Convergent synthesis of homogeneous Glc1Man9GlcNAc2-protein and derivatives as ligands of molecular chaperones in protein quality control.
Convergent synthesis of homogeneous Glc1Man9GlcNAc2-protein and derivatives as ligands of molecular chaperones in protein quality control.
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DOI：
10.1021/ja204831z
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发表时间：
2011-09-14
影响因子：
15
通讯作者：
Wang, Lai-Xi
中科院分区：
文献类型：
--
作者：
Amin, Mohammed N.;Huang, Wei;Mizanur, Rahman M.;Wang, Lai-Xi
由于缺乏明确的均一糖蛋白探针，对伴侣辅助蛋白质质量控制的分子机制的详细了解常常受到阻碍。在这里，我们描述了一种高度收敛的化学酶法合成牛核糖核酸酶(RNase)的单糖形式，作为凝集素样伴蛋白(CNX)和钙网蛋白(CRT)的特定配体，这两种配体已知识别蛋白质折叠中糖蛋白的单糖化高甘露糖寡糖成分。合成了一种选择性修饰的糖型Gal1Glc1Man9GlcNAc2-RNase，它是通过化学合成一个大的N-糖恶唑啉，然后在糖合成酶的催化下与GlcNAc-RNase连接而完成的。用β-半乳糖苷酶选择性去除末端半乳糖，以极高的产率得到了Glc1Man9GlcNAc2-RNase糖体。圆二色谱分析和RNA水解分析表明，合成的RNase糖体与天然牛核糖核酸酶B基本保持相同的全局构象，并且具有完全的活性。SPR结合实验表明，Glc1Man9GlcNAc2-RNase与凝集素CRT有较高的亲和力，而合成的Man9GlcNAc2-RNase糖体和天然RNase B不显示CRT结合活性。这些结果证实了葡萄糖部分在伴侣分子识别中的重要作用。有趣的是，半乳糖掩蔽的糖型Gal1Glc1Man9GlcNAc2-RNase也显示出与凝集素CRT的显著亲和力，这表明连接到关键葡萄糖部分的半乳糖β-1，4-不显著阻断凝集素的结合。这些人工合成的同质糖蛋白探针对于详细研究分子伴侣如何在蛋白质质量控制周期中区分错误折叠和折叠的糖蛋白是有价值的。
A detailed understanding of the molecular mechanism of chaperone-assisted protein quality control is often hampered by the lack of well-defined homogeneous glycoprotein probes. We describe here a highly convergent chemoenzymatic synthesis of the monoglucosylated glycoforms of bovine ribonuclease (RNase) as specific ligands of lectin-like chaperones calnexin (CNX) and calreticulin (CRT) that are known to recognize the monoglucosylated high-mannose oligosaccharide component of glycoproteins in protein folding. The synthesis of a selectively modified glycoform Gal1Glc1Man9GlcNAc2-RNase was accomplished by chemical synthesis of a large N-glycan oxazoline and its subsequent enzymatic ligation to GlcNAc-RNase under the catalysis of a glycosynthase. Selective removal of the terminal galactose by a β-galactosidase gave the Glc1Man9GlcNAc2-RNase glycoform in excellent yield. CD spectroscopic analysis and RNA-hydrolyzing assay indicated that the synthetic RNase glycoforms maintained essentially the same global conformations and were fully active as the natural bovine ribonuclease B. SPR binding studies revealed that the Glc1Man9GlcNAc2-RNase had high affinity to lectin CRT, while the synthetic Man9GlcNAc2-RNase glycoform and natural RNase B did not show CRT-binding activity. These results confirmed the essential role of the glucose moiety in the chaperone molecular recognition. Interestingly, the galactose-masked glycoform Gal1Glc1Man9GlcNAc2-RNase also showed significant affinity to lectin CRT, suggesting that a galactose β-1,4-linked to the key glucose moiety does not significantly block the lectin binding. These synthetic homogeneous glycoprotein probes should be valuable for a detailed mechanistic study on how molecular chaperones work in concert to distinguish between mis-folded and folded glycoproteins in the protein quality control cycle.
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影响因子：
3.2
作者：
Huang, Wei;Groothuys, Stan;Heredia, Alonso;Kuijpers, Brian H. M.;Rutjes, Floris P. J. T.;van Delft, Floris L.;Wang, Lai-Xi
通讯作者：
Wang, Lai-Xi
影响因子：
3.2
作者：
Arai, MA;Matsuo, I;Ito, Y
通讯作者：
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影响因子：
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作者：
Kapoor, M;Srinivas, H;Surolia, A
通讯作者：
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影响因子：
3.2
作者：
Parsons, Thomas B.;Patel, Mitul K.;Fairbanks, Antony J.
通讯作者：
Fairbanks, Antony J.
影响因子：
15
作者：
Huang, Wei;Wang, Denong;Yamada, Masao;Wang, Lai-Xi
通讯作者：
Wang, Lai-Xi