins with common characteristics, forming a superfamily of >10 enzymes. Here, we report an essential component of glycosylphosphatidylinositolmannosyltransferase I (GPI-MT-I), which transfers the first of the four mannoses in the GPI-anchor precursors. We isolated a Chinese hamster ovary (CHO) cell mutant defective in GPI-MT-I but not its catalytic component PIG-M. The mutant gene, termed phosphatidylinositolglycan-class X (PIG-X), encoded a 252-amino acid ER-resident type I transmembrane protein with a large lumenal domain. PIG-X and PIG-M formed a complex, and PIG-M expression was <10% in the absence of PIG-X, indicating that PIG-X stabilizes PIG-M. We found that Saccharomyces cerevisiae Pbn1p/YCL052Cp, which was previously reported to be involved in autoprocessing of proproteinase B, is the functional homologue of PIG-X; Pbn1p is critical for Gpi14p/YJR013Wp function, the yeast homologue of PIG-M. This is the first report of an essential subcomponent of glycosyltransferases using dolichol-phosphate-monosaccharide.
  • A new subspecies of Episcaphula matsumurai Chujo (Coleoptera: Erotylidae) from the Yaeyama group in the Southern Ryukyus, Southwestern Japan
    Narukawa N; Ashida H
    Ent. Rev. Japan 60 59 - 62 2005 [Refereed]
  • The complex of Trechiama kosugei (Coleoptera, Trechinae) from Hyôgo Prefecture, West Japan. (I) A remarkable new species from the western periphery of its distributional range
    Ashida H
    Elytra, Tokyo 33 659 - 664 2005 [Refereed]
  • L Deng; ZJ Liu; H Ashida; SC Li; YT Li; P Horanyi; W Tempel; J Rose; BC Wang
    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY BLACKWELL MUNKSGAARD 60 537 - 538 0907-4449 2004/03 [Refereed]
     
    The unique clostridial endo-beta-galactosidase (Endo-beta-Gal(GnGa)) capable of releasing the disaccharide GlcNAcalpha1,4Gal from O-glycans expressed in the gastric gland mucous cell-type mucin has been crystallized. The crystal belongs to space group P6(3), with unit-cell parameters a=160.4, c=86.1 Angstrom. Under cryocooled conditions and using a synchrotron X-ray source, the crystals diffract to 1.82 Angstrom resolution. The asymmetric unit contains two or three molecules.
  • A record of Kusumia septentrionalis S. Ueno et Okuda (Coleoptera, Trechinae) from Nara Prefecture, Central Japan.
    Ashida H; Kitayama K
    Elytra, Tokyo 32 27  2004 [Refereed]
  • K Nagamune; K Ohishi; H Ashida; YC Hong; J Hino; K Kangawa; N Inoue; Y Maeda; T Kinoshita
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA NATL ACAD SCIENCES 100 (19) 10682 - 10687 0027-8424 2003/09 [Refereed]
     
    Glycosylphosphatidylinositol (GPI) anchor is a membrane attachment mechanism for cell surface proteins widely used in eukaryotes. GPIs are added to proteins posttranslationally by a complex enzyme, GPI transamidase. Previous studies have shown that human and Saccharomyces cerevisiae GPI transamidases are similar and consist of five homologous components: GAA1, GP18, PIG-S, PIG-T, and PIG-U in humans and Gaa1p, Gpi8p, Gpi17p, Gpi16p, and Cdc91p in S. cerevisiae. We report that GPI transamidase of Trypanosoma brucei (Tb), a causative agent of African sleeping sickness, shares only three components (TbGAA1, TbGPl8 and TbGP116) with humans and S. cerevisiae but has two other specific components, trypanosomatid transamidase 1 (TTA1) and TTA2. GPI transamidases of both bloodstream form (growing in mammalian blood) and procyclic form (growing in tsetse fly vector) of the parasite have the same five components. Homologues of TTA1 and TTA2 are present in Leishmania and Trypanosoma cruzi but not in mammals, yeasts, flies, nematodes, plants, or malaria parasites, suggesting that these components may play unique roles in attachment of GPI anchors in trypanosomatid parasites and provide good targets for antitrypanosome drugs.
  • K Totani; T Kubota; T Kuroda; T Murata; KIPJ Hidari; T Suzuki; Y Suzuki; K Kobayashi; H Ashida; K Yamamoto; T Usui
    GLYCOBIOLOGY OXFORD UNIV PRESS INC 13 (5) 315 - 326 0959-6658 2003/05 [Refereed]
     
    Highly water-soluble glycopolymers with poly(alpha-L-glutamic acid) (PGA) backbones carrying multivalent sialyl oligosaccharides units were chemoenzymatically synthesized as polymeric inhibitors of infection by human influenza viruses. p-Aminophenyl disaccharide glycosides were coupled with gamma-carboxyl groups of PGA side chains and enzymatically converted to Neu5Acalpha2-3Galbeta1-4GlcNAcbeta-, Neu5Acalpha2-6Galbeta1-4GlcNAcbeta-, Neu5Acalpha2-3Galbeta1-3GalNAcalpha-, and Neu5Acalpha2-3Galbeta1-3GalNAcbeta- units, respectively, by alpha2,3- or alpha2,6-sialytransferases. The glycopolymers synthesized were used for neutralization of human influenza A and B virus infection as assessed by measurement of the degree of cytopathic inhibitory effect in virus-infected MDCK cells. Among the glycopolymers tested, alpha2,6-sialo-PGA with a high molecular weight (260 kDa) most significantly inhibited infection by an influenza A virus, strain A/Memphis/1/71 (H3N2), which predominantly binds to alpha2-6 Neu5Ac residue. The alpha2,6-sialo-PGA also inhibited infection by an influenza B virus, B/Lee/40. The binding preference of viruses to terminal sialic acids was affected by core determinants of the sugar chain, Galbeta1-4GlcNAcbeta- or Galbeta1-3GalNAcalpha/beta- units. Inhibition of infection by viruses was remarkably enhanced by increasing the molecular weight and sialic acid content of glycopolymers.
  • Ashida H; Kitayama K
    Elytra, Tokyo 31 221 - 229 2003 [Refereed]
  • H Ashida; K Maskos; SC Li; YT Li
    BIOCHEMISTRY AMER CHEMICAL SOC 41 (7) 2388 - 2395 0006-2960 2002/02 [Refereed]
     
    In contrast to the beta-linked GlcNAc, the alpha-linked GlcNAc has not been commonly found in glycoconjugates. We have recently revealed the presence of an unusual endo-beta-galactosidase (Endo-beta-Gal(GnGa)) in Clostridium perfringens capable of releasing GlcNAcalpha1 --> 4Gal from glycans expressed in the gastric mucous cell-type mucin [Ashida, H., Anderson, K., Nakayama, J., Maskos, K., Chou, C.-W., Cole, R. B., Li, S.-C., and Li, Y.-T. (2001) J. Biol Chem. 276,28226-28232]. To characterize Endo-beta-Gal(GnGa), we have cloned its gene, gngC, from the genomic DNA library prepared from C. perfringens ATCC 10543. The gene encodes 420 amino acid residues including a 17-residue signal peptide at the N-terminus. Using pUC18, we were able to prepare 25 mg of the fully active and pure recombinant Endo-beta-Gal(GnGa) from 1 L of Escherichia coli DHalpha culture, which was 170 times higher than that produced by the original clostridial strain. Endo-beta-Gal(GnGa) shares a low but significant sequence similarity with two other endo-beta-galactosidases (16-21% amino acid identity). It also shows some similarity with bacterial 1,3-1,4-beta-glucan 4-glucanohydrolases of the glycoside hydrolase family 16. Endo-beta-Gal(GnGa) was found to contain the EXDX(X)E sequence (Glu-168 to Glu-173), that has been identified as the catalytic motif of families 16 and 7 retaining glycoside hydrolases. We have used site-directed mutagenesis to show that Glu-168 and Glu-173 were essential for the Endo-beta-Gal(GnGa) activity. By NMR spectroscopy, Endo-beta-Gal(GnGa) was found to act as a retaining enzyme.
  • H Ashida; K Anderson; J Nakayama; K Maskos; CW Chou; RB Cole; SC Li; YT Li
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 276 (30) 28226 - 28232 0021-9258 2001/07 [Refereed]
     
