Molecular cloning and expression of endo-beta-N-acetylglucosaminidase D, which acts on the core structure of complex type asparagine-linked oligosaccharides

J Biochem. 2001 Jun;129(6):923-8. doi: 10.1093/oxfordjournals.jbchem.a002938.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Asparagine / metabolism*
  • Bacillus / enzymology
  • Base Sequence
  • Cloning, Molecular
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / chemistry
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / genetics*
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / metabolism*
  • Molecular Sequence Data
  • Oligosaccharides / chemistry*
  • Oligosaccharides / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Streptococcus pneumoniae / enzymology*
  • Substrate Specificity

Substances

  • Oligosaccharides
  • Recombinant Proteins
  • Asparagine
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase

Associated data

  • GENBANK/AB055806