Publications by authors named "Yusuke Tomabechi"

16 Publications

  • Page 1 of 1

Enzymatically synthesized exopolysaccharide of a probiotic strain NTM048 shows adjuvant activity to promote IgA antibody responses.

Gut Microbes 2021 Jan-Dec;13(1):1949097

Management Office, Noster Inc, Kyoto, Japan.

strain NTM048 produces an exopolysaccharide (EPS; glucose polymers 94% and fructose polymers 6%) with adjuvanticity for mucosal vaccination. Strain NTM048 includes three putative EPS-synthesizing genes, and for synthesizing glucose polymers, and for synthesizing fructose polymer. To elucidate the key polymer structure for adjuvanticity, two genes, and , which were annotated as glycoside hydrolase family 70 enzyme genes, were expressed in . Glycosyl-linkage composition analysis and NMR analysis showed that the recombinant enzyme Gtf1 produced a soluble form of α-1,6-glucan, whereas the recombinant enzyme Gtf2 produced glucans with approximately equal percentages of α-1,6- and α-1,3-glucose residues both in the supernatant (S-glucan) and as a precipitate (P-glucan). Comparison of polysaccharides synthesized by Gtf1, Gtf2, and LvnS revealed that Gtf2-S-glucan, which was produced in the supernatant by Gtf2 and formed particles of 7.8 µm, possessed 1.8-fold higher ability to stimulate IgA production from murine Peyer's patch cells than native NTM048 EPS. Evaluation of adjuvanticity by intranasal administration of mice with an antigen (ovalbumin) and Gtf2-S-glucan or NTM048 EPS showed that Gtf2-S-glucan induced the production of higher antigen-specific antibodies in the airway mucosa and plasma, suggesting a pivotal role of Gtf2-S-glucan in the adjuvanticity of NTM048 EPS.
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http://dx.doi.org/10.1080/19490976.2021.1949097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8550178PMC
December 2021

Conformationally supple glucose monomers enable synthesis of the smallest cyclodextrins.

Science 2019 05 11;364(6441):674-677. Epub 2019 Apr 11.

School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337, Japan.

Cyclodextrins (CDs) are cyclic oligomers of α-1,4-d-glucopyranoside and are known mainly as hexamers to octamers. The central cavities of CDs can retain small molecules, enabling diverse applications. The smallest members, CD3 and CD4, have ring sizes too small to permit the most stable conformations of glucopyranose and have not been accessible synthetically. In this study, we present methods to chemically synthesize both CD3 and CD4. The main factor in the successful synthesis is the creation of a glucopyranose ring conformationally counterbalanced between equatorial- and axial-rich forms. This suppleness is imparted by a bridge between O-3 and O-6 of glucose, which enables the generation of desirable, albeit deformed, conformers when synthesizing the cyclic trimer and tetramer.
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http://dx.doi.org/10.1126/science.aaw3053DOI Listing
May 2019

Sharing of human milk oligosaccharides degradants within bifidobacterial communities in faecal cultures supplemented with Bifidobacterium bifidum.

Sci Rep 2018 09 18;8(1):13958. Epub 2018 Sep 18.

Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.

Gut microbiota of breast-fed infants are generally rich in bifidobacteria. Recent studies show that infant gut-associated bifidobacteria can assimilate human milk oligosaccharides (HMOs) specifically among the gut microbes. Nonetheless, little is known about how bifidobacterial-rich communities are shaped in the gut. Interestingly, HMOs assimilation ability is not related to the dominance of each species. Bifidobacterium longum susbp. longum and Bifidobacterium breve are commonly found as the dominant species in infant stools; however, they show limited HMOs assimilation ability in vitro. In contrast, avid in vitro HMOs consumers, Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis, are less abundant in infant stools. In this study, we observed altruistic behaviour by B. bifidum when incubated in HMOs-containing faecal cultures. Four B. bifidum strains, all of which contained complete sets of HMO-degrading genes, commonly left HMOs degradants unconsumed during in vitro growth. These strains stimulated the growth of other Bifidobacterium species when added to faecal cultures supplemented with HMOs, thereby increasing the prevalence of bifidobacteria in faecal communities. Enhanced HMOs consumption by B. bifidum-supplemented cultures was also observed. We also determined the complete genome sequences of B. bifidum strains JCM7004 and TMC3115. Our results suggest B. bifidum-mediated cross-feeding of HMOs degradants within bifidobacterial communities.
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http://dx.doi.org/10.1038/s41598-018-32080-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6143587PMC
September 2018

