Probing the Glycan Biosynthetic Machinery of Campylobacter Jejuni.

描述（由申请方提供）：N-连接蛋白糖基化，即寡糖和天冬酰胺侧链之间形成糖酰胺键，是细胞表面受体、离子通道和溶酶体酶的功能、折叠和路由所需的基本蛋白质修饰。N-连接糖基化在真核生物和古细菌中已被发现，最近在革兰氏阴性肠道病原体空肠弯曲菌（C.空肠）。C.空肠是与胃肠炎有关的人肠道粘膜病原体，并且是北美食源性疾病的主要原因。此外，C.空肠感染与周围神经病如格林-巴利综合征（一种急性免疫介导的神经病）的发展有关。酶PgIC、A、J和H参与C的寡糖供体组装。空肠N-连接糖基化。编码PglH的基因的缺失导致C.空肠存活力，或人和鸡细胞定殖的丧失以及因此的致病性。因此，了解这种酶的活性可能会导致潜在的治疗C。空肠感染PglH是一种聚合酶，其催化三个N-乙酰基-半乳糖胺残基转移至十一异戊二烯基二磷酸连接的聚糖。本提案中概述的研究重点是PglH反应的表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的序列分析和初步动力学研究，我们假设PglH催化的糖基转移反应发生在酶上的不同催化位点，并且特定的氨基酸残基催化每个转移反应。为了测试这个模型，我们将利用一系列的诱变和生物物理学研究来研究这种酶催化多个糖基转移步骤的机制。2)聚糖组装酶需要高度的效率和保真度。我们假设，这种效率和保真度的一些是由Pgl途径中的连续酶之间的直接相互作用得到的。将利用一系列免疫共沉淀、双标记和发光共振能量转移实验来研究Pgl蛋白彼此相互作用的能力以及这种相互作用对通过PglJ和PglH反应的底物/产物通量的影响。

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.