Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity.
Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity.
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DOI：
10.1038/nchembio.1609
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发表时间：
2014-10
影响因子：
14.8
通讯作者：
DeLisa MP
中科院分区：
文献类型：
--
作者：
Ollis AA;Zhang S;Fisher AC;DeLisa MP
空肠弯曲杆菌蛋白糖基化位点 (pgl) 编码天冬酰胺连接（N 连接）糖基化机制，并作为细菌 N-糖基化的原型。该机制已被功能性地转移到大肠杆菌中，从而能够在这种遗传上易处理的宿主中方便地机械解剖 N-糖基化过程。在这里，我们试图鉴定寡糖转移酶 PglB 中的序列决定簇，将其特异性限制为仅那些在相对于天冬酰胺的-2位置含有带负电残基的聚糖受体位点。这涉及创建一种名为 gluSNAP（分泌型 N 连接受体蛋白的糖基化）的基因检测方法，该检测方法有助于对大肠杆菌中的糖表型进行高通量筛选。使用该测定，我们分离了几种空肠弯曲菌PglB变体，它们能够糖基化一系列非规范受体序列，包括真核N-糖蛋白中的一个。总的来说，这些结果强调了 gluSNAP 的实用性，可以帮助人们了解 N-糖基化的一些鲜为人知的方面，并为工程设计师 N-糖基化生物催化剂提供帮助。
The Campylobacter jejuni protein glycosylation locus (pgl) encodes machinery for asparagine-linked (N-linked) glycosylation and serves as the archetype for bacterial N-glycosylation. This machinery has been functionally transferred into Escherichia coli, thereby enabling convenient mechanistic dissection of the N-glycosylation process in this genetically tractable host. Here, we sought to identify sequence determinants in the oligosaccharyltransferase PglB that restrict its specificity to only those glycan acceptor sites containing a negatively charged residue at the −2 position relative to asparagine. This involved creation of a genetic assay named glycoSNAP (glycosylation of secreted N-linked acceptor proteins) that facilitates high-throughput screening of glycophenotypes in E. coli. Using this assay, we isolated several C. jejuni PglB variants that were capable of glycosylating an array of noncanonical acceptor sequences including one in a eukaryotic N-glycoprotein. Collectively, these results underscore the utility of glycoSNAP for shedding light on poorly understood aspects of N-glycosylation and for engineering designer N-glycosylation biocatalysts.