    We found that commercially available sialidases prepared from Clostridium perfringens ATCC10543 were contaminated with an endoglycosidase capable of releasing the disaccharide GlcNAc alpha1->4Gal from glycans expressed in the gastric gland mucous cell-type mucin. We have isolated this enzyme in electrophoretically homogeneous form from the culture supernatant of this organism by ammonium sulfate precipitation followed by affinity chromatography using a Sephacryl S-200 HR column. The enzyme was specifically retained by and eluted from the column with methyl-alpha -Glc. By spectroscopy, the structure of the disaccharide released from porcine gastric mucin by this enzyme was established to be GlcNAc alpha1-->4Gal. The specificity of this enzyme as an endo-beta -galactosidase was established by analyzing the liberation of GlcNAc alpha1-->4Gal from GlcNAc alpha1--->4Gal beta1-->4GlcNAc beta1-->6(GlcNAc alpha1-->4Gal beta1-3)GalNAc-ol by mass spectrometry. Because this novel endo-beta -galactosidase specifically releases the GlcNAc alpha1-->4Gal moiety from porcine gastric mucin, we propose to call this enzyme a GlcNAc alpha1-->4Gal-releasing endo-beta -galactosidase (Endo-beta -Gal(GnGa)). Endo-beta -Gal(GnGa) was found to remove the GlcNAc alpha1-->4Gal epitope expressed in gastric adenocarcinoma AGS cells transfected with alpha1,4-N-acetylglucosaminyltransferase cDNA. Endo-beta -Gal(GnGa) should become useful for studying the structure and function of glycoconjugates containing the terminal GlcNAc alpha1-->4Gal epitope.
  • H Ashida; K Yamamoto; H Kumagai
    CARBOHYDRATE RESEARCH ELSEVIER SCI LTD 330 (4) 487 - 493 0008-6215 2001/02 [Refereed]
     
    Thomsen-Friedenreich antigen (T antigen) disaccharide, beta -D-galactose-(1 --> 3)-alpha -N-acetyl-D-galactosamine (beta -D-Gal-(1 --> 3)-alpha -D-GalNAc), containing glycolipid mimicry was synthesized using the transglycosylation activity of endo-alpha -N-acetylgalactosaminidase from Bacillus sp. This enzyme could transfer the disaccharide from a p- nitrophenyl substrate to water-soluble 1-alkanols and other alcohols at a transfer ratio of 70% or more. Although the transfer ratios were lower for water-insoluble than water-soluble alcohols, they were shown to increase by adding sodium cholate to the reaction mixtures. The enzyme also transferred the disaccharide directly from asialofetuin to l-alkanols. The anomeric bond between the disaccharide and l-alkanols of the transglycosylation product is in the alpha configuration as determined by sequential digestion of jack bean beta -galactosidase and Acremonium alpha -N- acetylgalactosaminidase. Since the transglycosylation product, beta -D-Gal-(1--> 3)-alpha -D-GalNAc-(1 -->O)-hexyl, efficiently inhibits the binding of anti-T antigen monoclonal antibody to asialofetuin, it has potential as an agent for blocking T antigen-mediated cancer metastasis. (C) 2001 Elsevier Science Ltd. All rights reserved.
  • A new species of the group of Trechiama oni (Coleoptera, Trechinae) from Okayama Prefecture, western Honshu, West Japan
    Ashida H
    Elytra, Tokyo 29 481 - 485 2001 [Refereed]
  • H Ashida; H Tamaki; T Fujimoto; K Yamamoto; H Kumagai
    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS ACADEMIC PRESS INC 384 (2) 305 - 310 0003-9861 2000/12 [Refereed]
     
    Alpha-N-acetylgalactosaminidase (alpha -GaLNAc-ase; EC 3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal alpha -linked N-acetylgalactosamine in various sugar chains. The cDNA, nagA, encoding alpha -GalNAc-ase from Acremonium sp. was cloned, sequenced, and expressed in yeast Saccharomyces cerevisiae. The nagA contains an open reading frame which encodes for 547 amino acid residues including 21 residues of a signal peptide in its N-terminal. The calculated molecular mass of mature protein from the deduced amino acid sequence of nagA is 57260 Da, which corresponds to the value obtained from SDS-PAGE of native and recombinant enzymes treated with endo-beta -N-acetyl-glucosaminidase ii. The amino acid sequence of NagA showed significant similarity to those of eukaryotic alpha -GalNAc-ases and alpha -galactosidases (alpha -Gal-ases), particularly alpha -Gal-ase A (AglA) from Aspergillus niger. Phylogenetic analysis revealed that NagA does not belong to the cluster of vertebrate alpha -GalNAc-ase and alpha -Gal-ase but forms another cluster with AglA and yeast alpha -Gal-ases. Thus, the evolutionary origin of the fungal alpha -GalNAc-ase is suggested to be different from that of vertebrate alpha -GalNAc-ase. This is the first report of a microbial alpha -GalNAc-ase gene. (C) 2000 Academic Press.
  • H Ashida; K Yamamoto; H Kumagai
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY TAYLOR & FRANCIS LTD 64 (10) 2266 - 2268 0916-8451 2000/10 [Refereed]
     
    The effects of bovine fetuin O-glycans on its trypsin inhibitory activity were examined. De-sialylated (asialo-) and de-O-glycosylated fetuin were prepared from native fetuin using Arthrobacter neuraminidase and the mixture of it and Bacillus endo-alpha -N-acetylgalactosaminidase, respectively. De-sialylation and de-O-glycosylation from fetuin were confirmed with SDS-PAGE followed by western blotting using anti-human Thomsen-Friedenreich antigen (T antigen) antibody which recognizes O-linked galactosyl beta1,3 N-acetylgalactosamine (Gal beta1-->3GalNAc). Native fetuin completely inhibited the trypsin activity at about a 1:1 molar ratio. In contrast, the trypsin inhibitory activity of asialo- and de-O-glycosylated fetuin decreased about a half and one-third of that of native fetuin, respectively.
  • A new record of Thalassoduvalius masidai masidai S. Ueno (Coleoptera, Trechinae) from Kanmuri-jima Island, Kyoto Prefecture, Central Japan
    Ashida H; Kitayama K; Araya K
    Elytra, Tokyo 28 37 - 38 2000 [Refereed]
  • Ashida H
    Elytra, Tokyo 28 241 - 245 2000 [Refereed]
  • H Ashida; K Yamamoto; T Murata; T Usui; H Kumagai
    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS ACADEMIC PRESS INC 373 (2) 394 - 400 0003-9861 2000/01 [Refereed]
     
