Publications by authors named "Kaoru Takegawa"

166 Publications

Microbial α-L-Rhamnosidases of Glycosyl Hydrolase Families GH78 and GH106 Have Broad Substrate Specificities toward α-L-Rhamnosyl- and α-L-Mannosyl-Linkages.

J Appl Glycosci (1999) 2020 3;67(3):87-93. Epub 2020 Sep 3.

1 Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University.

α-L-Rhamnosidases (α-L-Rha-ases, EC 3.2.1.40) are glycosyl hydrolases (GHs) that hydrolyze a terminal α-linked L-rhamnose residue from a wide spectrum of substrates such as heteropolysaccharides, glycosylated proteins, and natural flavonoids. As a result, they are considered catalysts of interest for various biotechnological applications. α-L-rhamnose (6-deoxy-L-mannose) is structurally similar to the rare sugar α-L-mannose. Here we have examined whether microbial α-L-Rha-ases possess α-L-mannosidase activity by synthesizing the substrate 4-nitrophenyl α-L-mannopyranoside. Four α-L-Rha-ases from GH78 and GH106 families were expressed and purified from cells. All four enzymes exhibited both α-L-rhamnosyl-hydrolyzing activity and weak α-L-mannosyl-hydrolyzing activity. SpRhaM, a GH106 family α-L-Rha-ase from FP2001, was found to have relatively higher α-L-mannosidase activity as compared with three GH78 α-L-Rha-ases. The α-L-mannosidase activity of SpRhaM showed pH dependence, with highest activity observed at pH 7.0. In summary, we have shown that α-L-Rha-ases also have α-L-mannosidase activity. Our findings will be useful in the identification and structural determination of α-L-mannose-containing polysaccharides from natural sources for use in the pharmaceutical and food industries.
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http://dx.doi.org/10.5458/jag.jag.JAG-2020_0005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8132073PMC
September 2020

Substrate specificities of α1,2- and α1,3-galactosyltransferases and characterization of Gmh1p and Otg1p in Schizosaccharomyces pombe.

Glycobiology 2021 Sep;31(8):1037-1045

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 819-0395, Japan.

In the fission yeast Schizosaccharomyces pombe, α1,2- and α1,3-linked D-galactose (Gal) residues are transferred to N- and O-linked oligosaccharides of glycoproteins by galactosyltransferases. Although the galactomannans are important for cell-cell communication in S. pombe (e.g., in nonsexual aggregation), the mechanisms underlying galactosylation in cells remain unclear. Schizosaccharomyces pombe has 10 galactosyltransferase-related genes: seven belonging to glycosyltransferase (GT) family 34 and three belonging GT family 8. Disruption of all 10 α-galactosyltransferases (strain Δ10GalT) has been shown to result in a complete lack of α-Gal residues. Here, we have investigated the function and substrate specificities of galactosyltransferases in S pombe by using strains expressing single α-galactosyltransferases in the Δ10GalT background. High-performance liquid chromatography (HPLC) analysis of pyridylaminated O-linked oligosaccharides showed that two GT family 34 α1,2-galactosyltransferases (Gma12p and Gmh6p) and two GT family 8 α1,3-galactosyltransferases (Otg2p and Otg3p) are involved in galactosylation of O-linked oligosaccharide. Moreover, 1H-NMR of N-glycans revealed that three GT family 34 α1,2-galactosyltransferases (Gmh1p, Gmh2p and Gmh3p) are required for the galactosylation of N-linked oligosaccharides. Furthermore, HPLC and lectin-blot analysis revealed that Otg1p showed α1,3-galactosyltransferase activity under conditions of co-expression with Gmh6p, indicating that α-1,2-linked galactose is required for the galactosylation activity of Otg1p in S. pombe. In conclusion, eight galactosyltransferases have been shown to have activity in S. pombe with different substrate specificities. These findings will be useful for genetically tailoring the galactosylation of both N- and O-glycans in fission yeast.
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http://dx.doi.org/10.1093/glycob/cwab028DOI Listing
September 2021

The fission yeast gmn2 gene encodes an ERD1 homologue of Saccharomyces cerevisiae required for protein glycosylation and retention of luminal endoplasmic reticulum proteins.

J Gen Appl Microbiol 2021 Jun 3;67(2):67-76. Epub 2021 Feb 3.

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University.

The gmn2 mutant of Schizosaccharomyces pombe has previously been shown to exhibit defects in protein glycosylation of N-linked oligosaccharides (Ballou, L. and Ballou, CE., Proc. Natl. Acad. Sci. USA, 92, 2790-2794 (1995)). Like most glycosylation-defective mutants, the S. pombe gmn2 mutant was found to be sensitive to hygromycin B, an aminoglycoside antibiotic. As a result of complementation analysis, the gmn2 gene was found to be a single open reading frame that encodes a polypeptide of 373 amino acids consisting of multiple membrane-spanning regions. The Gmn2 protein shares sequence similarity with Kluyveromyces lactis and Saccharomyces cerevisiae Erd1 proteins, which are required for retention of luminal endoplasmic reticulum (ER) proteins. Although disruption of the gmn2 gene is not lethal, the secreted glycoprotein showed a significant glycosylation defect with destabilization of the glycosyltransferase responsible for N-glycan elongation. It was also shown that a significant amount of BiP was missorted to the cell surface according to ADEL receptor destabilization. Fluorescent microscopy revealed that the functional Gmn2-EGFP fusion protein is mainly localized in the Golgi membrane. These results indicate that the Gmn2 protein is required for protein glycosylation and for retention of ER-resident proteins in S. pombe cells.
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http://dx.doi.org/10.2323/jgam.2020.07.002DOI Listing
June 2021

Stm1 is a vacuolar PQ-loop protein involved in the transport of basic amino acids in Schizosaccharomyces pombe.

