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12. Metabolic Mechanism of Mannan in a Ruminal Bacterium, Ruminococcus albus, Involving Two Mannoside Phosphorylases and Cellobiose 2-Epimerase: DISCOVERY OF A NEW CARBOHYDRATE PHOSPHORYLASE, β-1,4-MANNOOLIGOSACCHARIDE PHOSPHORYLASE*

Author

Kawahara, Ryosuke, Saburi, Wataru, Odaka, Rei, Taguchi, Hidenori, Ito, Shigeaki, Mori, Haruhide, and Matsui, Hirokazu

Subjects
Enzyme Kinetics, Glycoside Hydrolases, Cell-Free System, Phosphorylases, Mannooligosaccharide Phosphorylase, Phosphorylase, Enzyme Purification, Ruminococcus albus, Phosphorolysis, Mannan, Bacteroides fragilis, Isoenzymes, Mannans, Bacterial Proteins, Ruminococcus, Enzymology, Carbohydrate Metabolism, Glycoside Hydrolase Family 130, Carbohydrate Epimerases
Abstract

Ruminococcus albus is a typical ruminal bacterium digesting cellulose and hemicellulose. Cellobiose 2-epimerase (EC 5.1.3.11, CE), which converts cellobiose to 4-O-β-D-glucosyl-D-mannose, is a particularly unique enzyme in R. albus, but its physiological function is unclear. Recently, a new metabolic pathway of mannan involving CE was postulated for another CE producing bacterium, Bacteroides fragilis. In this pathway, β-1,4-mannobiose is epimerized to 4-O-β-D-mannosyl-D-glucose (Man-Glc) by CE, and Man-Glc is phosphorolyzed to α-D-mannosyl 1-phosphate (Man1P) and D-glucose by Man-Glc phosphorylase (EC 2.4.1.281, MP). Ruminococcus albus NE1 showed intracellular MP activity, and two MP isozymes, RaMP1 and RaMP2, were obtained from the cell-free extract. These enzymes were highly specific for the mannosyl residue at the non-reducing end of the substrate and catalyzed the phosphorolysis and synthesis of Man-Glc through a sequential bi bi mechanism. In a synthetic reaction, RaMP1 showed high activity only towards D-glucose and 6-deoxy-D-glucose in the presence of Man1P, while RaMP2 showed acceptor specificity significantly different from RaMP1. RaMP2 acted on D-glucose derivatives at the C2- and C3-positions including deoxy- and deoxyfluoro-analogues and epimers, but not on those substituted at the C6-position. Furthermore, RaMP2 had high synthetic activity toward the following oligosaccharides: β-linked glucobioses, maltose, N, N'-diacetylchitobiose, and β-1,4-mannooligosaccharides. Particularly, β-1,4-mannooligosaccharides served as significantly better acceptor substrates for RaMP2 than D-glucose. In the phosphorolytic reactions, RaMP2 had weak activity towards β-1,4-mannobiose but efficiently degraded β-1,4-mannooligosaccharides longer than β-1,4-mannobiose. Consequently, RaMP2 is thought to catalyze the phosphorolysis of β-1,4-mannooligosaccharides longer than β-1,4-mannobiose to produce Man1P and β-1,4-mannobiose.

Published
2012

13. A 3D epithelial-mesenchymal co-culture model of human bronchial tissue recapitulates multiple features of airway tissue remodeling by TGF-β1 treatment.

Author

Shinkichi Ishikawa, Kanae Ishimori, Shigeaki Ito, Ishikawa, Shinkichi, Ishimori, Kanae, and Ito, Shigeaki

Subjects
AIRWAY (Anatomy), COLLAGEN, FIBROBLASTS, TRANSFORMING growth factors, MESENCHYMAL stem cells
Abstract

