Department of Chemistry & Biochemistry, California State University Long Beach, Long Beach, CA 90840, USA.
Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, Berkeley, CA 94720, USA.
Biochem Biophys Res Commun. 2014 Jul 18;450(1):124-8. doi: 10.1016/j.bbrc.2014.05.071. Epub 2014 May 24.
Apolipoprotein E3 (apoE3) is an anti-atherogenic apolipoprotein with the ability to exist in lipid-free and lipoprotein-associated states. During atherosclerosis, its function in promoting cholesterol efflux from macrophages via the ATP-binding cassette transporter A1 (ABCA1) takes a prominent role, leading to generation of nascent high density lipoprotein (nHDL) particles. The objective of this study is to understand the conformation adopted by apoE3 in macrophage-generated nHDL using a fluorescence spectroscopic approach involving pyrene. Pyrene-labeled recombinant human apoE3 displayed a robust ability to stimulate ABCA1-mediated cholesterol efflux from cholesterol-loaded J774 macrophages (which do not express apoE), comparable to that elicited by unlabeled apoE3. The nHDL recovered from the conditioned medium revealed the presence of apoE3 by immunoblot analysis. A heterogeneous population of nHDL bearing exogenously added apoE3 was generated with particle size varying from ∼12 to ∼19 nm in diameter, corresponding to molecular mass of ∼450 to ∼700 kDa. The lipid: apoE3 ratio varied from ∼60:1 to 10:1. A significant extent of pyrene excimer emission was noted in nHDL, indicative of spatial proximity between Cys112 on neighboring apoE3 molecules similar to that noted in reconstituted HDL. Cross-linking analysis using Cys-specific cross-linkers revealed the predominant presence of dimers. Taken together the data indicate a double belt arrangement of apoE molecules on nHDL. A similar organization of the C-terminal tail of apoE on nHDL was noted when pyrene-apoEA277C(201-299) was used as the cholesterol acceptor. These studies open up the possibility of using exogenously labeled apoE3 to generate nHDL for structural and conformational analysis.
载脂蛋白 E3（apoE3）是一种抗动脉粥样硬化的载脂蛋白，能够以无脂质和脂蛋白结合的状态存在。在动脉粥样硬化过程中，它通过 ATP 结合盒转运体 A1（ABCA1）促进巨噬细胞中胆固醇流出的功能起着重要作用，导致新生高密度脂蛋白（nHDL）颗粒的产生。本研究旨在使用涉及芘的荧光光谱方法来了解巨噬细胞产生的 nHDL 中 apoE3 采用的构象。芘标记的重组人 apoE3 显示出从负载胆固醇的 J774 巨噬细胞（不表达 apoE）中刺激 ABCA1 介导的胆固醇流出的强大能力，与未标记的 apoE3 相当。免疫印迹分析表明，从条件培养基中回收的 nHDL 存在 apoE3。生成了具有不同大小的异质 nHDL 群体，直径约为 12 至 19nm，分子量约为 450 至 700kDa。脂质与 apoE3 的比例从 60:1 到 10:1 不等。在 nHDL 中观察到显著的芘激基发射，表明相邻 apoE3 分子上 Cys112 之间的空间接近程度与在重组 HDL 中观察到的相似。使用半胱氨酸特异性交联剂进行交联分析表明，主要存在二聚体。总之，数据表明 apoE 分子在 nHDL 上呈双带排列。当使用芘-apoEA277C（201-299）作为胆固醇受体时，nHDL 上的 apoE 尾部 C 端也注意到了类似的组织排列。这些研究为使用外源性标记的 apoE3 生成 nHDL 进行结构和构象分析开辟了可能性。