    Endo-alpha-N-acetylgalactosaminidase was purified to homogeneity from the culture fluid of Bacillus sp. isolated from soil and characterized, The molecular mass of the enzyme was estimated as 110 kDa. The enzyme was stable at pH 4.0-10.0, up to 55 degrees C, and was most active at pH 5.0. The substrate specificity of the enzyme was strict for the disaccharide, galactosyl beta 1, 3 N-acetyl-D-galactosamine, bound to aglycone in alpha configuration. On the other hand, the specificity of the enzyme for the aglycone structure was fairly relaxed, The enzyme could transfer the disaccharide from para-nitrophenyl substrate to various accepters, such as monosaccharides, disaccharides, and sugar alcohols. Using this transglycosylation activity of the endoglycosidase, it may be possible to synthesize neo-oligosaccharides. (C) 2000 Academic Press.
  • A new species of Dacne (Coleoptera, Erotylidae) from Chejudo Island off South Korea
    Ashida H; Kim CG
    Elytra, Tokyo 27 381 - 385 1999 [Refereed]
  • Rediscovery of Callytron inspeculare from Hyogo Prefecture
    Ashida H; Kitayama K
    Ent. Rev. Japan 53 15 - 16 1998 [Refereed]
  • Two new anophthalmic Trechiama (Coleoptera, Trechinae) from Kyoto Prefecture, Central Japan
    Ashida H
    Elytra, Tokyo 26 289 - 295 1998 [Refereed]
  • R Iwata; F Yamada; H Kato; H Makihara; K Araya; H Ashida; M Takeda
    PAN-PACIFIC ENTOMOLOGIST PACIFIC COAST ENTOMOL SOC 73 (4) 213 - 224 0031-0603 1997/10 [Refereed]
     
    Spatial distributions and shapes of ''whirl-like'' scars on the trunks, made by gallery formation of mature larvae of Xylotrechus villioni (Villard) (Coleoptera: Cerambycidae), a primary borer of Abies and Picea coniferous trees in Japan, were investigated at an Abies firma Sieb. et Zucc. plantation in Hachioji, Tokyo Pref., an A. firma natural stand in Miyama, Kyoto Pref. and an A. sachalinensis (Fr. Schm.) Mast. plantation in Imakane, Hokkaido. Although all the forests investigated showed cumulative ''whirl-like'' scars on the tree trunks, a low density of existing larvae was inferred from the analyses of the locations and shapes of these scars. Mortality throughout the larval stages, as well as between the final phase of larva and the adult emergence, was suggested. Trunk analysis of a damaged A. firma tree showed that a ''whirl-like'' scar can remain on the trunk surface for as long as 27 years after the formation of the larval gallery. The most susceptible class of Abies trees had a diameter at the breast height of 35-45cm. ''Whirl-like'' scars were distributed more densely in the lower part of the trunks.
  • H ASHIDA; S HAYASHI; Y SAKAMOTO; Y TSUJI; K YAMAMOTO; H KUMAGAI; T TOCHIKURA
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY TAYLOR & FRANCIS LTD 59 (11) 2028 - 2032 0916-8451 1995/11 [Refereed]
     
    The actinomycete strain Streptomyces sp. H37 produces a novel glycosphingolipid-degrading enzyme. This strain was capable of converting ganglioside GM1 to lyso-GM1. After cultivation for 5 days in medium containing GM1, peptone, and detergent, GM1 was found to be almost completely converted to lyso-GM1. The product was purified on a DEAE-Sephadex A-25 column and thin layer chromatographies. The purified lyso-GM1 was hydrolyzed by endoglycoceramidase, and the released oligosaccharide moiety was identified as that of GM1 by HPLC using the pyridylaminoderivative method. The counterpart sphingosine moiety was confirmed with TLC, Moreover, the structure of lyso-GM1 was ascertained by H-1-NMR analysis, The maximum formation of lyso-GM1 was found in 50 mM potassium phosphate buffer (pH 7.5) containing 0.1% glycodeoxycholate. Various lyso-glycoshingolipids, including those of ganglio-, neolacto-, and globe-types, were formed from their parent glycosphingolipids using this strain.
  • Ashida H; Tsuji Y; Yamamoto K; Kumagai H; Tochikura T
    Arch. Biochem. Biophys. 305 559 - 562 1993 [Refereed]
  • H ASHIDA; K YAMAMOTO; H KUMAGAI; T TOCHIKURA
    EUROPEAN JOURNAL OF BIOCHEMISTRY SPRINGER VERLAG 205 (2) 729 - 735 0014-2956 1992/04 [Refereed]
     
    Endoglycoceramidase catalyzes the hydrolysis of the linkage between oligosaccharides and ceramides of various glycosphingolipids. We found that a bacterial strain Corynebacterium sp., isolated from soil, produced endoglycoceramidase both intracellularly and extracellularly. The intracellular enzyme bound to the cell membrane was solubilized with 1% Triton X-100 and purified to homogeneity about 170-fold with 60% recovery. The molecular mass of the enzyme was approximately 65 kDa. The enzyme is most active at pH 5.5-6.5 and stable at pH 3.5-8.0. Various neutral and acidic glycosphingolipids were hydrolyzed by the enzyme in the presence of 0.1% Triton X-100. Ganglio- and lacto-type glycosphingolipids were readily hydrolyzed, but globo-type glycosphingolipids were hydrolyzed slowly

Books etc

  • Toshihiko Katoh; Hisashi Ashida (ContributorBacterial Enzyme Assay for Mucin Glycan Degradation)Humana Press 2024/02 9781071636695
  • 糖鎖の新機能開発・応用ハンドブック〜創薬・医療から食品開発まで〜
    梅川碧里; 芦田久; 山本憲二 (Contributorエンド型グリコシダーゼを利用した糖鎖合成)株式会社 エヌ・ティー・エス 2015/08
  • バイオ医薬品開発における糖鎖技術
    梅川碧里; 芦田久; 山本憲二 (ContributorエンドM酵素による糖鎖の効率的な転移付加と均一化)シーエムシー出版 2011/11
  • Comprehensive Glycoscience Vol. 3
    Hisashi Ashida; Toshihiko Kato; Kenji Yamamoto (ContributorBiochemistry of Glycoconjugate Glycans - Degradation of Glycoproteins)Elsevier 2007
  • Endoglycosidases -Biochemistry, Biotechnology, Application-
    Hisashi Ashida; Su-Chen Li; Yu-Teh Li (ContributorAn unusual GlcNAcα1-4Gal releasing endo-β-galactosidase)Kodansha/Springer 2006
  • Method in Enzymology
    Method in Enzymology 2006
  • 糖鎖科学の新展開
    芦田 久; 木下タロウ (ContributorGPIアンカーの生合成と関連疾患)NTS出版 2005
  • 京都府レッドデータブック 普及版
    芦田 久 (Joint work)サンライズ出版株式会社 2003
  • 京都府自然環境目録
    芦田 久 (Joint work)京都府 2002
  • 京都の昆虫
    芦田 久 (Joint work)京都新聞社 1991

Conference Activities & Talks

  • Bifidobacterium bifidum degrades sulfated mucin O-glycans via a sulfatase-independent pathway  [Not invited]
    Toshihiko Katoh; Chihaya Yamada; Michael D. Wallace; Ayako Yoshida; Aina Gotoh; Moe Arai; Takako Maeshibu; Toma Kashima; Arno Hagenbeek; Miriam N. Ojima; Hiromi Takada; Mikiyasu Sakanaka; Hidenori Shimizu; Keita Nishiyama; Masanori Yamaguchi; Hisashi Ashida; Junko Hirose; Maria Suarez-Diez; Makoto Nishiyama; Ikuo Kimura; Keith A. Stubbs; Shinya Fushinobu; Takane Katayama
    GLYCO26  2023/08
  • Bifunctional sialidase/β-N-acetylgalactosaminidase from bifidobacteria acting on Sda antigen/GM2  [Not invited]
    Shogo Kataoka; Junya Kawasaki; Keijiro Kamio; Toshihiko Katoh; Takane Katayama; Hisashi Ashida
    Sialoglyco 2022  2022/09
  • かぶらずしに由来するプトレッシン産生乳酸菌Lactobacillus curvatus KP3-4の分離  [Not invited]
    本田涼将; 白澤秀斗; 河田明輝; 平野里佳; 小栁 喬; 芦田 久; 栗原 新
    日本乳酸菌学会 2020年度大会  2020/11
  • 消化管ミューシンを介した宿主と腸内細菌の共生機構  [Invited]
    芦田 久
    日本応用糖質科学会 近畿支部会  2020/11
  • ムチン糖鎖による宿主と腸内細菌の共生機構の解明
    芦田 久
    日本応用糖質科学会2020年度大会(第69回)  2020/09
  • 南高梅梅干しによるマウス腸内細菌フローラ調節と脂質代謝改善効果
    芦田 久
    令和元年度ウメ研究成果発表会  2020/09
  • ビフィズス菌 Bifidobacterium longum subsp. longum の特異なアラビノキシラン分解系
    芦田 久; 吉原 侑希; 米野 雅大
    日本農芸化学会大会2020年度大会  2020/03
  • アラビノオリゴ糖のプレバイオティック効果に関わるビフィズス菌の酵素系の解析
    吉原侑希; 米野雅大; 芦田 久
    近畿大学大学院サイエンスネットワーク2019 第9回 院生サミット  2019/10
  • ビフィズス菌由来GH43マルチドメイン酵素の解析
    吉原侑希; 鍋島 航; 米野雅大; 芦田 久
    日本応用糖質科学会大会  2019/09
  • D-Glucosamine functions as calorie restriction mimetics and extends nematode lifespan via autophagy induction
    Tomoya Shintani; Yuhei Kosuge; Hideya Shintani; Sato Masashi; Hisashi Ashida
    41st ESPEN  2019/08
  • Bifidobacterium longum subsp. infantis のα-L-フコシダーゼについて
    芦田 久
    第2回糖質分解酵素研究会  2019/08
  • ビフィズス菌の植物性多糖分解機構
    吉原侑希; 芦田 久
    先端技術総合研究所オープン・ラボ  2019/06
  • ビフィズス菌のα-アラビノフラノシダーゼ遺伝子群の解析
    吉原侑希; 鍋島 航; 米野雅大; 芦田 久
    関西グライコサイエンスフォーラム  2019/05
  • マクロファージにおいてTLR4シグナルはAKT/mTORを介してオートファジーを調節することで炎症反応の減弱を制御する
    米野雅大; 白木琢磨; 芦田 久
    日本農芸化学会2019年度大会  2019/03
  • アラビノース含有糖鎖の分解に関わるBifidobacterium longum subsp. longum由来のGH51酵素の解析
    吉原侑希; 宮原祐貴; 米野雅大; 芦田 久
    日本農芸化学会2019年度大会  2019/03
  • Bifidobacterium bifidum由来スルフォグリコシダーゼBbhIIのX線結晶構造解析
    加藤紀彦; 山田千早; 前渋貴子; 芦田 久; 伏信進矢; 片山高嶺
    日本農芸化学会2019年度大会  2019/03
  • 酸化還元ストレスによる酸応答性センサー EvgS の活性化制御
    稲田慎也; 内海龍太郎; 芦田久; 江口陽子
    第41回日本分子生物学会年会  2018/11
  • ムチン資化性菌 Bifidobacterium bifidum が産生するスルフォグリコシダーゼの機能解析
    前渋貴子; 加藤紀彦; 後藤愛那; 山口真範; 芦田久; 山本憲二; 片山高嶺
    日本応用糖質科学会 第44回近畿支部会  2018/11
  • 和歌山県産の食品素材による腸内細菌制御と脂質代謝改善
    芦田 久
    近畿大学生物理工学部 産学官交流会  2018/10
  • 果実成分によるオートファジー誘導を介した炎症抑制メカニズムの解析
    米野雅大; 白木琢磨; 芦田 久
    第16回 果実酒・果実飲料と健康に関する研究会  2018/10
  • 梅干しによる腸内細菌フローラ調節と脂質代謝改善
    芦田 久
    第16回 果実酒・果実飲料と健康に関する研究会  2018/10
  • グルコサミンはオートファジーを誘導して線虫C. elegansの寿命を延伸する
    新谷知也; 小菅雄平; 佐藤正資; 芦田 久
    日本農芸化学会中四国支部大会  2018/09
  • オートファジーを介した新規の炎症抑制メカニズムの解明
    米野雅大; 白木琢磨; 芦田 久
    近畿大学大学院 サイエンスネットワーク2018 第8回院生サミット  2018/09
  • 酸化還元ストレスによる大腸菌酸応答性センサー EvgS の活性化制御
    稲田慎也; 江口陽子; 芦田 久
    近畿大学大学院 サイエンスネットワーク2018 第8回院生サミット  2018/09
  • ビフィズス菌由来の糖質加水分解酵素による植物性バイオマス分解
    吉原侑希; 芦田 久
    近畿大学大学院 サイエンスネットワーク2018 第8回院生サミット  2018/09
  • Akkermansia muciniphila JCM 30893におけるフコシル化糖鎖分解系の解析
    米野雅大; 伊藤はるか; 吉原侑希; 加藤紀彦; 片山高嶺; 芦田 久
    日本応用糖質科学会平成30年度大会(第67回)  2018/09
  • ヘミセルロースの分解に関わるBifidobacterium longum subsp. longum由来のGH51酵素の解析
    吉原侑希; 宮原祐貴; 米野雅大; 芦田 久
    日本応用糖質科学会平成30年度大会(第67回)  2018/09
  • 梅干し廃液を用いて抽出した魚軟骨プロテオグリカンの腸内細菌フローラ改善効果
    芦田 久; 伊藤あずさ; 大東夏海; 吉原侑希; 米野雅大; 山口実沙子; 山口真範
    日本応用糖質科学会平成30年度大会(第67回)  2018/09
  • 梅干しの脂質代謝改善効果
    芦田 久; 伊藤あずさ; 大東夏海; 吉原侑希; 米野雅大; 永井宏平; 石島智子; 阿部啓子; 岡田晋治; 鈴木利雄
    第70回 日本生物工学会大会  2018/09
  • 腸内細菌はもはやあなたの臓器
    芦田 久
    近畿大学生物理工学部オープンキャンパス  2018/09
  • 柿ポリフェノールの脂質代謝改善効果とそのメカニズム解明
    鈴木利雄; 大東夏海; 伊藤あずさ; 吉原侑希; 米野雅大; 永井宏平; 米谷 俊; 石島智子; 阿部啓子; 岡田晋治; 森口仁文; 芦田 久
    日本食品化学工学会 第65回大会  2018/08
  • 和歌山県産果実成分の腸内細菌フローラ調節による脂質代謝改善効果
    芦田 久
    第1回糖質分解酵素研究会  2018/08
  • マクロファージはオートファジーを介して炎症反応を制御する
    米野雅大、白木琢磨、芦田久
    日本農芸化学会2018年度大会  2018/03
  • ヒスチジンキナーゼセンサーEvgSの活性化に対する酸化還元状態の影響
    稲田慎也、芦田 久、内海龍太郎、江口陽子
    日本農芸化学会2018年度大会  2018/03
  • 関西のバイオ拠点〜和歌山県産特産農産物を活用した健康産業イノベーション推進地域
    芦田 久
    メディカルジャパン2018  2018/02
  • マクロファージはオートファジーを活性化することで炎症反応を終結に導く
    米野雅大; 芦田 久
    ConBio2017  2017/12
  • ビフィズス菌由来ヒト糖鎖分解酵素の基質認識と触媒機構
    伏信進矢; 山田千早; 佐藤真与; 後藤愛那; 荒川孝俊; 芦田 久; 片山高嶺
    ConBio2017  2017/12
  • 大腸菌ヒスチジンキナーゼセンサー EvgS の活性化機構
    稲田 慎也; 内海 龍太郎; 芦田 久; 江口 陽子
    ConBio2017  2017/12
  • 大腸菌ヒスチジンキナーゼセンサー EvgS 活性化機構  [Not invited]
    稲田慎也; 江口陽子; 芦田 久
    近畿大学大学院サイエンスネットワーク2017・第7回院生サミット  2017/09
  • オートファジーは炎症シグナル分子を分解することで炎症を抑制する  [Not invited]
    米野雅大; 芦田 久
    近畿大学大学院サイエンスネットワーク2017・第7回院生サミット  2017/09
  • 梅ポリフェノールのマウス腸内細菌フローラ改善効果  [Not invited]
    芦田 久
    石川県立大学腸内細菌共生機構学寄附講座(IFO)シンポジウム  2017/08
  • Bifodobacterium longum のアラビナン分解系の解析  [Not invited]
    米野雅大; 芦田 久
