In Vitro Activity of Neisseria meningitidis PglL O-Oligosaccharyltransferase with Diverse Synthetic Lipid Donors and a UDP-activated Sugar
In Vitro Activity of Neisseria meningitidis PglL O-Oligosaccharyltransferase with Diverse Synthetic Lipid Donors and a UDP-activated Sugar
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DOI：
10.1074/jbc.m112.432815
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发表时间：
2013-04-12
影响因子：
4.8
通讯作者：
Feldman, Mario F.
中科院分区：
文献类型：
--
作者：
Musumeci, Matias A.;Hug, Isabelle;Feldman, Mario F.
寡糖基转移酶（OTase）是催化寡糖从脂质载体转移到受体分子（通常是蛋白质）的酶。根据糖基化反应的性质，OTase分为N-OTase和O-OTase。在称为N-连接糖基化的反应中，N-OTase催化聚糖转移到天冬酰胺中的酰胺基团。0-OTase负责蛋白质0-连接的糖基化，其涉及聚糖与丝氨酸或苏氨酸残基的羟基的连接。这些酶表现出宽松的特异性，并且能够将各种聚糖结构转移到不同的蛋白质受体。这种性质赋予OTase巨大的生物技术潜力，因为这些酶可以产生与制药工业相关的糖缀合物。此外，OTase被认为参与发病机制。OTase功能的几个方面尚未完全理解。在这项工作中，我们开发了一种新的方法来进行动力学研究的PglL，O-OTase从脑膜炎奈瑟菌。我们通过使用携带相同聚糖结构但不同酰基部分的合成底物测试糖基化反应的效率来研究脂质聚糖供体底物的酰基部分对PglL的功能性的重要性。我们发现PglL可以与许多脂质作为聚糖供体起作用，尽管脂质部分的长度和构象显著影响催化效率。有趣的是，PglL也能够转移单糖采用其核苷酸活化形式，作为Leloir糖基转移酶。这些结果为寡糖基转移酶的功能和进化提供了新的见解。
Oligosaccharyltransferases (OTases) are enzymes that catalyze the transfer of an oligosaccharide from a lipid carrier to an acceptor molecule, commonly a protein. OTases are classified as N-OTases and O-OTases, depending on the nature of the glycosylation reaction. The N-OTases catalyze the glycan transfer to amide groups in asparagines in a reaction named N-linked glycosylation. The O-OTases are responsible for protein O-linked glycosylation, which involves the attachment of glycans to hydroxyl groups of serine or threonine residues. These enzymes exhibit a relaxed specificity and are able to transfer a variety of glycan structures to different protein acceptors. This property confers OTases with great biotechnological potential as these enzymes can produce glycoconjugates relevant to the pharmaceutical industry. Furthermore, OTases are thought to be involved in pathogenesis mechanisms. Several aspects of the functionality of OTases are not fully understood. In this work, we developed a novel approach to perform kinetic studies on PglL, the O-OTase from Neisseria meningitidis. We investigated the importance of the acyl moiety of the lipid glycan donor substrate on the functionality of PglL by testing the efficiency of glycosylation reactions using synthetic substrates carrying the same glycan structure but different acyl moieties. We found that PglL can function with many lipids as glycan donors, although the length and the conformation of the lipid moiety significantly influenced the catalytic efficiency. Interestingly, PglL was also able to transfer a monosaccharide employing its nucleotide-activated form, acting as a Leloir glycosyltransferase. These results provide new insights on the function and the evolution of oligosaccharyltransferases.