APOLIPOPROTEIN-A-I CONFORMATION IN DISCOIDAL PARTICLES - EVIDENCE FOR ALTERNATE STRUCTURES
APOLIPOPROTEIN-A-I CONFORMATION IN DISCOIDAL PARTICLES - EVIDENCE FOR ALTERNATE STRUCTURES
复制标题
DOI：
10.1021/bi00076a023
复制
发表时间：
1993-06-29
期刊：
影响因子：
2.9
通讯作者：
MARCEL, YL
中科院分区：
文献类型：
--
作者：
CALABRESI, L;MENG, QH;MARCEL, YL
为了确定盘状颗粒中载脂蛋白 A-I 的构象，在无脂 apoA-I 和脂结合形式中评估了沿 apoA-I 序列分布的一系列表位的免疫反应性。为此，用棕榈酰油酰磷脂酰胆碱、胆固醇和 apoA-I 制备了重构的盘状脂蛋白，此处称为 LpA-I，并由每个颗粒的 apoA-I 数量（例如 Lp2A-I）来定义。已获得并研究了四个 LpA-1：两个属于 Lp2A-I 类，直径为 7.8 和 9.6 nm，两个属于 Lp3A-I 类，直径为 10.8 和 13.4 nm。与不含脂质的 apoA-I 相比，LpA-I 颗粒中所有测试表位的免疫反应性均显着不同，这表明与脂质的结合会导致 apoA-I 构象发生巨大变化。一级序列中的特定结构域变得高度暴露，而其他结构域则被掩盖。尽管不同 LpA-I 之间表位的免疫反应性差异并不显着，但在每一类（Lp2A-I 或 Lp3A-I）内的小颗粒与大颗粒中的许多抗体（Lp2A-I 或 Lp3A-I）中观察到计算的 ED50 值存在显着差异，表明颗粒大小可以调节 apoA-I 构象。此外，当分析单克隆抗体对之间的竞争以了解表位的相对位置时，观察到作为每一类中粒径的函数的高度显着差异。特别是，识别 apoA-I 中心区域表位的 mAb 之间的竞争在大颗粒中比在小颗粒中更大。这一看似对立的发现可以通过引入替代结构来合理化，例如由两个相邻的反平行α螺旋组成的铰链结构域，可能位于残基99-143，或取决于颗粒大小的不同构象。
To define the conformation of apolipoprotein A-I in discoidal particles, the immunoreactivity of a series of epitopes distributed along the apoA-I sequence has been evaluated in lipid-free apoA-I and in lipid-bound form. To this end, reconstituted discoidal lipoproteins, here called LpA-I, and defined by number of apoA-I per particle (e.g., Lp2A-I), have been prepared with palmitoyloleoylphosphatidylcholine, cholesterol, and apoA-I. Four LpA-1 have been obtained and studied: two in the Lp2A-I class, 7.8 and 9.6 nm in diameter, and two in the Lp3A-I class, 10.8 and 13.4 nm. The immunoreactivity of all the epitopes tested was significantly different in LpA-I particles compared to lipid-free apoA-I, demonstrating that binding to lipids produces a drastic change in apoA-I conformation. Specific domains in the primary sequence become highly exposed while others are masked. Although the variation in immunoreactivity of the epitopes between various LpA-I was not drastic, significant differences in the calculated ED50 values were observed for a number of antibodies in small versus large particles within each class (Lp2A-I or Lp3A-I), indicating that particle size can modulate apoA-I conformation. In addition, when the competition between pairs of mAbs was analyzed in order to understand the relative position of epitopes, highly significant differences were observed as a function of particle size within each class. In particular, the competition between mAbs recognizing epitopes in the central region of apoA-I was greater in the large particles than in their small counterparts. This seemingly antithetic finding can be rationalized with the introduction of alternate structures, such as a hinged domain constituted by two adjacent antiparallel alpha-helices, probably at residues 99-143, or distinct conformations dependent upon particle size.