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ASHIDA Hisashi


FacultyDepartment of Science and Technology on Food Safety / Graduate School of Biology-Oriented Science and Technology
Commentator Guide
Last Updated :2020/09/02

Education and Career


  •  - 1988 , Kyoto University, Faculty of Agriculture
  •   1983 04  - 1988 03 , Kyoto University, Faculty of Agriculture

Academic & Professional Experience

  •   2012 04 ,  - 現在, Professor, Faculty of Biology-Oriented Science and Technology, Kinki University
  •   2008 06 ,  - 2012 03 , Graduate School of Biostudies, Kyoto University
  •   2006 01 ,  - 2008 05 , Graduate School of Biostudies, Kyoto University
  •   2004 04 ,  - 2005 12 , Research Institute for Microbial Diseases, Osaka University
  •   2001 09 ,  - 2004 03 , Research Institute for Microbial Diseases, Osaka University
  •   2000 07 ,  - 2001 08 , Post doctoral fellow, Tulane University,
  •   1997 04 ,  - 2000 07 , Graduate School of Agriculture, Kyoto University

Research Activities

Research Areas

  • Life sciences, Applied molecular and cellular biology
  • Life sciences, Food sciences
  • Life sciences, Applied biochemistry
  • Life sciences, Applied microbiology

Research Interests

  • Biodiversity, Food Science and Technology, Applied Microbiology, Cell Biology, Glycobiology

Published Papers

  • 1,6-α-L-Fucosidases from Bifidobacterium longum subsp. infantis ATCC 15697 Involved in the Degradation of Core-fucosylated N-Glycan, Ashida Hisashi, Fujimoto Taku, Kurihara Shin, Nakamura Masayuki, Komeno Masahiro, Huang Yibo, Katayama Takane, Kinoshita Takashi, Takegawa Kaoru, Journal of Applied Glycoscience, Journal of Applied Glycoscience, 67(1), 23 - 29, Feb. 2020 , 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.