    第36回日本糖質学会年会  2017/07
  • 健康寿命のキーワード:オートファジーと腸内細菌  [Not invited]
    芦田 久
    平成29年度 第1回 BOST Science Cafe  2017/06
  • ムチンの硫酸化糖鎖に作用するビフィズス菌由来スルフォグリコシダーゼ  [Not invited]
    加藤紀彦; 前渋貴子; 吉川慶一; 後藤愛那; 山口真範; 芦田 久; 山本憲二; 片山高嶺
    第18回 関西グライコサイエンスフォーラム  2017/05
  • GH129 α-N-アセチルガラクトサミニダーゼの金属結合サイトと阻害剤を用いた解析  [Not invited]
    佐藤真与; LIEBSCHNER Dorothee; 山田悠介; 松垣直宏; 千田俊哉; 荒川孝俊; 芦田 久; 伏信進矢
    日本農芸化学会2017年度大会  2017/03
  • ムチン糖鎖に作用するビフィズス菌由来スルフォグリコシダーゼの同定と機能解析  [Not invited]
    加藤紀彦; 前渋貴子; 吉川慶一; 後藤愛那; 苫米地祐輔; 芦田 久; 山本憲二; 片山高嶺
    日本農芸化学会2017年度大会  2017/03
  • Isolation and identification of lactic acid bacteria from fermented fish and screening of bacteriocin-producing bacteria  [Not invited]
    Chaiwangsri; Koyanagi T; Ashida H; Matsuzaki C; Katayama T
    The 2nd joint seminar. Core to Core Program A. Advanced Research Networks.  2016/11
  • 食品成分によるオートファジー誘導と健康寿命伸長の可能性  [Not invited]
    芦田 久
    第12回 日本食品免疫学会 学術大会  2016/11
  • GH129ファミリー α-N-アセチルガラクトサミニダーゼのX線結晶構造解析  [Not invited]
    佐藤 真与; Dorothee Liebschner; 荒川 孝俊; 山田 悠介; 千田 俊哉; 芦田 久; 伏信 進矢
    日本応用糖質科学会平成28年度大会  2016/09
  • Bifidobacterium longumのアラビノ多糖・オリゴ糖資化メカニズムと関連する酵素群の解析  [Not invited]
    米野 雅大; 芦田 久
    日本応用糖質科学会平成28年度大会  2016/09
  • Sda抗原/GM2に作用するビフィズス菌由来のバイファンクショナル酵素  [Not invited]
    米野 雅大; 鈴木 里奈; 芦田 久
    第35回日本糖質学会年会  2016/09
  • アラビナン分解に関わる Bifidobacterium longum subsp. longumの2つの新奇 α-L-arabinofuranosidase  [Not invited]
    米野雅大; 速水穂乃香; 芦田 久
    日本乳酸菌学会2016年度大会  2016/07
  • Bifidobacterium longum subsp. longum のヘミセルロース分解酵素遺伝子クラスターの解析  [Not invited]
    芦田 久; 米野 雅大; 速水 穂乃香
    日本農芸化学会2016年度大会  2016/03
  • 梅酢中の糖質に作用するビフィズス菌のグリコシダーゼの解析  [Not invited]
    米野 雅大; 速水 穂乃香; 芦田 久
    地域イノベーション戦略支援プログラム 平成27年度シンポジウム  2016/01
  • 梅ポリフェノールの腸内細菌フローラ改善効果  [Not invited]
    島田良美; 香川昴雅; 晋家崇史; 堀西朝子; 尾﨑嘉彦; 芦田 久
    地域イノベーション戦略支援プログラム 平成27年度シンポジウム  2016/01
  • 梅酢に含まれる糖質のプレバイオティック効果  [Not invited]
    向井麻衣; 守田美咲; 前田直樹; 福永悟史; 島田良美; 芦田 久
    第64回 日本応用糖質科学会大会  2015/09
  • Bifidobacterium longum longum がもつ新規のアラビノース含有糖鎖分解酵素遺伝子クラスターの解析  [Not invited]
    米野雅大; 速水穂乃香; 芦田 久
    第64回 日本応用糖質科学会大会  2015/09
  • 腸内における共生に関わるビフィズス菌の酵素群  [Not invited]
    芦田 久
    第4回 石川県立大学 腸内細菌共生機構学特別セミナー  2015/09
  • 食品による健康寿命伸長の可能性  [Invited]
    芦田 久
    IUFoST-Japan 2015年度公開シンポジウム  2015/08
  • Bifidobacterium longum subsp. longum JCM 1217がもつ新規のヘミセルロース分解酵素遺伝子クラスターの解析  [Not invited]
    米野 雅大; 芦田 久
    第5回 近畿大学大学院 院生サミット  2015/07
  • 和歌山県産本なれ鮓の発酵に関わる細菌フローラ解析とプロバイオティック乳酸菌の探索  [Not invited]
    福永 悟史; 芦田 久
    第5回 近畿大学大学院 院生サミット  2015/07
  • ヒトとの共生に関わるビフィズス菌のグリコシダーゼ  [Invited]
    芦田 久
    第16回 関西グライコサイエンスフォーラム  2015/05
  • 和歌山県産本なれ鮓の菌叢解析と有用乳酸菌の探索  [Not invited]
    福永 悟史; 中林 直也; 島田 良美; 芦田 久
    2015年度 乳酸菌学会泊まり込みセミナー  2015/05
  • Bifidobacterium longum subsp. longum JCM 1217がもつ新規のアラビノース含有糖鎖分解酵素遺伝子クラスターの解析  [Not invited]
    米野 雅大; 速水 穂乃香; 芦田 久
    2015年度 乳酸菌学会泊まり込みセミナー  2015/05
  • Sda抗原を分解するビフィズス菌由来のバイファンクショナル酵素  [Not invited]
    芦田 久; 鈴木里奈
    日本農芸化学会2015年度大会  2015/03
  • 梅ポリフェノールの経口投与によるマウス腸内細菌叢改善効果  [Not invited]
    島田良美; 香川昂雅; 普家崇史; 堀西朝子; 尾﨑嘉彦; 芦田 久
    日本農芸化学会2015年度大会  2015/03
  • 梅ポリフェノールの腸内細菌叢改善効果  [Not invited]
    芦田 久
    地域イノベーション戦略支援プログラム事業 平成26年度成果報告会  2015/03
  • 梅ポリフェノールのマウス腸内細菌に及ぼす影響  [Not invited]
    島田良美; 香川昴雅; 晋家崇史; 尾﨑嘉彦; 芦田 久
    第12回 果実酒・果実飲料と健康に関する研究会  2014/11
  • ムチン型糖鎖に作用するグリコシダーゼ ~土壌微生物から腸内細菌へ~  [Invited]
    芦田 久
    日本応用糖質科学会 第39回近畿支部会・講演会  2014/11
  • ムチンの糖鎖に作用するビフィズス菌の新奇グリコシダーゼ  [Not invited]
    芦田 久
    食品酵素化学研究会第14回学術講演会  2014/08
  • Isolation of lactic acid bacteria isolated from fermented foods  [Not invited]
    Thida Chaiwangsri; Darunee Namuangrak; Takashi Koyanagi; Hisashi Ashida; Takane Katayama
    The 1st Joint Seminar. New Core to Core Program. A. Advanced Research Networks  2014/08
  • Screening and analysis of useful glycosidases for production of bifidogenic factors  [Not invited]
    Hisashi Ashida; Yoshimi Shimada; Yoshihisa Funeno; Masayuki Kubota; Thida Chaiwangsri; Toshihiko Katoh; Takashi Koyanagi; Hisanori Tamaki; Kenji Yamamoto; Takane Katayama
    The 1st Joint Seminar. New Core to Core Program. A. Advanced Research Networks  2014/08
  • Microbiota analyses on Japanese fermented foods with 16S rDNA-pyrosequencing approach  [Not invited]
    Takashi Koyanagi; Masashi Kiyohara; Hiroshi Matsui; Hisashi Ashida; Thida Chaiwangsri; Toshihiko Katoh; Hisanori Tamaki; Kenji Yamamoto; Takane Katayama; Hidehiko Kumagai
    The 1st Joint Seminar. New Core to Core Program. A. Advanced Research Networks  2014/08
  • ビフィズス菌由来の新規ラクト-N-ビオシダーゼの同定と機能解析  [Not invited]
    櫻間晴子; 清原正志; 芦田 久; 北岡本光; 高橋里美; 山本憲二; 片山高嶺
    第15回 関西グライコサイエンスフォーラム  2014/05
  • 食品による健康寿命伸長の可能性  [Not invited]
    芦田 久
    平成26年度第1回 近畿大学生物理工学部公開講座 Bost Science Cafe  2014/05
  • 果実由来成分の腸内細菌フローラへの影響  [Not invited]
    芦田 久
    地域イノベーション戦略支援プログラム事業 平成25年度成果報告会  2014/03
  • 食品由来オートファジー誘導物質の寿命への効果  [Invited]
    芦田 久
    第11回果実酒・果実飲料と健康に関する研究会  2013/12
