FUNCTION OF LIPOPROTEIN LIPASE PROTEOGLYCAN INTERACTION

脂蛋白脂肪酶（LPL）是负责水解血浆中富含甘油三酯的脂蛋白的主要酶。该酶是决定血浆脂蛋白的组成和浓度的关键因素，包括富含甘油三酯的脂蛋白、低密度脂蛋白和高密度脂蛋白。因此，了解这种酶是如何被调节的，将有助于了解动脉粥样硬化的机制。LPL的一个主要特性是其活性二聚体形式与硫酸肝素（HS）链结合的亲和力高。这种特性对调节肝外组织中的酶活性和肝脏中的脂蛋白受体活性有影响。我们提出验证HS链所连接的核心蛋白类型（syndecans与glyypicans）决定了该酶在分泌或降解途径中的细胞靶向性的假设。我们将利用四环素诱导的义和反义构建体的表达，在不同分化阶段的3T3-F442细胞中过表达或抑制glypican-4或syndecan-4来验证这一假设。在这些细胞中，LPL、syndecan- 4和glypican-4的转换将通过脉冲追踪方案来确定。通过Cre-loxP系统表征syndecan- 4或glypican-4在脂肪组织中有条件消失的小鼠的表型，也将评估特定硫酸肝素蛋白聚糖核心蛋白在LPL翻转和脂质稳态中的功能。携带“floxed”glypican-4或syndecan-4基因的小鼠将在ap2启动子的控制下与过表达Cre重组酶的转基因小鼠杂交。第二个假设将检验apoE、LPL、肝脂肪酶（HL）与LDL受体相关蛋白（LRP）的相互作用在与脂蛋白表面和硫酸肝素链相互作用时显著增强的概念。这一假设将在一系列实验中进行评估，利用生物传感器和表面等离子体共振（SPR）测量来确定动力学常数，以确定富含apoE、HL或LPL的β - VLDL与含有不同摩尔比的高纯度肝糖甘肽、辛德甘肽和LRP的各种组合的重建表面的相互作用。

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.