  • Two Novel α-L-Arabinofuranosidases from Bifidobacterium longum subsp. longum Belonging to Glycoside Hydrolase Family 43 Cooperatively Degrade Arabinan., Komeno M, Hayamizu H, Fujita K, Ashida H, Applied and Environmental Microbiology, Applied and Environmental Microbiology, 85, e02582-18, Jan. 2019 , Refereed
  • Calorie Restriction Mimetics: Upstream-Type Compounds for Modulating Glucose Metabolism., Shintani H, Shintani T, Ashida H, Sato M, Nutrients, Nutrients, 10(12), 1821, Nov. 2018 , Refereed
  • Bifunctional properties and characterization of a novel sialidase with esterase activity from Bifidobacterium bifidum., Ashida H, Tanigawa K, Kiyohara M, Katoh T, Katayama T, Yamamoto K, Bioscience, Biotechnology, and Biochemistry, Bioscience, Biotechnology, and Biochemistry, 82(11), 2030 - 2039, Nov. 2018 , Refereed
  • Ume polyphenol: prebiotic effects on diet-induced obesity mice and growth-promoting mechanism for bifidobacteria, Masahiro Komeno, Azusa Ito, Natsumi Ohigashi, Yuki Yoshihara, Toshio Suzuki, Kouhei Nagai, Hisashi Ashida, Mem Faculty B.O.S.T., Kindai University, Mem Faculty B.O.S.T., Kindai University, (42), 1 - 12, Oct. 2018 , Refereed
  • Glucosamine extends the lifespan of Caenorhabditis elegans via autophagy induction, Shintani T, Kosuge Y, Ashida H, Journal of Applied Glycoscience, Journal of Applied Glycoscience, 65(3), 37 - 43, Aug. 2018 , Refereed
  • Application study of 1,2-alpha-L-fucosynthase: introduction of Fuc alpha 1-2Gal disaccharide structures on N-glycan, ganglioside, and xyloglucan oligosaccharide, Yuta Sugiyama, Toshihiko Katoh, Yuji Honda, Aina Gotoh, Hisashi Ashida, Shin Kurihara, Kenji Yamamoto, Takane Katayama, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 81(2), 283 - 291, Feb. 2017 , Refereed
    Summary: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.
  • Introduction of H-antigens into oligosaccharides and sugar chains of glycoproteins using highly efficient 1,2-alpha-L-fucosynthase, Yuta Sugiyama, Aina Gotoh, Toshihiko Katoh, Yuji Honda, Erina Yoshida, Shin Kurihara, Hisashi Ashida, Hidehiko Kumagai, Kenji Yamamoto, Motomitsu Kitaoka, Takane Katayama, GLYCOBIOLOGY, GLYCOBIOLOGY, 26(11), 1235 - 1247, Nov. 2016 , Refereed
    Summary: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.
  • Microbial diversity of ‘narezushi’ from Wakayama Prefecture, western Japan, Shimada Y, Ashida H, Mem. Faculty. B.O.S.T. Kindai University, Mem. Faculty. B.O.S.T. Kindai University, (38), 1 - 10, Oct. 2016
  • Effect of orally administered ume polyphenol on the cecal microflora of mice, Yoshimi Shimada, Takamasa Kagawa, Takafumi Fuke, Asako Horinishi, Yoshihiko Ozaki, Hisashi Ashida, Mem. Faculty. B.O.S.T. Kindai University, Mem. Faculty. B.O.S.T. Kindai University, (37), 1 - 10, May 2016
  • Novel substrate specificities of two lacto-N-biosidases towards beta-linked galacto-N-biose-containing oligosaccharides of globo H, Gb5, and GA1, Aina Gotoh, Toshihiko Katoh, Yuta Sugiyama, Shin Kurihara, Yuji Honda, Haruko Sakurama, Taiho Kambe, Hisashi Ashida, Motomitsu Kitaoka, Kenji Yamamoto, Takane Katayama, CARBOHYDRATE RESEARCH, CARBOHYDRATE RESEARCH, 408, 18 - 24, May 2015 , Refereed
    Summary: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.
  • alpha-N-Acetylglucosaminidase from Bifidobacterium bifidum specifically hydrolyzes alpha-linked N-acetylglucosamine at nonreducing terminus of O-glycan on gastric mucin, Yoshimi Shimada, Yuka Watanabe, Takura Wakinaka, Yoshihisa Funeno, Masayuki Kubota, Thida Chaiwangsri, Shin Kurihara, Kenji Yamamoto, Takane Katayama, Hisashi Ashida, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 99(9), 3941 - 3948, May 2015 , Refereed
    Summary: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.
  • Glycosidases: inborn errors of glycosphingolipid catabolism., Ashida H, Li YT, Advances in Neurobiology, Advances in Neurobiology, 9, 463 - 484, Jul. 2014 , Refereed
  • beta-Glucuronidase from Lactobacillus brevis useful for baicalin hydrolysis belongs to glycoside hydrolase family 30, Haruko Sakurama, Shigenobu Kishino, Yoshie Uchibori, Yasunori Yonejima, Hisashi Ashida, Keiko Kita, Satomi Takahashi, Jun Ogawa, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 98(9), 4021 - 4032, May 2014 , Refereed
    Summary: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.
  • Deficiency of alpha-glucosidase I alters glycoprotein glycosylation and lifespan in Caenorhabditis elegans, Toshihiko Katoh, Juri Takase, Yasushi Tani, Ryuta Amamoto, Naofumi Aoshima, Michael Tiemeyer, Kenji Yamamoto, Hisashi Ashida, GLYCOBIOLOGY, GLYCOBIOLOGY, 23(10), 1142 - 1151, Oct. 2013 , Refereed
    Summary: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.
  • Identification and characterization of endo-β-N-acetylglucosaminidase from methylotrophic yeast Ogataea minuta., Satoshi Murakami, Yuki Takaoka, Hisashi Ashida, Kenji Yamamoto, Hisashi Narimatsu, Yasunori Chiba, Glycobiology, Glycobiology, 23(6), 736 - 44, Jun. 2013 , Refereed
    Summary: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.
  • Bifidobacterial alpha-galactosidase with unique carbohydrate-binding module specifically acts on blood group B antigen, Takura Wakinaka, Masashi Kiyohara, Shin Kurihara, Akiko Hirata, Thida Chaiwangsri, Takayuki Ohnuma, Tamo Fukamizo, Takane Katayama, Hisashi Ashida, Kenji Yamamoto, GLYCOBIOLOGY, GLYCOBIOLOGY, 23(2), 232 - 240, Feb. 2013 , Refereed
    Summary: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.
  • Differences in the Substrate Specificities and Active-Site Structures of Two alpha-L-Fucosidases (Glycoside Hydrolase Family 29) from Bacteroides thetaiotaomicron, Haruko Sakurama, Erika Tsutsumi, Hisashi Ashida, Takane Katayama, Kenji Yamamoto, Hidehiko Kumagai, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 76(5), 1022 - 1024, May 2012 , Refereed
    Summary: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.
  • 1,3-1,4-alpha-L-Fucosynthase That Specifically Introduces Lewis a/x Antigens into Type-1/2 Chains, Haruko Sakurama, Shinya Fushinobu, Masafumi Hidaka, Erina Yoshida, Yuji Honda, Hisashi Ashida, Motomitsu Kitaoka, Hidehiko Kumagai, Kenji Yamamoto, Takane Katayama, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 287(20), 16709 - 16719, May 2012 , Refereed
