Mutational studies on endo-beta-N-acetylglucosaminidase D which hydrolyzes core portion of asparagine-linked complex type oligosaccharides

Glycoconj J. 2005 Feb;22(1-2):35-42. doi: 10.1007/s10719-005-0847-7.

Abstract

Endo-beta-N-acetylglucosaminidase D (Endo D) produced by Streptococcus pneumoniae hydrolyzes the di-N-acetylchitobiose structure in the core of complex-type asparagine-linked oligosaccharides, and has a molecular weight of 180 kDa. A truncated Endo D of 102 kDa in which 134 N-terminal amino acids and 599 C-terminal amino acids were deleted, still retained the enzymatic activity. The truncated Endo D has specificity indistinguishable from the intact enzyme, and also acted on the core structure of asparagine-linked oligosaccharides attached to intact IgG. Because of its lower molecular weight, the truncated enzyme may be useful as a tool for protein deglycosylation. The entire region of the truncated Endo D had 32% sequence identity to endo- beta-N-acetylglucosaminidase BH (Endo BH) from Bacillus halodurans, which acted on high-mannose type oligosaccharides. Chimeric constructs of the truncated Endo D and Endo BH showed no activity. Glutamic acid 324 (E 324) in Endo D is conserved in Endo BH and Endo M, and is an essential amino acid in Endo M. Mutation of E324 abolished Endo D activity. The specificity of Endo D for complex type oligosaccharides is probably defined by multiple domains in the Endo D structure.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Asparagine / chemistry*
  • Bacillus / genetics
  • Genome, Bacterial
  • Hydrolysis
  • Immunoglobulin G / chemistry
  • Mannose / chemistry
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / chemistry*
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / genetics*
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / isolation & purification
  • Molecular Sequence Data
  • Mutation
  • Oligosaccharides / chemistry*

Substances

  • Immunoglobulin G
  • Oligosaccharides
  • Asparagine
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
  • Mannose