FUNCTION OF LIPOPROTEIN LIPASE PROTEOGLYCAN INTERACTION

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LPL is the major enzyme responsible for the hydrolysis of triglyceride rich lipoproteins. The enzyme plays a major role in determining the concentration and composition of plasma lipoproteins including the LDL and HDL fractions. Therefore, understanding mechanistic aspects of LPL regulation will contribute to an understanding of atherosclerosis. In cells producing LPL a large fraction of the synthesized enzyme is degraded and degradation rate determines the net efflux of enzyme. The current proposal focuses on the molecular aspects of the function of heparan sulfate proteoglycans(HSPGs) and the low density lipoprotein receptor related protein (LRP) in the degradation of LPL> It is hypothesized that LPL on the surface of cells binds and is internalized by both HSPGs and LRP. In adipocytes, the HSPG mediated degradation predominates. We will test the notion that the core proteins of HSPGs plays a signaling function in targeting HS chains to which LPL is bound to endocytic pathways or to export to the extracellular fluid. It is also hypothesized that the LRP mediated pathway is dependent for maximal flux on HSPG binding to both LPL and LRP. Adipocyte HSPGs and tightly associated proteins will be purified, cloned, and antibodies prepared. The identification of the LPL binding domains will be completed by site directed mutagenesis, chemical modification of HSPG protected basic residues and with monoclonal antibody probes. The fraction of LPL degraded via LRP, and specific HSPGs will be quantitatively determined in pulse chase studies. RAP (Receptor-Associated Protein), X-ch-LPL-3-6a, a monoclonal antibody which inhibits LPL binding to HSPGs, phosphatidylinositol specific phospholipase C and cells devoid of HS chains will be utilized to inhibit selectively pathways of LPL degradation. Rate constants (Kon and Koff) of wild type or mutant LPL binding to LRP in the presence or absence of HS chains on cell surfaces will be determined to evaluate the function of HSPGs in the LPL/LR P interaction.