Mutational studies on endo-beta-N-acetylglucosaminidase D which hydrolyzes core portion of asparagine-linked complex type oligosaccharides.
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.
Yamamoto
Satoshi
S
Department of Biochemistry, Nagoya University Graduate School of Medicine, Showa-ku, Aichi, Japan.
Muramatsu
Hisako
H
Muramatsu
Takashi
T
D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry*; D001407:Bacillus / Q000235:genetics; D016680:Genome, Bacterial; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000737:chemistry; D008358:Mannose / Q000737:chemistry; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry*
D007074:Immunoglobulin G; D009844:Oligosaccharides; D001216:Asparagine; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008358:Mannose