Biosynthesis of Phosphorylcholine Oligosaccharides

描述（由申请人提供）：丝状线虫通过调节或抑制宿主的免疫系统感染大约1.5亿人和动物。先前的研究表明，含磷胆碱寡糖蛋白的抗炎作用在线虫体内而非宿主体内发挥重要作用。这项高风险/高影响的R21资助计划的具体目的是克隆秀丽隐杆线虫的磷酸胆碱寡糖合成酶(s)，这种线虫的完整基因组序列是已知的，由于其易于操作和缺乏致病性，应该是研究这些分子在丝状线虫中的生物合成的一个有吸引力的模型。为了实现上述目标，我们建议采取两种互补的方法。基于最近克隆的鞘磷脂合成酶和磷酸胆碱寡糖合成酶之间的推测机制相似性，我们将表达秀丽隐杆线虫编码酿酒酵母鞘磷脂合成酶已知或相关基因的cdna。转化酵母的提取物不含内源性鞘磷脂合成酶，将用于合成磷胆碱低聚糖。我们推测线虫鞘磷脂合成酶可能具有两种酶的活性，或者相关基因可能具有磷酸胆碱寡糖合成酶的活性。如果上述方法不成功，那么将从秀丽隐杆线虫中获得一个高纯度的含有合成酶活性的部分，并在秀丽隐杆线虫全基因组的基础上进行蛋白质组学分析和检查。候选基因将在酿酒酵母中表达，并测定其磷酸化胆碱寡糖合成酶的活性。这些研究如果成功，将为以下工作奠定基础：(a)尝试克隆包括马来布鲁贾线虫、pahangi布鲁贾线虫和vitae Achanthocheilonema在内的丝状线虫的磷酸化胆碱寡糖合成酶；(b)从秀丽线虫中对合成酶进行生化表征，包括鉴定可能的抑制剂。在宿主中缺乏这种反应使得鉴定潜在的抑制剂有吸引力的治疗靶点。

DESCRIPTION (provided by applicant): Filarial nematodes infect approximately 150 million people and animals by modulating or suppressing the immune system of the host. Previous studies have suggested that the anti-inflammatory action of phosphorylcholine oligosaccharide -containing proteins, occurring in nematodes, but not in the hosts, play an important role in this process. The specific aim of this R21, high risk/high impact, grant proposal is to clone the phosphorylcholine oligosaccharide synthase(s) of C. elegans, a nematode whose complete genome sequence is known and because of its ease of manipulation and lack of pathogenicity should be an attractive model to study the biosynthesis of such molecules in filarial nematodes. For the above goal we propose to pursue two complementary approaches. Based on the putative mechanistic similarities between the recently cloned sphingomyelin synthases and phosphorylcholine oligosaccharide synthases, we will express C. elegans cDNAs, encoding either known or related genes of sphingomyelin synthases in S. cerevisiae. Extracts from the transformed yeast, which don't have endogenous sphingomyelin synthases, will be assayed for synthesis of phosphorylcholine oligosaccharides. We hypothesize that sphingomyelin synthases of nematodes may have both enzymatic activities or that the related genes may have phosphorylcholine oligosaccharide synthase activity. Should the above approach not succeed then a highly purified fraction containing synthase activity from C. elegans will be obtained and subjected to proteomic analysis and examination on the basis of the complete genome of C. elegans. Candidate genes will be expressed in S. cerevisiae and assayed for phosphorylcholine oligosaccharide synthase activity. These studies, if successful, will constitute the basis for (a) attempts to clone the phosphorylcholine oligosaccharide synthase from filarial nematodes including Brugia malayi, Brugia pahangi and Achanthocheilonema vitae and (b) biochemical characterization of the synthase from C. elegans including identification of possible inhibitors. The absence of this reaction in hosts makes identification of potential inhibitors attractive therapeutic targets.