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性病原体，对因艾滋病或其他疾病而免疫功能低下的个体造成威胁生命的疾病。目前对隐球菌病的治疗是不够的。真菌聚糖结构为特定的化疗提供了有吸引力的潜在靶点，因为它们与宿主生物中的糖结构有很大的不同。新形态C.中的碳水化合物结构修饰蛋白质和脂质，构成大部分细胞壁，并形成围绕细胞壁的精细多糖胶囊，这是毒性所必需的。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，而细胞壁已经被证实是药物靶点。我们发现了一种新的糖基化酶，它将木糖磷酸从udp -木糖转移到甘露糖残基。这种活动在本质上是前所未有的。在这个R21申请中，我们建议继续假设该酶在隐球菌生物学中很重要，并探索其生化活性，着眼于新功能和开发筛选方法。正如Aim I中提出的那样，蛋白质的生化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分析。该酶的细胞背景将通过Aim II中定位和潜在结合伙伴的研究来确定。最后，这种独特的蛋白质在生物学中的作用将通过基因工程细胞来消除这种活性。这些将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。方法将包括生化分析、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，并为真菌生物学和聚糖合成提供基本的理解。

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.