Probing the substrate specificity of Golgi alpha-mannosidase II by use of synthetic oligosaccharides and a catalytic nucleophile mutant.
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.
Zhong
Wei
W
Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, Georgia 30602, USA.
Kuntz
Douglas A
DA
Ember
Brian
B
Singh
Harminder
H
Moremen
Kelley W
KW
Rose
David R
DR
Boons
Geert-Jan
GJ
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D004330:Drosophila / Q000201:enzymology / Q000235:genetics; D006031:Glycosylation; D008358:Mannose / Q000737:chemistry / Q000378:metabolism; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013865:Thioglycosides / Q000737:chemistry / Q000378:metabolism
D009844:Oligosaccharides; D013865:Thioglycosides; D008361:Mannosidases; C038829:mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase; D008358:Mannose