    Summary: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 and alpha-L-fucosidase (EC 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.
  • Bifidobacterium longum subsp infantis uses two different beta-galactosidases for selectively degrading type-1 and type-2 human milk oligosaccharides, Erina Yoshida, Haruko Sakurama, Masashi Kiyohara, Masahiro Nakajima, Motomitsu Kitaoka, Hisashi Ashida, Junko Hirose, Takane Katayama, Kenji Yamamoto, Hidehiko Kumagai, GLYCOBIOLOGY, GLYCOBIOLOGY, 22(3), 361 - 368, Mar. 2012 , Refereed
    Summary: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.
  • A Selected Probiotic Strain of Lactobacillus fermentum CM33 Isolated from Breast-Fed Infants as a Potential Source of beta-Galactosidase for Prebiotic Oligosaccharide Synthesis, Wattana Sriphannam, Saisamorn Lumyong, Piyanuch Niumsap, Hisashi Ashida, Kenji Yamamoto, Chartchai Khanongnuch, JOURNAL OF MICROBIOLOGY, JOURNAL OF MICROBIOLOGY, 50(1), 119 - 126, Feb. 2012 , Refereed
    Summary: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.
  • alpha-N-Acetylgalactosaminidase from Infant-associated Bifidobacteria Belonging to Novel Glycoside Hydrolase Family 129 Is Implicated in Alternative Mucin Degradation Pathway, 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, JOURNAL OF BIOLOGICAL CHEMISTRY, 287(1), 693 - 700, Jan. 2012 , Refereed
    Summary: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, African Journal of Biotechnology, 11(51), 11242 - 11251, Jan. 2012 , Refereed
  • Crystallography of enzymes in the unique sugar metabolism of Bifidobacteria, Masafumi Hidaka, Ryuichiro Suzuki, Takane Katayama, Motomitsu Kitaoka, Takayoshi Wakagi, Hirofumi Shoun, Hisashi Ashida, Kenji Yamamoto, Shinya Fushinobu, Photon Factory Activity Report 2009, Photon Factory Activity Report 2009, 27(B), 250 - 250, Dec. 2010
  • Cooperation of beta-galactosidase and beta-N-acetylhexosaminidase from bifidobacteria in assimilation of human milk oligosaccharides with type 2 structure, Mika Miwa, Tomohiro Horimoto, Masashi Kiyohara, Takane Katayama, Motomitsu Kitaoka, Hisashi Ashida, Kenji Yamamoto, GLYCOBIOLOGY, GLYCOBIOLOGY, 20(11), 1402 - 1409, Nov. 2010 , Refereed
    Summary: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.
  • Efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline., 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, Biochim. Biophys. Acta General Subjects, 1800, 1203 - 1209, Nov. 2010 , Refereed
  • Crystallographic and mutational analyses of substrate recognition of endo-alpha-N-acetylgalactosaminidase from Bifidobacterium longum., Suzuki R, Katayama T, Kitaoka M, Kumagai H, Wakagi T, Shoun H, Ashida H, Yamamoto K, Fushinobu S, J. Biochem. (Tokyo), J. Biochem. (Tokyo), 146(3), 389 - 398, Sep. 2009 , Refereed
  • Crystallographic analysis of novel sugar metabolic enzymes from Bifidobacteria, Ryuichiro Suzuki, Jun Wada, Takane Katayama, Shinya Fushinobu, Takayoshi Wakagi, Hirofumi Shoun, Motomitsu Kitaoka, Hidehiko Kumagai, Hisashi Ashida, Kenji Yamamoto, Photon Factory Activity Report 2007, Photon Factory Activity Report 2007, 25(B), 235 - 235, Dec. 2008
  • PGAP2 is essential for correct processing and stable expression of GPI-anchored proteins., Tashima Y, Taguchi R, Murata C, Ashida H, Kinoshita T, Maeda Y, Mol. Biol. Cell, Mol. Biol. Cell, 17(3), 1410 - 1420, Mar. 2006 , Refereed
  • DPM1, the catalytic subunit of dolichol-phosphate mannose synthase, is tethered to and stabilized on the endoplasmic reticulum membrane by DPM3., Ashida H, Maeda Y, Kinoshita T, J. Biol. Chem., J. Biol. Chem., 281(2), 896 - 904, Jan. 2006 , Refereed
  • CHO glycosylation mutants: GPI anchor., Maeda Y, Ashida H, Kinoshita T, Methods Enz., Methods Enz., 416, 182 - 205, 2006 , Refereed
  • A clostridial endo-beta-galactosidase that cleaves both blood group A and B glycotopes: the first member of a new glycoside hydrolase family, GH98., Anderson KM, Ashida H, Maskos K, Dell A, Li SC, Li YT, J. Biol. Chem., J. Biol. Chem., 280(9), 7720 - 7728, Mar. 2005 , Refereed
  • Mammalian PIG-X and yeast Pbn1p are the essential components of glycosylphosphatidylinositol-mannosyltransferase I., Ashida H, Hong Y, Murakami Y, Shishioh N, Sugimoto N, Kim YU, Maeda Y, Kinoshita T, Mol. Biol. Cell, Mol. Biol. Cell, 16, 1439 - 1448, Mar. 2005 , Refereed
  • Characterization of endo-alpha-N-acetylgalactosaminidase from Bacillus sp. and syntheses of neo-oligosaccharides using its transglycosylation activity, Ashida H, Yamamoto K, Murata T, Usui T, Kumagai H, Arch. Biochem. Biophys., Arch. Biochem. Biophys., 373(394), 400, 2000 , Refereed
  • Trypsin inhibitory activity of bovine fetuin de-O-glycosylated by endo-alpha-N-acetylgalactosaminidase, Ashida H, Yamamoto K, Kumagai H, Biosci. Biotech. Biochem., Biosci. Biotech. Biochem., 64, 2066 - 2068, 2000 , Refereed
  • Enzymatic Adaptation of Bifidobacterium bifidum to Host Glycans, Viewed from Glycoside Hydrolyases and Carbohydrate-Binding Modules, Toshihiko Katoh, Miriam N. Ojima, Mikiyasu Sakanaka, Hisashi Ashida, Aina Gotoh, Takane Katayama, Microorganisms, Microorganisms, 8(4), 481 - 481, Mar. 28 2020 , Refereed
    Summary: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.
  • Chemo-enzymatic synthesis of the glucagon containing N-linked oligosaccharide and its characterization, Takayuki Higashiyama, Midori Umekawa, Masaya Nagao, Toshihiko Katoh, Hisashi Ashida, Kenji Yamamoto, Carbohydrate Research, Carbohydrate Research, 455, 92 - 96, Jan. 02 2018 , Refereed
    Summary: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.
  • The first crystal structure of a family 129 glycoside hydrolase from a probiotic bacterium reveals critical residues and metal cofactors, 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, JOURNAL OF BIOLOGICAL CHEMISTRY, 292(29), 12126 - 12138, Jul. 2017 , Refereed
    Summary: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.
