Substitute sweeteners: diverse bacterial oligosaccharyltransferases with unique N-glycosylation site preferences.
Substitute sweeteners: diverse bacterial oligosaccharyltransferases with unique N-glycosylation site preferences.
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DOI：
10.1038/srep15237
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发表时间：
2015-10-20
影响因子：
4.6
通讯作者：
DeLisa MP
中科院分区：
文献类型：
--
作者：
Ollis AA;Chai Y;Natarajan A;Perregaux E;Jaroentomeechai T;Guarino C;Smith J;Zhang S;DeLisa MP
空肠弯曲菌天冬酰胺连接的糖基化途径的中心酶是寡糖转移酶(OST)，它将预先组装的糖链转移到靶蛋白中特定的天冬酰胺残基上。虽然空肠弯曲菌PglB(CjPglB)可以转移多种不同的糖链结构，但它识别的受体主要局限于那些在−2位相对于天冬酰胺有带负电荷残基的受体。在这里，我们调查了23个具有自然序列变异的同源物与CjPglB相比的受体位点偏好。通过在糖基化能力强的大肠杆菌中对CjPglB功能的异位反式互补实验，我们证明了16个候选Ost的体内活性。有趣的是，与CjPglB相比，来自结肠弯曲菌、上游弯曲菌、硫化硫弧菌、长硫硫弧菌和普通硫弧菌的Ost对−2位置的特异性显著放松。这些酶在多个靶点上糖基化最小的N-X-T基序，每个都遵循独特的、尚不清楚的规则来控制受体位置的偏好。一个值得注意的例子是D.Gigas PglB，它是唯一一种糖基化人免疫球蛋白G的Fc结构域的细菌，它的天然‘QYNST’序列。总体而言，我们发现细菌Ost的子集遵循它们自己的受体位点特异性规则，从而扩展了糖工程工具箱，具有以前不可用的生物催化多样性。
The central enzyme in the Campylobacter jejuni asparagine-linked glycosylation pathway is the oligosaccharyltransferase (OST), PglB, which transfers preassembled glycans to specific asparagine residues in target proteins. While C. jejuni PglB (CjPglB) can transfer many diverse glycan structures, the acceptor sites that it recognizes are restricted predominantly to those having a negatively charged residue in the −2 position relative to the asparagine. Here, we investigated the acceptor-site preferences for 23 homologs with natural sequence variation compared to CjPglB. Using an ectopic trans-complementation assay for CjPglB function in glycosylation-competent Escherichia coli, we demonstrated in vivo activity for 16 of the candidate OSTs. Interestingly, the OSTs from Campylobacter coli, Campylobacter upsaliensis, Desulfovibrio desulfuricans, Desulfovibrio gigas, and Desulfovibrio vulgaris, exhibited significantly relaxed specificity towards the −2 position compared to CjPglB. These enzymes glycosylated minimal N-X-T motifs in multiple targets and each followed unique, as yet unknown, rules governing acceptor-site preferences. One notable example is D. gigas PglB, which was the only bacterial OST to glycosylate the Fc domain of human immunoglobulin G at its native ‘QYNST’ sequon. Overall, we find that a subset of bacterial OSTs follow their own rules for acceptor-site specificity, thereby expanding the glycoengineering toolbox with previously unavailable biocatalytic diversity.