Exploring the fundamentals of bacterial protein O-mannosylation

从细菌到人类都观察到了蛋白质甘露糖化，这似乎是一种高度保守的蛋白质修饰，在真核生物中是必不可少的。负责蛋白质甘露糖化的酶属于高度保守的糖基转移酶家族(GT39)，它们以单拷贝基因存在于1300多种细菌中，其中大部分属于放线杆菌类群。放线杆菌物种中的蛋白质甘露糖化最初是在40年前被描述的，然而我们仍然缺乏对这些细菌为什么进行这种翻译后修饰的基本了解。我们对从全细胞裂解液中提取的甘露糖化蛋白进行了有针对性的浓缩，使用刀豆蛋白A的凝集素层析，我们能够鉴定出各种各样的甘露糖化蛋白。除了已知的分泌型甘露糖化纤维素酶外，我们还发现细胞质和膜相关蛋白也可以甘露糖化。我们的数据表明了纤维单胞菌中蛋白质甘露糖化的一般性质，就像在棒状杆菌和分枝杆菌中所看到的那样。然而，糖基化的生物化学和这种常见修饰的生物学意义还没有对任何这些承担修饰的蛋白质进行充分的描述。为了充分了解这个重要的(和保守的)翻译后修饰系统的生物学作用，我们需要利用过去的全球敲除表型，并以更密切的方式与选定的靶点探讨这一点，以了解碳水化合物如何影响蛋白质功能。这项建议将通过重组表达GT39酶来检测糖基化途径的生物化学，以确定其在体内的膜拓扑结构和结构功能。我们还将表达保守的甘露糖化糖蛋白作为目标，以便更彻底地分析糖基化途径本身。我们的计划试图提供一个更深入的分析分子基础的修饰反应本身，以及该修饰的后果使用保守的蛋白质参与基本生长和新陈代谢放线杆菌。我们对细胞糖蛋白的调查将巩固在细菌生理学中具有不可或缺作用的保守蛋白的名单。研究不同物种中保守蛋白质子集的这种能量密集型修饰将有助于我们破译控制蛋白质相互作用的蛋白质-碳水化合物相互作用，这反过来又控制细胞的生长和新陈代谢。将这一知识应用于工业用放线菌种类的菌种改良，可以帮助产品产量，或者可能扩大它们可以提供的产品范围。也有可能确定可能成为致病放线菌新的治疗靶点的过程。

Protein mannosylation has been observed from bacteria to humans and appears to be a highly conserved protein modification that is essential in eukaryotes. The enzyme responsible for the protein mannosylation belongs to the highly conserved glycosyltransferase family (GT39), which are present as single copy genes in over 1300 species of bacteria, most of them in the actinobacterial group. Protein mannosylation in actinobacterial species was originally described 40 years ago, however we still lack a basic understanding of why these bacteria make this post-translational modification. We undertook a targeted enrichment of mannosylated proteins extracted from whole cell lysates using lectin chromatography with Concanavalin A and we were able to identify wide variety of mannosylated proteins. In addition to the known secreted mannosylated cellulases, we discovered that cytoplasmic and membrane associated proteins could also be mannosylated. Our data indicate the general nature of the protein mannosylation in Cellulomonas, as has been seen in Corynebacterium and Mycobacteria. However, the biochemistry of glycosylation and the biological significance of this common modification has not been adequately described for any of those proteins that bear the modification. To fully understand the biological role of this important (and conserved) post-translation modification system, we need to take this past global knock out phenotypes and probe this is in a more intimate way with selected targets to see how the carbohydrate influences protein function.    This proposal will examine the biochemistry of the glycosylation pathway using recombinant expression of the GT39 enzyme to determine its membrane topology, and structure-function in vivo. We will also express conserved mannosylated glycoproteins as targets for a more thorough analysis of the glycosylation pathway itself. Our program seeks to provide a more in-depth analysis of the molecular basis for the modification reaction itself, and the consequences of that modification using conserved proteins involved in basic growth and metabolism of Actinobacteria. Our survey of cellular glycoproteins will solidify the list of conserved proteins which have integral roles in bacterial physiology. Investigating this energy intensive modification of a subset of conserved proteins in diverse species will help us decipher the protein - carbohydrate interactions that control protein interactions, which in turn control cell growth and metabolism. Application of this knowledge to strain improvement of the industrially employed actinobacterial species could help product yields or perhaps expand the range of products they can provide. There is also potential to identify processes which could become new therapeutic targets in the pathogenic Actinobacteria.