Lipoprotein lipase (LPL) is the major enzyme responsible for hydrolysis of triglyceride molecules that circulate as a component of plasma lipoproteins. LPL is synthesized by a variety of tissues, but it functions bound to surface glycosaminoglycans (GAGS) on the luminal side of capillary endothelial cells (EC). Control of the activity of LPL is in many ways more complex than that of most secretory proteins and may be exercised at several loci. After synthesis and secretion LPL must be transported to an appropriate binding site on EC, a distant cell type. The overall objective of this proposal is to understand the pathways required for LPL to move from its site of synthesis to its site of physiologic action. Transport of LPL across EC will be explored in four specific aims. 1. To study the route of LPL transport from basolateral to apical (luminal) surfaces of endothelial cells. Prior to its arrival on the capillary lumen, LPL must be transported through or around EC. The transport of LPL from the basolateral to apical side of cultured EC monolayers will be studied using biochemical and histochemical methods. 2. To determine factors which regulate the transport of LPL across endothelial cell monolayers. LPL transport will be studied with alterations of three different factors: a) the metabolism of EC, b) GAGs or other molecules important for LPL interaction with the basolateral surface of EC, and c) the structure of LPL molecules and their affinity for interaction with EC. 3. To study the interaction of LPL with the surface of adipocytes and the transport of adipocyte LPL across EC monolayers. Like EC, adipocytes have LPL bound to the cell surface which is released by treatment of the cells with heparin. LPL release from adipocyte surfaces and transport of adipocyte LPL across EC monolayers will be assessed. 4. To characterize the LPL binding site on the surface of endothelial cells. Using a combination of affinity chromatography and gel filtration techniques, LPL binding proteoglycans on EC membranes will be isolated and their core proteins and the size and substructure of their GAG chains determined. The specificity of these molecules for LPL versus other proteoglycan binding proteins will be assessed. The information derived from the experiments in this proposal will increase our understanding of regulation of LPL, an important factor determining plasma levels of atherogenic and anti-atherogenic lipoproteins. In addition, insights may be provided into extracellular processing events affecting other membrane-bound proteins.
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