  • Identification and characterization of a sulfoglycosidase from Bifidobacterium bifidum implicated in mucin glycan utilization, 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, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 81(10), 2018 - 2027, 2017 , Refereed
    Summary: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.
  • Glycan region of GPI anchored-protein is required for cytocidal oligomerization of an anticancer parasporin-2, Cry46Aa1 protein, from Bacillus thuringiensis strain A1547, Yuich Abe, Hiroshi Inoue, Hisashi Ashida, Yusuke Maeda, Taroh Kinoshita, Sakae Kitada, JOURNAL OF INVERTEBRATE PATHOLOGY, JOURNAL OF INVERTEBRATE PATHOLOGY, 142, 71 - 81, Jan. 2017 , Refereed
    Summary: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.
  • Structural analysis of cerebrosides from Aspergillus fungi: the existence of galactosylceramide in A. oryzae, Yasushi Tani, Yasunori Amaishi, Tori Funatsu, Masahiro Ito, Saki Itonori, Yoji Hata, Hisashi Ashida, Kenji Yamamoto, BIOTECHNOLOGY LETTERS, BIOTECHNOLOGY LETTERS, 36(12), 2507 - 2513, Dec. 2014 , Refereed
    Summary: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.
  • Lacto-N-biosidase Encoded by a Novel Gene of Bifidobacterium longum Subspecies longum Shows Unique Substrate Specificity and Requires a Designated Chaperone for Its Active Expression, 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, JOURNAL OF BIOLOGICAL CHEMISTRY, 288(35), 25194 - 25206, Aug. 2013 , Refereed
    Summary: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.
  • Crystal structures of a glycoside hydrolase family 20 lacto-N-biosidase from Bifidobacterium bifidum, 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, Journal of Biological Chemistry, 288(17), 11795 - 11806, Apr. 26 2013 , Refereed
    Summary: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.
  • Identification of a second catalytically active trans-sialidase in Trypanosoma brucei, Fumiki Nakatani, Yasu S. Morita, Hisashi Ashida, Kisaburo Nagamune, Yusuke Maeda, Taroh Kinoshita, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 415(2), 421 - 425, Nov. 2011 , Refereed
    Summary: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.
  • Physiology of Consumption of Human Milk Oligosaccharides by Infant Gut-associated Bifidobacteria, Sadaki Asakuma, Emi Hatakeyama, Tadasu Urashima, Erina Yoshida, Takane Katayama, Kenji Yamamoto, Hidehiko Kumagai, Hisashi Ashida, Junko Hirose, Motomitsu Kitaoka, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 286(40), 34583 - 34592, Oct. 2011 , Refereed
    Summary: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.
  • Longevity in Mice Is Promoted by Probiotic-Induced Suppression of Colonic Senescence Dependent on Upregulation of Gut Bacterial Polyamine Production, Mitsuharu Matsumoto, Shin Kurihara, Ryoko Kibe, Hisashi Ashida, Yoshimi Benno, PLOS ONE, PLOS ONE, 6(8), e23652, Aug. 2011 , Refereed
    Summary: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.
  • An exo-alpha-sialidase from bifidobacteria involved in the degradation of sialyloligosaccharides in human milk and intestinal glycoconjugates, Masashi Kiyohara, Kana Tanigawa, Thida Chaiwangsri, Takane Katayama, Hisashi Ashida, Kenji Yamamoto, GLYCOBIOLOGY, GLYCOBIOLOGY, 21(4), 437 - 447, Apr. 2011 , Refereed
    Summary: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.
  • Glycoside Hydrolase Family 89 alpha-N-acetylglucosaminidase from Clostridium perfringens Specifically Acts on GlcNAc alpha 1,4Gal beta 1R at the Non-reducing Terminus of O-Glycans in Gastric Mucin, 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, JOURNAL OF BIOLOGICAL CHEMISTRY, 286(8), 6479 - 6489, Feb. 2011 , Refereed
    Summary: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.
  • Efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline, Midori Umekawa, Takayuki Higashiyama, Yurie Koga, Tomonari Tanaka, Masato Noguchi, Atsushi Kobayashi, Shin-ichiro Shoda, Wei Huang, Lai-Xi Wang, Hisashi Ashida, Kenji Yamamoto, BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1800(11), 1203 - 1209, Nov. 2010 , Refereed
    Summary:Background: An efficient method for synthesizing homogenous glycoproteins is essential for elucidating the structural and functional roles of glycans of glycoproteins. We have focused on the transglycosylation activity of endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) as a tool for glycoconjugate syntheses, since it can transfer en bloc the oligosaccharide of not only high-mannose type but also complex-ype N-glycan onto various acceptors having an N-acetylglucosamine residue. However, there are two major bottlenecks for its practical application: the low yield of the transglycosylation product and the difficulty to obtain the activated sugar oxazoline substrate, especially the slab-complex type one. Methods: We carried out the transglycosylation using a glycosynthase-like N175Q mutant of Endo-M, which was found to possess enhanced transglycosylation activity with sugar oxazoline as a donor substrate, in combination with an easy preparation of the sialo-complex-type sugar oxazoline from natural sialoglycopeptide in egg yolk. Results: Endo-M-N175Q showed efficient transglycosylation toward sialo-complex-type sugar oxazoline onto bioactive peptides and bovine ribonuclease B, and each sialylated compound was obtained in significantly high yield. Conclusions: Highly efficient and simple chemo-enzymatic syntheses of various sialylated compounds were enabled, by a combination of a simple synthesis of sialo-complex-type sugar oxazoline and the Endo-M-175Q catalyzed transglycosylation. General significance: Our method would be very useful for a practical synthesis of biologically important glycopeptides and glycoproteins. (C) 2010 Elsevier B.V. All rights reserved.
  • Crystal Structures of Phosphoketolase THIAMINE DIPHOSPHATE-DEPENDENT DEHYDRATION MECHANISM, Ryuichiro Suzuki, Takane Katayama, Byung-Jun Kim, Takayoshi Wakagi, Hirofumi Shoun, Hisashi Ashida, Kenji Yamamoto, Shinya Fushinobu, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 285(44), 34279 - 34287, Oct. 2010 , Refereed
    Summary: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.
