Probing the substrate specificity of Golgi alpha-mannosidase II by use of synthetic oligosaccharides and a catalytic nucleophile mutant

J Am Chem Soc. 2008 Jul 16;130(28):8975-83. doi: 10.1021/ja711248y. Epub 2008 Jun 18.


Inhibition of Golgi alpha-mannosidase II (GMII), which acts late in the N-glycan processing pathway, provides a route to blocking cancer-induced changes in cell surface oligosaccharide structures. To probe the substrate requirements of GMII, oligosaccharides were synthesized that contained an alpha(1,3)- or alpha(1,6)-linked 1-thiomannoside. Surprisingly, these oligosaccharides were not observed in X-ray crystal structures of native Drosophila GMII (dGMII). However, a mutant enzyme in which the catalytic nucleophilic aspartate was changed to alanine (D204A) allowed visualization of soaked oligosaccharides and led to the identification of the binding site for the alpha(1,3)-linked mannoside of the natural substrate. These studies also indicate that the conformational change of the bound mannoside to a high-energy B 2,5 conformation is facilitated by steric hindrance from, and the formation of strong hydrogen bonds to, Asp204. The observation that 1-thio-linked mannosides are not well tolerated by the catalytic site of dGMII led to the synthesis of a pentasaccharide containing the alpha(1,6)-linked Man of the natural substrate and the beta(1,2)-linked GlcNAc moiety proposed to be accommodated by the extended binding site of the enzyme. A cocrystal structure of this compound with the D204A enzyme revealed the molecular interactions with the beta(1,2)-linked GlcNAc. The structure is consistent with the approximately 80-fold preference of dGMII for the cleavage of substrates containing a nonreducing beta(1,2)-linked GlcNAc. By contrast, the lysosomal mannosidase lacks an equivalent GlcNAc binding site and kinetic analysis indicates oligomannoside substrates without non-reducing-terminal GlcNAc modifications are preferred, suggesting that selective inhibitors for GMII could exploit the additional binding specificity of the GlcNAc binding site.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Binding Sites
  • Catalysis
  • Crystallography, X-Ray
  • Drosophila / enzymology
  • Drosophila / genetics
  • Glycosylation
  • Mannose / chemistry
  • Mannose / metabolism
  • Mannosidases / chemistry
  • Mannosidases / genetics
  • Mannosidases / metabolism*
  • Models, Molecular
  • Mutation
  • Oligosaccharides / chemical synthesis
  • Oligosaccharides / chemistry
  • Oligosaccharides / metabolism*
  • Structure-Activity Relationship
  • Substrate Specificity
  • Thioglycosides / chemistry
  • Thioglycosides / metabolism


  • Oligosaccharides
  • Thioglycosides
  • Mannosidases
  • mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase
  • Mannose