Hamilton Ryan T, Asatryan Liana, Nilsen Jon T, Isas Jose M, Gallaher Timothy K, Sawamura Tatsuya, Hsiai Tzung K
Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA.
Arch Biochem Biophys. 2008 Nov 1;479(1):1-14. doi: 10.1016/j.abb.2008.07.026. Epub 2008 Aug 7.
Oxidatively- or enzymatically-modified low-density lipoprotein (LDL) is intimately involved in the initiation and progression of atherosclerosis. The in vivo modified LDL is electro-negative (LDL(-)) and consists of peroxidized lipid and unfolded apoB-100 protein. This study was aimed at establishing specific protein modifications and conformational changes in LDL(-) assessed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and circular dichroism analyses, respectively. The functional significance of these chemical modifications and structural changes were validated with binding and uptake experiments to- and by bovine aortic endothelial cells (BAEC). The plasma LDL(-) fraction showed increased nitrotyrosine and lipid peroxide content as well as a greater cysteine oxidation as compared with native- and total-LDL. LC/MS/MS analyses of LDL(-) revealed specific modifications in the apoB-100 moiety, largely involving nitration of tyrosines in the alpha-helical structures and beta(2) sheet as well as cysteine oxidation to cysteic acid in beta(1) sheet. Circular dichroism analyses showed that the alpha-helical content of LDL(-) was substantially lower ( approximately 25%) than that of native LDL ( approximately 90%); conversely, LDL(-) showed greater content of beta-sheet and random coil structure, in agreement with unfolding of the protein. These results were mimicked by treatment of LDL subfractions with peroxynitrite (ONOO(-)) or SIN-1: similar amino acid modifications as well as conformational changes (loss of alpha-helical structure and gain in beta-sheet structure) were observed. Both LDL(-) and ONOO(-)-treated LDL showed a statistically significant increase in binding and uptake to- and by BAEC compared to native LDL. We further found that most binding and uptake in control-LDL was through LDL-R with minimal oxLDL-R-dependent uptake. ONOO(-)-treated LDL was significantly bound and endocytosed by LOX-1, CD36, and SR-A with minimal contribution from LDL-R. It is suggested that lipid peroxidation and protein nitration may account for the mechanisms leading to apoB-100 protein unfolding and consequential increase in modified LDL binding and uptake to and by endothelial cells that is dependent on oxLDL scavenger receptors.
氧化或酶修饰的低密度脂蛋白（LDL）与动脉粥样硬化的发生和发展密切相关。体内修饰的LDL带负电（LDL(-)），由过氧化脂质和未折叠的载脂蛋白B-100蛋白组成。本研究旨在分别通过液相色谱/串联质谱（LC/MS/MS）和圆二色性分析确定LDL(-)中特定的蛋白质修饰和构象变化。通过与牛主动脉内皮细胞（BAEC）的结合和摄取实验验证了这些化学修饰和结构变化的功能意义。与天然LDL和总LDL相比，血浆LDL(-)部分的硝基酪氨酸和脂质过氧化物含量增加，半胱氨酸氧化程度更高。对LDL(-)的LC/MS/MS分析揭示了载脂蛋白B-100部分的特定修饰，主要包括α-螺旋结构和β(2)折叠中酪氨酸的硝化以及β(1)折叠中半胱氨酸氧化为半胱氨酸。圆二色性分析表明，LDL(-)的α-螺旋含量（约25%）显著低于天然LDL（约90%）；相反，LDL(-)的β-折叠和无规卷曲结构含量更高，这与蛋白质的展开一致。用过氧亚硝酸盐（ONOO(-)）或SIN-1处理LDL亚组分可模拟这些结果：观察到类似的氨基酸修饰以及构象变化（α-螺旋结构丧失和β-折叠结构增加）。与天然LDL相比，LDL(-)和经ONOO(-)处理的LDL与BAEC的结合和摄取在统计学上均显著增加。我们进一步发现，对照LDL中的大多数结合和摄取是通过LDL受体进行的，而oxLDL受体依赖性摄取最少。经ONOO(-)处理的LDL被凝集素样氧化型低密度脂蛋白受体-1（LOX-1）、CD36和清道夫受体A（SR-A）显著结合并内吞，LDL受体的贡献最小。提示脂质过氧化和蛋白质硝化可能是导致载脂蛋白B-100蛋白展开以及修饰LDL与内皮细胞结合和摄取增加的机制，这种增加依赖于oxLDL清除受体。
