Biosynthesis of Phosphorylcholine Oligosaccharides

描述（由申请方提供）：丝虫通过调节或抑制宿主的免疫系统感染约1.5亿人和动物。先前的研究表明，在线虫体内而非宿主体内，含有磷酸胆碱寡糖的蛋白质的抗炎作用在这一过程中起重要作用。这项R21高风险/高影响拨款提案的具体目标是克隆C. elegans是一种线虫，其完整的基因组序列是已知的，并且由于其易于操作和缺乏致病性，应该是研究丝虫线虫中这些分子的生物合成的有吸引力的模型。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酶和磷酰胆碱寡糖酶之间的假定机制相似性，我们将表达C。elegans中已知或相关鞘磷脂酶基因的cDNA。啤酒。将测定不具有内源性鞘磷脂酰胆碱脱氢酶的转化酵母的提取物的磷酸胆碱寡糖的合成。我们推测线虫的鞘磷脂酰胆碱酯酶可能同时具有两种酶活性，或者相关基因可能具有磷酸胆碱寡糖合成酶活性。如果上述方法不成功，则从C.将获得秀丽隐杆线虫，并基于C.优雅候选基因将在S.酿酒酵母中的磷酸胆碱寡糖合酶活性进行测定。这些研究如果成功，将构成（a）试图从包括马来丝虫（Brugia malayi）、帕汉丝虫（Brugia pahangi）和Achanthocheilonema vitae的丝虫中克隆磷酸胆碱寡糖合酶和（B）从C.包括鉴定可能的抑制剂。宿主中不存在这种反应使得鉴定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.