PDF(Pubmed)
Abstract：
The application of bacterial oligosaccharyltransferases (OSTs) such as the Campylobacter jejuni PglB for glycoengineering has attracted considerable interest in glycoengineering and glycoconjugate vaccine development. However, PglB has limited specificity for glycans that can be transferred to candidate proteins, which along with other factors is dependent on the reducing end sugar of glycans. In this study, we developed a cell-free glycosylation assay that offers the speed and simplicity of a \'yes\' or \'no\' determination. Using the assay, we tested the activity of eleven PglBs from Campylobacter species and more distantly related bacteria. The following assorted glycans with diverse reducing end sugars were tested for transfer, including Streptococcus pneumoniae capsule serotype 4, Salmonella enterica serovar Typhimurium O antigen (B1), Francisella tularensis O antigen, Escherichia coli O9 antigen and Campylobacter jejuni heptasaccharide. Interestingly, while PglBs from the same genus showed high activity, whereas divergent PglBs differed in their transfer of glycans to an acceptor protein. Notably for glycoengineering purposes, Campylobacter hepaticus and Campylobacter subantarcticus PglBs showed high glycosylation efficiency, with C. hepaticus PglB potentially being useful for glycoconjugate vaccine production. This study demonstrates the versatility of the cell-free assay in rapidly assessing an OST to couple glycan/carrier protein combinations and lays the foundation for future screening of PglBs by linking amino acid similarity to glycosyltransferase activity.
摘要：
细菌寡糖转移酶(OST)如空肠弯曲杆菌PglB用于糖工程的应用在糖工程和糖缀合物疫苗开发中引起了相当大的兴趣。然而,PglB对可以转移到候选蛋白的聚糖具有有限的特异性，与其他因素一起依赖于聚糖的还原端糖。在这项研究中,我们开发了一种无细胞糖基化测定法，它提供了“是”或“否”确定的速度和简单性。使用该测定法，我们测试了来自弯曲杆菌和更远相关细菌的11种PglB的活性。测试了以下具有不同还原端糖的各种聚糖的转移，包括肺炎链球菌胶囊血清型4，沙门氏菌血清O型鼠伤寒O抗原（B1），图拉氏菌O抗原，大肠杆菌O9抗原和空肠弯曲杆菌七糖。有趣的是,而来自同一属的PGLBs显示出高活性，而不同的PglB在将聚糖转移到受体蛋白方面有所不同。值得注意的是，出于糖工程的目的，肝弯曲菌和亚北极弯曲菌PglBs显示出较高的糖基化效率，肝梭菌PglB可能可用于糖缀合物疫苗的生产。这项研究证明了无细胞测定在快速评估OST以偶联聚糖/载体蛋白组合方面的多功能性，并通过将氨基酸相似性与糖基转移酶活性联系起来为将来筛选PglB奠定了基础。
