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. ? ? ?