Biochim Biophys Acta Biomembr 2021 02 12;1863(2):183507. Epub 2020 Nov 12.

Laboratory of Molecular Physiology and Genetics, Department of Bioscience, Graduate School of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan; Division of Cell-Free Life Science, Proteo-Science Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan. Electronic address:

The stm1 (SPAC17C9.10) gene of Schizosaccharomyces pombe is closely related to genes encoding vacuolar PQ-loop proteins, Ypq1, Ypq2, and Ypq3, of Saccharomyces cerevisiae. When stm1 fused with GFP was expressed in fission or budding yeast, Stm1-GFP localized at the vacuolar membrane. Isolated vacuolar membrane vesicles from S. cerevisiae cells overexpressing stm1 exhibited stm1-dependent arginine and lysine uptake activity. Exchange activity of arginine and histidine/arginine, as observed for Ypq2 of S. cerevisiae, was also detected in the vesicles expressing stm1. The expression levels of stm1 in S. pombe cells significantly affected the vacuolar contents of lysine, histidine, and arginine. These results suggest that Stm1 is a vacuolar PQ-loop protein involved in the transport of basic amino acids across the vacuolar membrane.
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http://dx.doi.org/10.1016/j.bbamem.2020.183507DOI Listing
February 2021

Single-Molecule FISH Reveals Subcellular Localization of α-Amylase and Actin mRNAs in the Filamentous Fungus .

Front Microbiol 2020 22;11:578862. Epub 2020 Sep 22.

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.

The machinery for mRNA localization is one of crucial molecular structures allowing cellular spatiotemporal organization of protein synthesis. Although the molecular mechanisms underlying mRNA localization have been thoroughly investigated in unicellular organisms, little is known about multicellular and multinuclear filamentous fungi. Here, we conducted single-molecule fluorescence hybridization (smFISH) to first visualize the mRNA molecules of α-amylase, which are encoded by , and which are thought to be abundantly secreted from the hyphal tips of the industrially important fungus . Consistent with previous biochemical studies, fluorescein amidite (FAM) fluorescence derived from expression was observed in hyphae cultured in a minimal medium containing maltose instead of glucose as the sole carbon source. Moreover, after more than 1 h incubation with fresh maltose-containing medium, the fluorescence of mRNAs was observed throughout the cells, suggesting α-amylase secretion potentially from each cell, instead of the hyphal tip only. Furthermore, in cultures with complete medium containing maltose, mRNAs were excluded from the tip regions, where no nuclei exist. In contrast, mRNAs of actin, encoded by , were localized mainly to the tip, where actin proteins also preferentially reside. Collectively, our smFISH analyses revealed distinct localization patterns of α-amylase and actin mRNAs in hyphal cells.
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http://dx.doi.org/10.3389/fmicb.2020.578862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536267PMC
September 2020

Identification and characterization of β-d-galactofuranosidases from Aspergillus nidulans and Aspergillus fumigatus.

J Biosci Bioeng 2021 Jan 1;131(1):1-7. Epub 2020 Oct 1.

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan. Electronic address:

Although β-d-galactofuranosidases (Galf-ases) that hydrolyze β-d-galactofuranose (Galf)-containing oligosaccharides have been characterized in various organisms, to date no Galf-specific Galf-ase-encoding genes have been reported in Aspergillus fungi. Based on the amino acid sequences of previously identified bacterial Galf-ases, here we found two candidate Galf-specific Galf-ase genes AN2395 (gfgA) and AN3200 (gfgB) in the genome of Aspergillus nidulans. Indeed, recombinant GfgA and GfgB proteins exhibited Galf-specific Galf-ase activity, but no detectable α-l-arabinofuranosidase (Araf-ase) activity. Phylogenetic analysis of GfgA and GfgB orthologs indicated that there are two types of Aspergillus species: those containing one ortholog each for GfgA and GfgB; and those containing only one ortholog in total, among which Aspergillus fumigatus there is a representative with a single ortholog Galf-ase Afu2g14520. Unlike GfgA and GfgB, the recombinant Afu2g14520 protein showed higher Araf-ase activity than Galf-ase activity. An assay of substrate specificity revealed that although GfgA and GfgB are both exo-type Galf-ases and hydrolyze β-(1,5) and β-(1,6) linkages, GfgA hydrolyzes β-(1,6)-linked Galf-oligosaccharide more effectively as compared with GfgB. Collectively, our findings indicate that Galf-ases in Aspergillus species may have a role in cooperatively degrading Galf-containing oligosaccharides depending on environmental conditions.
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http://dx.doi.org/10.1016/j.jbiosc.2020.09.006DOI Listing
January 2021

Golgi localization of glycosyltransferases requires Gpp74p in Schizosaccharomyces pombe.

