Exploring the fundamentals of bacterial protein O-mannosylation

蛋白质甘露糖基化已经从细菌到人类被观察到，并且似乎是真核生物中必需的高度保守的蛋白质修饰。负责蛋白质甘露糖基化的酶属于高度保守的糖基转移酶家族（GT 39），其作为单拷贝基因存在于超过1300种细菌中，其中大多数在放线菌群中。放线菌物种中的蛋白质甘露糖基化最初是在40年前描述的，但是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理解。我们进行了有针对性的富集甘露糖基化的蛋白质提取全细胞裂解液使用凝集素层析与伴刀豆球蛋白A，我们能够识别各种各样的甘露糖基化的蛋白质。除了已知的分泌型甘露糖基化纤维素酶外，我们发现细胞质和膜相关蛋白也可以被甘露糖基化。我们的数据表明了纤维单胞菌属中蛋白质甘露糖基化的一般性质，如在棒状杆菌和分枝杆菌中所见。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充分描述任何这些蛋白质携带的修饰。为了充分理解这个重要的（和保守的）翻译后修饰系统的生物学作用，我们需要采取过去的全局敲除表型，并以更亲密的方式与选定的靶点进行探测，以了解碳水化合物如何影响蛋白质功能。 该提案将使用GT39酶的重组表达来检查糖基化途径的生物化学，以确定其膜拓扑结构和体内结构-功能。我们还将表达保守的甘露糖基化糖蛋白，作为对糖基化途径本身进行更彻底分析的靶点。我们的计划旨在为修饰反应本身提供更深入的分子基础分析，以及使用参与放线菌基本生长和代谢的保守蛋白质进行修饰的后果。我们对细胞糖蛋白的研究将巩固在细菌生理学中起重要作用的保守蛋白的列表。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，从而控制细胞生长和代谢。将这些知识应用于工业上使用的放线菌物种的菌株改良可以帮助产品产量或可能扩大它们可以提供的产品范围。也有可能确定的过程，可能成为新的治疗目标的致病放线菌。

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.