Microbial production of novel sulphated alkaloids for drug discovery.

Sci Rep 2018 05 22;8(1):7980. Epub 2018 May 22.

Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi, Ishikawa, 921-8836, Japan.

Natural products from plants are useful as lead compounds in drug discovery. Plant benzylisoquinoline alkaloids (BIAs) exhibit various pharmaceutical activities. Although unidentified BIAs are expected to be of medicinal value, sufficient quantities of such BIAs, for biological assays, are sometimes difficult to obtain due to their low content in natural sources. Here, we showed that high productivity of BIAs in engineered Escherichia coli could be exploited for drug discovery. First, we improved upon the previous microbial production system producing (S)-reticuline, an important BIA intermediate, to obtain yields of around 160 mg/L, which was 4-fold higher than those of the previously reported highest production system. Subsequently, we synthesised non-natural BIAs (O-sulphated (S)-reticulines) by introducing human sulphotransferases into the improved (S)-reticuline production system. Analysis of human primary cells treated with these BIAs demonstrated that they affected a biomarker expression in a manner different from that by the parent compound (S)-reticuline, suggesting that simple side-chain modification altered the characteristic traits of BIA. These results indicated that highly productive microbial systems might facilitate the production of scarce or novel BIAs and enable subsequent evaluation of their biological activities. The system developed here could be applied to other rare natural products and might contribute to the drug-discovery process as a next-generation strategy.
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http://dx.doi.org/10.1038/s41598-018-26306-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964154PMC
May 2018

Identification and characterization of a sulfoglycosidase from Bifidobacterium bifidum implicated in mucin glycan utilization.

Biosci Biotechnol Biochem 2017 Oct 17;81(10):2018-2027. Epub 2017 Aug 17.

a Graduate School of Biostudies , Kyoto University , Kyoto , Japan.

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-β-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.
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http://dx.doi.org/10.1080/09168451.2017.1361810DOI Listing
October 2017

Structural characterization of the immunostimulatory exopolysaccharide produced by Leuconostoc mesenteroides strain NTM048.

Carbohydr Res 2017 Aug 15;448:95-102. Epub 2017 Jun 15.

Nitto Pharmaceutical Industries, Ltd., Kamiueno, Muko, Kyoto 617-0006, Japan.

The exopolysaccharide (EPS) produced by probiotic Leuconostoc mesenteroides subsp. mesenteroides strain NTM048 has been reported to be an immunostimulant that enhances mucosal IgA production. In this study, we found that intranasal administration of mice with the EPS and an antigen (ovalbumin) resulted in secretion of antigen-specific IgA and IgG in the airway mucosa and the serum, suggesting that the EPS has the adjuvant activity for use with mucosal vaccination. Methylation analysis coupled to GC-MS, and 1D and 2D NMR spectroscopy revealed that 94% of the EPS consists of an α-(1 → 6) glucan containing 4% of 1→3-linked α-glucose branches. To determine structures of minor components, we enzymatically digested the glucan with dextranase and used 2D NMR spectroscopy to identify the remaining polymer as a fructan (or fructans), containing both β-(2 → 6)- and β-(2 → 1)-linked fructofuranose residues. These residues may either enter into separate polymers of each linkage type or form a mixed fructan containing both linkage types.
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http://dx.doi.org/10.1016/j.carres.2017.06.004DOI Listing
August 2017

Chemo-enzymatic synthesis of a glycosylated peptide containing a complex N-glycan based on unprotected oligosaccharides by using DMT-MM and Endo-M.

