Dong J, Peters-Libeu C A, Weisgraber K H, Segelke B W, Rupp B, Capila I, Hernáiz M J, LeBrun L A, Linhardt R J
Gladstone Institute of Cardiovascular Disease, Cardiovascular Research Institute, and Department of Pathology, University of California, San Francisco, California 94941-9100, USA.
Biochemistry. 2001 Mar 6;40(9):2826-34. doi: 10.1021/bi002417n.
Apolipoprotein E (apoE) is an important lipid-transport protein in human plasma and brain. It has three common isoforms (apoE2, apoE3, and apoE4). ApoE is a major genetic risk factor in heart disease and in neurodegenerative disease, including Alzheimer's disease. The interaction of apoE with heparan sulfate proteoglycans plays an important role in lipoprotein remnant uptake and likely in atherogenesis and Alzheimer's disease. Here we report our studies of the interaction of the N-terminal domain of apoE4 (residues 1-191), which contains the major heparin-binding site, with an enzymatically prepared heparin oligosaccharide. Identified by its high affinity for the N-terminal domain of apoE4, this oligosaccharide was determined to be an octasaccharide of the structure DeltaUAp2S(1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNpS6S by nuclear magnetic resonance spectroscopy, capillary electrophoresis, and polyacrylamide gel electrophoresis. Kinetic analysis of the interaction between the N-terminal apoE4 fragment and immobilized heparin by surface plasmon resonance yielded a K(d) of 150 nM. A similar binding constant (K(d) = 140 nM) was observed for the interaction between immobilized N-terminal apoE4 and the octasaccharide. Isothermal titration calorimetry revealed a K(d) of 75 nM for the interaction of the N-terminal apoE fragment and the octasaccharide with a binding stoichiometry of approximately 1:1. Using previous studies and molecular modeling, we propose a binding site for this octasaccharide in a basic residue-rich region of helix 4 of the N-terminal fragment. From the X-ray crystal structure of the N-terminal apoE4, we predicted that binding of the octasaccharide at this site would result in a change in intrinsic fluorescence. This prediction was confirmed experimentally by an observed increase in fluorescence intensity with octasaccharide binding corresponding to a K(d) of approximately 1 microM.
载脂蛋白E（apoE）是人体血浆和大脑中一种重要的脂质转运蛋白。它有三种常见的异构体（apoE2、apoE3和apoE4）。ApoE是心脏病和神经退行性疾病（包括阿尔茨海默病）的主要遗传风险因素。ApoE与硫酸乙酰肝素蛋白聚糖的相互作用在脂蛋白残粒摄取中起重要作用，可能在动脉粥样硬化和阿尔茨海默病中也起作用。在此，我们报告了对apoE4 N端结构域（第1至191位氨基酸残基）与酶法制备的肝素寡糖相互作用的研究。该寡糖因其对apoE4 N端结构域具有高亲和力而被鉴定，通过核磁共振光谱、毛细管电泳和聚丙烯酰胺凝胶电泳确定其为结构为ΔUAp2S(1-->4)-α-D-GlcNpS6S(1-->4)-α-L-IdoAp2S(1-->4)-α-D-GlcNpS6S的八糖。通过表面等离子体共振对apoE4 N端片段与固定化肝素之间的相互作用进行动力学分析，得到解离常数（K(d)）为150 nM。对于固定化的apoE4 N端与八糖之间的相互作用，观察到类似的结合常数（K(d)=140 nM）。等温滴定量热法显示apoE N端片段与八糖相互作用的K(d)为75 nM，结合化学计量比约为1:1。利用先前的研究和分子建模，我们在N端片段螺旋4富含碱性残基的区域提出了该八糖的一个结合位点。根据apoE4 N端的X射线晶体结构，我们预测该位点上八糖的结合会导致固有荧光发生变化。通过观察到八糖结合时荧光强度增加，解离常数约为1 μM，这一预测在实验中得到了证实。
