title: Transglycosylation Activity of Glycosynthase Mutants of Endo-β-N-Acetylglucosaminidase from Coprinopsis cinerea.
abstract: Endo-β-N-acetylglucosaminidase (ENGase), which catalyzes hydrolysis of N-linked oligosaccharides, is a useful tool for analyzing oligosaccharide contents of glycoproteins. However, there are only a few known ENGases that can catalyze the hydrolysis of human complex type oligosaccharides, and although commercially available, they are expensive. Here, we report the cloning of two ENGase encoding cDNAs from the basidiomycete fungus Coprinopsis cinerea, Endo-CC1 and Endo-CC2. We successfully expressed recombinant His6-tagged Endo-CC1 and Endo-CC2 in Escherichia coli and purified them for enzymatic characterization. Both Endo-CC1 and Endo-CC2 showed hydrolytic activity on high-mannose and complex type oligosaccharides. Since Endo-CC1 could be prepared more easily than Endo-CC2 from E. coli cultures, we examined the enzymatic properties of Endo-CC1 in detail. Our results showed that Endo-CC1 acted on both N-linked high-mannose type and sialobiantennary type complex oligosaccharides of glycoproteins RNase B and human transferrin, respectively, but not on the sialotriantennary type complex oligosaccharide of glycoprotein fetuin. Examination of the transglycosylation activity of Endo-CC1 revealed that the wild-type Endo-CC1 could not transfer the sialobiantennary type complex oligosaccharide onto the deglycosylated RNase B. To obtain an Endo-CC1 mutant with desired transglycosylation activity, we performed mutation analysis of the active site residue Asn 180 (N180), known to be important for catalysis, by individually replacing it with the remaining 19 amino acid residues. Transglycosylation analyses of these mutants led us to identify one mutant, namely Endo-CC1N180H, which exhibited the desired transglycosylation activity. Taken together, we suggest that Endo-CC1 would potentially be a valuable tool for analyzing oligosaccharides on glycoproteins, as large quantities of it could be made available more easily and less expensively than the currently used enzyme, Endo-M.
authors.affiliation: Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka, Japan.
authors.forename: Yasunari
authors.initials: Y
authors.lastname: Eshima
authors.affiliation: Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka, Japan.
authors.forename: Yujiro
authors.initials: Y
authors.lastname: Higuchi
authors.affiliation: Fushimi Pharmaceutical Co. Ltd., Marugame, Kagawa, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kinoshita
authors.affiliation: Department of Functional Glycomics, Life Science Research Center, Kagawa University, Miki-cho, Kagawa, Japan.
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Nakakita
authors.affiliation: Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka, Japan.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Takegawa
mesh_terms: D000118:Acetylglucosaminidase / Q000235:genetics / Q000378:metabolism*; D001487:Basidiomycota / Q000201:enzymology*; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D006868:Hydrolysis; D008358:Mannose / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism*; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D016415:Sequence Alignment; D014168:Transferrin / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014168:Transferrin; D006867:Hydrolases; D012260:Ribonucleases; C029600:ribonuclease B; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008358:Mannose