Glycoconj J 2017 08 18;34(4):481-487. Epub 2017 May 18.

Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa, 921-8836, Japan.

For chemo-enzymatic synthesis of a glycosylated peptide, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) was used for the synthesis of a N-acetylglucosaminyl peptide and a pseudoglycopeptide by solid-phase peptide synthesis without the requirement of protecting groups on the carbohydrate. We also performed transglycosylation of an N-glycan to the N-acetylglucosaminyl peptide using endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) to synthesize a glycopeptide containing a complex N-glycan.
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http://dx.doi.org/10.1007/s10719-017-9770-yDOI Listing
August 2017

Generation of a Mutant Mucor hiemalis Endoglycosidase That Acts on Core-fucosylated N-Glycans.

J Biol Chem 2016 10 14;291(44):23305-23317. Epub 2016 Sep 14.

the Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, Japan, and.

Endo-β-N-acetylglucosaminidase M (Endo-M), an endoglycosidase from the fungus Mucor hiemalis, is a useful tool for chemoenzymatic synthesis of glycoconjugates, including glycoprotein-based therapeutics having a precisely defined glycoform, by virtue of its transglycosylation activity. Although Endo-M has been known to act on various N-glycans, it does not act on core-fucosylated N-glycans, which exist widely in mammalian glycoproteins, thus limiting its application. Therefore, we performed site-directed mutagenesis on Endo-M to isolate mutant enzymes that are able to act on mammalian-type core-α1,6-fucosylated glycans. Among the Endo-M mutant enzymes generated, those in which the tryptophan at position 251 was substituted with alanine or asparagine showed altered substrate specificities. Such mutant enzymes exhibited increased hydrolysis of a synthetic α1,6-fucosylated trimannosyl core structure, whereas their activity on the afucosylated form decreased. In addition, among the Trp-251 mutants, the W251N mutant was most efficient in hydrolyzing the core-fucosylated substrate. W251N mutants could act on the immunoglobulin G-derived core-fucosylated glycopeptides and human lactoferrin glycoproteins. This mutant was also capable of transferring the sialyl glycan from an activated substrate intermediate (sialyl glyco-oxazoline) onto an α1,6-fucosyl-N-acetylglucosaminyl biotin. Furthermore, the W251N mutant gained a glycosynthase-like activity when a N175Q substitution was introduced and it caused accumulation of the transglycosylation products. These findings not only give insights into the substrate recognition mechanism of glycoside hydrolase family 85 enzymes but also widen their scope of application in preparing homogeneous glycoforms of core-fucosylated glycoproteins for the production of potent glycoprotein-based therapeutics.
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http://dx.doi.org/10.1074/jbc.M116.737395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087746PMC
October 2016

Laboratory-scale production of (S)-reticuline, an important intermediate of benzylisoquinoline alkaloids, using a bacterial-based method.

Biosci Biotechnol Biochem 2017 Feb 14;81(2):396-402. Epub 2016 Oct 14.

a Research Institute for Bioresources and Biotechnology , Ishikawa Prefectural University , Nonoichi , Japan.

Benzylisoquinoline alkaloids (BIAs) are a group of plant secondary metabolites that have been identified as targets for drug discovery because of their diverse pharmaceutical activities. Well-known BIAs are relatively abundant in plants and have therefore been extensively studied. However, although unknown BIAs are also thought to have valuable activities, they are difficult to obtain because the raw materials are present at low abundance in nature. We have previously reported the fermentative production of an important intermediate (S)-reticuline from dopamine using Escherichia coli. However, the yield is typically limited. Here, we improved production efficiency by combining in vivo tetrahydropapaveroline production in E. coli with in vitro enzymatic synthesis of (S)-reticuline. Finally, 593 mg of pure (S)-reticuline was obtained from 1 L of the reaction mixture. Because this bacterial-based method is simple, it could be widely used for production of (S)-reticuline and related BIAs, thereby facilitating studies of BIAs for drug discovery.
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http://dx.doi.org/10.1080/09168451.2016.1243985DOI Listing
February 2017

Convergent chemo-enzymatic synthesis of mannosylated glycopeptides; targeting of putative vaccine candidates to antigen presenting cells.

