The long-term objective of the proposed project is to understand the mechanisms that govern the uptake of lipids by insect oocytes. This will fill a gap in our knowledge on insect development, which may lead to the design of novel and effective ways to control the insects that play a role in the transmission of diseases like malaria, yellow fever, river blindness, Chagas disease, etc. The proposed project will focus on a recently identified lipoprotein lipase from mature eggs of the hawkmoth Manduca sexta. The observed hydrolysis of lipoprotein-transported diacylglycerol by this lipoprotein lipase indicate a critical role of the enzyme in the uptake of lipids by the developing oocyte.
The specific aims of the project are: 1. To determine the biochemical characteristics of the lipoprotein lipase. Using standardized lipoprotein lipase assays, various lipid substrates and inhibitors of lipase activity, the enzyme will be characterized and compared with known vertebrate lipases. 2. To determine the site of synthesis and the subcellular site of activity of the lipase. Analyzing the synthesis of lipoprotein lipase and its mRNA in different tissues, combined with histological and immunocytochemical localization of the enzyme, will reveal sites at which the uptake of lipids by the oocyte is controlled. 3. To develop molecular probes of the lipoprotein lipase. Molecular cloning and sequencing of lipoprotein lipase cDNA will reveal the degree of homology with vertebrate lipases and will provide valuable probes for developmental studies. 4. To determine the possible role of other protein factors in lipid transport to the oocyte. In addition to the role of lipoprotein lipase, the possible involvement and relative importance of putative lipoprotein receptors, lipid transfer proteins and fatty acid binding proteins in the uptake of lipids by insect oocytes will be assessed.