  • Overexpression, crystallization and preliminary X-ray analysis of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from Bifidobacterium breve, 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, ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 66, 941 - 943, Aug. 2010 , Refereed
    Summary: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.
  • One-step synthesis of efficient binding-inhibitor for influenza virus through multiple addition of sialyloligosaccharides on chitosan, Myco Umemura, Yutaka Makimura, Masae Itoh, Takeshi Yamamoto, Toshiki Mine, Seiji Mitani, Ichiro Simizu, Hisashi Ashida, Kenji Yamamoto, CARBOHYDRATE POLYMERS, CARBOHYDRATE POLYMERS, 81(2), 330 - 334, Jun. 2010
    Summary: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.
  • Novel neogala-series glycosphingolipids with terminal mannose and glucose residues from Hirsutella rhossiliensis, an aureobasidin A-resistant ascomycete fungus, Yasushi Tani, Tori Funatsu, Hisashi Ashida, Masahiro Ito, Saki Itonori, Mutsumi Sugita, Kenji Yamamoto, GLYCOBIOLOGY, GLYCOBIOLOGY, 20(4), 433 - 441, Apr. 2010 , Refereed
    Summary: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.
  • Syntheses of mucin-type O-glycopeptides and oligosaccharides using transglycosylation and reverse-hydrolysis activities of Bifidobacterium endo-alpha-N-acetylgalactosaminidase, Hisashi Ashida, Hayato Ozawa, Kiyotaka Fujita, Shun'ichi Suzuki, Kenji Yamamoto, GLYCOCONJUGATE JOURNAL, GLYCOCONJUGATE JOURNAL, 27(1), 125 - 132, Jan. 2010 , Refereed
    Summary: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.
  • Efficient Glycosynthase Mutant Derived from Mucor hiemalis Endo-beta-N-acetylglucosaminidase Capable of Transferring Oligosaccharide from Both Sugar Oxazoline and Natural N-Glycan, Midori Umekawa, Cishan Li, Takayuki Higashiyama, Wei Huang, Hisashi Ashida, Kenji Yamamoto, Lai-Xi Wang, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 285(1), 511 - 521, Jan. 2010 , Refereed
    Summary: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.
  • Glucosamine induces autophagy via an mTOR-independent pathway, 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, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 391(4), 1775 - 1779, Jan. 2010 , Refereed
    Summary: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.
  • Two distinct alpha-l-fucosidases from Bifidobacterium bifidum are essential for the utilization of fucosylated milk oligosaccharides and glycoconjugates, Hisashi Ashida, Akiko Miyake, Masashi Kiyohara, Jun Wada, Erina Yoshida, Hidehiko Kumagai, Takane Katayama, Kenji Yamamoto, GLYCOBIOLOGY, GLYCOBIOLOGY, 19(9), 1010 - 1017, Sep. 2009 , Refereed
    Summary: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.
  • Deficiency of Dol-P-Man Synthase Subunit DPM3 Bridges the Congenital Disorders of Glycosylation with the Dystroglycanopathies, 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, AMERICAN JOURNAL OF HUMAN GENETICS, 85(1), 76 - 86, Jul. 2009 , Refereed
    Summary: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.
  • Prebiotic Effect of Lacto-N-biose I on Bifidobacterial Growth, Masashi Kiyohara, Asaki Tachizawa, Mamoru Nishimoto, Motomitsu Kitaoka, Hisashi Ashida, Kenji Yamamoto, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 73(5), 1175 - 1179, May 2009 , Refereed
    Summary: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.
  • Characterization of two different endo-alpha-N-acetylgalactosaminidases from probiotic and pathogenic enterobacteria, Bifidobacterium longum and Clostridium perfringens, Hisashi Ashida, Riichi Maki, Hayato Ozawa, Yasushi Tani, Masashi Kiyohara, Masaya Fujita, Akihiro Imamura, Hideharu Ishida, Makoto Kiso, Kenji Yamamoto, GLYCOBIOLOGY, GLYCOBIOLOGY, 18(9), 727 - 734, Sep. 2008 , Refereed
    Summary: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.
  • Design of a sialylglycopolymer with a chitosan backbone having efficient inhibitory activity against influenza virus infection, Myco Umemura, Masae Itoh, Yutaka Makimura, Kohji Yamazaki, Midori Umekawa, Ayano Masui, Yoshiharu Matahira, Mari Shibata, Hisashi Ashida, Kenji Yamamoto, JOURNAL OF MEDICINAL CHEMISTRY, JOURNAL OF MEDICINAL CHEMISTRY, 51(15), 4496 - 4503, Aug. 2008 , Refereed
    Summary: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.
  • Bifidobacterium bifidum lacto-N-biosidase, a critical enzyme for the degradation of human milk oligosaccharides with a type 1 structure, Jun Wada, Takuro Ando, Masashi Kiyohara, Hisashi Ashida, Motomitsu Kitaoka, Masanori Yamaguchi, Hidehiko Kumagai, Takane Katayama, Kenji Yamamoto, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 74(13), 3996 - 4004, Jul. 2008 , Refereed
    Summary: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.
  • Structural and thermodynamic analyses of solute-binding protein from Bifidobacterium longum specific for core 1 disaccharide and lacto-N-biose I, 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, JOURNAL OF BIOLOGICAL CHEMISTRY, 283(19), 13165 - 13173, May 2008 , Refereed
    Summary: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.
  • Mutants of Mucor hiemalis endo-beta-N-acetylglucosaminidase show enhanced transglycosylation and glycosynthase-like activities, Midori Umekawa, Wei Huang, Bing Li, Kiyotaka Fujita, Hisashi Ashida, Lai-Xi Wang, Kenji Yamamoto, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 283(8), 4469 - 4479, Feb. 2008 , Refereed
    Summary: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.
  • Purification, crystallization and preliminary X-ray analysis of the galacto-N-biose-/lacto-N-biose I-binding protein (GL-BP) of the ABC transporter from Bifidobacterium longum JCM1217, 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, ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, 63, 751 - 753, Sep. 2007 , Refereed
    Summary: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.
  • Purification, crystallization and preliminary X-ray analysis of the galacto-N-biose-/lacto-N-biose I-binding protein (GL-BP) of the ABC transporter from Bifidobacterium longum JCM1217, 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 and Crystallization Communications, Acta Crystallographica Section F: Structural Biology and Crystallization Communications, 63(9), 751 - 753, Aug. 10 2007 , Refereed
