Glycoprotein Processing in Bacteria

我们身体中的大多数蛋白质都装饰着糖--这些糖是蛋白质功能所必需的。因此，出生时糖路有缺陷的儿童会患上先天性糖基化障碍，这种疾病会影响许多器官系统，通常是致命的。细菌还向蛋白质中添加糖，是理解这一过程的极好模型，并被用作制造新糖蛋白的工厂。我们在细菌蛋白质糖基化方面处于世界领先地位。本研究的主要目的是了解空肠弯曲菌中N-连接糖蛋白及其中间体的质量控制和回收利用。我们还将开发在细胞内跟踪细菌糖蛋白的新方法，这些方法可以应用于检测任何细菌N或O连接的糖蛋白途径，并改进大肠杆菌中的糖工程努力。自从十年前我们在空肠弯曲菌中发现了一条N-连接的糖蛋白通路以来，不同的研究小组一直在努力了解糖蛋白的生物合成。我们已经证明了该途径的中心酶PglB能够将糖添加到蛋白质中，并将这些糖从脂质载体中以游离寡糖(FOS)的形式释放到周质中。最近，我们证明了整个弯曲杆菌属(29个已知物种)都能够进行蛋白糖基化和FOS释放，而其他组在其他Delta和表蛋白细菌中也发现了类似的途径。重要的是，这些N-糖基化途径可以功能性地转移到大肠杆菌中，创造了在细菌中设计糖结合疫苗的新能力。为了进一步了解和利用细菌的N-糖基化，有必要研究控制细胞糖蛋白命运和脂质循环的潜在机制。我们假设，特定的周质伴侣可以帮助蛋白质折叠或降解，这取决于细胞感受到的环境线索。弯曲杆菌和大肠杆菌一样，也拥有一种蛋白酶体样降解复合体的同系物，用于处理不需要的蛋白质。然而，目前还不清楚N-葡聚糖在这个过程中是被酶释放还是被降解。同样，目前还不清楚FOS释放后，肽聚糖生物合成所需的通用脂质载体是如何循环回到细胞质中的。初步结果表明，已经建立了防止有害中间体积聚的机制。我们将使用已经开发的FOS和糖蛋白产生的定量分析结合几种生化和显微技术来跟踪空肠弯曲菌的N-糖基化途径。这些发现将有助于了解细菌和高等生物中糖蛋白的降解和循环途径，并为如何优化糖蛋白生产以用于工业应用提供线索。

Most proteins in our body are decorated with sugars - and these sugars are essential for protein function. Therefore, children born with defects in their sugar pathways develop congenital disorders of glycosylation that influence many organ systems and are typically fatal. Bacteria also add sugars to proteins and serve as an excellent model for understanding this process and being used as factories to make new glycoproteins. We are the world leaders in bacterial protein glycosylation. The main objective of this study is to understand the quality control and recycling of N-linked glycoproteins and their intermediates in Campylobacter jejuni. We will also develop new methods to follow bacterial glycoproteins intracellularly that can be applied to examine any bacterial N- or O-linked glycoprotein pathway and to improve glycoengineering efforts in Escherichia coli. Since we identified an N-linked glycoprotein pathway in C. jejuni a decade ago, there have been extensive efforts by various groups to understand glycoprotein biosynthesis. We have shown that the central enzyme of the pathway, PglB, has the ability to add sugars to protein and release these sugars from their lipid carrier as free oligosaccharides (fOS) into the periplasm. Recently we demonstrated that the entire Campylobacter genus (29 known species) is capable of protein glycosylation and fOS release, while other groups have identified similar pathways in other delta and epsilonproteobacteria. Importantly, these N-glycosylation pathways can be functionally transferred into E. coli creating a new capacity to engineer glycoconjugate vaccines in bacteria. To further understand and exploit bacterial N-glycosylation, it is necessary to examine the underlying mechanisms controlling the fate of cellular glycoproteins and lipid recycling. We hypothesize that specific periplasmic chaperones can either assist in protein folding or degradation depending on environmental cues sensed by the cell. Campylobacters, like E. coli, also possess homologues of a proteasome-like degradation complex for processing unwanted proteins. However, it is unclear whether the N-glycans are enzymatically released or degraded during this process. Similarly, it is unclear how the universal lipid carrier, that is also required for peptidoglycan biosynthesis, is recycled back into the cytoplasm after fOS release. Preliminary results suggest that there are mechanisms in place to prevent the build-up of harmful intermediates. We will use already developed quantitative assays for fOS and glycoprotein production coupled with several biochemical and microscopic techniques to follow the N-glycosylation pathway in C. jejuni. These findings will help to understand the glycoprotein degradation and recycling pathway in bacteria and higher organisms and also provide clues on how to optimize glycoprotein production for industrial applications.