  • 酵母由来endo-β-N-acetylglucosaminidaseの遺伝子クローニングおよび諸性質の解析  [Not invited]
    村上智史; 高橋佳江; 高岡友紀; 芦田 久; 山本憲二; 成松 久; 千葉靖典
    第32回日本糖質学会年会  2013/08
  • 健康寿命を伸ばす食品の秘密  [Not invited]
    芦田 久
    平成25年度第9回 近畿大学生物理工学部公開講座  2013/08
  • グルコサミンによるオートファジー誘導と寿命への効果  [Invited]
    芦田 久
    平成25年度 日本応用糖質科学会東日本支部シンポジウム  2013/07
  • Identification and characterization of endo-β-N-acetylglucosaminidase from methylotrophic yeast Ogataea minuta  [Not invited]
    Satoshi Murakami; Yuki Takaoka; Hisashi Ashida; Kenji Yamamoto; Hisashi Narimatsu; Yasunori Chiba
    Glyco 22  2013/06
  • Glucosamine as an Autophagy Inducer  [Invited]
    ASHIDA Hisashi
    13th International Conference of Functional Food Center  2013/05
  • 植物性多糖に作用するBifidobacterium longum由来の酵素の解析  [Not invited]
    芦田 久; 北村有里奈; 竹内涼子
    平成25年度日本農芸化学会大会  2013/03
  • 酵母由来のEndo-β-N-acetylglucosaminidaseのクローニングと性状解析  [Not invited]
    村上智史; 高岡友紀; 芦田 久; 山本憲二; 千葉靖典
    平成25年度日本農芸化学会大会  2013/03
  • 果実成分によるオートファジー誘導の可能性  [Not invited]
    芦田 久
    地域イノベーション戦略支援プログラム事業 平成24年度報告会  2013/03
  • 和歌山県特産農産物を活用した健康産業イノベーション  [Not invited]
    芦田 久
    和歌山県・近畿大学 包括的連携協定締結セミナー  2012/12
  • 酵母由来Endo-β-N-acetylglucosaminidaseのクローニングと特性解析  [Not invited]
    村上智史; 高岡友紀; 芦田 久; 山本憲二; 千葉靖典
    第85回 日本生化学会大会  2012/12
  • Bifidobacterium longum subsp. longum JCM 1217 由来の酵素GH43グリコシダーゼの解析  [Not invited]
    北村有里奈; 福澤秀哉; 芦田 久
    第85回 日本生化学会大会  2012/12
  • Bifidobacterium longum subsp. longum JCM 1217 由来GH31グリコシダーゼの解析  [Not invited]
    竹内涼子; 福澤秀哉; 芦田 久
    第85回 日本生化学会大会  2012/12
  • Analyses of metabolic pathway of oligosaccharides and its related enzymes in useful lactic acid bacteria  [Not invited]
    Thida Chaiwangsri; Masashi Kiyohara; Hisashi Ashida; Takane Katayama; Kenji Yamamoto
    The final joint seminar of Asian Core Program  2012/11
  • Application of glycosidase from lactic acid bacteria isolated from healthy infant faecies  [Not invited]
    Chartchai Khanongnuch; Wattana Sriphannam; Kenji Yamamoto; Hisashi Ashida; Goro Takata
    The final joint seminar of Asian Core Program  2012/11
  • 健康長寿を指向した和歌山県産果実由来の消化管機能改善成分の探索と機能解明  [Not invited]
    芦田 久
    地域イノベーション戦略支援プログラム キックオフセミナー  2012/10
  • 糸状菌由来エンドグリコシダーゼ(Endo-M)のグライコシンターゼ様変異体酵素を活用したシアロ糖タンパク質の化学-酵素合成  [Not invited]
    梅川碧里; 芦田 久; Lai-Xi Wang; 山本憲二
    応用糖質科学シンポジウム  2012/09
  • バクテロイデス菌由来α-N-Acetylglucosaminidaseへの糖鎖結合モジュール(CBM)付与効果  [Not invited]
    土田明子; 藤田雅也; 森 昌子; 後藤浩太朗; 弘瀬友理子; 大隅賢二; 野口真人; 正田晋一郎; 芦田 久; 水野真盛
    日本糖質学会 第31回年会  2012/09
  • Bifidobacterium longum subsp. infantis由来1,3-1,4-α-L-フコシダーゼが有するユニークな基質特異性の構造学的基盤  [Not invited]
    櫻間晴子; 伏信進矢; 北岡本光; 日高將文; 芦田 久; 山本憲二; 熊谷英彦; 片山高嶺
    第16回腸内細菌学会  2012/06
  • 良い腸内細菌は健康の要  [Not invited]
    芦田 久
    平成24年度 第3回近畿大学生物理工学部公開講座  2012/06
  • A fucosynthase specifically introducing histo-blood group antigens Lewis a/x into type-1/2 chains  [Not invited]
    Haruko Sakurama; Shinya Fushinobu; Erina Yoshida; Yuji Honda; Masafumi Hidaka; Hisashi Ashida; Motomitsu Kitaoka; Takane Katayama; Kenji Yamamoto; Hidehiko Kumagai
    The 12th Japan-China-Korea Joint Symposium on Enzyme Engineering  2012/05
  • ユニークな糖結合モジュールCBM51を含むビフィズス菌のGH110 α-ガラクトシダーゼ  [Not invited]
    脇中琢良; 清原正志; 大沼貴之; 深溝 慶; 山本憲二; 芦田 久
    第13回 関西グライコサイエンスフォーラム  2012/05
  • ビフィズス菌の共生戦略  [Not invited]
    片山高嶺; 芦田 久; 熊谷英彦; 山本憲二
    日本農芸化学会2012年度大会  2012/03
  • 抗肝繊維化物質テトランドリンによる肝星細胞でのマクロリポファジー制御  [Not invited]
    宮前友策; 仲井奈緒美; 翁長彰子; 韓畯奎; 磯田博子; 芦田 久; 神戸大朋; 入江一浩; 永尾雅哉
    日本農芸化学会2012年度大会  2012/03
  • 1,3-1,4-α-L-フコシダーゼの結晶構造解析に基づく基質特異性の構造学的基盤  [Not invited]
    櫻間晴子; 伏信進矢; 北岡本光; 日高将文; 芦田 久; 片山高嶺; 山本憲二; 熊谷英彦
    日本農芸化学会2012年度大会  2012/03
  • Glucosamine-induced autophagy is mediated by release of ammonia and promotes longevity in C. elegans  [Not invited]
    Yuhei Kosuge; Hiroki Sakai; Tomoya Shintani; Hisashi Ashida
    The 10th International Student Seminar  2012/03
  • GH110 α-galactosidase from bifidobacteria specifically acts on blood group B antigens of gastrointestinal mucin  [Not invited]
    Takura Wakinaka; Masashi Kiyohara; Kenji Yamamoto; Hisashi Ashida
    The 10th International Student Seminar  2012/03
  • オートファジー誘導物質としてのグルコサミン  [Invited]
    芦田 久
    第8回 グルコサミン研究会学術集会  2012/01
  • A novel glycosides from infant-associated bifidobacteria involved in mucin degradation pathway  [Invited]
    ASHIDA Hisashi
    U.P. Special Lectures on Probiotic Lactic Acid Bacteria  2011/12
  • ビフィズス菌のユニークなアミノ糖含有ヘテロオリゴ糖代謝  [Invited]
    芦田 久
    第4回アグリバイオセミナー  2011/11
  • Induction of autophagy by chitobiose and its derivatives  [Not invited]
    Hisashi Ashida; Hiroki Sakai; Yuhei Kosuge; Tomoya Shintani
    The 31st Naito Conference on Glycan Expression and Regulation  2011/09
  • 2種のビフィズス菌の異なるフコシルオリゴ糖利用戦略  [Not invited]
    芦田 久; 藤本 卓; 片山高嶺; 山本憲二
    日本乳酸菌学会2011年度大会  2011/07
  • ビフィズス菌の巧みなオリゴ糖利用戦略  [Invited]
    芦田 久
    京都大学微生物科学寄附研究部門主催シンポジウム  2011/06

