The overall goal of this project is to understand the extracellular events which regulate the physiological actions of lipoprotein lipase (LPL), the major enzyme responsible for hydrolysis of triglyceride molecules that circulate as a component of plasma lipoproteins. LPL is one of a number of heparin-binding proteins that are associated with cell surfaces by protein-glycosaminoglycan and protein-protein interactions. The molecules that mediate LPL binding to endothelial cells and adipocytes, however, remain to be defined. By modulating LPL specific activity, these molecules are likely to regulate LPL actions. During the previous funding period two endothelial cell surface proteins that bind to LPL were identified. One is a membrane spanning heparan sulfate proteoglycan. Recently, the specific oligosaccharide sequence that associates with LPL was also isolated and a three dimensional model of this decasaccharide was constructed. The second non-proteoglycan LPL binding protein has homology to or may be a fragment of apolipoprotein B. The interactions of LPL and these two proteins will be studied in four specific aims. l. To determine if and how the amino-terminal apoB-like protein and heparan sulfate proteoglycans cooperate to bind LPL to the surface of endothelial cells. The individual and cooperative actions of the LPL-binding molecules will be determined. 2. To define the molecular determinants of LPL and apoB which allow their interaction. Biochemical, immunological, and molecular biological techniques will be used to determine the domains of LPL and apoB that mediate their binding. 3. To assess the molecular mechanisms of LPL a) transport from adipocytes to endothelial cells, and b) transcytosis across endothelial cells. The factors that dissociate LPL from adipocyte surfaces and stabilize its activity as it transfers to and transverses across endothelial cells will be determined. 4. To study the endothelial cell production of the two LPL- binding molecules. The origin of the apoB-like protein will be resolved by assessing endothelial cell mRNA and protein. In addition, the distribution of the LPL - binding decasaccharide amongst endothelial proteoglycans will be determined. We expect that information derived from the experiments in this proposal will increase our understanding of LPL regulation, protein-proteoglycan interactions, and extracellular protein trafficking. Because abnormalities of LPL actions are associated with dyslipidemias and increased risk for atherosclerosis, we are hopeful that our data will lead to new therapeutic targets for these disorders.
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