A minimal sequon sufficient for O-linked glycosylation by the versatile oligosaccharyltransferase PglS.
A minimal sequon sufficient for O-linked glycosylation by the versatile oligosaccharyltransferase PglS.
复制标题
DOI：
10.1093/glycob/cwab043
复制
发表时间：
2021-09-20
期刊：
影响因子：
4.3
通讯作者：
Harding CM
中科院分区：
文献类型：
--
作者：
Knoot CJ;Robinson LS;Harding CM
生物缀合物疫苗由连接到载体蛋白的多糖组成，在称为生物缀合的过程中使用原核糖基化系统酶促产生。生物缀合的关键是一组被称为寡糖基转移酶（OTase）的酶，其将多糖转移到含有被称为序列子的保守氨基酸序列的工程化载体蛋白。最近发现的OTase，PglS，已被证明具有最广泛的底物范围，转移许多不同类型的细菌聚糖，包括在还原端具有葡萄糖的那些。然而，PglS目前在它识别的序列方面是最不了解的。PglS是一种菌毛蛋白特异性O-连接OTase，其天然地使单一蛋白质ComP糖基化。除了ComP之外，我们先前证明了含有ComP的大片段的工程化载体蛋白也被PglS糖基化。在此，我们试图鉴定足以用于PglS糖基化的最小ComP序列子。我们测试了>100个不同的单独融合到铜绿假单胞菌外毒素A（EPA）的ComP片段，导致鉴定出足以通过PglS进行稳健糖基化的11个氨基酸序列。我们还证明了ComP序列子在载体蛋白上的放置对于稳定性和随后的糖基化是至关重要的。此外，我们确定了新的网站上的EPA的表面上，是服从ComP序列插入，并发现，交叉反应性材料197融合到ComP片段也糖基化。这些结果代表了糖工程工具箱的显著扩展以及我们对细菌O-连接序列的理解。
Bioconjugate vaccines, consisting of polysaccharides attached to carrier proteins, are enzymatically generated using prokaryotic glycosylation systems in a process termed bioconjugation. Key to bioconjugation are a group of enzymes known as oligosaccharyltransferases (OTases) that transfer polysaccharides to engineered carrier proteins containing conserved amino acid sequences known as sequons. The most recently discovered OTase, PglS, has been shown to have the broadest substrate scope, transferring many different types of bacterial glycans including those with glucose at the reducing end. However, PglS is currently the least understood in terms of the sequon it recognizes. PglS is a pilin-specific O-linking OTase that naturally glycosylates a single protein, ComP. In addition to ComP, we previously demonstrated that an engineered carrier protein containing a large fragment of ComP is also glycosylated by PglS. Here we sought to identify the minimal ComP sequon sufficient for PglS glycosylation. We tested >100 different ComP fragments individually fused to Pseudomonas aeruginosa exotoxin A (EPA), leading to the identification of an 11-amino acid sequence sufficient for robust glycosylation by PglS. We also demonstrate that the placement of the ComP sequon on the carrier protein is critical for stability and subsequent glycosylation. Moreover, we identify novel sites on the surface of EPA that are amenable to ComP sequon insertion and find that Cross-Reactive Material 197 fused to a ComP fragment is also glycosylated. These results represent a significant expansion of the glycoengineering toolbox as well as our understanding of bacterial O-linking sequons.
登录
查看更多内容
影响因子：
5.5
作者：
Hatz, Christoph F. R.;Bally, Bettina;Fonck, Veronica Gambillara
通讯作者：
Fonck, Veronica Gambillara
影响因子：
5.8
作者：
Kay EJ;Yates LE;Terra VS;Cuccui J;Wren BW
通讯作者：
Wren BW
影响因子：
56.3
作者：
Huttner, Angela;Hatz, Christoph;Fonck, Veronica Gambillara
通讯作者：
Fonck, Veronica Gambillara
DOI：
10.1073/pnas.0500044102
发表时间：
2005-02-22
影响因子：
11.1
作者：
Feldman, MF;Wacker, M;Aebi, M
通讯作者：
Aebi, M
79种囊菌类型Klebsiella spp中囊膜多糖合成基因簇的遗传分析。
影响因子：
4.6
作者：
Pan YJ;Lin TL;Chen CT;Chen YY;Hsieh PF;Hsu CR;Wu MC;Wang JT
通讯作者：
Wang JT