    Summary: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 C2221, with unit-cell parameters a = 106.3, b = 143.6, c = 114.6 Å for the lacto-N-biose I complex and a = 106.4, b = 143.4, c = 115.5 Å for the galacto-N-biose complex, and diffracted to 1.85 and 1.99 Å resolution, respectively. © International Union of Crystallography 2007.
  • Unique peptide : N-glycanase of Caenorhabditis elegans has activity of protein disulphide reductase as well as of deglycosylation, Toshihiko Kato, Akihito Kawahara, Hisashi Ashida, Kenji Yamamoto, JOURNAL OF BIOCHEMISTRY, JOURNAL OF BIOCHEMISTRY, 142(2), 175 - 181, Aug. 2007 , Refereed
    Summary: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.
  • Both mammalian PIG-M and PIG-X are required for growth of GPI14-disrupted yeast, Youn Uck Kim, Hisashi Ashida, Kenichiro Mori, Yusuke Maeda, Yeongjin Hong, Taroh Kinoshita, JOURNAL OF BIOCHEMISTRY, JOURNAL OF BIOCHEMISTRY, 142(1), 123 - 129, Jul. 2007 , Refereed
    Summary: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.
  • Free oligosaccharides in the cytosol of Caenorhabditis elegans are generated through endoplasmic reticulum-Golgi trafficking, Toshihiko Kato, Kumiko Kitamura, Megumi Maeda, Yoshinobu Kimura, Takane Katayama, Hisashi Ashida, Kenji Yamamoto, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 282(30), 22080 - 22088, Jul. 2007 , Refereed
    Summary: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.
  • Degradation of Glycoproteins, H. Ashida, T. Kato, K. Yamamoto, Comprehensive Glycoscience: From Chemistry to Systems Biology, Comprehensive Glycoscience: From Chemistry to Systems Biology, 3-4, 151 - 170, Jan. 01 2007 , Refereed
  • Removal or maintenance of inositol-linked acyl chain in glycosylphosphatidylinositol is critical in trypanosome life cycle, Y Hong, K Nagamune, YS Morita, F Nakatani, H Ashida, Y Maeda, T Kinoshita, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 281(17), 11595 - 11602, Apr. 2006 , Refereed
    Summary: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.
  • TbGPI16 is an essential component of GPI transamidase in Trypanosoma brucei, YC Hong, K Nagamune, K Ohishi, YS Morita, H Ashida, Y Maeda, T Kinoshita, FEBS LETTERS, FEBS LETTERS, 580(2), 603 - 606, Jan. 2006 , Refereed
    Summary: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.
  • Three-dimensional structure of GlcNAc alpha 1-4Gal releasing Endo-beta-galactosidase from clostridium perfringens, 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, PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS, 59(1), 141 - 144, Apr. 2005 , Refereed
  • PIG-V involved in transferring the second mannose in glycosylphosphatidylinositol, JY Kang, YJ Hong, H Ashida, N Shishioh, Y Murakami, YS Morita, Y Maeda, T Kinoshita, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 280(10), 9489 - 9497, Mar. 2005 , Refereed
    Summary: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.
  • GPI7 is the second partner of PIG-F and involved in modification of glycosylphosphatidylinositol, N Shishioh, YJ Hong, K Ohishi, H Ashida, Y Maeda, T Kinoshita, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 280(10), 9728 - 9734, Mar. 2005 , Refereed
    Summary: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.
  • Crystallization and preliminary X-ray analysis of GlcNAc alpha 1,4Gal-releasing endo-gamma-galactosidase from Clostridium perfringens, L Deng, ZJ Liu, H Ashida, SC Li, YT Li, P Horanyi, W Tempel, J Rose, BC Wang, ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 60, 537 - 538, Mar. 2004 , Refereed
    Summary: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.
  • GPI transamidase of Trypanosoma brucei has two previously uncharacterized (trypanosomatid transamidase 1 and 2) and three common subunits, 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, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 100(19), 10682 - 10687, Sep. 2003 , Refereed
    Summary: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.
  • Chemoenzymatic synthesis and application of glycopolymers containing multivalent sialyloligosaccharides with a poly(L-glutamic acid) backbone for inhibition of infection by influenza viruses, K Totani, T Kubota, T Kuroda, T Murata, KIPJ Hidari, T Suzuki, Y Suzuki, K Kobayashi, H Ashida, K Yamamoto, T Usui, GLYCOBIOLOGY, GLYCOBIOLOGY, 13(5), 315 - 326, May 2003 , Refereed
    Summary: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.
  • Characterization of a novel endo-beta-galactosidase specific for releasing the disaccharide GlcNAc alpha1-4 Gal from glycoconjugates, H Ashida, K Maskos, SC Li, YT Li, BIOCHEMISTRY, BIOCHEMISTRY, 41(7), 2388 - 2395, Feb. 2002 , Refereed
    Summary: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.
  • A novel endo-beta-galactosidase from Clostridium perfringens that liberates the disaccharide GlcNAc alpha1-4 Gal from glycans specifically expressed in the gastric gland mucous cell-type mucin, H Ashida, K Anderson, J Nakayama, K Maskos, CW Chou, RB Cole, SC Li, YT Li, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 276(30), 28226 - 28232, Jul. 2001 , Refereed
    Summary: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.
  • Enzymatic syntheses of T antigen-containing glycolipid mimicry using the transglycosylation activity of endo-alpha-N-acetylgalactosaminidase, H Ashida, K Yamamoto, H Kumagai, CARBOHYDRATE RESEARCH, CARBOHYDRATE RESEARCH, 330(4), 487 - 493, Feb. 2001 , Refereed
    Summary: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.
  • Molecular cloning of cDNA encoding alpha-N-acetylgalactosaminidase from Acremonium sp and its expression in yeast, H Ashida, H Tamaki, T Fujimoto, K Yamamoto, H Kumagai, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 384(2), 305 - 310, Dec. 2000 , Refereed
    Summary:Alpha-N-acetylgalactosaminidase (alpha -GaLNAc-ase; EC 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.
    Summary: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.
    Summary: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