Appl Microbiol Biotechnol 2020 Oct 12;104(20):8897-8909. Epub 2020 Sep 12.

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan.

The majority of Golgi glycosyltransferases are type II membrane proteins with a small cytosolic tail at their N-terminus. Several mechanisms for localizing these glycosyltransferases to the Golgi have been proposed. In Saccharomyces cerevisiae, the phosphatidylinositol-4-phosphate-binding protein ScVps74p interacts with the cytosolic tail of a Golgi glycosyltransferase and contributes to its localization. In this study, we investigated whether a similar mechanism functions in the fission yeast Schizosaccharomyces pombe. First, we identified gpp74 (GPP34 domain-containing Vps74 homolog protein), a gene encoding the S. pombe homolog of S. cerevisiae Vps74p. Deletion of the gpp74 gene resulted in the missorting of three Golgi glycosyltransferases, SpOch1p, SpMnn9p, and SpOmh1p, to vacuoles, but not SpAnp1p, indicating Gpp74p is required for targeting some glycosyltransferases to the Golgi apparatus. Gpp74p with an N-terminal GFP-tag localized to both the Golgi apparatus and the cytosol. Golgi localization of Gpp74p was dependent on the phosphatidylinositol 4-kinase SpPik1p. Site-directed mutagenesis of hydrophobic and basic amino acids in the cytosolic tails of SpOch1p and SpMnn9p resulted in their missorting to vacuoles, indicating these cytosolic N-terminal residues are important for localization in the Golgi. Unexpectedly, no prominent alternations in protein glycosylation were observed in S. pombe gpp74Δ cells, probably due to the residual Golgi localization of some SpOch1p and SpMnn9p in these cells. Collectively, these results demonstrate that both Gpp74p-dependent and Gpp74p-independent mechanisms are responsible for the Golgi localization of glycosyltransferases to the Golgi in S. pombe. KEY POINTS: • Gpp74p is involved in the localization of glycosyltransferases to the Golgi. • The cytosolic tails of glycosyltransferases are important for Golgi localization. • Gpp74p localizes to the Golgi in a SpPik1p-dependent manner.
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http://dx.doi.org/10.1007/s00253-020-10881-9DOI Listing
October 2020

Characterization and functional analysis of ERAD-related AAA+ ATPase Cdc48 in Aspergillus oryzae.

Fungal Biol 2020 09 24;124(9):801-813. Epub 2020 Jul 24.

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 744 Motooka, Fukuoka, 819-0395, Japan.

Aspergillus oryzae can secrete large amounts of enzymes. However, the production of abundant secretory proteins triggers the unfolded protein response (UPR) in the endoplasmic reticulum (ER), and it is not clear how ER-associated protein degradation (ERAD) contributes to bulk protein production in A. oryzae. Here we identified AoCdc48, the sole A. oryzae ortholog of Saccharomyces cerevisiae AAA+ ATPase Cdc48, a component of the ERAD machinery. We found that AoCdc48 localizes in both nuclei and cytoplasm. Generation of an Aocdc48 conditional mutant showed that Aocdc48 repression leads to reduced cell growth and aberrant hyphal morphology. When Aocdc48-repressed cells were cultured on starch-containing plates, the α-amylase-encoding gene amyB was about 1.3-fold higher expressed. Indeed, a halo produced by secreted amylase was seen on potato starch-containing plates even when there was almost no growth under Aocdc48 repression. Fluorescence microscopy revealed that although AmyB seemed to be secreted, various organelle distributions were aberrant in Aocdc48-repressed cells. We found that D1 AAA domain is crucial for cell viability. Finally, we show that Aocdc48-overexpression also causes defects of cell growth, colonial morphology and conidial formation. Collectively, our results suggest that AoCdc48 is essential for growth and organelle distribution but dispensable for amylase secretion.
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http://dx.doi.org/10.1016/j.funbio.2020.06.004DOI Listing
September 2020

SpMnn9p and SpAnp1p form a protein complex involved in mannan synthesis in the fission yeast Schizosaccharomyces pombe.

J Biosci Bioeng 2020 Oct 7;130(4):335-340. Epub 2020 Jul 7.

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan. Electronic address:

The cell walls of yeast cells possess a large mannan structure mainly comprising of a linear α1,6-linked mannose oligomer on the N-linked glycans. The biosynthesis of the mannan is initiated by ScOch1p α1,6-mannosyltransfease, and elongated by the mannan polymerase complexes M-Pol I and II in the Golgi of Saccharomyces cerevisiae. Here, we functionally characterized SpMnn9 and SpAnp1 proteins in the fission yeast Schizosaccharomyces pombe; these proteins are homologs of S. cerevisiae M-Pol II complex proteins ScMnn9p and ScAnp1p. Cells harboring disruptions in Spmnn9 and Spanp1 genes showed slower growth at 37°C and an increased sensitivity to hygromycin B, characteristic of a glycosylation defect. Results obtained from the acid phosphatase assay and high-performance liquid chromatography analysis of N-linked glycans in Spmnn9Δ and Spanp1Δ mutants suggested that the mannan structure in S. pombe is synthesized sequentially by the α-mannosyltransferases in the order of SpOch1p, SpMnn9p and SpAnp1p. Immunoprecipitation and split YFP analyses demonstrated that SpMnn9p and SpAnp1p form the M-Pol-II like complex. Together, these results provided an improved understanding of the mechanism of mannan synthesis by SpMnn9p and SpAnp1p in S. pombe.
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http://dx.doi.org/10.1016/j.jbiosc.2020.06.003DOI Listing
October 2020

Author Correction: Characterization of novel endo-β-N-acetylglucosaminidases from Sphingobacterium species, Beauveria bassiana and Cordyceps militaris that specifically hydrolyze fucose-containing oligosaccharides and human IgG.

