Lipoprotein lipase(LPL) is the major enzyme responsible for the hydrolysis of plasma triglyceride rich lipoproteins. The enzyme is a key factor in determining the compositions and concentrations of plasma lipoproteins including triglyceride rich lipoproteins, LDL and HDL. Therefore understanding how this enzyme is regulated should contribute to a mechanistic understanding of atherosclerosis. A major property of LPL is its high affinity in its active dimeric form to bind to heparan sulfate(HS) chains. This property has an impact in modulating enzyme activity in extra-hepatic tissues and lipoprotein receptor activity in the liver. We propose to test the hypothesis that the type of core proteins to which the HS chains are attached (syndecans versus glypicans) determines the cellular targeting of the enzyme to secretion or degradation pathways. This hypothesis will be tested by overexpressing or extinguishing glypican-4 or syndecan-4 in 3T3-F442 cells at different stages of differentiation by utilizing tetracycline-inducible expression of sense and anti-sense constructs. In these cells, LPL, syndecan- 4, and glypican-4 turn-over will be determined by pulse-chase protocols. The function of specific heparan sulfate proteoglycan core proteins in LPL turn-over and lipid homeostasis will also be evaluated by characterizing the phenotypes of mice with syndecan- 4 or glypican-4 extinguished conditionally in adipose tissue by the Cre-loxP system. Mice with """"""""floxed"""""""" glypican-4 or syndecan-4 genes will be crossed with transgenic mice overexpressing Cre recombinase under the control of the ap2 promoter. The second hypothesis will test the notion that the interaction of apoE, LPL, hepatic lipase(HL) with the LDL receptor related protein (LRP) is enhanced dramatically when interacting with both the lipoprotein surface and heparan sulfate chains. This hypothesis will be evaluated in a series of experiments utilizing a biosensor and surface plasmon resonance (SPR) measurement to determine the kinetic constants defining the interaction of beta- VLDL enriched with apoE, HL or LPL with reconstituted surfaces containing, various combinations of highly purified liver glypican, syndecan, and LRP in various molar ratios.
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