Structure and Function of Apolipoprotein A-IV

描述（由申请人提供）：载脂蛋白（apo） a - iv是一种46 Kd的糖蛋白，由哺乳动物肠上皮细胞在脂质吸收过程中合成，并分泌到乳糜微粒表面的淋巴中。尽管载脂蛋白a - iv具有广泛的生理功能，但大量证据表明其主要作用是在肠道脂质吸收中。载脂蛋白a - iv具有较低的界面排斥压力，因此其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，这种行为使载脂蛋白a - iv充当气压调节器，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，我们发现载脂蛋白A-IV在扩展界面上表现出独特的弹性，载脂蛋白A-IV的c端突变改变了其界面活性、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在富甘油三酯颗粒组装过程中与载脂蛋白b相互作用，并改变脂蛋白的运输和分泌。因此，我们提出载脂蛋白a - iv独特的动态界面特性在富含甘油三酯的颗粒组装中发挥特殊作用，具体来说，载脂蛋白a - iv在内质网和高尔基体中作用，调节新生乳糜微粒表面的界面张力和脂质堆积，从而控制脂化和膨胀、细胞内运输和分泌动力学，并最终控制肠道脂质吸收的效率。为了实现这一假设，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位点定向突变体，以阐明其界面弹性和与脂质和载脂蛋白B相互作用的结构决定因素；2)我们将转染具有生物物理特征的载脂蛋白A-IV突变体的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)我们将使用apo A-IV基因敲除和突变型apo A-IV转基因小鼠来检测肠道apo A-IV表达对脂肪酸吸收效率和肠道脂蛋白大小和组成的影响。我们相信这些研究将为载脂蛋白A-IV在脂蛋白组装复杂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.