Kim Kyung-Tae, Choi Han-Ho, Steinmetz Michel O, Maco Bohumil, Kammerer Richard A, Ahn So Young, Kim Hak-Zoo, Lee Gyun Min, Koh Gou Young
Biomedical Research Center and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon.
J Biol Chem. 2005 May 20;280(20):20126-31. doi: 10.1074/jbc.M500292200. Epub 2005 Mar 15.
Angiopoietin-1 (Ang1) is an essential molecule for blood vessel formation; however, little is known about the structure-function relationships of Ang1 with its receptor, Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2'). In this study, we generated several Ang1 and angiopoietin-2 (Ang2) variants to define the role of the superclustering and oligomerization domains of the Ang1 protein. Then we analyzed the molecular structure of the variants with SDS-PAGE and rotary metal-shadowing transmission electron microscopy (RMSTEM) and determined the effects of these variants on the binding and activation of Tie2. Ang1 exists as heterogeneous multimers with basic trimeric, tetrameric, and pentameric oligomers, whereas Ang2 exists as trimeric, tetrameric, and pentameric oligomers. The variant Ang1C265S, consisting of trimers, tetramers, and pentamers without multimeric forms of Ang1, yielded less Tie2 activation than did Ang1, whereas monomeric Ang1 (Ang1/FD), dimeric Ang1 variants (Ang1D2, and Ang1D3), and dimeric and trimeric Ang1 variant (Ang1D1) dramatically lost their ability to bind and activate Tie2. An Ang1 protein in which two cysteines (amino acids 41 and 54) were replaced with serines (Ang1C41S/C54S) formed mostly dimers and trimers that were not able to bind and activate Tie2. In addition, improper creation of a new cysteine in Ang2 (Ang2S263C) dramatically induced Ang2 aggregation without activating Tie2. In conclusion, proper oligomerization of Ang1 having at least four subunits by the intermolecular disulfide linkage involving cysteines 41 and 54 is critical for Tie2 binding and activation. Thus, our data shed a light on the structure-function relationships of Ang1 with Tie2.
血管生成素1（Ang1）是血管形成的必需分子；然而，关于Ang1与其受体Tie2（具有免疫球蛋白和表皮生长因子同源结构域-2'的酪氨酸激酶）的结构-功能关系却知之甚少。在本研究中，我们构建了几种血管生成素1和血管生成素2（Ang2）变体，以确定血管生成素1蛋白超聚集和寡聚结构域的作用。然后，我们用SDS-PAGE和旋转金属阴影透射电子显微镜（RMSTEM）分析了这些变体的分子结构，并确定了这些变体对Tie2结合和激活的影响。血管生成素1以异质性多聚体形式存在，包括基本的三聚体、四聚体和五聚体寡聚体，而血管生成素2以三聚体、四聚体和五聚体寡聚体形式存在。由没有血管生成素1多聚体形式的三聚体、四聚体和五聚体组成的变体血管生成素1C265S，其激活Tie2的能力比血管生成素1弱，而单体血管生成素1（血管生成素1/FD）、二聚体血管生成素1变体（血管生成素1D2和血管生成素1D3）以及二聚体和三聚体血管生成素1变体（血管生成素1D1）显著丧失了结合和激活Tie2的能力。一种将两个半胱氨酸（第41和54位氨基酸）替换为丝氨酸的血管生成素1蛋白（血管生成素1C41S/C54S）主要形成二聚体和三聚体，无法结合和激活Tie2。此外，血管生成素2中一个新半胱氨酸的不当产生（血管生成素2S263C）显著诱导了血管生成素2聚集，但未激活Tie2。总之，通过涉及第41和54位半胱氨酸的分子间二硫键形成至少具有四个亚基的血管生成素1的适当寡聚化对于Tie2的结合和激活至关重要。因此，我们的数据揭示了血管生成素1与Tie2的结构-功能关系。
