Structural insights from random mutagenesis of Campylobacter jejuni oligosaccharyltransferase PglB
Structural insights from random mutagenesis of Campylobacter jejuni oligosaccharyltransferase PglB
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DOI：
10.1186/1472-6750-12-67
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发表时间：
2012-09-24
影响因子：
3.5
通讯作者：
Thoeny-Meyer, Linda
中科院分区：
文献类型：
--
作者：
Ihssen, Julian;Kowarik, Michael;Thoeny-Meyer, Linda
背景：蛋白质糖基化在许多生物系统中至关重要。细菌中 N-糖基化的发现以及空肠弯曲杆菌 N-寡糖转移酶 PglB 在大肠杆菌中的功能表达使得工程化糖蛋白的生产和潜在分子机制的研究成为可能。重组细菌中蛋白质糖基化的一个特别有前途的应用是生产有效的结合疫苗，其中病原菌的多糖抗原与免疫原性载体蛋白共价结合。结果：在这项研究中，临床相关病原体金黄色葡萄球菌血清型 5 (CP5) 的荚膜多糖在大肠杆菌中表达，并在体内与铜绿假单胞菌外毒素的解毒版本连接（美国环保局）。我们使用新建立的 96 孔筛选系统研究了 PglB 周质结构域的哪些氨基酸对于糖基化反应至关重要，该系统能够通过酶联免疫吸附测定对糖蛋白进行相对定量。通过易错 PCR 生成随机突变体文库，并筛选失活氨基酸取代。除了先前已知的 N-寡糖基转移酶严格保守的 WWDYG 基序内有 15 个氨基酸发生变化的失活变体之外，还有 8 个失活突变映射到紧邻受体天冬酰胺酰胺氮原子的柔性环，如同源酶 C. lari PglB 的晶体结构中所揭示的那样。通过定点诱变证实了保守环残基 H479 对于糖基化的重要性，而相邻非保守 L480 的丙氨酸变化则没有影响。此外，我们还研究了细菌 N-寡糖转移酶的所谓 MIV 基序的功能要求。假设与受体肽相互作用的氨基酸残基I571和V575进行盒式饱和诱变。除 I571C 外，在活性变体中仅发现疏水残基。变体 I571V 的表现与野生型相同，相同位置的半胱氨酸略微降低了糖蛋白产量，而苯丙氨酸的变化使活性降低了三倍。 结论：这项研究为空肠弯曲杆菌 N-寡糖转移酶 PglB 的周质结构域提供了新的结构-功能关系，并描述了在工程大肠杆菌系统中生成和筛选寡糖转移酶突变体文库的程序。
Background: Protein glycosylation is of fundamental importance in many biological systems. The discovery of N-glycosylation in bacteria and the functional expression of the N-oligosaccharyltransferase PglB of Campylobacter jejuni in Escherichia coli enabled the production of engineered glycoproteins and the study of the underlying molecular mechanisms. A particularly promising application for protein glycosylation in recombinant bacteria is the production of potent conjugate vaccines where polysaccharide antigens of pathogenic bacteria are covalently bound to immunogenic carrier proteins.Results: In this study capsular polysaccharides of the clinically relevant pathogen Staphylococcus aureus serotype 5 (CP5) were expressed in Escherichia coli and linked in vivo to a detoxified version of Pseudomonas aeruginosa exotoxin (EPA). We investigated which amino acids of the periplasmic domain of PglB are crucial for the glycosylation reaction using a newly established 96-well screening system enabling the relative quantification of glycoproteins by enzyme-linked immunosorbent assay. A random mutant library was generated by error-prone PCR and screened for inactivating amino acid substitutions. In addition to 15 inactive variants with amino acid changes within the previously known, strictly conserved WWDYG motif of N-oligosaccharyltransferases, 8 inactivating mutations mapped to a flexible loop in close vicinity of the amide nitrogen atom of the acceptor asparagine as revealed in the crystal structure of the homologous enzyme C. lari PglB. The importance of the conserved loop residue H479 for glycosylation was confirmed by site directed mutagenesis, while a change to alanine of the adjacent, non-conserved L480 had no effect. In addition, we investigated functional requirements in the so-called MIV motif of bacterial N-oligosaccharyltransferases. Amino acid residues I571 and V575, which had been postulated to interact with the acceptor peptide, were subjected to cassette saturation mutagenesis. With the exception of I571C only hydrophobic residues were found in active variants. Variant I571V performed equally well as the wild type, cysteine at the same position reduced glycoprotein yield slightly, while a change to phenylalanine reduced activity by a factor of three.Conclusions: This study provides novel structure-function relationships for the periplasmic domain of the Campylobacter jejuni N-oligosaccharyltransferase PglB and describes procedures for generating and screening oligosaccharyltransferase mutant libraries in an engineered E. coli system.