Lookene A, Savonen R, Olivecrona G
Department of Medical Biochemistry and Biophysics, Umeå University, Sweden.
Biochemistry. 1997 Apr 29;36(17):5267-75. doi: 10.1021/bi962699k.
Interaction of different classes of lipoproteins with heparan sulfate, heparin, and lipoprotein lipase was studied by a surface plasmon resonance based technique on a BIAcore. The proteoglycans were covalently attached to sensor chips as previously described [Lookene, A., Chevreuil, O., Ostergaard, P., & Olivecrona, G. (1996) Biochemistry 35, 12155-12163]. Binding of all lipoproteins, except for beta-VLDL, to endothelial heparan sulfate was low. Binding of chylomicrons (from rat lymph) and of human VLDL was much increased by the presence of lipoprotein lipase. With human LDL, binding was low in the absence of lipase or at low lipase concentrations. For efficient binding, 2-4 lipase dimers per LDL particle were necessary, indicating cooperativity in the interaction. In contrast, HDL did not bind under any conditions. Heparin had higher binding capacity for lipoproteins than heparan sulfate. This was due to a higher number of binding sites on the heparin chains. Binding of LDL, VLDL, and chylomicrons to heparan sulfate-covered surfaces, both in the presence and in the absence of lipoprotein lipase, was characterized by high values for association rate constants (10(4)-10(5) M(-1) s(-1)) and low values for dissociation rate constants (10(-4)-10(-5) M(-1) s(-1)). In some experiments, rabbit beta-VLDL were directly immobilized to the sensor chips. Binding of lipoprotein lipase to these surfaces was characterized by a very high association rate constant (10(6) M(-1) s(-1)). The dissociation of triacylglycerol-rich lipoproteins was more rapid with catalytically active lipase than with active site-inhibited lipase. It was also markedly increased in the presence of free heparin, suggesting fast exchange kinetics at the surface. Based on that, we propose that lipoproteins are relatively mobile at heparan sulfate covered surfaces. Our study emphasizes the important role of lipoprotein lipase, or molecules with similar properties (apolipoprotein E, hepatic lipase), as mediators for binding of lipoproteins to proteoglycans. It also demonstrates the great potential for the use of biosensors for studies of lipoprotein interactions.
利用BIAcore表面等离子体共振技术研究了不同类型脂蛋白与硫酸乙酰肝素、肝素及脂蛋白脂肪酶之间的相互作用。蛋白聚糖如前文所述[Lookene, A., Chevreuil, O., Ostergaard, P., & Olivecrona, G. (1996) Biochemistry 35, 12155 - 12163]共价连接到传感器芯片上。除β-VLDL外，所有脂蛋白与内皮硫酸乙酰肝素的结合力较低。脂蛋白脂肪酶的存在显著增加了乳糜微粒（来自大鼠淋巴）和人极低密度脂蛋白（VLDL）的结合。对于人低密度脂蛋白（LDL），在无脂肪酶或脂肪酶浓度较低时结合力较低。为实现有效结合，每个LDL颗粒需要2 - 4个脂肪酶二聚体，这表明相互作用中存在协同性。相比之下，高密度脂蛋白（HDL）在任何条件下均不结合。肝素对脂蛋白的结合能力高于硫酸乙酰肝素。这是由于肝素链上的结合位点数量更多。在有和无脂蛋白脂肪酶的情况下，LDL、VLDL和乳糜微粒与硫酸乙酰肝素覆盖表面的结合，其结合速率常数（10⁴ - 10⁵ M⁻¹ s⁻¹）较高，解离速率常数（10⁻⁴ - 10⁻⁵ M⁻¹ s⁻¹）较低。在一些实验中，兔β-VLDL直接固定在传感器芯片上。脂蛋白脂肪酶与这些表面的结合，其结合速率常数非常高（10⁶ M⁻¹ s⁻¹）。富含三酰甘油的脂蛋白与具有催化活性的脂肪酶的解离比与活性位点被抑制的脂肪酶更快。在游离肝素存在时解离也显著增加，表明表面存在快速交换动力学。基于此，我们提出脂蛋白在硫酸乙酰肝素覆盖的表面相对可移动。我们的研究强调了脂蛋白脂肪酶或具有相似性质的分子（载脂蛋白E、肝脂肪酶）作为脂蛋白与蛋白聚糖结合的介质的重要作用。它还证明了生物传感器在脂蛋白相互作用研究中的巨大应用潜力。
