A NOVEL GLYCAN SYNTHETIC ACTIVITY IN CRYPTOCOCCUS NEOFORMANS

描述（由申请人提供）：新型隐球菌是一种机会致病菌，可导致因艾滋病或其他疾病而免疫功能低下的个体发生危及生命的疾病。目前隐球菌病的治疗是不够的。真菌聚糖结构为特异性化疗提供了有吸引力的潜在靶点，因为它们与宿主生物体中发现的聚糖结构有很大不同。C.碳水化合物结构新变型改造蛋白质和脂质，构成细胞壁的大部分，并形成包围细胞壁的精心制作的多糖胶囊，这是毒力所必需的。值得注意的是，胶囊和细胞壁是哺乳动物细胞中完全不存在的关键结构，并且细胞壁已经被验证为药物靶标。我们在C. neoformans，其将木糖-磷酸从UDP-木糖转移到甘露糖残基。这一活动是前所未有的。在这个R21的应用程序中，我们建议追求的假设，这种酶是重要的隐球菌生物学，并探讨其生化活性，着眼于新的功能和筛选试验的发展。如目标I中所提议的，蛋白质的生物化学研究将解决所观察到的活性的反应物和动力学，以及关键残留物和改进的测定。酶的细胞环境将通过研究目标II中的定位和潜在结合伴侣来确定。最后，这种独特的蛋白质在生物学中的作用将使用基因工程细胞来解决。将这些菌株与亲本菌株进行比较，以评估Aim III中糖基化和相关表型的变化。方法将包括生化测定、聚糖分析、表位标记、成像、代谢放射性标记和肽分析。这种多方面的策略将阐明一种独特蛋白质的活性，并提供对真菌生物学和聚糖合成的基本理解。 公共卫生相关性：这项研究与公共卫生高度相关，因为正在研究的生物体会导致严重的人类疾病，目前的治疗方法不足以治疗这些疾病。此外，这项工作将有助于基础科学知识，这将影响病原微生物和其他生物学领域的研究。

DESCRIPTION (provided by applicant): Cryptococcus neoformans is an opportunistic pathogen, responsible for life-threatening disease in individuals who are immunocompromised due to AIDS or other conditions. Current therapy for cryptococcosis is inadequate. Fungal glycan structures offer appealing potential targets for specific chemotherapy, because they differ substantially from those found in host organisms. Carbohydrate structures in C. neoformans modify proteins and lipids, compose most of the cell wall, and form an elaborate polysaccharide capsule that surrounds the cell wall and is required for virulence. Notably, the capsule and cell wall are critical structures that are completely absent from mammalian cells, and the cell wall has already been validated as a drug target. We have discovered a novel glycosylating enzyme in C. neoformans, which transfers xylose-phosphate from UDP-xylose to mannose residues. This activity is unprecedented in nature. In this R21 application, we propose to pursue the hypothesis that this enzyme is important in cryptococcal biology, and to explore its biochemical activity with an eye to novel function and the development of screening assays. As proposed in Aim I, biochemical studies of the protein will address the reactants and kinetics of the observed activity, as well as critical residues and improved assays. The cellular context of the enzyme will be determined by studies of localization and potential binding partners in Aim II. Finally, the role of this unique protein in biology will be addressed using cells genetically engineered to lack the activity. These will be compared to parental strains to assess alterations in glycosylation and related phenotypes in Aim III. Methods will include biochemical assays, glycan analysis, epitope-tagging, imaging, metabolic radiolabeling, and peptide analysis. This multi-faceted strategy will elucidate the activity of a unique protein and provide fundamental understanding of fungal biology and glycan synthesis. PUBLIC HEALTH RELEVANCE: This research is highly relevant to public health because the organism being investigated causes serious human illness, for which current therapies are not adequate. Further, this work will contribute to basic science knowledge, which will impact the study of pathogenic microbes and other areas of biology.