  • Comprehensive Glycoscience Vol. 3, Hisashi Ashida, Toshihiko Kato, Kenji Yamamoto, Contributor, Biochemistry of Glycoconjugate Glycans - Degradation of Glycoproteins, Elsevier,   2007
  • Endoglycosidases -Biochemistry, Biotechnology, Application-, Hisashi Ashida, Su-Chen Li, Yu-Teh Li, Contributor, An unusual GlcNAcα1-4Gal releasing endo-β-galactosidase, Kodansha/Springer,   2006
  • Method in Enzymology, Method in Enzymology,   2006

Conference Activities & Talks

  • Isolation and identification of lactic acid bacteria from fermented fish and screening of bacteriocin-producing bacteria, Chaiwangsri, Koyanagi T, Ashida H, Matsuzaki C, Katayama T, The 2nd joint seminar. Core to Core Program A. Advanced Research Networks.,   2016 11 14
  • Isolation of lactic acid bacteria isolated from fermented foods, 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 10
  • Screening and analysis of useful glycosidases for production of bifidogenic factors, 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 10
  • Microbiota analyses on Japanese fermented foods with 16S rDNA-pyrosequencing approach, 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 10
  • Identification and characterization of endo-β-N-acetylglucosaminidase from methylotrophic yeast Ogataea minuta, Satoshi Murakami, Yuki Takaoka, Hisashi Ashida, Kenji Yamamoto, Hisashi Narimatsu, Yasunori Chiba, Glyco 22,   2013 06 23
  • Glucosamine as an Autophagy Inducer, ASHIDA Hisashi, 13th International Conference of Functional Food Center,   2013 05 12 , 招待有り
  • Analyses of metabolic pathway of oligosaccharides and its related enzymes in useful lactic acid bacteria, Thida Chaiwangsri, Masashi Kiyohara, Hisashi Ashida, Takane Katayama, Kenji Yamamoto, The final joint seminar of Asian Core Program,   2012 11 20
  • Application of glycosidase from lactic acid bacteria isolated from healthy infant faecies, Chartchai Khanongnuch, Wattana Sriphannam, Kenji Yamamoto, Hisashi Ashida, Goro Takata, The final joint seminar of Asian Core Program,   2012 11 19
  • A fucosynthase specifically introducing histo-blood group antigens Lewis a/x into type-1/2 chains, 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 29
  • Glucosamine-induced autophagy is mediated by release of ammonia and promotes longevity in C. elegans, Yuhei Kosuge, Hiroki Sakai, Tomoya Shintani, Hisashi Ashida, The 10th International Student Seminar,   2012 03 05
  • GH110 α-galactosidase from bifidobacteria specifically acts on blood group B antigens of gastrointestinal mucin, Takura Wakinaka, Masashi Kiyohara, Kenji Yamamoto, Hisashi Ashida, The 10th International Student Seminar,   2012 03 05
  • A novel glycosides from infant-associated bifidobacteria involved in mucin degradation pathway, ASHIDA Hisashi, U.P. Special Lectures on Probiotic Lactic Acid Bacteria,   2011 12 15 , 招待有り
  • Induction of autophagy by chitobiose and its derivatives, Hisashi Ashida, Hiroki Sakai, Yuhei Kosuge, Tomoya Shintani, The 31st Naito Conference on Glycan Expression and Regulation,   2011 09 14