Sci Rep 2020 Apr 29;10(1):7413. Epub 2020 Apr 29.

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka, 812-8581, Japan.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-63704-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190819PMC
April 2020

Yeast Flocculin: Methods for Quantitative Analysis of Flocculation in Yeast Cells.

Methods Mol Biol 2020 ;2132:437-444

Department of Bioscience & Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.

Flocculation, the clump forming property of yeast, has long been appreciated in breweries and utilized as an off-cost method to enable the reuse of yeast cells. Members of the flocculin protein family were identified as the adherent proteins on the cell surface responsible for flocculation, and their properties have been investigated. Crystal structures of the adhesion domain of flocculins revealed their unique mode of ligand binding where a calcium ion is located in the middle of the interface between flocculin and the interacting sugar. Here we describe the most commonly used flocculation assay. The method is simple and easy, yet it is the most direct and reliable assay to evaluate the flocculation cellular phenotype.
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http://dx.doi.org/10.1007/978-1-0716-0430-4_42DOI Listing
March 2021

Characterization of N- and O-linked galactosylated oligosaccharides from fission yeast species.

J Biosci Bioeng 2020 Aug 4;130(2):128-136. Epub 2020 Apr 4.

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan. Electronic address:

The N- and O-linked oligosaccharides from fission yeast Schizosaccharomyces pombe not only contain large amounts of d-mannose (Man) but also contain large amounts of d-galactose (Gal). Although the galactomannans of S. pombe are mainly composed of α1,2- or α1,3-linked Gals, some of the terminal α1,2-linked Gals are found to be linked to pyruvylated β1,3-linked galactose (PvGal). We have determined the structural characteristics of the N-glycans and O-glycans in three Schizosaccharomyces species (S. japonicus, S. octosporus, and S. cryophilus) using lectin blot, H NMR spectroscopy, and size-fractionation high performance liquid chromatography (HPLC), and found that the galactosylation of oligosaccharides was a common feature in fission yeasts. In addition, each of the terminal Galα1,2-, Galβ1,3- and non-substituted Man residues exhibited distinct characteristics. A BLAST search of gene databases in Schizosaccharomyces identified genes homologous to pvg1 encoding pyruvyltransferase of S. pombe. These genes, when expressed in an S. pombe pvg1Δ strains, led to the pyruvylation of non-reducing terminal β-linked Gal, suggesting the biosynthetic pathway of PvGal-containing oligosaccharides is highly conserved in fission yeasts.
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http://dx.doi.org/10.1016/j.jbiosc.2020.03.008DOI Listing
August 2020

The endogenous galactofuranosidase GlfH1 hydrolyzes mycobacterial arabinogalactan.

J Biol Chem 2020 04 27;295(15):5110-5123. Epub 2020 Feb 27.

Univ. Lille, CNRS, UMR8576 - UGSF - Unit[c33c]zpi;● de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France

Despite impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the tubercle bacillus adapts its cell wall structure and composition to various environmental conditions, especially during infection, remain poorly understood. Being the central portion of the mAGP complex, arabinogalactan (AG) is believed to be the constituent of the mycobacterial cell envelope that undergoes the least structural changes, but no reports exist supporting this assumption. Herein, using recombinantly expressed mycobacterial protein, bioinformatics analyses, and kinetic and biochemical assays, we demonstrate that the AG can be remodeled by a mycobacterial endogenous enzyme. In particular, we found that the mycobacterial GlfH1 (Rv3096) protein exhibits exo-β-d-galactofuranose hydrolase activity and is capable of hydrolyzing the galactan chain of AG by recurrent cleavage of the terminal β-(1,5) and β-(1,6)-Galf linkages. The characterization of this galactosidase represents a first step toward understanding the remodeling of mycobacterial AG.
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http://dx.doi.org/10.1074/jbc.RA119.011817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152746PMC
April 2020

1,6-α-L-Fucosidases from subsp. ATCC 15697 Involved in the Degradation of Core-fucosylated -Glycan.

J Appl Glycosci (1999) 2020 20;67(1):23-29. Epub 2020 Feb 20.

3 Faculty of Agriculture, Kyushu University.

subsp. 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 : 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 -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- -acetylglucosaminidase. We demonstrated that Blon_0426 can de-fucosylate Fucα1-6GlcNAc-IgG prepared from Rituximab using Endo-CoM from . To generate homogenous non-fucosylated -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 . Collectively, our results strongly suggest that Blon_0426 and Blon_0248 are useful for glycoprotein glycan remodeling.
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http://dx.doi.org/10.5458/jag.jag.JAG-2019_0016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8367633PMC
February 2020

Author Correction: Identification and characterization of a novel β-D-galactosidase that releases pyruvylated galactose.

Sci Rep 2020 Feb 13;10(1):2873. Epub 2020 Feb 13.

