title: Molecular characterization of acidic peptide:N-glycanase from the dimorphic yeast Yarrowia lipolytica.
abstract: Peptide:N-glycanase (PNGase) A is used preferentially to cleave the glycans from plant and insect glycopeptides. Although many putative PNGase A homologous genes have been found in the plant and fungus kingdoms through sequence similarity analyses, only several PNGases from plants and one from a filamentous fungus have been characterized. In this study, we identified and characterized a PNGase A-like enzyme, PNGase Yl, in the dimorphic yeast Yarrowia lipolytica. The corresponding gene was cloned and recombinantly expressed in Pichia pastoris. The purified enzyme cleaved glycans from glycopeptides with the maximum activity at pH 5. No metal ions were required for full activity, and rather it was repressed by three metal ions (Fe(3+), Cu(2+) and Zn(2+)). Using glycopeptide substrates, PNGase Yl was shown to release various types of N-glycans including high-mannose and complex-type glycans as well as glycans containing core-linked α(1,3)-fucose that are frequently found in plants and insects. Moreover, in comparison with PNGase A, PNGase Yl was able to cleave with higher efficiency the glycans from some denatured glycoproteins. Taken together, our results suggest that PNGase Yl, the first biochemically characterized yeast PNGase A homologue, can be developed through protein engineering as a useful deglycosylation tool for N-glycosylation study.
authors.affiliation: Biochemicals and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-Gu, Daejeon 305-806, Korea; Department of Medicine, Chung-Ang University, Seoul 156-756, Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Korea; Department of Biological Engineering, Inha University, Incheon 402-751, Korea; and College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea Biochemicals and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-Gu, Daejeon 305-806, Korea; Department of Medicine, Chung-Ang University, Seoul 156-756, Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Korea; Department of Biological Engineering, Inha University, Incheon 402-751, Korea; and College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea.
authors.forename: Kyung Jin
authors.initials: KJ
authors.lastname: Lee
authors.affiliation: Biochemicals and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-Gu, Daejeon 305-806, Korea; Department of Medicine, Chung-Ang University, Seoul 156-756, Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Korea; Department of Biological Engineering, Inha University, Incheon 402-751, Korea; and College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea.
authors.forename: Jin Young
authors.initials: JY
authors.lastname: Gil
authors.affiliation: Biochemicals and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-Gu, Daejeon 305-806, Korea; Department of Medicine, Chung-Ang University, Seoul 156-756, Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Korea; Department of Biological Engineering, Inha University, Incheon 402-751, Korea; and College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea.
authors.forename: Sang-Yoon
authors.initials: SY
authors.lastname: Kim
authors.affiliation: Biochemicals and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-Gu, Daejeon 305-806, Korea; Department of Medicine, Chung-Ang University, Seoul 156-756, Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Korea; Department of Biological Engineering, Inha University, Incheon 402-751, Korea; and College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea Biochemicals and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-Gu, Daejeon 305-806, Korea; Department of Medicine, Chung-Ang University, Seoul 156-756, Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Korea; Department of Biological Engineering, Inha University, Incheon 402-751, Korea; and College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea.
authors.forename: Ohsuk
authors.initials: O
authors.lastname: Kwon
authors.affiliation: Biochemicals and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-Gu, Daejeon 305-806, Korea; Department of Medicine, Chung-Ang University, Seoul 156-756, Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Korea; Department of Biological Engineering, Inha University, Incheon 402-751, Korea; and College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea.
authors.forename: Kisung
authors.initials: K
authors.lastname: Ko
authors.affiliation: Biochemicals and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-Gu, Daejeon 305-806, Korea; Department of Medicine, Chung-Ang University, Seoul 156-756, Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Korea; Department of Biological Engineering, Inha University, Incheon 402-751, Korea; and College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea.
authors.forename: Dong-Il
authors.initials: DI
authors.lastname: Kim
authors.affiliation: Biochemicals and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-Gu, Daejeon 305-806, Korea; Department of Medicine, Chung-Ang University, Seoul 156-756, Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Korea; Department of Biological Engineering, Inha University, Incheon 402-751, Korea; and College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea.
authors.forename: Dae Kyong
authors.initials: DK
authors.lastname: Kim
authors.affiliation: Biochemicals and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-Gu, Daejeon 305-806, Korea; Department of Medicine, Chung-Ang University, Seoul 156-756, Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Korea; Department of Biological Engineering, Inha University, Incheon 402-751, Korea; and College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea.
authors.forename: Ha Hyung
authors.initials: HH
authors.lastname: Kim
authors.affiliation: Biochemicals and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-Gu, Daejeon 305-806, Korea; Department of Medicine, Chung-Ang University, Seoul 156-756, Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Korea; Department of Biological Engineering, Inha University, Incheon 402-751, Korea; and College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea Biochemicals and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-Gu, Daejeon 305-806, Korea; Department of Medicine, Chung-Ang University, Seoul 156-756, Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Korea; Department of Biological Engineering, Inha University, Incheon 402-751, Korea; and College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea dboh@kribb.re.kr.
authors.forename: Doo-Byoung
authors.initials: DB
authors.lastname: Oh
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D006020:Glycopeptides / Q000737:chemistry / Q000235:genetics*; D006031:Glycosylation; D008358:Mannose / Q000235:genetics; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011134:Polysaccharides / Q000096:biosynthesis / Q000737:chemistry; D025062:Yarrowia / Q000201:enzymology*
chemical_list: D006020:Glycopeptides; D011134:Polysaccharides; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D008358:Mannose
keywords: Yarrowia lipolytica; acidic peptide:N-glycanse; glycan analysis; glycopeptide; glycoprotein