title: Discovery and characterization of a novel extremely acidic bacterial N-glycanase with combined advantages of PNGase F and A.
abstract: Peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidases [PNGases (peptide N-glycosidases), N-glycanases, EC 3.5.1.52] are essential tools in the release of N-glycans from glycoproteins. We hereby report the discovery and characterization of a novel bacterial N-glycanase from Terriglobus roseus with an extremely low pH optimum of 2.6, and annotated it therefore as PNGase H+. The gene of PNGase H+ was cloned and the recombinant protein was successfully expressed in Escherichia coli. The recombinant PNGase H+ could liberate high mannose-, hybrid- and complex-type N-glycans including core α1,3-fucosylated oligosaccharides from both glycoproteins and glycopeptides. In addition, PNGase H+ exhibited better release efficiency over N-glycans without core α1,3-fucose compared with PNGase A. The facile expression, non-glycosylated nature, unusual pH optimum and broad substrate specificity of this novel type of N-glycanase makes recombinant PNGase H+ a versatile tool in N-glycan analysis.
authors.affiliation: *Glycomics and Glycan Bioengineering Research Center, College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.
authors.forename: Ting
authors.initials: T
authors.lastname: Wang
authors.affiliation: *Glycomics and Glycan Bioengineering Research Center, College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.
authors.forename: Zhi P
authors.initials: ZP
authors.lastname: Cai
authors.affiliation: *Glycomics and Glycan Bioengineering Research Center, College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.
authors.forename: Xiao Q
authors.initials: XQ
authors.lastname: Gu
authors.affiliation: †Department of Plant Pathology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.
authors.forename: Hong Y
authors.initials: HY
authors.lastname: Ma
authors.affiliation: *Glycomics and Glycan Bioengineering Research Center, College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.
authors.forename: Ya M
authors.initials: YM
authors.lastname: Du
authors.affiliation: *Glycomics and Glycan Bioengineering Research Center, College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.
authors.forename: Kun
authors.initials: K
authors.lastname: Huang
authors.affiliation: *Glycomics and Glycan Bioengineering Research Center, College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.
authors.forename: Josef
authors.initials: J
authors.lastname: Voglmeir
authors.affiliation: *Glycomics and Glycan Bioengineering Research Center, College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Liu
mesh_terms: D061271:Acidobacteria / Q000201:enzymology* / Q000235:genetics; D000143:Acids / Q000737:chemistry; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006020:Glycopeptides / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008358:Mannose / Q000378:metabolism; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000143:Acids; D001426:Bacterial Proteins; D006020:Glycopeptides; D006023:Glycoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D008358:Mannose