title: Molecular cloning and expression of endo-beta-N-acetylglucosaminidase D, which acts on the core structure of complex type asparagine-linked oligosaccharides.
abstract: Endo-beta-N-acetylglucosaminidase D (Endo D) produced by Streptococcus pneumoniae cleaves the di-N-acetylchitobiose structure in asparagine-linked oligosaccharides. The enzyme generally acts on complex type oligosaccharides after removal of external sugars by neuraminidase, beta-galactosidase, and beta-N-acetylglucosaminidase. We cloned the gene encoding the enzyme and expressed it as a periplasm enzyme in Escherichia coli. The first 37 amino acids in the predicted sequence are removed in the mature enzyme, yielding a protein with a molecular mass of 178 kDa. The substrate specificity of the recombinant enzyme is indistinguishable from the enzyme produced by S. pneumoniae. Endo-beta-N-acetylglucosaminidase A (Endo A) from Arthrobacter protophormiae, the molecular mass of which is 72 kDa, had 32% sequence identity to Endo D, starting from the N-terminal sides of both enzymes, although Endo A hydrolyzes high-mannose-type oligosaccharides and does not hydrolyze complex type ones. Endo D is not related to endo-beta-N-acetylglucosaminidases H, F(1), F(2), or F(3), which share common structural motifs. Therefore, there are two distinct groups of endo-beta-N-acetylglucosaminidases acting on asparagine-linked oligosaccharides. The C-terminal region of Endo D shows homology to beta-galactosidase and beta-N-acetylglucosaminidase from S. pneumoniae and has an LPXTG motif typical of surface-associated proteins of Gram-positive bacteria. It is possible that Endo D is located on the surface of the bacterium and, together with other glycosidases, is involved in virulence.
authors.affiliation: Department of Biochemistry, Nagoya University School of Medicine, Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Muramatsu
authors.forename: H
authors.initials: H
authors.lastname: Tachikui
authors.forename: H
authors.initials: H
authors.lastname: Ushida
authors.forename: X
authors.initials: X
authors.lastname: Song
authors.forename: Y
authors.initials: Y
authors.lastname: Qiu
authors.forename: S
authors.initials: S
authors.lastname: Yamamoto
authors.forename: T
authors.initials: T
authors.lastname: Muramatsu
mesh_terms: D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology; D001483:Base Sequence; D003001:Cloning, Molecular; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D009844:Oligosaccharides; D011994:Recombinant Proteins; D001216:Asparagine; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase