Biosynthesis of Phosphorylcholine Oligosaccharides

磷酰胆碱寡糖的生物合成

基本信息

批准号：
7126266
负责人：
CARLOS Benjamin HIRSCHBERG
金额：
$ 20.31万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-01 至 2008-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7126266
关键词：
Caenorhabditis elegans Mycobacterium RNA interference Saccharomyces cerevisiae complementary DNA enzyme activity green fluorescent proteins helminth genetics inhibitor /antagonist mutant oligosaccharides phosphorylcholine protein localization protein purification protein structure function proteomics sphingomyelin phosphodiesterase transfection /expression vector

项目摘要

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.

描述（由申请人提供）：丝虫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，在线虫中而非宿主中存在的含磷酸胆碱寡糖的蛋白质的抗炎作用在此过程中发挥重要作用。该 R21 高风险/高影响拨款提案的具体目标是克隆秀丽隐杆线虫的磷酸胆碱寡糖合酶，线虫是一种线虫，其完整基因组序列已知，并且由于其易于操作且缺乏致病性，应该成为研究丝虫线虫中此类分子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酶和磷酸胆碱寡糖合酶之间假定的机制相似性，我们将表达秀丽隐杆线虫cDNA，编码酿酒酵母中鞘磷脂合酶的已知或相关基因。转化酵母的提取物不具有内源性鞘磷脂合酶，将用于分析磷酸胆碱寡糖的合成。我们推测线虫的鞘磷脂合酶可能同时具有这两种酶活性，或者相关基因可能具有磷酸胆碱寡糖合酶活性。如果上述方法不成功，则将获得来自秀丽隐杆线虫的含有合酶活性的高度纯化的级分，并基于秀丽隐杆线虫的完整基因组进行蛋白质组分析和检查。候选基因将在酿酒酵母中表达并测定磷酸胆碱寡糖合酶活性。这些研究如果成功，将构成以下基础：(a) 尝试从丝虫线虫（包括马来丝虫、彭亨丝虫和 Achanthocheilonema vitae）克隆磷酸胆碱寡糖合酶；以及 (b) 线虫合酶的生化表征，包括鉴定可能的抑制剂。宿主体内不存在这种反应使得潜在抑制剂的鉴定成为有吸引力的治疗靶点。