Structure and Function of Apolipoprotein A-IV

描述(申请人提供)：载脂蛋白(Apo)A-IV是一种46kD的糖蛋白，由哺乳动物肠道细胞在脂肪吸收过程中合成，并分泌到乳糜粒表面的淋巴中。虽然已经提出了载脂蛋白A-IV的广泛的生理功能，但大多数证据表明，它的主要作用是在肠道脂肪吸收中。载脂蛋白A-IV具有低的界面排斥压力，因此它与脂蛋白的结合对其表面的物理状态很敏感。我们已经提出，这一行为使载脂蛋白A-IV能够起到稳压的作用，将脂蛋白表面张力和脂质堆积维持在脂质转移反应所需的临界范围内。利用新的表面化学技术，我们发现apo A-IV在扩展的界面上显示出独特的弹性，并且apo A-IV的C端突变改变了它的界面活性、弹性和调节脂质交换反应的能力。在COS、MCA-RH7777和IPEC细胞中的初步研究表明，载脂蛋白A-IV在富含甘油三酯的颗粒组装过程中与载脂蛋白B相互作用，改变脂蛋白的运输和分泌。因此，我们认为载脂蛋白A-IV独特的动态界面性质在富含甘油三酯的颗粒组装中起着特殊的作用，具体地说，载脂蛋白A-IV作用于内质网和高尔基体，调节界面张力和新生乳胶粒表面的脂质堆积，从而控制脂肪的膨胀、细胞内的运输和分泌动力学，最终控制肠道脂质的吸收效率。为了探索这一假说，我们提出了三个具体目标：1)我们将利用光谱和表面化学技术研究信息丰富的载脂蛋白A-IV定点突变体，以阐明其界面弹性以及与脂质和载脂蛋白B相互作用的结构决定因素；2)我们将用生物物理特征的载脂蛋白A-IV突变体转染COS、MCA-RH7777和IPEC细胞，以研究载脂蛋白A-IV的界面行为如何调节富含甘油三酯的脂蛋白的组装、运输和分泌；3)利用载脂蛋白A-IV基因敲除和突变的载脂蛋白A-IV转基因小鼠，检测肠道载脂蛋白A-IV的表达对脂肪酸吸收效率和肠道脂蛋白的大小和组成的影响。我们相信，这些研究将为载脂蛋白A-IV在复杂的脂蛋白组装过程中的功能提供新的知识，这些过程与饮食控制动脉粥样硬化性心血管疾病和肥胖有关。

DESCRIPTION (provided by applicant): Apolipoprotein (apo) A-IV is a 46 Kd glycoprotein that is synthesized by the intestinal enterocytes of mammals during lipid absorption and secreted into lymph on the surface of chylomicrons. Although a broad spectrum of physiologic functions has been proposed for apo A-IV, a preponderance of evidence suggests that its primary role is in intestinal lipid absorption. Apo A-IV has a low interfacial exclusion pressure, and thus its binding to lipoproteins is sensitive to the physical state of their surface. We have proposed that this behavior enables apo A-IV to act as a barostat that maintains lipoprotein surface tension and lipid packing within a critical range required for lipid transfer reactions. Using novel surface chemistry techniques, we have found that apo A-IV displays unique elasticity at expanding interfaces, and that C-terminal mutations in apo A-IV alter its interfacial activity, elasticity, and ability to modulate lipid exchange reactions. Preliminary studies in COS, McA-RH7777, and IPEC cells suggest that apo A-IV interacts with apoB during triglyceride-rich particle assembly and alters lipoprotein trafficking and secretion. Thus, we propose that the unique dynamic interfacial properties of apo A-IV play a specific role in triglyceride-rich particle assembly, specifically, that apo A-IV acts in the endoplasmic reticulum and Golgi to modulate interfacial tension and lipid packing at the nascent chylomicron surface, thereby controlling lipidation and expansion, intracellular trafficking and secretion kinetics and, ultimately, the efficiency of intestinal lipid absorption. To pursue this hypothesis we propose three specific aims: 1) we will study informative apo A-IV site-directed mutants using spectroscopic and surface chemistry techniques to elucidate the structural determinants of its interfacial elasticity and interaction with lipid and apo B; 2) we will transfect COS, McA-RH7777, and IPEC cells with biophysically characterized apo A-IV mutants to examine how the interfacial behavior of apo A-IV modulates assembly, trafficking, and secretion of triglyceride-rich lipoproteins; 3) we will use apo A-IV knockout and mutant apo A-IV transgenic mice to examine the effect of intestinal apo A-IV expression on the efficiency of fatty acid absorption and the size and composition of intestinal lipoproteins. We believe that these studies will provide new knowledge on the function of apo A-IV in the complex process of lipoprotein assembly relevant to the dietary control of atherosclerotic cardiovascular disease and obesity.