Background: The collagen gel contraction assay measures gel size to assess the contraction of cells embedded in collagen gel matrices. Using the assay with lung fibroblasts is useful in studying the lung tissue remodeling process in wound healing and disease development. However, the involvement of bronchial epithelial cells in this process should also be investigated.Methods: We applied a layer of mucociliary differentiated bronchial epithelial cells onto collagen gel matrices with lung fibroblasts. This co-culture model enables direct contact between epithelial and mesenchymal cells. We stimulated the culture with transforming growth factor (TGF) β1 as an inducer of tissue remodeling for 21 days, and measured gel size, histological changes, and expression of factors related to extracellular matrix homeostasis.Results: TGF-β1 exerted a concentration-dependent effect on collagen gel contraction and on contractile myofibroblasts in the mesenchymal collagen layer. TGF-β1 also induced expression of the mesenchymal marker vimentin in the basal layer of the epithelium, suggesting the induction of epithelial-mesenchymal transition. In addition, the expression of various genes encoding extracellular matrix proteins was upregulated. Fibrotic tenascin-C accumulated in the sub-epithelial region of the co-culture model.Conclusion: Our findings indicate that TGF-β1 can affect both epithelial and mesenchymal cells, and induce gel contraction and structural changes. Our novel in vitro co-culture model will be a useful tool for investigating the roles of epithelial cells, fibroblasts, and their interactions in the airway remodeling process. [ABSTRACT FROM AUTHOR]

Published
2017
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16. Identification of the Cellobiose 2-Epimerase Gene in the Genome of Bacteroides fragilis NCTC 9343.

Author

Senoura, Takeshi, Taguchi, Hidenori, Ito, Shigeaki, Hamada, Shigeki, Matsui, Hirokazu, Fukiya, Satoru, Yokota, Atsushi, Watanabe, Jun, Wasaki, Jun, and Ito, Susumu

Subjects
GENOMICS, CATALYSIS, BACTEROIDES, GENOMES, THIN layer chromatography, ENZYME analysis
Abstract

The article focuses on a study regarding gene sequence and catalytic properties of celloboise 2-epiramese in Barcteroides fragilis. Computer analysis, enzyme assay and thin-layer chromatography (TLC) are some of the methods used by the researchers. Results show that B. fragilis cells possessed CE activity and that it exhibited 44-63% identities to N-acyl-D-glucosamine 2-epimerase-like hypothetical proteins of unknown function in bacterial genome sequences.

Published
2009
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17. Identification of the Cellobiose 2-Epimerase Gene in the Genome of Bacteroides fragilisNCTC 9343

Author

SENOURA, Takeshi, TAGUCHI, Hidenori, ITO, Shigeaki, HAMADA, Shigeki, MATSUI, Hirokazu, FUKIYA, Satoru, YOKOTA, Atsushi, WATANABE, Jun, WASAKI, Jun, and ITO, Susumu

Abstract

Cellobiose 2-epimerase (CE, EC 5.1.3.11) catalyzes the reversible epimerization of cellobiose to 4-O-β-D-glucopyranosyl-D-mannose. In this study, we found a CE gene in the genome sequence of non-cellulolytic Bacteroides fragilisNCTC 9343. The recombinant enzyme, expressed in Escherichia colicells, catalyzed a hydroxyl stereoisomerism at the C-2 positions of the reducing terminal glucose and at the mannose moiety of cello-oligosaccharides, lactose, β-mannobiose (4-O-β-D-mannopyranosyl-D-mannose), and globotriose [O-α-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl-(1→4)-D-glucose]. The CE from B. fragilisshowed less than 40% identity to reported functional CEs. It exhibited 44–63% identities to N-acyl-D-glucosamine 2-epimerase-like hypothetical proteins of unknown function in bacterial genome sequences of the phyla Firmicutes, Bacteroidetes, Proteobacteria, Chloroflexi, and Verrucomicrobia. On the other hand, it showed less than 26% identity to functional N-acyl-D-glucosamine 2-epimerases. Based on the amino acid homology and phylogenetic positions of the functional epimerases, we emphasize that many genes for putative N-acyl-D-glucosamine 2-epimerases and related hypothetical proteins of unknown function reported to date in the bacterial genomes should be annotated as CE-like proteins or putative CEs.

Published
2009
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