Chem Sci 2015 Aug 19;6(8):4636-4642. Epub 2015 May 19.

Department of Chemistry , University of Canterbury , Private Bag 4800 , Christchurch , 8140 , New Zealand . Email:

The combination of solid phase peptide synthesis and -β--acetylglucosaminidase (ENGase) catalysed glycosylation is a powerful convergent synthetic method allowing access to glycopeptides bearing full-length -glycan structures. Mannose-terminated -glycan oligosaccharides, produced by either total or semi-synthesis, were converted into oxazoline donor substrates. A peptide from the human cytomegalovirus (CMV) tegument protein pp65 that incorporates a well-characterised T cell epitope, containing -acetylglucosamine at specific Asn residues, was accessed by solid phase peptide synthesis, and used as an acceptor substrate. High-yielding enzymatic glycosylation afforded glycopeptides bearing defined homogeneous high-mannose -glycan structures. These high-mannose containing glycopeptides were tested for enhanced targeting to human antigen presenting cells (APCs), putatively mediated the mannose receptor, and for processing by the APCs for presentation to human CD8+ T cells specific for a 9-mer epitope within the peptide. Binding assays showed increased binding of glycopeptides to APCs compared to the non-glycosylated control. Glycopeptides bearing high-mannose -glycan structures at a single site outside the T cell epitope were processed and presented by the APCs to allow activation of a T cell clone. However, the addition of a second glycan within the T cell epitope resulted in ablation of T cell activation. We conclude that chemo-enzymatic synthesis of mannosylated glycopeptides enhances uptake by human APCs while preserving the immunogenicity of peptide epitopes within the glycopeptides, provided those epitopes are not themselves glycosylated.
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http://dx.doi.org/10.1039/c5sc00952aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500846PMC
August 2015

Direct thiophenylation accompanying orthoester-cleavage of 1,2,4-O-orthoacetyl-3,6-O-(o-xylylene)glucopyranose.

Carbohydr Res 2015 Jan 20;402:118-23. Epub 2014 Oct 20.

School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337, Japan. Electronic address:

The 3,6-O-(o-xylylene) bridge locks the conformation of glucopyranose to an axial-rich form. Although the conformational lock induces complete β-selectivity in a glycosylation reaction, the leaving group of the glycosyl donor is limited to fluorine. On the other hand, the bridge confers the furanose-preferred property to glucose, which makes synthesis of corresponding pyranosyl derivatives that equip various leaving groups difficult. This problem was solved through direct phenylthio glucosidation of 3,6-O-(o-xylylene)-1,2,4-O-orthoacetylglucose accompanying cleavage of the orthoester moiety. This paper describes the process of establishing direct thiophenylation. This process reduced the synthetic steps for the known glucopyranosyl fluoride and will expand application of conformationally locked glycosyl donors.
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http://dx.doi.org/10.1016/j.carres.2014.10.004DOI Listing
January 2015

Convergent chemoenzymatic synthesis of a library of glycosylated analogues of pramlintide: structure-activity relationships for amylin receptor agonism.

Org Biomol Chem 2014 Nov 17;12(41):8142-51. Epub 2014 Jul 17.

The School of Chemical Sciences, University of Auckland, 23 Symonds St, Auckland 1010, New Zealand.