  • Hexoses with calorie-restriction mimetic effect, Shintani Tomoya, ASHIDA Hisashi, SATO Masashi, Kagaku to Seibutsu, 56, 12, 777, 778,   2018 12
  • 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, 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, 日本食品科学工学会大会講演集, 65th, 115,   2018 08 22 ,
  • 抗老化効果を有する機能性ヘキソース, 新谷知也, 佐藤正資, 芦田久, 応用糖質科学, 8, 3, (57),   2018 08 20 ,
  • Interaction between gut microbes and host through intestinal mucin, ASHIDA Hisashi, Kagaku to Seibutsu, 54, 12, 901, 908,   2016 12 , 招待有り, 10.1271/kagakutoseibutsu.54.901
  • 梅ポリフェノールのマウス腸内細菌叢に及ぼす影響, 島田良美, 香川昴雅, 晋家崇史, 尾﨑嘉彦, 芦田 久, 和歌山医学, 66, 3, 93,   2015 09
  • 食品由来オートファジー誘導物質の寿命への効果, 芦田 久, 和歌山医学, 66, 3, 91, 92,   2015 09
  • Lactobacillus sakei の学名について, 芦田 久, 乳酸菌学会誌, 25, 2, 141,   2014
  • Glucosamine as an Autophagy Inducer, ASHIDA Hisashi, Functional and Medical Foods with Bioactive Compounds: Science and Practical Application, 13, 58, 59,   2013 05
  • オートファジー誘導物質としてのグルコサミン, 芦田 久, グルコサミン研究, 8, 6, 11,   2012 07
  • グリコシダーゼの機能改変によるプレバイオティックオリゴ糖の効率的合成法に関する研究, 芦田 久, 三島海雲記念財団研究報告書, 48, 51-55,   2011 11 ,
  • ミルクオリゴ糖をめぐる3種のビフィズス菌の異なる戦略, 芦田 久, 生物工学会誌, 89, 1, 29,   2011 01 ,
  • グルコサミンによるmTOR非依存性のオートファジー誘導, 芦田久, 新谷知也, 酒井宏樹, 小菅雄平, 青島直史, 戸谷一英, 山本憲二, J Appl Glycosci, 57, Suppl., 49, 94,   2010 07 20 , 10.11541/jsag.2010.0.94.0,
  • 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, 芦田久, 新谷知也, 山崎文義, 加藤紀彦, 梅川碧里, 又平芳春, 垣塚彰, 戸谷一英, 山本憲二, 日本農芸化学会大会講演要旨集, 2010, 121,   2010 03 05 ,
  • 2Cp20 Enhancement of Activity of Influenza Virus Binding Inhibitor with Neuraminidase Inhibitors, MITANI Seiji, UMEMURA Myco, MAKIMURA Yutaka, ITOH Masae, ASHIDA HISASHI, YAMAMOTO Kenji, 日本生物工学会大会講演要旨集, 21,   2009 ,
  • シアロ糖鎖を活用したインフルエンザウイルス捕捉型感染阻害剤のデザイン, 梅村舞子, 伊藤正恵, 牧村裕, 芦田久, 山本憲二, 日本農芸化学会関西支部講演会講演要旨集, 458th, 17,   2009 ,
  • Enzymes involved in generation and degradation of the free oligosaccharides in the cytosol of Caenorhabditis elegans, Ashida H, Kato T, Kawahara A, Tanaka Y, Umekawa M, Yamamoto K, J. Appl. Glycosci., 56, 137, 143,   2009 , 10.5458/jag.56.137
  • Crystal structure of GH101 endo-alpha-N-acetylgalactosaminidase from Bifidobacterium longum., Suzuki R, Katayama T, Fushinobu S, Kitaoka M, Kumagai H, Wakagi T, Shoun H, Ashida H, Yamamoto K, J. Appl. Glycosci., 56, 105-110, 105, 110,   2009 , 10.5458/jag.56.105
  • Trechiama ion (Coleoptera, Trechinae), a remarkable new species from Chûgoku District, West Japan, Ashida H, Souma A, Elytra, Tokyo, 36, 381, 386,   2008 12
  • 天然型のシアロ糖鎖を結合したインフルエンザウイルス捕捉型感染阻害剤の新規な製造法, 梅村舞子, 伊藤正恵, 牧村裕, 芦田久, 山本憲二, 日本農芸化学会大会講演要旨集, 2008, 179,   2008 03 05 ,
  • Functions of novel glycosidases isolated from bifidobacteria, Katayama T, Wada J, Fujita K, Kiyohara M, Ashida H, Yamamoto K, J. Appl. Glycosci., 55, 101, 109,   2008 , 10.5458/jag.55.101
  • インフルエンザウイルスの捕捉に最適なキトサン鎖を持つシアロ糖鎖含有阻害剤の探索, 梅村舞子, 梅川碧里, 牧村裕, 増井彩乃, 又平芳春, 伊藤正恵, 芦田久, 山本憲二, 日本糖質学会年会要旨集, 27th, 183,   2007 07 10 ,
  • Occurrence of the Trechiama yamajii complex of the group of Trechiama oni (Coleoptera, Trechinae) in the northwestern corner of Hyogo Prefecture, West Japan, Ashida H, J. speleol. Soc. Japan, 32, 1, 8,   2007 , Refereed
  • 3I12-3 Optimization of the length of chitosan backbone in chemoenzymatically synthesized binding-inhibitor against influenza virus, UMEMURA Maiko, UMEKAWA Midori, MAKIMURA Yutaka, MASUI Ayano, MATAHIRA Yoshiharu, ITOH Masae, ASHIDA Hisashi, YAMAMOTO Kenji, 日本生物工学会大会講演要旨集, 19,   2007 ,
  • 1K14-5 Multivalent effect of sugar chains in chemo-enzymatically synthesized inhibitor against receptor-binding of influenza virus, UMEMURA Maiko, UMEKAWA Midori, MAKIMURA Yutaka, ITOH Masae, ASHIDA Hisashi, YAMAMOTO Kenji, 日本生物工学会大会講演要旨集, 18,   2006 ,
  • 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 fujitai (Coleoptera, Trechinae) from Hyogo Prefecture, West Japan (II) -Two new species and several new records from the Ibo-gawa drainage area-, Ashida H, Elytra, Tokyo, 32, 259, 263,   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
  • The complex of Trechiama fujitai (Coleoptera: Trechinae) from Hyôgo Prefecture, West Japan (III) -A new relative of Trechiama latilobatus Ashida-, Ashida H, Ent. Rev. Japan, 60, 17, 21,   2005 , Refereed
  • 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
  • A new Stygiotrechus (Coleoptera, Trechinae) from near the northern end of the Daiko Mountains in the Kii Peninsula, Central Japan., Ashida H, Kitayama K, Elytra, Tokyo, 32, 23, 27,   2004 , Refereed
  • An additional species belonging to the Trechiama notoi complex (Coleoptera, Trechinae) from the southern part of the Tajima area in Hyogo Prefecture, Central Japan., Ashida H, Elytra, Tokyo, 32, 259, 263,   2004 , Refereed
  • The group of Stygiotrechus ohtanii (Coleoptera, Trechinae) from the Kii Peninsula, Central Japan., Ashida H, Kitayama K, Elytra, Tokyo, 31, 221, 229,   2003 , Refereed
  • The complex of Trechiama fujitai (Coleoptera, Trechinae) from Hyogo Prefecture, West Japan (I) -Two new species from the Maruyama-gawa drainage area-., Ashida H, Elytra, Tokyo, 31, 431, 438,   2003 , Refereed
  • Occurrence of a new Stygiotrechus (Coleoptera, Trechinae) in the Takanawa Peninsula of northwestern Shikoku, Southwest Japan., Ueno S-I, Ashida H, Elytra, Tokyo, 31, 409, 414,   2003 , Refereed
  • Two new anophthalmic species of the group of Trechiama oni (Coleoptera, Trechinae) from the Tajima area, Central Japan, Ashida H, Elytra, Tokyo, 30, 49, 56,   2002 , Refereed
  • A distinct species-complex of Trechiama notoi (Coleoptera, Trechinae) mainly distributed in the Tajima area, Central Japan, Ashida H, Elytra, Tokyo, 30, 385, 397,   2002 , Refereed
  • 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
  • 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
  • An additional new species of the genus Kusumia (Coleoptera, Trechinae), Ashida H, Elytra, Tokyo, 28, 241, 245,   2000 , Refereed
  • A new species of Dacne (Coleoptera, Erotylidae) from Chejudo Island off South Korea, Ashida H, Kim CG, Elytra, Tokyo, 27, 381, 385,   1999 , Refereed
  • A new species of the group of Trechiama oni (Coleoptera, Trechinae) from the Kii Peninsula, Central Japan, Ashida H, Elytra, Tokyo, 27, 605, 610,   1999 , Refereed
  • 擬態分子で宿主に接着する病原性微生物, 芦田 久, 化学と生物, 37, 114, 115,   1999 , 10.1271/kagakutoseibutsu1962.37.114
  • 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
  • Guidelines for the use and interpretation of assays for monitoring autophagy., Daniel J Klionsky, Hisashi Ashida, et al., Autophagy, 8, 4, 445, 544,   2012 04 , Refereed, 10.4161/auto.19496
    Summary:In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
  • Bifidobacterium bifidum Lacto-N-Biosidase, a Critical Enzyme for the Degradation of Human Milk Oligosaccharides with a Type 1 Structure (vol 74, pg 3996, 2009), 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, 6414,   2009 10 , 10.1128/AEM.01847-09
  • Generation and Metabolism of Cytosolic Free Oligosaccharides in Caenorhabditis elegans, Toshihiko Katoh, Hisashi Ashida, Kenji Yamamoto, TRENDS IN GLYCOSCIENCE AND GLYCOTECHNOLOGY, 21, 119, 163, 177,   2009 05 , Refereed, 10.4052/tigg.21.163
    Summary:Free oligosaccharides (FOSs) found in the cytosol of eukaryotic cells are produced by the enzymatic degradation of dolichol-linked intermediates of N-linked glycosylation and/or by the action of cytosolic peptide:N-glycanases that are involved in the process of endoplasmic reticulum-associated degradation (ERAD) of glycoproteins. FOSs are subsequently trimmed by cytosolic endo-beta-N-acetylglucosaminidase and a-mannosidase and then ultimately transferred to the lysosome for degradation into monosaccharides. In this minireview, we describe the formation, catabolism, and possible physiological roles of FOSs and present the results of our study on the structure and function of enzymes associated with the generation of FOSs in the nematode Caenorhabditis elegans.
  • PIG-V transfers the second mannose to glycosylphosphaddylinositol., JY Kang, YJ Hong, N Shishioh, H Ashida, Y Maeda, T Kinoshita, GLYCOBIOLOGY, 14, 11, 1188, 1189,   2004 11
  • Functional association of PGAP2 with glycosylphosphatidylinositol remodeling, Y Maeda, Y Tashima, H Ashida, C Murata, R Taguchi, T Kinoshita, MOLECULAR BIOLOGY OF THE CELL, 15, 186A, 186A,   2004 11
  • Mammalian PIG-X and yeast Pbn1p are the regulatory components of ER-resident GPI-mannosyltransferase I, H Ashida, YJ Hong, N Sugimoto, Y Murakami, Y Maeda, T Kinoshita, GLYCOBIOLOGY, 14, 11, 1184, 1185,   2004 11
  • Expression of GPI-anchored proteins: Events post-attachment of the anchor en route to the cell surface, T Kinoshita, S Tanaka, H Ashida, Y Tashima, Y Maeda, GLYCOBIOLOGY, 13, 11, 836, 836,   2003 11
  • Cloning and expression of a novel endo-beta-galactosidase that liberates GlcNAc alpha1-4 Gal from the gastric gland mucous cell-type mucin, H Ashida, SC Li, YT Li, GLYCOBIOLOGY, 11, 10, 918, 919,   2001 10
  • Nature of galleries, durability of boring scars, and density of Xylotrechus villioni (Villard) larvae (Coleoptera: Cerambycidae), on coniferous tree trunks, R Iwata, F Yamada, H Kato, H Makihara, K Araya, H Ashida, M Takeda, PAN-PACIFIC ENTOMOLOGIST, 73, 4, 213, 224,   1997 10 , Refereed
    Summary: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.

Awards & Honors

  •   2012 03 , JSBBA, Most-Cited Paper Award, Prebiotic Effect of Lacto-N-biose I on Bifidobacterial Growth
  •   2010 03 , JSBBA, Topics Award

Research Grants & Projects

  • Novel function of chitobiose and chitooligosaccharides
  • Application of a mutant endoglycosidase on syntheses of glycomedicines
  • Isolation and application of bifidobacterial glycosidases acting on human milk oligosaccharides