Probing the Glycan Biosynthetic Machinery of Campylobacter Jejuni.

探索空肠弯曲杆菌的聚糖生物合成机制。

• 批准号: 7540686
• 负责人: JERRY M TROUTMAN
• 金额: $ 4.96万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-12 至 2010-08-11
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7540686
• 关键词: Acute; Amino Acids; Anabolism; Asparagine; Binding; Campylobacter jejuni; Catalytic Domain; Cell Surface Receptors; Cells; Co-Immunoprecipitations; Dependence; Development; Diphosphates; Enzyme Interaction; Enzymes; Galactosamine; Gastroenteritis; Gene Deletion; Guillain-Barré Syndrome; Human; Immune; Infection; Ion Channel; Kinetics; Label; Lead; Link; Lipids; Mediating; Modeling; Mutagenesis; Neuropathy; North America; Object Attachment; Oligosaccharides; Organism; Pathogenicity; Pathway interactions; Peripheral Nervous System Diseases; Polymerase; Polysaccharides; Post-Translational Protein Processing; Protein Glycosylation; Proteins; Reaction; Route; Sequence Analysis; Series; Side; Testing; base; enzyme activity; foodborne illness; glycosylation; link protein; luminescence resonance energy transfer; pathogen; prenyl; research study

DESCRIPTION (provided by applicant): N-linked protein glycosylation, the formation of a glycosylamide bond between an oligosaccharide and an asparagine side chain, is an essential protein modification required for the function, folding and routing of cell surface receptors, ion channels and lysosomal enzymes. N-linked glycosylation has been identified in eukaryotic and archaeal organisms, and has recently been discovered in the gram-negative enteropathogen Campylobacter jejuni (C. jejuni). C. jejuni is a human-gut mucosal pathogen implicated in gastroenteritis, and is the leading cause of food-borne illness in North America. In addition, C. jejuni infection is associated with the development of peripheral neuropathies such as Guillain-Barre syndrome, an acute immune-mediated neuropathy. The enzymes PgIC, A, J and H are involved in oligosaccharide donor assembly for C. jejuni N-linked glycosylation. Deletion of the gene encoding PglH leads to either loss of C. jejuni viability, or a loss in human and chick cell colonization and hence pathogenicity. Therefore, understanding the activity of this enzyme may lead to potential treatments for C. jejuni infections. PglH is a polymerase enzyme, which catalyzes the transfer of three N-acetyl-galactosamine residues to an undecaprenyl diphosphate-linked glycan. The studies outlined in this proposal focus on the characterization of the PglH reaction and the potential interactions of this enzyme with other proteins in the oligosaccharide donor biosynthesis (or Pgl) pathway. 1) Based on sequence analysis and preliminary kinetic studies of PglH, we hypothesize that the PglH-catalyzed glycosyl transfer reactions occur at different catalytic sites on the enzyme, and that specific amino acid residues catalyze each transfer reaction. In order to test this model, we will utilize a series of mutagenesis and biophysical studies to investigate the mechanism by which this enzyme catalyzes multiple glycosyl transfer steps. 2) The glycan assembling enzymes require a high degree of efficiency and fidelity. We hypothesize that some of this efficiency and fidelity is derived by direct interactions between sequential enzymes in the Pgl pathway. A series of co-immunoprecipitation, dual labeling and luminescence resonance energy transfer experiments will be utilized to investigate the ability of the Pgl proteins to interact with one another and the influence of such interactions on substrate/product flux through the PglJ and PglH reactions.

说明(申请人提供)：N-连接蛋白糖基化，在寡糖和天冬酰胺侧链之间形成糖基酰胺键，是细胞表面受体、离子通道和溶酶体酶的功能、折叠和路线选择所必需的蛋白质修饰。N-连接糖基化在真核生物和古生物中已被发现，最近在革兰氏阴性肠病原菌空肠弯曲杆菌(C.jejui)中发现。空肠弯曲菌是一种与胃肠炎有关的人肠道粘膜病原体，是北美食源性疾病的主要原因。此外，空肠弯曲菌感染与周围神经疾病的发展有关，如格林-巴利综合征，一种急性免疫介导性神经疾病。PGIC、A、J和H酶参与了空肠弯曲菌N-连接糖基化的寡糖供体组装。PglH编码基因的缺失会导致空肠弯曲菌活力的丧失，或者导致人和鸡细胞定植的丧失，从而导致致病性的丧失。因此，了解这种酶的活性可能会导致对空肠弯曲菌感染的潜在治疗。PglH是一种聚合酶，催化三个N-乙酰半乳糖胺残基转移到十一碳烯基二磷酸连接的糖链上。这项建议中概述的研究集中在PglH反应的特征以及该酶与寡糖供体生物合成(或PGL)途径中其他蛋白质的潜在相互作用。1)基于PglH的序列分析和初步的动力学研究，我们假设PglH催化的糖基转移反应发生在酶的不同催化位置，每个转移反应都由特定的氨基酸残基催化。为了验证这一模型，我们将利用一系列的诱变和生物物理研究来研究该酶催化多个糖基转移步骤的机制。2)聚糖酶的效率和保真度要求很高。我们假设，这种效率和保真度中的一部分是由PGL途径中连续酶之间的直接相互作用得出的。我们将利用一系列的免疫共沉淀、双重标记和发光共振能量转移实验来研究PGL蛋白相互作用的能力，以及这种相互作用通过PglJ和PglH反应对底物/产物通量的影响。