Galactomannan-based targeting of glycoproteins for secretion and cell wall incorporation

该项目解决了真菌细胞如何能够正确组装其细胞壁并将酶分泌到其环境中的问题。真菌通过引起食物变质、破坏材料和建筑物以及成为植物和动物疾病的媒介对社会产生负面影响。它们也被工业用于生产柠檬酸盐和一些重要的蛋白质产品。了解真菌细胞壁形成的机制将有助于在许多实际情况下控制真菌的生长。更好地了解真菌的分泌过程也可以促进工业蛋白的生产。该研究项目的一个重要方面是为博士后研究人员和从事科研工作的本科生提供培训机会。此外，还有一个项目让布法罗地区的高中科学教师参与到研究中来。该项目将定义模型丝状真菌，粗神经孢子菌，如何正确地将细胞壁蛋白靶向细胞壁并将蛋白质分泌到环境中。这两组糖蛋白都是由真菌分泌到细胞壁空间的，必须是被纳入细胞壁或释放到环境中的目标。先前的工作已经证明了n -连接半乳甘露聚糖结构对细胞壁蛋白结合到细胞壁中的重要性。该项目测试了一种假设，即对n链低聚糖的修饰用于引导细胞壁蛋白进入细胞壁，并允许分泌的糖蛋白释放到环境中。细胞壁和分泌蛋白上的n链修饰将在许多突变株中使用糖生物学技术来鉴定和表征半乳甘露聚糖结构。DFG-5 α -1,6-甘露聚糖酶在生成糖蛋白进入细胞壁所需的半乳甘露聚糖结构中起着关键作用，本研究将表征DFG-5的酶活性以及DFG-5与细胞壁糖蛋白之间的相互作用。这些实验将使用his6标记版本的DFG-5来纯化酶。DFG-5酶活性将通过底物半乳甘露聚糖进行评估，蛋白质/蛋白质相互作用将通过针对细胞壁和分泌糖蛋白的抗体进行共纯化和共免疫沉淀实验来表征。研究者小组先前的研究表明，地衣素（一种具有重复-1,4-葡萄糖- -1,4-葡萄糖- -1,3-葡萄糖三糖结构的葡聚糖）用于将细胞壁蛋白附着到细胞壁上，该项目将鉴定和表征地衣素合酶。这将通过分子遗传学来鉴定推测的地衣素合成酶和针对地衣素的抗体来证明地衣素的合成。结合已发表的文献，该研究将提供真菌细胞壁生物发生的全面图景，并将回答真菌细胞如何区分分泌和细胞壁糖蛋白并将其靶向到正确的细胞外位置的问题。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project addresses the question of how fungal cells are able to correctly assemble their cell walls and secrete enzymes into their environment. Fungi negatively impact society by causing food spoilage, damaging materials and buildings, and being agents of plant and animal diseases. They are also used by industry for producing citrate and a number of important protein products. An understanding of the mechanisms involved in fungal cell wall formation will facilitate efforts to control the growth of fungi in many practical situations. A better understanding of the fungal secretion process could also facilitate increased production of industrial proteins. An important aspect of this research project is to provide training opportunities for postdoctoral researchers and undergraduate students pursuing careers as research scientists. Additionally, there is a program to involve high school science teachers in the Buffalo area in the research.The project will define how the model filamentous fungus, Neurospora crassa, correctly targets cell wall proteins to the cell wall and secreted proteins to the environment. Both groups of glycoproteins are secreted by the fungi into the cell wall space and must be targeted either for incorporation into the wall or for release to the environment. Previous work has demonstrated the importance of an N-linked galactomannan structure for the incorporation of cell wall proteins into the cell wall. The project tests the hypothesis that modifications to the N-linked oligosaccharides are used to direct cell wall proteins into the wall and to allow secreted glycoproteins to be released into the environment. The N-linked modifications present on cell wall and secreted proteins will be examined in a number of mutant isolates using techniques from glycobiology to identify and characterize galactomannan structures. The DFG-5 alpha-1,6-mannanase plays a critical role in generating the galactomannan structure needed for incorporation of glycoproteins into the wall, and the research will characterize the enzymatic activity of DFG-5 and the interactions between DFG-5 and cell wall glycoproteins. These experiments will utilize a HIS6-tagged version of DFG-5 to purify the enzyme. DFG-5 enzymatic activity will be assessed using substrate galactomannans, and protein/protein interactions will be characterized in co-purification and co-immunoprecipitation experiments using antibodies directed against cell wall and secreted glycoproteins. Previous research from the investigator’s groups demonstrated that lichenin (a glucan with a repeating beta-1,4-glucose-beta-1,4-glucose-beta-1,3-glucose trisaccharide structure) is used to attach cell wall proteins into the cell wall and the project will identify and characterize lichenin synthases. This will be done using molecular genetics to identify putative lichenin synthases and anti-bodies directed against lichenin to demonstrate the synthesis of lichenin. In conjunction with the published literature, the research will provide a comprehensive picture of fungal cell wall biogenesis and will answer the question of how fungal cells distinguish between secreted and cell wall glycoproteins and target them to their correct extracellular locations.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.