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka, 812-8581, Japan.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-60002-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016099PMC
February 2020

Biosynthesis of β-(1→5)-Galactofuranosyl Chains of Fungal-Type and -Mannose-Type Galactomannans within the Invasive Pathogen Aspergillus fumigatus.

mSphere 2020 01 15;5(1). Epub 2020 Jan 15.

Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto, Japan

The pathogenic fungus contains galactomannans localized on the surface layer of its cell walls, which are involved in various biological processes. Galactomannans comprise α-(1→2)-/α-(1→6)-mannan and β-(1→5)-/β-(1→6)-galactofuranosyl chains. We previously revealed that GfsA is a β-galactofuranoside β-(1→5)-galactofuranosyltransferase involved in the biosynthesis of β-(1→5)-galactofuranosyl chains. In this study, we clarified the biosynthesis of β-(1→5)-galactofuranosyl chains in Two paralogs exist within : GfsB and GfsC. We show that GfsB and GfsC, in addition to GfsA, are β-galactofuranoside β-(1→5)-galactofuranosyltransferases by biochemical and genetic analyses. GfsA, GfsB, and GfsC can synthesize β-(1→5)-galactofuranosyl oligomers at up to lengths of 7, 3, and 5 galactofuranoses within an established highly efficient assay of galactofuranosyltransferase activity. Structural analyses of galactomannans extracted from Δ, Δ, Δ, and Δ strains revealed that GfsA and GfsC synthesized all β-(1→5)-galactofuranosyl residues of fungal-type and -mannose-type galactomannans and that GfsB exhibited limited function in The loss of β-(1→5)-galactofuranosyl residues decreased the hyphal growth rate and conidium formation ability and increased the abnormal hyphal branching structure and cell surface hydrophobicity, but this loss is dispensable for sensitivity to antifungal agents and virulence toward immunocompromised mice. β-(1→5)-Galactofuranosyl residues are widely distributed in the subphylum Pezizomycotina of the phylum Ascomycota. Pezizomycotina includes many plant and animal pathogens. Although the structure of β-(1→5)-galactofuranosyl residues of galactomannans in filamentous fungi was discovered long ago, it remains unclear which enzyme is responsible for biosynthesis of this glycan. Fungal cell wall formation processes are complicated, and information concerning glycosyltransferases is essential for understanding them. In this study, we showed that GfsA and GfsC are responsible for the biosynthesis of all β-(1→5)-galactofuranosyl residues of fungal-type and -mannose-type galactomannans. The data presented here indicate that β-(1→5)-galactofuranosyl residues are involved in cell growth, conidiation, polarity, and cell surface hydrophobicity. Our new understanding of β-(1→5)-galactofuranosyl residue biosynthesis provides important novel insights into the formation of the complex cell wall structure and the virulence of the members of the subphylum Pezizomycotina.
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http://dx.doi.org/10.1128/mSphere.00770-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6968653PMC
January 2020

Secretory production of N-glycan-deleted glycoprotein in Aspergillus oryzae.

J Biosci Bioeng 2020 May 7;129(5):573-580. Epub 2020 Jan 7.

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.

The pharmaceutical industry has a high demand for glycoprotein production. The glycoform of glycoproteins is crucial for pharmacological activity. However, in general, cells produce glycoproteins with a heterologous glycoform, which is unfavorable for making uniform, efficacious therapeutic proteins. Here, to produce more glycoproteins with N-glycan uniformity, we applied the GlycoDelete strategy, in which endo-β-N-acetylglucosaminidase (ENGase) from the fungus Hypocrea jecorina (EndoT) is expressed at the Golgi membrane to cleave N-glycan from secretory glycoproteins, to Aspergillus oryzae cells. First, we selected candidate transmembrane domains to target EndoT to the Golgi membrane in A. oryzae cells, generated constructs for expressing the transmembrane-fused EndoT proteins and produced four potential AoGlycoDelete strains. We then confirmed that these strains produced α-amylase with a molecular weight lower than that of native α-amylase without an effect on growth. To test whether the A. oryzae α-amylase proteins had been cleaved by EndoT, we expressed and purified HA-tagged α-amylase AmyB and glucoamylase GlaA proteins from the AoGlycoDelete strain. MS and N-glycan analyses of the intact proteins confirmed neither AmyB-HA nor GlaA-HA produced from the AoGlycoDelete strain contained N-glycan. Lastly, we determined the enzymatic activities of the amylases produced by the AoGlycoDelete strain, which showed that the lack of N-glycan did not affect their activity under the conditions tested. Collectively, our findings demonstrate successful generation of an AoGlycoDelete strain that might be a good candidate for producing pharmaceutical glycoproteins with a uniform N-glycan structure.
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http://dx.doi.org/10.1016/j.jbiosc.2019.12.006DOI Listing
May 2020

Identification and characterization of a novel, versatile sialidase from a Sphingobacterium that can hydrolyze the glycosides of any sialic acid species at neutral pH.

Biochem Biophys Res Commun 2020 03 27;523(2):487-492. Epub 2019 Dec 27.

