Biosynthesis of Phosphorylcholine Oligosaccharides

描述(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，磷胆碱寡糖蛋白的抗炎作用在这一过程中发挥了重要作用，这些蛋白存在于线虫中，但不存在于宿主中。这项R21，高风险/高影响，拨款提案的具体目的是克隆线虫的磷胆碱低聚糖合成酶(S)，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，应该是研究丝状线虫这种分子的生物合成的一个有吸引力的模型。为实现上述目标，我们建议采取两种相辅相成的办法。基于最近克隆的鞘磷脂合成酶和磷胆碱低聚糖合成酶在机制上的相似性，我们将在酿酒酵母中表达编码鞘磷脂合成酶已知或相关基因的线虫cDNAs。从转化的酵母中提取的不含内源性鞘磷脂合成酶的提取物将被检测以合成磷胆碱低聚糖。我们推测线虫的鞘磷脂合成酶可能同时具有酶活性，或者相关基因可能具有磷胆碱寡糖合成酶活性。如果上述方法不成功，则将从线虫中获得含有合酶活性的高纯度组分，并根据线虫的全基因组进行蛋白质组学分析和检测。候选基因将在酿酒酵母中表达，并检测磷胆碱低聚糖合成酶的活性。如果这些研究成功，将构成(A)尝试从丝虫线虫(包括马来丝虫、帕汉氏丝虫和棘轮线虫)中克隆磷胆碱低聚糖合成酶的基础，以及(B)线虫合成酶的生化特征，包括鉴定可能的抑制物。宿主中没有这种反应，这使得识别潜在的抑制剂成为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.