Kleinman Y, Krul E S, Burnes M, Aronson W, Pfleger B, Schonfeld G
Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110.
J Lipid Res. 1988 Jun;29(6):729-43.
To assess the effects of perturbing the surface of low density lipoprotein (LDL) on the conformation of apoB-100, LDL (d 1.030-1.050 g/ml) isolated from normal subjects were treated with phospholipase A2 (PL-A2) for 0.5 to 15 min. The resulting P-LDL and concurrent control LDL (C-LDL) incubated without PL-A2 were isolated by gel permeation chromatography. Approximately 50% of LDL-phosphatidylcholine was hydrolyzed in 2 min and approximately 85% in 5 min. Lysophosphatidylcholine compounds (LPC) and free fatty acids (FFA) accumulated during lipolysis but most of the LPC and all of FFA could be removed by adding FFA-free albumin to the lipolysis mixtures. Immunoreactivities of P-LDL and C-LDL were evaluated in competitive radioimmunoassays, using a library of anti-human LDL monoclonal antibodies directed against the major regions of apoB-100 (the T4, T3, and T2 thrombin fragments). One epitope defined by monoclonal antibody 465B6C3 and localized near the carboxyl end of the apoB-100 molecule became less immunoreactive (ED 50s increased); three other epitopes on the T2 fragment near the LDL receptor recognition site and four epitopes localized towards the middle (T3) and amino terminal (T4) regions did not change. Altered immunoreactivities were not related to LPC and FFA contents. Thus, the conformation of apoB-100 was selectively altered by phospholipolysis. The interactions of P-LDL with cultured fibroblasts were grossly altered: P-LDL were bound nonspecifically to fibroblasts of both normal and homozygous familial hypercholesterolemic subjects and P-LDL were not degraded. LPC and FFA retained in LDL did not explain these alterations, nor did changes of epitope expression near the LDL receptor recognition site. It is likely that the apoB-100 aberrant cell interaction is due to loss of surface phospholipids and "uncovering" of core lipids that react nonspecifically with cell surface components.
为评估扰动低密度脂蛋白（LDL）表面对载脂蛋白B-100（apoB-100）构象的影响，将从正常受试者分离的LDL（密度为1.030 - 1.050 g/ml）用磷脂酶A2（PL-A2）处理0.5至15分钟。通过凝胶渗透色谱法分离得到的磷脂化LDL（P-LDL）和未用PL-A2孵育的同期对照LDL（C-LDL）。约50%的LDL-磷脂酰胆碱在2分钟内被水解，5分钟内约85%被水解。溶血磷脂酰胆碱化合物（LPC）和游离脂肪酸（FFA）在脂解过程中积累，但通过向脂解混合物中添加无FFA白蛋白可去除大部分LPC和所有FFA。使用针对apoB-100主要区域（T4、T3和T2凝血酶片段）的抗人LDL单克隆抗体库，通过竞争性放射免疫测定评估P-LDL和C-LDL的免疫反应性。由单克隆抗体465B6C3定义并位于apoB-100分子羧基末端附近的一个表位免疫反应性降低（半数有效剂量增加）；LDL受体识别位点附近T2片段上的其他三个表位以及位于中间（T3）和氨基末端（T4）区域的四个表位未发生变化。免疫反应性的改变与LPC和FFA含量无关。因此，apoB-100的构象因磷脂水解而选择性改变。P-LDL与培养的成纤维细胞的相互作用发生了显著改变：P-LDL与正常和纯合子家族性高胆固醇血症受试者的成纤维细胞非特异性结合，且P-LDL未被降解。LDL中保留的LPC和FFA无法解释这些改变，LDL受体识别位点附近表位表达的变化也不能解释。apoB-100异常的细胞相互作用可能是由于表面磷脂的丢失和核心脂质的“暴露”，后者与细胞表面成分发生非特异性反应。