Bioscience and Biotechnology Center, Nagoya University, Nagoya, 464-8601, Japan; Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan; Program for Leading Graduate Schools, Integrative Graduate Education and Research Program in Green Natural Sciences, Nagoya University, Nagoya, 464-8601, Japan. Electronic address:

Bacterial sialidases are widely used to remove sialic acid (Sia) residues from glycans. Most of them cleave the glycosides of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) under acidic pHs; however, currently available bacterial sialidases had no activity to the glycosides of deaminoneuraminic acid (Kdn). In this study, we found a novel sialidase from Sphingobacterium sp. strain HMA12 that could cleave any of the glycosides of Neu5Ac, Neu5Gc, and Kdn. It also had a broad linkage specificity, i.e., α2,3-, α2,6-, α2,8-, and α2,9-linkages, and the optimal pH at neutral ranges, pH 6.5-7.0. These properties are particularly important when sialidases are applied for in vivo digestion of the cell surface sialosides under physiological conditions. Interestingly, 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (Neu5Ac2en), which is a transition state analog-based inhibitor, competitively inhibited the enzyme-catalyzed reaction for Kdn as well as for Neu5Ac, suggesting that the active site is common to the Neu5Ac and Kdn residues. Taken together, this sialidase is versatile and useful for the in vivo research on sialo-glycoconjugates.
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http://dx.doi.org/10.1016/j.bbrc.2019.12.079DOI Listing
March 2020

Structural basis for the specific cleavage of core-fucosylated -glycans by endo-β--acetylglucosaminidase from the fungus .

J Biol Chem 2019 11 23;294(45):17143-17154. Epub 2019 Sep 23.

Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

-Linked glycans play important roles in various cellular and immunological events. Endo-β--acetylglucosaminidase (ENGase) can release or transglycosylate -glycans and is a promising tool for the chemoenzymatic synthesis of glycoproteins with homogeneously modified glycans. The ability of ENGases to act on core-fucosylated glycans is a key factor determining their therapeutic utility because mammalian -glycans are frequently α-1,6-fucosylated. Although the biochemistries and structures of various ENGases have been studied extensively, the structural basis for the recognition of the core fucose and the asparagine-linked GlcNAc is unclear. Herein, we determined the crystal structures of a core fucose-specific ENGase from the caterpillar fungus (Endo-CoM), which belongs to glycoside hydrolase family 18. Structures complexed with fucose-containing ligands were determined at 1.75-2.35 Å resolutions. The fucose moiety linked to GlcNAc is extensively recognized by protein residues in a round-shaped pocket, whereas the asparagine moiety linked to the GlcNAc is exposed to the solvent. The -glycan-binding cleft of Endo-CoM is Y-shaped, and several lysine and arginine residues are present at its terminal regions. These structural features were consistent with the activity of Endo-CoM on fucose-containing glycans on rituximab (IgG) and its preference for a sialobiantennary substrate. Comparisons with other ENGases provided structural insights into their core fucose tolerance and specificity. In particular, Endo-F3, a known core fucose-specific ENGase, has a similar fucose-binding pocket, but the surrounding residues are not shared with Endo-CoM. Our study provides a foothold for protein engineering to develop enzymatic tools for the preparation of more effective therapeutic antibodies.
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http://dx.doi.org/10.1074/jbc.RA119.010842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851319PMC
November 2019

Galactofuranosidase from JHA 19 Streptomyces sp.: subcloning and biochemical characterization.

Carbohydr Res 2019 Jul 28;480:35-41. Epub 2019 May 28.

ICOA UMR CRNS 7311, Université d'Orléans, Rue de Chartres, BP 6759, 45067, Orléans Cedex 2, France. Electronic address:

Despite the crucial role of the rare galactofuranose (Galf) in many pathogenic micro-organisms and our increased knowledge of its metabolism, there is still a lack of recombinant and efficient galactofuranoside hydrolase available for chemo-enzymatic synthetic purposes of specific galactofuranosyl-conjugates. Subcloning of the Galf-ase from JHA 19 Streptomyces sp. and its further overexpression lead us to the production of this enzyme with a yield of 0.5 mg/L of culture. It exhibits substrate specificity exclusively towards pNP β-d-Galf, giving a K value of 250 μM, and the highest enzymatic efficiency ever observed of 14 mM  s. It proved to be stable to temperature up to 60 °C and to at least 4 freeze-thaw's cycles. Thus, Galf-ase demonstrated to be an efficient and stable biocatalyst with greatly improved specificity toward the galactofuranosyl entity, thus paving the way to the further development of transglycosylation and thioligation reactions.
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http://dx.doi.org/10.1016/j.carres.2019.05.011DOI Listing
July 2019

Catechol O-methyltransferase homologs in Schizosaccharomyces pombe are response factors to alkaline and salt stress.

Appl Microbiol Biotechnol 2019 Jun 3;103(12):4881-4887. Epub 2019 May 3.

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan.

