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.

中文翻译：

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一个足以通过多功能寡糖基转移酶 PglS 进行 O-连接糖基化的最小序列

生物偶联疫苗由附着在载体蛋白上的多糖组成，是在称为生物偶联的过程中使用原核糖基化系统酶促产生的。生物偶联的关键是一组称为寡糖基转移酶 (OTase) 的酶，它们将多糖转移到工程载体蛋白上，该载体蛋白含有称为序列子的保守氨基酸序列。最近发现的 OTase PglS 已被证明具有最广泛的底物范围，可以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，就其识别的序列而言，PglS 目前是最不了解的。PglS 是一种菌毛特异性O-连接 OTase，天然糖基化单一蛋白质 ComP。除了 ComP，我们之前还证明了含有大片段 ComP 的工程化载体蛋白也被 PglS 糖基化。在这里，我们试图确定足以进行 PglS 糖基化的最小 ComP 序列。我们测试了超过 100 个不同的 ComP 片段，它们分别与铜绿假单胞菌融合外毒素 A (EPA)，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。我们还证明了在载体蛋白上放置 ComP 序列对稳定性和随后的糖基化至关重要。此外，我们确定了 EPA 表面上适合 ComP 序列插入的新位点，并发现与 ComP 片段融合的交叉反应材料 197 也被糖基化。这些结果代表了糖工程工具箱的显着扩展以及我们对细菌O连接序列的理解。