Probing the Substrate Specificity of Golgi α-Mannosidase II by Use of Synthetic Oligosaccharides and a Catalytic Nucleophile Mutant

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• 作者：Wei Zhong ; Douglas A. Kuntz ; Brian Ember ; Harminder Singh ; Kelley W. Moremen ; David R. Rose ; Geert-Jan Boons
• 刊名：Journal of the American Chemical Society
• 出版年：2008
• 出版时间：July 16, 2008
• 年：2008
• 卷：130
• 期：28
• 页码：8975 - 8983
• 全文大小：1966K
• 年卷期：v.130,no.28(July 16, 2008)
• ISSN：1520-5126

文摘

Inhibition of Golgi α-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 α(1,3)- or α(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 α(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 α(1,6)-linked Man of the natural substrate and the β(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 β(1,2)-linked GlcNAc. The structure is consistent with the ~80-fold preference of dGMII for the cleavage of substrates containing a nonreducing β(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.