MISC

Industrial Property Rights

  • エンドグリコセラミダーゼおよびその製造方法
    特開平4-126076
  • リゾ糖脂質の製造方法
    特開平7-107988
  • カリウムイオン測定用試薬組成物および試験片
    特許第3493411
  • 体液中のマグネシウム測定用試験片
    特許第3809484
  • ガングリオシドの定量方法
    特許第3985021
  • 特願2019-149688:腸内細菌フローラにおけるクロストリジウムクラスター抑制用組成物  
    芦田久, 山口真範  近畿大学、和歌山大学
  • 特開2012-050428:消化管ムチンの糖鎖に特異的に結合するポリペプチド  
    藤田 雅也, 土田 明子, 芦田 久

Awards & Honors

  • 2020/09 日本応用糖質科学会 令和2年度 日本応用糖質科学会 学会賞
     ムチン糖鎖による宿主と腸内細菌の共生機構の解明 
    受賞者: 芦田 久
  • 2012/03 JSBBA Most-Cited Paper Award
     Prebiotic Effect of Lacto-N-biose I on Bifidobacterial Growth 
    受賞者: Masashi KIYOHARA;Asaki TACHIZAWA;Mamoru NISHIMOTO;Motomitsu KITAOKA;Hisashi ASHIDA;Kenji YAMAMOTO
  • 2010/03 JSBBA Topics Award
     キチン加水分解物より見出したオートファジー誘導物質 japan_society 
    受賞者: ASHIDA Hisashi

Research Grants & Projects

  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2022/04 -2026/03 
    Author : 正木 秀幸; 芦田 久; 東 慶直
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2023/06 -2025/03 
    Author : 栗原 新; 松岡 悠美; 芦田 久; 岡本 成史
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2022/04 -2025/03 
    Author : Hisashi Ashida
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2020/04 -2023/03 
    Author : 栗原 新; 小柳 喬; 芦田 久; 松本 光晴
     
    ポリアミンは、末端にアミノ基を持つ脂肪族炭化水素で、生命活動に不可欠な物質である。ポリアミンの摂取により寿命が延びるなど、動物においてポリアミンが健康増進効果を持つことが報告されている。食品からの高ポリアミン産生菌の同定は、新規プロバイオティクス候補の開発につながる可能性がある。 本研究では、食品からの高ポリアミン産生菌の同定を目指し、日本国内で生産される野菜や発酵食品から菌を分離・収集した。この結果、ポリアミンの一種であるプトレッシンを約200 μMの濃度で培養上清中に蓄積する乳酸菌として、石川県の伝統発酵食品である「かぶらずし」から分離したLatilactobacillus curvatus KP 3-4を取得することに成功した。L. curvatus KP 3-4のポリアミン合成能を、Japan Collection of Microorganismsから入手可能な代表的なプロバイオティクス乳酸菌およびL. curvatus株と比較したところ、L. curvatus KP 3-4のみが培養上清に多量のプトレッシンを放出できることが判明した。さらに本菌の全ゲノム解析から、プトレッシンはオルニチン脱炭酸酵素を介して合成されることが示唆された。L. curvatus KP 3-4 を無菌マウスに投与したところ、糞便中のプトレッシン濃度が上昇した。 同様の手法により、培養上清に高濃度(約500 μM)のプトレッシンを蓄積する能力を持つStaphylococcus epidermidis FB146を日本の発酵食品である味噌から単離した。S. epidermidis FB146に加え、代表的なブドウ球菌21種の基準株の培養上清と細胞中のポリアミンの存在を解析したところ、S. epidermidis FB146のみが高いプトレッシン生産性を有していた。
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2018/04 -2022/03 
    Author : MASAKI Hideyuki
     
    Zika virus (ZIKV) causes congenital abnormality, and has an aspect of sexually transmitted disease agent. Its major protective immunity - inducing antigen is the envelope (E) protein. Mucosal epithelium is the contact interface to infectious agents, and mucosal immunity consists of systemic immune network. In order to confirm whether effective protective immunity against ZIKV infection can be induced to mucosa of sexual organ by oral immunization with Saccharomyces or lactobacilli on which Zika virus E protein is expressed, we tried to establish the Saccharomyces and the lactobacillus strains which stably express Zika virus E protein on cell surface for the purpose of using as oral mucosal vaccine antigen, and those which express GFP on cell surface to use as control antigen. As results, we have succeeded in establishing the Saccharomyces strains which stably express Zika virus E protein or GFP on their cell surface. Establishing of the lactobacillus strains is however still under way.
  • 農産物を活用した腸内環境調節を介する新規機能性食品素材の開発とメカニズム解明
    日本学術振興会:科研費基盤研究(C)
    Date (from‐to) : 2018/04 -2021/03 
    Author : 芦田久
  • 伝統的な和歌山ダイコンに乳酸菌の機能性を加味した紀の川漬の開発
    和歌山県:先駆的産業技術研究開発支援補助金事業
    Date (from‐to) : 2018 -2021
  • 食品由来廃棄物を有効活用した新規ビフィズス菌増殖因子の創成
    日本学術振興会:科研費基盤研究(C)
    Date (from‐to) : 2015/04 -2018/03
  • 和歌山県特産農産物を活用した健康産業イノベーション推進地域
    文部科学省:地域イノベーション戦略支援プログラム
    Date (from‐to) : 2012 -2016
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2012/04 -2015/03 
    Author : FUSHINOBU Shinya; ASHIDA Hisashi; FUJITA Kiyotaka
     
