FAMILIAL DEFECTIVE APO-B100

家族性载脂蛋白（apo）B100（FDB）缺陷是一种遗传性疾病， 脂质代谢异常，导致中度至重度 高胆固醇血症 载脂蛋白B100的突变严重降低了 低密度脂蛋白（LDL）与LDL受体结合的能力。 因此，LDL在血浆中积累，因为它们的有效受体- 介导的catalysis被破坏。 有一种强烈的联想， Apo-B100突变（Arg 3500-> Gln）。 该突变 在美国和欧洲广泛传播，在某些人群中， 遗传异常是第一或第二个最普遍的遗传原因 高胆固醇血症和过早的动脉粥样硬化。 的一个主要 本申请的目的是证明Arg 3500-> Gln 突变导致FDB，并了解突变如何破坏LDL 受体结合 有和没有3500的全长载脂蛋白B小基因 突变将在培养的肝细胞和转基因小鼠中表达。 小鼠 将从细胞培养物中分离“LDL”或LDL样颗粒 培养基和转基因小鼠的血浆，表征和测试 低密度脂蛋白受体结合。 我们将检验3500人 突变位于apo-B的一个区域，该区域不直接与 LDL受体，但调节或改变结合的构象 绝佳的价钱 作为我们对FDB研究的一部分，apo-B100中的新突变 导致LDL结合缺陷的原因将通过检查 从高胆固醇血症受试者中分离的LDL与LDL结合 受体。 我们还将利用FDB突变来了解更多信息 关于正常VLDL、IDL、LDL和Lp（a）代谢。 第二主 目的是确定apo-B100的受体结合结构域， 确定哪些氨基酸对apo-B100与LDL结合至关重要 受体的 这个目标的一部分是确定LDL的亲和力 从八种脊椎动物的LDL受体对正常人 成纤维细胞 推定的受体结合结构域序列的比较 与LDL的受体结合能力应该高度揭示 保守的区域对这一功能至关重要， 用于诱变。 定点诱变将改变或删除位点 我们认为apo-B100的残基对LDL结合至关重要。 的 突变的apo-B100小基因将在培养的细胞中表达， 转基因小鼠，彻底表征，并检测受体 约束力 apo-B_（100）受体结合位点的解析 了解apo-B100的某些突变如何破坏LDL 结合将进一步加深我们对载脂蛋白相互作用的了解， B100与LDL受体及遗传的分子基础 导致高胆固醇血症和早产的异常 动脉粥样硬化

Familial defective apolipoprotein (apo) B100 (FDB) is a genetic abnormality of lipid metabolism that causes moderate to severe hypercholesterolemia. A mutation in apo-B100 severely diminishes the ability of low density lipoproteins (LDL) to bind to the LDL receptor. Therefore, LDL accumulate in the plasma because their efficient receptor- mediated catabolism is disrupted. There is a strong association of the mutation (Arg3500 -> Gln) in apo-B100 with FDB. This mutation is widespread in the United States and Europe and, in some populations, this genetic abnormality is the first or second most prevalent genetic cause of hypercholesterolemia and premature atherosclerosis. One of the main objectives of this application is to prove that the Arg3500 -> Gln mutation causes FDB and to understand how the mutation disrupts LDL receptor binding. A full-length apo-B minigene with and without the 3500 mutation will be expressed in hepatic cells in culture and in transgenic mice. The "LDL" or LDL-like particles will be isolated from cell culture media and from the plasma of transgenic mice, characterized, and tested for LDL receptor binding. We will test the hypothesis that the 3500 mutation is in a region of apo-B that does not interact directly with the LDL receptor, but modulates or alters the conformation of the binding site. As part of our investigations on FDB, new mutations in apo-B100 that cause defective LDL binding will be sought by examining the ability of LDL isolated from hypercholesterolemic subjects to bind to LDL receptors. We will also take advantage of the FDB mutation to learn more about normal VLDL, IDL, LDL and Lp(a) metabolism. The second main objective is to define the receptor binding domain of apo-B100 and determine which amino acids are crucial for apo-B100 binding to the LDL receptor. Part of this objective is to determine the affinity of LDL from eight vertebrate species for LDL receptors on normal human fibroblasts. Comparison of the putative receptor binding domain sequence with the receptor binding ability of the LDL should reveal highly conserved regions critical for this function which will then be targets for mutagenesis. Site-directed mutagenesis will alter or delete the site and residues of apo-B100 we believe are critical for LDL binding. The mutated apo-B100 minigene will be expressed in cultured cells and transgenic mice, characterized thoroughly, and tested for receptor binding. The elucidation of the receptor binding site of apo-B100 and the understanding of how certain mutations of apo-B100 disrupt LDL binding will further our growing knowledge about the interaction of apo- B100 with the LDL receptor and the molecular basis of genetic abnormalities that cause hypercholesterolemia and premature atherosclerosis.