Pramlintide (Symlin®), a synthetic analogue of the naturally occurring pancreatic hormone amylin, is currently used with insulin in adjunctive therapy for type 1 and type 2 diabetes mellitus. Herein we report a systematic study into the effect that N-glycosylation of pramlintide has on activation of amylin receptors. A highly efficient convergent synthetic route, involving a combination of solid phase peptide synthesis and enzymatic glycosylation, delivered a library of N-glycosylated variants of pramlintide bearing either GlcNAc, the core N-glycan pentasaccharide [Man3(GlcNAc)2] or a complex biantennary glycan [(NeuAcGalGlcNAcMan)2Man(GlcNAc)2] at each of its six asparagine residues. The majority of glycosylated versions of pramlintide were potent receptor agonists, suggesting that N-glycosylation may be used as a tool to optimise the pharmacokinetic properties of pramlintide and so deliver improved therapeutic agents for the treatment of diabetes and obesity.
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http://dx.doi.org/10.1039/c4ob01208aDOI Listing
November 2014

Endo-β-N-acetylglucosaminidase catalysed glycosylation: tolerance of enzymes to structural variation of the glycosyl amino acid acceptor.

Org Biomol Chem 2014 Feb;12(6):942-55

Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch, 8140, New Zealand.

Endo-β-N-Acetylglucosaminidases (ENGases) are highly useful biocatalysts that can be used to synthetically access a wide variety of defined homogenous N-linked glycoconjugates in a convergent manner. The synthetic efficiency of a selection of family GH85 ENGases was investigated as the structure of the acceptor substrate was varied. Several different GlcNAc-asparagine acceptors were synthesised, and used in conjunction with penta- and decasaccharide oxazoline donors. Different enzymes showed different tolerances of modification of the GlcNAc acceptor. Whilst none tolerated modification of either the 4- or 6-hydroxyl, both Endo M and Endo D tolerated modification of OH-3. For Endo D the achievable synthetic efficiency was increased by a factor of three by the use a 3-O-benzyl protected acceptor. The presence of a fucose at position-3 was not tolerated by any of the enzymes assayed.
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http://dx.doi.org/10.1039/c3ob42104jDOI Listing
February 2014

Glycosylation of pramlintide: synthetic glycopeptides that display in vitro and in vivo activities as amylin receptor agonists.

Chemistry 2013 Nov 7;19(45):15084-8. Epub 2013 Oct 7.

Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch 8140 (New Zealand).

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http://dx.doi.org/10.1002/chem.201303303DOI Listing
November 2013

Acceptor specificity in the transglycosylation reaction using Endo-M.

Carbohydr Res 2010 Nov 17;345(17):2458-63. Epub 2010 Sep 17.

Department of Applied Chemistry, School of Engineering, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan.

To determine the structural specificity of the glycosyl acceptor of the transglycosylation reaction using endo-β-N-acetylglucosaminidase (ENGase) (EC 3.2.1.96) from Mucor hiemalis (Endo-M), several acceptor derivatives were designed and synthesized. The narrow regions of the 1,3-diol structure from the 4- to 6-hydroxy functions of GlcNAc were found to be essential for the transglycosylation reaction using Endo-M. Furthermore, it was determined that Endo-M strictly recognizes a 1,3-diol structure consisting of primary and secondary hydroxyl groups.
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http://dx.doi.org/10.1016/j.carres.2010.08.022DOI Listing
November 2010

Chemo-enzymatic synthesis of glycosylated insulin using a GlcNAc tag.

Bioorg Med Chem 2010 Feb 16;18(3):1259-64. Epub 2009 Dec 16.

Department of Applied Chemistry, School of Engineering, Tokai University, 1117 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan.

Artificial insulin with an N-linked oligosaccharide was synthesized by a chemo-enzymatic method using endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M). GlcNAc-modified insulin was prepared by the reaction of the carboxymethyl glycoside of GlcNAc and 3 amino groups of bovine insulin using a dimethylphosphinothioic mixed anhydride (Mpt-MA) method. A transglycosylation reaction of the GlcNAc-modified insulin using Endo-M gave mono-transglycosylated insulin predominantly. We determined the transglycosylation site of the mono-transglycosylated insulin.
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http://dx.doi.org/10.1016/j.bmc.2009.12.031DOI Listing
February 2010
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