    We determined the crystal structure of GH127 beta-L-arabinofuranosidase (HypBA1). HypBA1 has a Cys residue at its active center and was the first "cysteine glycosidase". We found four new glycosidases from B. bifidum. Moreover, we revealed the transcription regulation mechanism of the beta-arabinooligosaccharide degradation system in B. longum and investigated characteristics of a HypBA1 homolog.
  • 健康と長寿を指向したアミノ糖含有ヘテロ糖鎖による腸内環境改善技術の開発
    日本学術振興会:科研費基盤研究(C)
    Date (from‐to) : 2012/04 -2015/03
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2010 -2011 
    Author : YAMAMOTO Kenji; ASHIDA Hisashi
     
    We have succeeded in simple syntheses of efficient binding-inhibitors for influenza virus, which are composed of sialyl saccharides of glycopeptides extracted from hen egg yolk. We modified alginate and chitosan with multiple sialyl saccharides using condensation reaction and reductive amination reaction, respectively. These binding-inhibitors utilize the carbohydrate recognition of influenza virus for sialic acid, thus they can prevent the virus from infection. The resulting inhibitors showed sufficient inhibitory activity against influenza virus infection in MDCK cells. Unlike the other binding-inhibitors of influenza virus, these virus inhibitors require simple step in their syntheses.
  • キチン二糖・オリゴ糖の新機能
    生研センター:イノベーション創出基礎的研究推進事業
    Date (from‐to) : 2008 -2010
  • 微生物のエンドグリコシダーゼの変異酵素を活用した糖鎖医薬品の開発と調製法の確立
    日本学術振興会:科研費基盤研究(B)
    Date (from‐to) : 2008 -2010
  • ミルクオリゴ糖を分解するビフィズス菌由来の酵素の探索と応用
    日本学術振興会:科研費基盤研究(B)
    Date (from‐to) : 2005 -2009
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2006 -2007 
    Author : YAMAMOTO Kenji; ASHIDA Hisashi; KATAYAMA Takane
     
    The purposes of this Scientific Research B is to enzymatically synthesize various bioactive glycoconjugates compounds rising transglycosylation activity of endoglycosidases and also to analyze the enzyme reaction in order to establish the preparation method. The followings are the results of this research. 1. Large preparation of the recombinant enzyme : We succeeded in the large production of recombinant Endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) by cloning the gene of Endo-M using Escherichia coil and its growing at low temperature without induction by IPTG. 2. Obtaining the mutant enzyme having higher transglycosylation activity : We obtained Y217F mutant enzyme having higher transglycosylation activity by site-directed mutagenesis of the residues in the putative catalytic region of Endo-M. 3. Obtaining mutant enzyme having glycosynthase-like activity : We obtained N175A mutant having glycosynthase-like activity by site-directed mutagenesis. This mutant shows no activities of hydrolysis and transglycosylation. However, the enzyme expressed the transglycosylation activity when the oxazoline form of sugar chain was added to the reaction mixture as donor of sugar chain in transglycosylation, and the increase of transglycosylation product continued without its hydrolysis. Using this mutant enzyme, we could synthesize the bioactive glycopeptide having anti-HIV activity with high yield. 4. Addition of mucin type sugar chain to serine or threonine residue of the peptide using the recombinant endo-α-N-acetylgalactosaminidase from Bifidobacterium: We succeeded in the addition of mucin type sugar chain (galactosylβ1,3N-acetylgalactosamine) to serine or threonine residue of Muc1a which is the peptide of a part of human mucin, in addition to free serine and threonine.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2005 -2006 
    Author : KINOSHITA Taroh; MORITA Yasuhiro; ASIDA Hisashi
     
    A protozoan parasite Trypanosoma brucei is coated by glycosylphosphatidylinositol (GPI) anchored proteins. During GPI biosynthesis, inositol in phosphatidylinositol becomes acylated. Inositol is deacylated prior to attachment to variant surface glycoproteins (VSG) in the bloodstream form whereas it remains acylated in procyclins in the procyclic form. We have cloned a T brucei GPl inositol deacylase (GPIdeAc2). In accordance with the acylation / deacylation profile, GPIdeAc2 was expressed at a 6-fold higher level in the bloodstream form than the procyclic form. Knockdown of GPIdeAc2 in the bloodstream form caused accumulation of an inositol-acylated GPI, a decreased VSG expression on the cell surface and slower growth, indicating the essential role of inositol-deacylation for growth of the bloodstream form. Overexpression of GPIdeAc2 in the procyclic form caused an accumulation of GPI lacking inositol-linked acyl chain and decreased cell surface procyclins due to release into the culture medium, indicating that overexpression of GPIdeAc2 is deleterious to the surface coat of the procyclic form. In the fly's intestine, the trypanosomes survive digestive and trypanocidal environments, proliferate and translocate into the salivary gland where they become infectious to the next mammalian host. For the successful survival in tsetse flies, the trypanosomes use trans-sialidase to transfer sialic acids, which they cannot synthesize, from host's glycoconjugates to the GPIs that are abundantly expressed on their surface. T.brucei has at least 8 trans-sialidase like genes and the enzyme activity has been shown in only one of them, TSB38p. We cloned other genes and found that two of them, termed TS550band TS290b, do not have the enzyme activity. One of them, termed TS270b, clearly had trans-sialidase activity. Co-transfection of soluble forms of TSB38p and TS270b into TbGPI8 knockout trypanosomes resulted in attachment of additive amounts of sialic acid onto the parasites, suggesting that the two isoforms had different substrate specificities.
  • 日本学術振興会:科学研究費助成事業
    Date (from‐to) : 2002 -2006 
    Author : 木下 タロウ; 前田 裕輔; 村上 良子; 芦田 久; 森田 康裕
     
    GPIアンカー生合成経路の酵素群の研究に関し、第1ステップに働くNアセチルグルコサミン転移酵素の新規構成成分PIG-Yをクローニングし、この酵素が7つのサブユニットからなる極めてユニークな単糖転移酵素であることを示した。 GPIアンカー欠損症で、後天性遺伝子病である発作性夜間血色素尿症の発症メカニズムに関し、GPIアンカー欠損クローンの拡大が良性腫瘍性に起こることを示唆する第2の遺伝子変異を証明した。 平成15年度の成果として報告したPGAP1は、タンパク質に結合直後のGPIアンカーのイノシトールから脂肪酸を除去する脱アシル化酵素である。PGAP1が機能しないとGPIアンカーは3つの脂肪鎖を持ったまま細胞表面に発現してしまう。このとき生体に何が起こるかを知るため、PGAP1ノックアウトマウスを作製した。ノックアウト個体の多くは周産期致死を示し、生き残った個体も成長が遅延した。雄は不妊であったので、不妊の機序を解析したところ、精子が卵子の透明体に付着できないことがわかった。ノックアウト個体の精子上のGPIアンカー型タンパク質のレベルを調べたところ、いくつかの蛋白質の量が正常より多かった。GPIアンカーの切断が、精子の妊性獲得に重要であるとの報告もあり、イノシトールからの脂肪酸除去不全がGPIアンカー型タンパク質の精子からの切断除去を阻害している可能性が考えられた。

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