How cells of the fission yeast Schizosaccharomyces pombe respond to alkaline stress is not well understood. Here, to elucidate the molecular mechanism underlying the alkaline stress response in S. pombe, we performed DNA microarray analysis. We found that a homolog of human catechol O-methyltransferase 2 (COMT2) is highly upregulated in S. pombe cells exposed to alkaline conditions. We designated the S. pombe homolog as cmt2 and also identified its paralog, cmt1, in the S. pombe genome. Reverse transcription PCR confirmed that both cmt1 and cmt2 are upregulated within 1 h of exposure to alkaline stress and downregulated within 30 min of returning to an acidic environment. Moreover, we verified that recombinant Cmt proteins exhibit catechol O-methyltransferase activity. To further characterize the expression of cmt1 and cmt2, we carried out an EGFP reporter assay using their promoter sequences, which showed that both genes respond not only to alkaline but also to salt stress. Collectively, our findings indicate that the cmt promoter might be an advantageous expression system for use in S. pombe under alkaline culture conditions.
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http://dx.doi.org/10.1007/s00253-019-09858-0DOI Listing
June 2019

Chemo-enzymatic synthesis of p-nitrophenyl β-D-galactofuranosyl disaccharides from Aspergillus sp. fungal-type galactomannan.

Carbohydr Res 2019 Feb 11;473:99-103. Epub 2019 Jan 11.

Graduate School of Environmental and Life Science, Okayama University, Tsushima-naka 1-1-1, Okayama, 700-8530, Japan. Electronic address:

β-d-Galactofuranose (Galf) is a component of polysaccharides and glycoconjugates. There are few reports about the involvement of galactofuranosyltransferases and galactofuranosidases (Galf-ases) in the synthesis and degradation of galactofuranose-containing glycans. The cell walls of filamentous fungi in the genus Aspergillus include galactofuranose-containing polysaccharides and glycoconjugates, such as O-glycans, N-glycans, and fungal-type galactomannan, which are important for cell wall integrity. In this study, we investigated the synthesis of p-nitrophenyl β-d-galactofuranoside and its disaccharides by chemo-enzymatic methods including use of galactosidase. The key step was selective removal of the concomitant pyranoside by enzymatic hydrolysis to purify p-nitrophenyl β-d-galactofuranoside, a promising substrate for β-d-galactofuranosidase from Streptomyces species.
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http://dx.doi.org/10.1016/j.carres.2019.01.005DOI Listing
February 2019

Draft Genome Sequence of Bacillus sp. HMA207, a Strain That Exhibits β-d-Galactosidase Activity To Release Pyruvylated Galactose.

Microbiol Resour Announc 2018 Sep 13;7(10). Epub 2018 Sep 13.

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.

The genome sequence of the Bacillus sp. strain HMA207, the culture supernatant of which exhibited β-d-galactosidase activity to release pyruvylated galactose (PvGal), was examined to identify a PvGal-ase-encoding gene. We report here the result of whole-genome shotgun sequencing, which revealed putative PvGal-ase genes.
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http://dx.doi.org/10.1128/MRA.01169-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6256595PMC
September 2018

Identification and characterization of a novel β-D-galactosidase that releases pyruvylated galactose.

Sci Rep 2018 08 13;8(1):12013. Epub 2018 Aug 13.

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka, 812-8581, Japan.

Pyruvyl modification of oligosaccharides is widely seen in both prokaryotes and eukaryotes. Although the biosynthetic mechanisms of pyruvylation have been investigated, enzymes that metabolize and degrade pyruvylated oligosaccharides are not well known. Here, we searched for a pyruvylated galactose (PvGal)-releasing enzyme by screening soil samples. We identified a Bacillus strain, as confirmed by the 16S ribosomal RNA gene analysis, that exhibited PvGal-ase activity toward p-nitrophenyl-β-D-pyruvylated galactopyranose (pNP-β-D-PvGal). Draft genome sequencing of this strain, named HMA207, identified three candidate genes encoding potential PvGal-ases, among which only the recombinant protein encoded by ORF1119 exhibited PvGal-ase activity. Although ORF1119 protein displayed broad substrate specificity for pNP sugars, pNP-β-D-PvGal was the most favorable substrate. The optimum pH for the ORF1119 PvGal-ase was determined as 7.5. A BLAST search suggested that ORF1119 homologs exist widely in bacteria. Among two homologs tested, BglC from Clostridium but not BglH from Bacillus showed PvGal-ase activity. Crystal structural analysis together with point mutation analysis revealed crucial amino acids for PvGal-ase activity. Moreover, ORF1119 protein catalyzed the hydrolysis of PvGal from galactomannan of Schizosaccharomyces pombe, suggesting that natural polysaccharides might be substrates of the PvGal-ase. This novel PvGal-catalyzing enzyme might be useful for glycoengineering projects to produce new oligosaccharide structures.
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http://dx.doi.org/10.1038/s41598-018-30508-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6090015PMC
August 2018

Genomic Sequence of Saccharomyces cerevisiae BAW-6, a Yeast Strain Optimal for Brewing Barley Shochu.

Genome Announc 2018 Apr 5;6(14). Epub 2018 Apr 5.

Research Laboratory, Sanwa Shurui Co., Ltd., Oita, Japan.

Here, we report the draft genome sequence of strain BAW-6, which is used for the production of barley shochu, a traditional Japanese spirit. This genomic information can be used to elucidate the genetic basis underlying the high alcohol production capacity and citric acid tolerance of shochu yeast.
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http://dx.doi.org/10.1128/genomeA.00228-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5887032PMC
April 2018

Draft Genome Sequence of Sphingobacterium sp. Strain HMA12, Which Encodes Endo-β--Acetylglucosaminidases and Can Specifically Hydrolyze Fucose-Containing Oligosaccharides.

Genome Announc 2018 Feb 22;6(8). Epub 2018 Feb 22.

