Ohki Izuru, Amida Hirokazu, Yamada Risato, Sugihara Mamoru, Ishigaki Tomoko, Tate Shin-ichi
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0198, Japan.
Biochim Biophys Acta. 2011 Feb;1814(2):345-54. doi: 10.1016/j.bbapap.2010.10.006. Epub 2010 Oct 28.
Lectin-like oxidized low-density lipoprotein (OxLDL) receptor 1 (LOX-1) is the major OxLDL receptor of vascular endothelial cells and is involved in an early step of atherogenesis. LOX-1 exists as a disulfide-linked homodimer on the cell surface, which contains a pair of the ligand-binding domains (CTLD; C-type lectin-like domain). Recent research using living cells has suggested that the clustered state of LOX-1 dimer on the cell is functionally required. These results questioned how LOX-1 exists on the cell to achieve OxLDL binding. In this study, we revealed the functional significance of the clustered organization of the ligand-binding domain of LOX-1 with surface plasmon resonance. Biotinylated CTLD was immobilized on a streptavidin sensor chip to make CTLD clusters on the surface. In this state, the CTLD had high affinity for OxLDL with a dissociation constant (K(D)) in the nanomolar range. This value is comparable to the K(D) measured for LOX-1 on the cell. In contrast, a single homodimeric LOX-1 extracellular domain had lower affinity for OxLDL in the supra-micromolar range of K(D). Monomeric CTLD showed marginal binding to OxLDL. In combination with the analyses on the loss-of-binding mutant W150A, we concluded that the clustered organization of the properly formed homodimeric CTLD is essential for the strong binding of LOX-1 to OxLDL.
凝集素样氧化型低密度脂蛋白（OxLDL）受体1（LOX-1）是血管内皮细胞的主要OxLDL受体，参与动脉粥样硬化发生的早期步骤。LOX-1以二硫键连接的同二聚体形式存在于细胞表面，其中包含一对配体结合结构域（CTLD；C型凝集素样结构域）。最近使用活细胞的研究表明，细胞上LOX-1二聚体的聚集状态在功能上是必需的。这些结果引发了关于LOX-1如何在细胞上存在以实现与OxLDL结合的疑问。在本研究中，我们通过表面等离子体共振揭示了LOX-1配体结合结构域聚集组织的功能意义。将生物素化的CTLD固定在链霉亲和素传感器芯片上，以在表面形成CTLD簇。在这种状态下，CTLD对OxLDL具有高亲和力，解离常数（K(D)）在纳摩尔范围内。该值与在细胞上测量的LOX-1的K(D)相当。相比之下，单个同二聚体LOX-1细胞外结构域对OxLDL的亲和力较低，K(D)在超微摩尔范围内。单体CTLD与OxLDL的结合很微弱。结合对结合缺失突变体W150A的分析，我们得出结论，正确形成的同二聚体CTLD的聚集组织对于LOX-1与OxLDL的强结合至关重要。
