Investigation of the Catalytic Mechanism of a Soluble N-glycosyltransferase Allows Synthesis of N-glycans at Noncanonical Sequons.
Investigation of the Catalytic Mechanism of a Soluble N-glycosyltransferase Allows Synthesis of N-glycans at Noncanonical Sequons.
复制标题
研究可溶性N-糖基转移酶的催化机制允许在非规范序列上合成N-聚糖。
DOI：
10.1021/jacsau.3c00214
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发表时间：
2023-08-28
期刊：
影响因子：
8
通讯作者：
Peng, Wenjie
中科院分区：
文献类型：
--
作者：
Hao, Zhiqiang;Guo, Qiang;Feng, Yuanyuan;Zhang, Zihan;Li, Tiantian;Tian, Zhixin;Zheng, Jianting;Da, Lin-Tai;Peng, Wenjie
胸膜肺炎放线杆菌可溶性N-糖基转移酶（ApNGT）能在Asn-Xaa-Ser/Thr共有序列的天冬酰胺残基上建立N-糖苷键，是生产N-糖蛋白最有前途的工具之一。在这里，通过整合计算和实验策略，我们揭示了底物识别的分子机制和后续的催化ApNGT。这些发现使我们能够确定ApNGT中负责肽底物识别的关键结构基序（215 DVYM 218）。此外，发现ApNGT的Y222和H371参与激活受体Asn。所构建的模型得到了进一步的晶体学研究的支持，并通过测量各种突变体对合成肽库的糖基化活性来验证所鉴定的残基的功能作用。有趣的是，对于特定的突变体，可以实现来自SARS-CoV-2刺突蛋白的天然多肽内的典型或非典型序列的位点选择性N-糖基化，其用于研究病毒进入期间膜融合中N-糖基化的生物学作用。因此，我们的研究提供了深入的分子机制的底物识别和催化ApNGT，导致以前未知的化学定义的N-糖蛋白的合成探索在特定位点的N-糖基化的生物学意义。
The soluble N-glycosyltransferase from Actinobacillus pleuropneumoniae (ApNGT) can establish an N-glycosidic bond at the asparagine residue in the Asn-Xaa-Ser/Thr consensus sequon and is one of the most promising tools for N-glycoprotein production. Here, by integrating computational and experimental strategies, we revealed the molecular mechanism of the substrate recognition and following catalysis of ApNGT. These findings allowed us to pinpoint a key structural motif (215DVYM218) in ApNGT responsible for the peptide substrate recognition. Moreover, Y222 and H371 of ApNGT were found to participate in activating the acceptor Asn. The constructed models were supported by further crystallographic studies and the functional roles of the identified residues were validated by measuring the glycosylation activity of various mutants against a library of synthetic peptides. Intriguingly, with particular mutants, site-selective N-glycosylation of canonical or noncanonical sequons within natural polypeptides from the SARS-CoV-2 spike protein could be achieved, which were used to investigate the biological roles of the N-glycosylation in membrane fusion during virus entry. Our study thus provides in-depth molecular mechanisms underlying the substrate recognition and catalysis for ApNGT, leading to the synthesis of previously unknown chemically defined N-glycoproteins for exploring the biological importance of the N-glycosylation at a specific site.