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka, Japan

The genome sequence of the soil bacterium sp. strain HMA12, the culture supernatant of which exhibited endo-β--acetylglucosaminidase (ENGase) activity, was examined for ENGase-encoding genes. Here, we report the characterization of new genes of ENGases, obtained by whole-genome shotgun sequencing, that are capable of specifically hydrolyzing fucose-containing oligosaccharides.
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http://dx.doi.org/10.1128/genomeA.01525-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5824009PMC
February 2018

Mutation in fission yeast phosphatidylinositol 4-kinase Pik1 is synthetically lethal with defect in telomere protection protein Pot1.

Biochem Biophys Res Commun 2018 02 2;496(4):1284-1290. Epub 2018 Feb 2.

Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Japan. Electronic address:

Fission yeast Pik1p is one of three phosphatidylinositol 4-kinases associated with the Golgi complex, but its function is not fully understood. Deletion of pot1 causes telomere degradation and chromosome circularization. We searched for the gene which becomes synthetically lethal with pot1Δ. We obtained a novel pik1 mutant, pik1-1, which is synthetically lethal with pot1Δ. We found phosphoinositol 4-phosphate in the Golgi was reduced in pik1-1. To investigate the mechanism of the lethality of the pot1Δ pik1-1 double mutant, we constructed the nmt-pot1-aid pik1-1 strain, where Pot1 function becomes low by drugs, which leads to telomere loss and chromosome circularization, and found pik1-1 mutation does not affect telomere resection and chromosome circularization. Thus, our results suggest that pik1 is required for the maintenance of circular chromosomes.
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http://dx.doi.org/10.1016/j.bbrc.2018.02.001DOI Listing
February 2018

Substrate specificity of Nudix hydrolases from Myxococcus xanthus.

J Gen Appl Microbiol 2018 05 25;64(2):94-98. Epub 2018 Jan 25.

Department of Bioscience and Biotechnology, Kyushu University.

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http://dx.doi.org/10.2323/jgam.2017.07.001DOI Listing
May 2018

Characterization of novel endo-β-N-acetylglucosaminidases from Sphingobacterium species, Beauveria bassiana and Cordyceps militaris that specifically hydrolyze fucose-containing oligosaccharides and human IgG.

Sci Rep 2018 01 10;8(1):246. Epub 2018 Jan 10.

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka, 812-8581, Japan.

Endo-β-N-acetylglucosaminidase (ENGase) catalyzes hydrolysis of N-linked oligosaccharides. Although many ENGases have been characterized from various organisms, so far no fucose-containing oligosaccharides-specific ENGase has been identified in any organism. Here, we screened soil samples, using dansyl chloride (Dns)-labeled sialylglycan (Dns-SG) as a substrate, and discovered a strain that exhibits ENGase activity in the culture supernatant; this strain, named here as strain HMA12, was identified as a Sphingobacterium species by 16S ribosomal RNA gene analysis. By draft genome sequencing, five candidate ENGase encoding genes were identified in the genome of this strain. Recombinant proteins, purified from Escherichia coli expressing candidate genes ORF1152, ORF1188, ORF3046 and ORF3750 exhibited fucose-containing oligosaccharides-specific ENGase activity. These ENGases exhibited optimum activities at very acidic pHs (between pH 2.3-2.5). BLAST searches using sequences of these candidate genes identified two fungal homologs of ORF1188, one in Beauveria bassiana and the other in Cordyceps militaris. Recombinant ORF1188, Beauveria and Cordyceps ENGases released the fucose-containing oligosaccharides residues from rituximab (immunoglobulin G) but not the high-mannose-containing oligosaccharides residues from RNase B, a result that not only confirmed the substrate specificity of these novel ENGases but also suggested that natural glycoproteins could be their substrates.
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http://dx.doi.org/10.1038/s41598-017-17467-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762919PMC
January 2018

Regulation of mating type switching by the mating type genes and RME1 in Ogataea polymorpha.

Sci Rep 2017 11 24;7(1):16318. Epub 2017 Nov 24.

Graduate School of Engineering, Osaka University, Osaka, Japan.

Saccharomyces cerevisiae and its closely related yeasts undergo mating type switching by replacing DNA sequences at the active mating type locus (MAT) with one of two silent mating type cassettes. Recently, a novel mode of mating type switching was reported in methylotrophic yeast, including Ogataea polymorpha, which utilizes chromosomal recombination between inverted-repeat sequences flanking two MAT loci. The inversion is highly regulated and occurs only when two requirements are met: haploidy and nutritional starvation. However, links between this information and the mechanism associated with mating type switching are not understood. Here we investigated the roles of transcription factors involved in yeast sexual development, such as mating type genes and the conserved zinc finger protein Rme1. We found that co-presence of mating type a1 and α2 genes was sufficient to prevent mating type switching, suggesting that ploidy information resides solely in the mating type locus. Additionally, RME1 deletion resulted in a reduced rate of switching, and ectopic expression of O. polymorpha RME1 overrode the requirement for starvation to induce MAT inversion. These results suggested that mating type switching in O. polymorpha is likely regulated by two distinct transcriptional programs that are linked to the ploidy and transmission of the starvation signal.
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http://dx.doi.org/10.1038/s41598-017-16284-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701183PMC
November 2017
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