From peptide to protein:: Comparative analysis of the substrate specificity of N-linked glycosylation in C-jejuni
From peptide to protein:: Comparative analysis of the substrate specificity of N-linked glycosylation in C-jejuni
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DOI：
10.1021/bi602633n
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发表时间：
2007-05-08
期刊：
影响因子：
2.9
通讯作者：
Imperiali, Barbara
中科院分区：
文献类型：
--
作者：
Chen, Mark M.;Glover, Kerney Jebrell;Imperiali, Barbara
最近发现，革兰氏阴性细菌空肠弯曲菌含有一般的N-连接蛋白糖基化途径。该途径的中心是PglB，其是真核寡糖基转移酶（OT）的Stt 3 p亚基的同源物，其参与寡糖从聚异戊二烯焦磷酸载体转移到保守糖基化位点D/E-X-1-N-X-2-S/T内的蛋白质的天冬酰胺侧链，其中X-1和X-2可以是除脯氨酸之外的任何氨基酸。使用肽底物库和基于定量放射性的体外测定，我们评估了共有糖基化序列的每个位置处的氨基酸对糖基化效率的影响，由此发现序列DQNAT是最佳受体底物。在全长折叠蛋白质的背景下，发现糖基化序列的变化之间的差异与从其肽基对应物观察到的趋势一致，尽管由于额外的影响而不那么显著。除了表征PglB的受体偏好之外，我们还评估了对聚糖供体的选择性。有趣的是，尽管最近报道了对聚糖供体的放松的选择性，但未发现PglB能够利用糖基供体如长羟焦磷酸-壳二糖，其是真核OT过程的最小底物。
The gram-negative bacterium Campylobacter jejuni was recently discovered to contain a general N-linked protein glycosylation pathway. Central to this pathway is PglB, a homologue of the Stt3p subunit of the eukaryotic oligosaccharyl transferase (OT), which is involved in the transfer of an oligosaccharide from a polyisoprenyl pyrophosphate carrier to the asparagine side chain of proteins within the conserved glycosylation sites D/E-X-1-N-X-2-S/T, where X-1 and X-2 can be any amino acids except proline. Using a library of peptide substrates and a quantitative radioactivity-based in vitro assay, we assessed the amino acids at each position of the consensus glycosylation sequence for their impact on glycosylation efficiency, whereby the sequence DQNAT was found to be the optimal acceptor substrate. In the context of a full-length folded protein, the differences between variations of the glycosylation sequences were found to be consistent with the trends observed from their peptidyl counterparts, though less dramatic because of additional influences. In addition to characterizing the acceptor preferences of PglB, we also assessed the selectivity toward the glycan donor. Interestingly, despite recent reports of relaxed selectivity toward the glycan donor, PglB was not found to be capable of utilizing glycosyl donors such as dolichyl-pyrophosphate-chitobiose, which is the minimum substrate for the eukaryotic OT process.