Glycoprotein Processing in Bacteria

我们体内的大多数蛋白质都是由糖修饰的，而这些糖对蛋白质的功能至关重要。因此，出生时糖通路有缺陷的儿童会发生先天性糖基化障碍，影响许多器官系统，通常是致命的。细菌还将糖添加到蛋白质中，并作为理解这一过程的极好模型，并被用作制造新糖蛋白的工厂。我们是细菌蛋白糖基化领域的世界领导者。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产物的质量控制和回收。我们还将开发新的方法来跟踪细菌糖蛋白的细胞内，可以应用于检查任何细菌N-或O-连接的糖蛋白途径，并提高大肠杆菌中的糖工程努力。由于我们在C. jejuni十年前，不同的研究小组进行了广泛的努力来了解糖蛋白的生物合成。我们已经表明，该途径的中心酶PglB具有将糖添加到蛋白质并将这些糖从其脂质载体释放为游离寡糖（fOS）进入周质的能力。最近，我们证明，整个弯曲杆菌属（29个已知物种）是能够蛋白质糖基化和fOS释放，而其他团体已经确定了类似的途径，在其他三角洲和epsilonproteobacteria。重要的是，这些N-糖基化途径可以功能性地转移到E.大肠杆菌创造了一种新的能力，在细菌中设计糖缀合物疫苗。为了进一步了解和利用细菌N-糖基化，有必要研究控制细胞糖蛋白命运和脂质再循环的潜在机制。我们推测，特定的周质分子伴侣可以帮助蛋白质折叠或降解取决于细胞所感知的环境线索。弯曲杆菌属，如E.大肠杆菌也具有蛋白酶体样降解复合物的同源物，用于加工不需要的蛋白质。然而，尚不清楚N-聚糖在该过程中是否被酶促释放或降解。类似地，尚不清楚肽聚糖生物合成所需的通用脂质载体在fOS释放后如何再循环回到细胞质中。初步结果表明，存在防止有害中间体积累的机制。我们将使用已经开发的fOS和糖蛋白产生的定量测定方法，结合几种生化和显微镜技术来跟踪C.空肠。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回收途径，并为如何优化糖蛋白生产提供线索。

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.